Preparation and application of bp / pcn-224 / fe3o4 nanoheterojunction

Abstract

The application discloses a preparation method of Bp / PCN-224 / Fe3O4 nano heterojunction, ultrasonic stripping of black phosphorus is dispersed in DMF to obtain Bp NSs dispersion; zirconium chloride is added into DMF and ultrasonic, and then the Bp NSs dispersion is added dropwise to obtain a first dispersion; meso-tetra(4-carboxyphenyl) porphyrin and benzoic acid are dissolved in DMF and ultrasonic, and then the first dispersion is added and stirred uniformly to obtain a first mixed solution; the first mixed solution is transferred into a reaction container, the obtained product is washed by DMF and then centrifuged to collect, and a Bp / PCN-224 dispersion is obtained; the Bp / PCN-224 dispersion is added dropwise into a DMF dispersion of Fe3O4 to obtain the Bp / PCN-224 / Fe3O4 nano heterojunction. The above steps are adopted to take the Bp NSs nanosheet as a substrate, grow PCN-224 on the surface of the substrate in situ to form the Bp / PCN-224 nano heterojunction, and greatly improve the generation efficiency of active oxygen.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology for tumor diagnosis and treatment, and in particular to the preparation and application of Bp / PCN-224 / Fe3O4 nanoheterostructures. Background Technology

[0002] Chemotherapy is one of the most effective treatments for cancer, and along with surgery and radiotherapy, it is considered one of the three major cancer treatments. However, traditional chemotherapy drugs have toxic side effects on normal cells and poor targeting of tumor sites, making it difficult for them to accumulate. Catalytic therapy uses safe and non-toxic catalysts to replace traditional drugs, achieving specific cancer treatment by generating therapeutic products in situ within tumor tissue and significantly reducing the side effects of cancer treatment. However, the limited catalytic efficiency of a single catalyst, the shielding effect of biological tissues on the energy source, and the complex inhibitory microenvironment of tumor tissues, such as hypoxia and high glutathione (GSH) levels, severely affect the treatment efficiency of traditional catalytic therapy.

[0003] In terms of improving catalytic efficiency, heterostructures have been shown to significantly inhibit excited electron-hole recombination. Developing nanomedicines based on heterostructures will provide new materials for catalytic therapy. By utilizing heterostructured nanomedicines, methods such as PDT and SDT can be initiated, thereby generating ROS or mechanical damage to effectively kill tumor cells.

[0004] However, both SDT and PDT are ROS-mediated treatments, and their efficacy is largely influenced by oxygen supply. The hypoxic environment of the tumor may limit the effectiveness of these two therapies. Compared to traditional tumor treatments, CDT has higher specificity and lower toxicity. Studies have found that only 0.7% of the injected dose of various nanoparticles can accumulate at the tumor site. Therefore, while researching ways to increase drug efficacy, effectively accumulating the drug at the tumor site is also a crucial issue. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing Bp / PCN-224 / Fe3O4 nanoheterojunctions. Using two-dimensional Bp NSs nanosheets as a substrate, PCN-224 is grown in situ on its surface to form Bp / PCN-224 nanoheterojunctions, which can achieve rapid separation of photogenerated carriers, effectively improve electrons and holes, and greatly increase the generation efficiency of reactive oxygen species.

[0006] To achieve the above objectives, this invention provides the preparation of Bp / PCN-224 / Fe3O4 nanoheterojunctions, comprising the following steps:

[0007] S1. Disperse the ground black phosphorus in N-methylpyrrolidone, exfoliate by ultrasonication, centrifuge and collect the supernatant, disperse the supernatant in the first solvent, stir magnetically and wash with DMF, and disperse in DMF to obtain BpNSs dispersion.

[0008] S2. Zirconium chloride is added to DMF and sonicated, then the Bp NSs dispersion from S1 is added dropwise and stirred to obtain the first dispersion;

[0009] S3. Dissolve methyl-tetra(4-carboxyphenyl)porphyrin and benzoic acid in DMF and sonicate. After sonication, add the mixture to the first dispersion in S2 and stir until homogeneous to obtain the first mixed solution.

[0010] S4. Transfer the first mixed solution from S3 to a reaction vessel, fill it with inert gas and seal it. React at 70-100℃ for 10-15h. The product obtained is washed with DMF and collected by centrifugation to obtain Bp / PCN-224 dispersion.

