Preparation of zr-based heterojunction sonosensitizer and its application in synergistic tumor therapy

Abstract

The application belongs to the technical field of biological medicine, and discloses a preparation of a Zr-based heterojunction sonosensitizer and a synergistic tumor treatment application thereof, and a preparation process thereof comprises the following steps: H2BDC and ZrOCl2.8H2O are dissolved in DMF, glacial acetic acid is added, and Zr-MOF is obtained after heating, centrifugation and washing; EDTA-FeNa powder is dissolved in deionized water, Fe-CDs are obtained after ultrasonic mixing, annealing, centrifugation and filtration; finally, Zr-MOF, DOX and Fe-CDs are mixed to prepare DOX / Zr-MOF / Fe-CDs. The DOX / Zr-MOF / Fe-CDs nano platform is formed by combining Zr-MOF and Fe-doped carbon dots (Fe-CDs), and has excellent sonodynamic and chemical dynamic performance. The nano platform generates a large amount of reactive oxygen species (ROS) under the action of ultrasound, indirectly consumes glutathione (GSH) through a Fenton reaction, and meanwhile, Zr-MOF serves as a protective layer for degradation behavior, so that the DOX / Zr-MOF / Fe-CDs has controllable degradation behavior. The application solves the problems of low ROS yield and easy consumption of GSH of the existing sonosensitizer, and is suitable for tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the preparation of a Zr-based heterojunction acoustic sensitizer and its synergistic application in tumor treatment. Background Technology

[0002] In recent years, treatment methods utilizing highly cytotoxic reactive oxygen species (ROS) to kill tumor cells based on exogenous stimulation or endogenous chemical reactions have been widely used to improve anti-tumor efficacy, including photodynamic therapy (PDT), sonodynamic therapy (SDT), and chemodynamic therapy (CDT). SDT uses ultrasound to activate sonosensitive agents to generate highly destructive ROS. Since ultrasound can penetrate to depths exceeding 10 cm, this therapy has shown promising efficacy against deep tumors. However, the effectiveness of SDT is hampered by inefficient sonosensitive agents and the complex tumor microenvironment (TME). The ineffectiveness of organic sonosensitive agents in SDT is due to their limited water solubility and photostability. Inorganic sonosensitive agents, represented by TiO2 nanostructures, possess better chemical stability and modifiable band gaps, but the tendency for electrons and holes generated within the structure to recombine severely limits their application prospects in SDT. Therefore, finding a narrow-bandgap, highly efficient sonosensitive agent that inhibits electron-hole recombination is essential to overcoming these obstacles and enhancing SDT.

[0003] Metal-organic frameworks (MOFs) are highly ordered porous crystals composed of metal ion clusters linked by organic binders. Their unique structure and properties make them suitable for applications in catalysis, drug delivery, and other fields. Zr-based UiO-66 is one type of MOF material, possessing outstanding characteristics such as large specific surface area, suitable size for EPR effects, good biocompatibility, and ease of handling, making it ideal for drug delivery applications. However, UiO-66 degrades and releases drugs under neutral conditions, causing strong off-target toxicity. Moreover, it degrades significantly within 4 minutes in acidic TMEs, limiting drug uptake by cells and penetration into deep tissues, ultimately leading to poor therapeutic effects and significant safety hazards to normal tissues. Furthermore, UiO-66 has poor sonodynamic properties and lacks Fenton-like reactivity, making it unsuitable as a good sonosensitizer and Fenton-like agent for SDT and CDT.

[0004] Therefore, it is necessary to explore an excellent multifunctional nanomaterial as a sonosensitizer and Fenton-like agent, which can enhance the sonokinetic and chemokinetic activity of UIO-66 through the construction of heterojunctions, and can also act as a protective layer to regulate the degradation behavior of UIO-66, so that it can only degrade and release drugs in acidic TME, thereby achieving tumor-specific chemotherapy-enhanced SDT and CDT. Summary of the Invention

[0005] The present invention aims to provide a method for preparing a Zr-based heterojunction acoustic sensitizer and its synergistic application in tumor therapy, in order to solve the problems of low ROS yield and easy consumption of ROS by endogenous GSH in current nano-acoustic sensitizers, and to enhance the acoustic dynamic tumor therapy effect through effective regulation of the tumor microenvironment.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a Zr-based heterojunction acoustic sensor includes the following steps: S1. At room temperature, H2BDC and ZrOCl2·8H2O were simultaneously dissolved in DMF, and then glacial acetic acid was added. The solution was then stored in an 80 °C oven for 4 hours, centrifuged at 3000 r / min for 5 min, washed with DMF and ethanol, and finally dried under vacuum to obtain solid Zr-MOF powder. S2. Dissolve EDTA-FeNa powder in deionized water and mix uniformly under US for 10 min. Grind the dried product into fine powder, load the powder into a quartz boat, and anneal in a tube furnace at 350 ℃ at a temperature of 5 ℃ / min under N2 atmosphere for 2 hours. Add the obtained product to anhydrous methanol and stir for 20 min. Centrifuge at 11000 r / min for 20 min. Filter the supernatant through a 0.22 μm oil film to remove carbon flakes, then remove the methanol solvent by rotation. Add DI water to convert to an aqueous phase, and then filter through a 0.22 μm oil film to obtain purified Fe-CDs. S3. Dissolve Zr-MOF and DOX in deionized water, stir for 24 h, centrifuge, wash with deionized water to remove excess DOX, suspend in deionized water, add Fe-CDs solution and stir for 24 h, finally centrifuge the solution, wash with deionized water to remove excess Fe-CDs, and obtain DOX / Zr-MOF / Fe-CDs.