[0011] S5. The Bp / PCN-224 dispersion in S4 was added dropwise to the DMF dispersion of Fe3O4, sonicated, shaken, and collected by centrifugation to obtain the Bp / PCN-224 / Fe3O4 nanoheterojunction.

[0012] Preferably, in S1, the first solvent is a DMF solution of polyvinylpyrrolidone.

[0013] Preferably, in S1, the power of ultrasonic ablation is 800-1000w, and the time is 8-12h.

[0014] The Bp / PCN-224 / Fe3O4 nanoheterojunction prepared by the above method is shown in the figure.

[0015] Applications of the aforementioned Bp / PCN-224 / Fe3O4 nanoheterostructures: Bp / PCN-224 / Fe3O4 nanoheterostructures are used in nanomedicine for tumor diagnosis and treatment.

[0016] Therefore, the beneficial effects of the present invention are as follows:

[0017] 1. Bp / PCN-224 / Fe3O4 achieves highly efficient treatment of deep tumors through the synergistic effects of PDT, SDT and CDT, while reducing the single dose and mitigating toxic side effects, making its advantages more obvious in clinical cancer treatment;

[0018] 2. Bp NSs and PCN-224NPs can form a Bp / PCN-224 nanostructure, which enables rapid separation of charge carriers stimulated by light and sound, improves the problem of easy recombination of electrons and holes, accelerates the flow of electrons in the composite system, and further improves the generation efficiency of ROS.

[0019] 3. Fe ions in Fe3O4 catalyze the decomposition of H2O2 to generate ·OH, thereby achieving CDT. At the same time, it exhibits excellent endogenous GSH consumption capacity, which amplifies the oxidative stress response of tumors and further enhances the therapeutic effect of CDT.

[0020] 4. Under 660nm illumination, PCN-224 can emit near-infrared light, while iron ions in Fe3O4 can endow the nanocomposite system with good T2-weighted MRI imaging capabilities.

[0021] 5. Nanomedicines prepared based on Bp / PCN-224 / Fe3O4 can not only improve the effective utilization efficiency of external excitation energy, but also contain multiple reactive sites, expand substrate selectivity, and significantly improve the effect in tumor treatment.

[0022] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0023] Figure 1 This is a combined atomic force microscope and Raman spectrum of the Bp NSs of this invention;

[0024] Figure 2 This is a combined SEM image of Bp NSs, PCN-224, Fe3O4 and Bp / PCN-224 / Fe3O4 in this invention;

[0025] Figure 3 These are the XRD patterns of Bp NSs, PCN-224, Bp / PCN-224, Fe3O4 and Bp / PCN-224 / Fe3O4 in this invention;

[0026] Figure 4 This is a combined diagram of photocatalysis and sonodynamic characterization in Example 2 of the present invention;

[0027] Figure 5 These are combined nuclear magnetic resonance imaging images of different concentrations of Bp / PCN-224 / Fe3O4 in Example 2 of this invention;

[0028] Figure 6 This is a bar chart of relative fluorescence intensity in Example 3 of the present invention;

[0029] Figure 7 These are cell-killing effect diagrams under different stimulation conditions in Example 3 of the present invention;

[0030] Figure 8 This is a statistical graph of the average fluorescence intensity of changes in intracellular ROS levels under different stimulating conditions.

[0031] Figure 9 This is a statistical graph of the average fluorescence intensity of changes in intracellular O2 levels;

[0032] Figure 10 This is a statistical graph showing changes in intracellular GSH levels;

[0033] Figure 11 This is a schematic flowchart of the preparation method provided by the present invention. Detailed Implementation

[0034] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0035] The present invention will be explained in more detail through the following embodiments. The purpose of disclosing the present invention is to protect all changes and modifications within the scope of the present invention. The present invention is not limited to the following embodiments.

[0036] Example 1

[0037] S1. The ground black phosphorus was dispersed in N-methylpyrrolidone and ultrasonically peeled using the ultrasonic probe of a cell disruptor. Then, it was placed in a constant temperature ultrasonic cleaner for ultrasonic peeling at 4°C for 72 hours. After centrifugation, the supernatant was collected. 10 mg of the supernatant was dispersed in 40 mL of DMF solution containing 20 mg of polyvinylpyrrolidone (PVP). After magnetic stirring for 12 hours, it was washed with DMF and dispersed in 5 mL of DMF to obtain the Bp NSs dispersion.

[0038] S2. Add 20 mg of zirconium chloride to 10 mL of DMF and sonicate for 10 min. Then add 5 mL of Bp NSs dispersion from S1 and stir for 12 h to obtain the first dispersion.

[0039] S3. Dissolve 20 mg of 4-tetra(4-carboxyphenyl)porphyrin TCPP and 0.56 g of benzoic acid in 5 mL of DMF and sonicate for 10 min. After sonication, add the solution to the first dispersion in S2 and stir until homogeneous to obtain the first mixed solution.

[0040] S4. Transfer the first mixed solution from S3 to a 100 mL single-necked flask, purge with inert gas and seal. React at 90 °C for 12 h. The resulting product is washed with DMF and collected by centrifugation to obtain the Bp / PCN-224 dispersion (B-2), the XRD pattern of which is shown below. Figure 2 As shown.

[0041] S5. Fe3O4 powder was added to 20 mL of DMF and ultrasonically dispersed for 1 h to prepare a 1 mM Fe3O4 DMF dispersion. The Bp / PCN-224 dispersion from S4 was added dropwise to the Fe3O4 DMF dispersion, ultrasonicated for 30 min, shaken for 12 h, and centrifuged to collect the Bp / PCN-224 / Fe3O4 nanoheterojunction (BPF).

[0042] Example 2

[0043] Characterization of Bp / PCN-224 / Fe3O4 nanoheterostructures and their constituent monomers

[0044] a. Preparation of Bp NSs

[0045] 0.2 g of black phosphorus was ground and dispersed in N-methylpyrrolidone, then ultrasonically exfoliated using the ultrasonic probe of a cell disruptor at an output power of 900 W for 10 h. The resulting product was placed in a constant-temperature ultrasonic cleaner and sonicated at 4 °C for another 72 h. After sonication, the resulting black phosphorus nanosheet suspension was centrifuged (5000 g, 5 min), and the supernatant was collected. The collected supernatant was then rinsed with an inert gas atmosphere and stored at 4 °C.

[0046] Images observed using an atomic force microscope, such as Figure 1 As shown in a, the thickness of the obtained Bp NSs is approximately 5 nm. Its Raman spectrum is shown below. Figure 1 As shown in b, under 532nm laser excitation at 300-500cm... -1 Three peaks (360.7, 435.7, and 464.1 cm⁻¹) appeared within the range. -1 ) respectively with black phosphorus A 1 g B 2g and A 2 g The peak positions are basically overlapping, which proves that we have successfully prepared black phosphorus nanosheets Bp NSs and no obvious structural transformation occurred during the preparation process.

[0047] b. Preparation of PCN-224

[0048] 20 mg of zirconium chloride, 20 mg of 4-tetra(4-carboxyphenyl)porphyrin (TCPP), and 0.56 g of benzoic acid were dissolved in 20 mL of DMF. The solution was sonicated for 5 min and magnetically stirred for 5 min. The solution was then transferred to a 100 mL single-necked flask and magnetically stirred in an oil bath at 90 °C for 12 h. After cooling to room temperature, the suspension was washed three times each with DMF and acetone by ultrasonic centrifugation. Finally, it was dried overnight in a vacuum oven at 120 °C to obtain PCN-224 powder.

[0049] c. Preparation of Fe3O4

[0050] 0.54 g of ferric chloride and 0.2 g of ferrous chloride were dissolved in 90 mL of oxygen-free water, followed by the addition of 12 mL of ammonia solution and magnetic stirring for 30 min. The mixture was then heated to 80 °C and reacted for 1 h. 100 μL of polydiallyldimethylammonium chloride (PDDA) was added and stirred for 1 h. After cooling to room temperature, the suspension was washed three times each by ultrasonic centrifugation with ultrapure water. Finally, it was dried overnight in a vacuum oven at 60 °C to obtain Fe3O4 powder.

[0051] d. SEM image

[0052] like Figure 2 As shown in a, Bp NSs exhibits a two-dimensional sheet structure with a particle size of approximately 200 nm.

[0053] like Figure 2 As shown in b, the PCN-224 nanoparticles are spherical with a crystal size of approximately 70 nm.

[0054] like Figure 2 As shown in c, the Fe3O4 nanoparticles are spherical with a crystal size of approximately 20 nm.

[0055] like Figure 2 As shown in d, the Bp / PCN-224 / Fe3O4 nanoheterostructure is a sheet with a crystal size of approximately 200 nm.

[0056] e. XRD patterns

[0057] The XRD patterns of Bp NSs, PCN-224, Fe3O4, Bp / PCN-224, and Bp / PCN-224 / Fe3O4 nanoheterostructures are as follows: Figure 3 As shown, by Figure 3 The results show that the characterization peaks of Bp / PCN-224 / Fe3O4 are consistent with the previously synthesized crystal structures of Bp NSs, PCN-224, Fe3O4, and Bp / PCN-224, confirming the successful synthesis of Bp / PCN-224 / Fe3O4 nanoheterojunctions.

[0058] f. Characterization of Bp / PCN-224 / Fe3O4 nanoheterostructures

[0059] The photocurrent effect can be used to evaluate the separation of photogenerated electrons and holes in the photoelectrode; the larger the photocurrent, the higher the separation efficiency. Electrochemical impedance (EIS) can be used to evaluate electron-hole recombination; the smaller the impedance, the lower the recombination efficiency.

[0060] like Figure 4As shown in Figure a, the photocurrent intensity of the Bp / PCN-224 / Fe3O4 nanoheterojunction is almost identical to that of Bp / PCN-224, both higher than that of the single material. This indicates that the electron-hole recombination efficiency of the Bp / PCN-224 / Fe3O4 nanoheterojunction is lower than that of PCN-224, while the photocatalytic ability is improved, indicating that the heterojunction was successfully constructed. By measuring the band gap and valence band of Bp NSs and PCN-224, the conduction band was calculated, and thus the band structure of the Bp / PCN-224 / Fe3O4 nanoheterojunction was obtained, as shown below. Figure 4 The value of 'b' indicates the successful construction of the Z-type heterostructure. Furthermore, the addition of Fe3O4 did not weaken the photocurrent intensity of Bp / PCN-224. Figure 4 (a) and reduced the point impedance value ( Figure 4 c) indicates that ROS generation will be further enhanced under light and ultrasonic stimulation conditions. Electron spin resonance spectroscopy (ESR) further confirms the successful construction of the Z-type heterostructure. Figure 4 (d)

[0061] g. Nuclear magnetic resonance imaging

[0062] To demonstrate the nuclear magnetic resonance (NMR) imaging effect of Bp / PCN-224 / Fe3O4 nanoheterojunctions, Bp / PCN-224 / Fe3O4 nanoheterojunctions were dispersed in 2% agarose gel at concentrations ranging from 0 to 1000 μg / mL. A GE 3.0T NMR spectrometer was used to obtain the following results: Figure 5 a and Figure 5 b. The results show that the Bp / PCN-224 / Fe3O4 nanoheterojunction has a certain degree of magnetism. The T2-weighted image gradually darkens with the increase of Bp / PCN-224 / Fe3O4 nanoheterojunction concentration, indicating that Bp / PCN-224 / Fe3O4 nanoheterojunction can be used as a T2 contrast agent.

[0063] Example 3

[0064] In vitro antitumor effects of Bp / PCN-224 / Fe3O4 nanoheterostructures

[0065] a) Cellular uptake of Bp / PCN-224 / Fe3O4 nanoheterostructures

[0066] Bp / PCN-224 / Fe3O4 nanoheterojunctions were co-incubated with mouse skin melanoma B16F10 cells under both magnetized and non-magnetized conditions (1, 2, 4, 6 h). Fluorescence images were recorded using a confocal microscope (CLSM). The red fluorescence intensified with increasing time. The relative fluorescence intensity change was calculated from the fluorescence images. Figure 6As shown, the group with magnets showed a significant difference compared to the group without magnets, indicating that nanoparticles can be rapidly taken up by cells under the influence of magnets.

[0067] b. Bp / PCN-224 / Fe3O4 nanoheterostructure cell killing

[0068] Cells were first seeded in 96-well plates (approximately 1 × 10⁶ cells per well). 4 (Number of cells) were incubated at 37°C for 12 hours, followed by treatment with the material for 4 hours. Then, the cells were irradiated with a 660nm laser (0.33W / cm²). 2 The samples were subjected to ultrasound treatment (US 1 MHz, 5 min, 50% duty) and then incubated for 20 hours. 20 μL of MTT solution was then added to each well, and incubation continued for 4 hours. Subsequently, the supernatant was removed, and the resulting crystals from each well were dissolved in 200 μL of DMSO. The absorbance of all samples at 490 nm was measured using a spectrophotometer.

[0069] like Figure 7 As shown, cell experiments were randomly divided into 7 groups: control group, H2O2 group (BPF-H2O2), 660nm laser irradiation group (BPF-660), US irradiation group (BPF-US), US irradiation + H2O2 group (BPF-US+H2O2), 660nm laser irradiation + US irradiation group (BPF-660+US), and 660nm laser irradiation + US irradiation + H2O2 group (BPF-660+US+H2O2). When cells were treated with US irradiation + H2O2, the cell viability was significantly lower than that of the H2O2 group and the US irradiation group, indicating that the reaction of Fe ions with H2O2 to produce O2 can enhance the effect of SDT. In addition, due to the synergistic effect of PDT and SDT, the 660nm laser irradiation + US irradiation group also showed strong cytotoxicity. Ultimately, as the Bp / PCN-224 / Fe3O4 nanoheterojunctions and stimulation conditions gradually increase, cell viability gradually decreases. Under external light and ultrasound stimulation, the Bp / PCN-224 / Fe3O4 nanoheterojunctions can generate a large amount of reactive oxygen species (ROS), thereby killing tumor cells.

[0070] c. Verify whether the Bp / PCN-224 / Fe3O4 nanoheterojunction generates ROS.

[0071] To demonstrate that Bp / PCN-224 / Fe3O4 nanoheterostructures can generate ROS in tumors under external stimuli, the ROS content in cells was measured using the DCFH-DA reactive oxygen species probe, and the results were recorded using an inverted fluorescence microscope.

[0072] like Figure 8As shown, the intracellular ROS level gradually increases with the increase of various stimulating conditions, indicating that the Bp / PCN-224 / Fe3O4 nanoheterostructure can effectively generate ROS under 660nm laser and ultrasound stimulation conditions, thereby killing tumors.

[0073] d. Verify whether Bp / PCN-224 / Fe3O4 nanoheterostructures can alleviate tumor hypoxia.

[0074] To demonstrate the alleviating effect of Bp / PCN-224 / Fe3O4 nanoheterostructures on tumor microenvironment hypoxia, [Ru(dpp)3] was used. 2+ The Cl2 oxygen indicator probe was used to measure the oxygen content in cells, and the results were recorded using CLSM.

[0075] Average fluorescence intensity as Figure 9 As shown, the oxygen level inside tumor cells gradually increases over time, indicating that nanomaterials can effectively generate O2 and alleviate hypoxia inside tumors.

[0076] e. Intracellular GSH levels in mouse skin melanoma cells

[0077] The glutathione oxidase-like activity (GSH-Ox) of Bp / PCN-224 / Fe3O4 nanoheterostructures reduces intracellular GSH levels. For example... Figure 10 As shown, the intracellular GSH concentration decreased with the addition of Fe3O4, indicating that BPF has good GSH-Ox activity in cells.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Preparation of Bp / PCN-224 / Fe304 nano-heterojunction, characterized by: The method comprises the following steps: S1, dispersing the ground black phosphorus in N-methyl pyrrolidone, ultrasonic exfoliation, centrifugation and collection of the supernatant, dispersing the supernatant in a first solvent, magnetic stirring and washing with DMF, and then dispersing in DMF to obtain a Bp NSs dispersion; S2, adding zirconium chloride into DMF and ultrasonic, then adding the Bp NSs dispersion in S1 dropwise and stirring to obtain a first dispersion; S3, dissolving meso-tetra(4-carboxyphenyl) porphyrin and benzoic acid in DMF and ultrasonic, then adding the first dispersion in S2 after ultrasonic to obtain a first mixed solution; S4, transferring the first mixed solution in S3 into a reaction container, filling with inert gas and sealing, reacting at 70-100℃ for 10-15 h, then washing the obtained product with DMF, centrifugation and collection to obtain a Bp / PCN-224 dispersion; S5, adding the Bp / PCN-224 dispersion in S4 into a DMF dispersion of Fe3O4, ultrasonic and shaking, then centrifugation and collection to obtain a Bp / PCN-224 / Fe3O4 nano-heterojunction.

2. The preparation of Bp / PCN-224 / Fe304 nano-heterojunction according to claim 1, characterized in that: In S1, the first solvent is a DMF solution of polyvinylpyrrolidone.

3. The preparation of Bp / PCN-224 / Fe304 nano-heterojunction according to claim 1, characterized in that: In S1, the power of ultrasonic exfoliation is 800-1000 w, and the time is 8-12 h.

4. The Bp / PCN-224 / Fe3O4 nano-heterojunction prepared by the method according to any one of claims 1-3.

5. The use of Bp / PCN-224 / Fe304 nano-heterojunction according to claim 4, characterized in that: The Bp / PCN-224 / Fe3O4 nano-heterojunction is applied to the preparation of a nano-drug for tumor diagnosis and treatment.

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