[0007] Further, in step S1, 498.4 mg H2BDC and 147.78 mg ZrOCl2·8H2O are simultaneously dissolved in 40 mL DMF, and then 18 mL glacial acetic acid is added.

[0008] Further, in step S2, 2 g of EDTA-FeNa powder is dissolved in 10 mL of deionized water and mixed, 1 g of powder is loaded into a quartz boat, and the annealed product is added to 80 mL of anhydrous methanol. 30 mL of DI water is added to convert it into an aqueous phase.

[0009] Further, in step S3, 30 mg Zr-MOF and 3 mg DOX are dissolved in 60 mL of deionized water. After removing excess DOX, the solution is suspended in 40 mL of deionized water and then 20 mL of Fe-CDs solution at 1.5 mg / mL is added.

[0010] Furthermore, the washing process in steps S1-S3 needs to be performed three times.

[0011] A method for preparing a Zr-based heterojunction sonosensitive agent yields a DOX / Zr-MOF / Fe-CDs product, which can be used in sonodynamic therapy and chemodynamic therapy to enhance antitumor efficacy.

[0012] A method for preparing a Zr-based heterojunction sonosensitive agent yields a DOX / Zr-MOF / Fe-CDs product, which is used as a sonosensitive agent and Fenton-like agent in sonodynamic and chemodynamic therapy.

[0013] The principle and beneficial effects of the technical solution: The product designed and prepared by this invention combines Fe-doped carbon dots (Fe-CDs) with excellent chemical and acoustic kinetic activity and Zr-MOF with easy degradation characteristics. In addition to its degradability, the suitable size of about 200 nm and large specific surface area endow Zr-MOF with excellent drug loading capacity, making the DOX loading rate as high as about 30%. Furthermore, Fe-CDs are loaded on the Zr-MOF surface to form a DOX / Zr-MOF / Fe-CDs nanoplatform. This nanoplatform has the following advantages for clinical translation: 1. The prepared DOX / Zr-MOF / Fe-CDs nanoplatform can serve as a heterojunction nanoplatform, which exhibits enhanced acoustic and chemical kinetic activity by inhibiting hole recombination and accelerating carrier transfer processes. 2. Fe-CDs can act as a protective layer controlling the degradation behavior of Zr-MOF, enabling DOX / Zr-MOF / Fe-CDs to exhibit controllable degradation behavior. While they cannot degrade under normal physiological conditions, after completing heterojunction-mediated enhanced SDT, they can specifically degrade and release Fe-CDs and DOX in response to acidic TME. 3. The loading of Fe-CDs also endows the heterojunction with GSH depletion capacity, thereby achieving a cascade amplification of ROS generation. 4. Tumor-specific DOX release can induce DNA damage and enhance chemotherapy. Based on these characteristics, by enhancing SDT and CDT with tumor-specific chemotherapy, satisfactory therapeutic effects of complete tumor eradication can be achieved. Attached Figure Description

[0014] Figure 1Images of DOX / Zr-MOF / Fe-CDs under TEM and HRTEM are shown. In the figure, a) is the TEM image of DOX / Zr-MOF / Fe-CDs, and b) is the HRTEM image of DOX / Zr-MOF / Fe-CDs. Figure 2 The figure shows the test results of Example 2, where a) represents the DOX / Zr-MOF / Fe-CDs. 1 O2 generation rate test; b) Comparison of generation rates between Zr-MOF and DOX / Zr-MOF / Fe-CDs; c) Comparison of ESR spectra between Zr-MOF and DOX / Zr-MOF / Fe-CDs; Figure 3 The figures show the test results for Example 2. In the figures, a) shows the •OH generation rate test of DOX / Zr-MOF / Fe-CDs; b) shows the comparison of the •OH generation rates of Zr-MOF and DOX / Zr-MOF / Fe-CDs; c) shows the comparison of the ESR spectra of Zr-MOF and DOX / Zr-MOF / Fe-CDs; and d) shows the performance test of DOX / Zr-MOF / Fe-CDs in consuming GSH. Figure 4 The image shows the test results for Example 3. Figure a) is a confocal image of reactive oxygen species generated by Zr-MOF and DOX / Zr-MOF / Fe-CDs; figures b and c) are images of DOX / Zr-MOF / Fe-CDs irradiated with US (50 kHz, 3.0 W cm⁻¹). -2 Comparison of cell survival rates (5 min) or without irradiation; Figure 5 The figure shows the test results of Example 4. In the figure, a) is the change in tumor volume after intravenous injection of DOX / Zr-MOF / Fe-CDs; b) is the survival time of mice after treatment; and c) is the change in weight of mice during tumor treatment. Detailed Implementation

[0015] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments: Example 1 A method for preparing heterojunction acoustic sensitizers (DOX / Zr-MOF / Fe-CDs) includes the following steps: 1. At room temperature, 498.4 mg H2BDC and 147.78 mg ZrOCl2·8H2O were simultaneously dissolved in 40 mL DMF, and then 18 mL glacial acetic acid was added. The solution was then stored in an 80 °C oven for 4 hours, centrifuged at 3000 r / min for 5 min, washed three times with DMF and ethanol, and finally dried under vacuum to obtain solid Zr-MOF powder.

[0016] 2. Dissolve 2 g of EDTA-FeNa powder in 10 mL of deionized water and mix evenly under US conditions for 10 min. Grind the dried product into a fine powder. Place 1 g of the powder into a quartz boat and anneal it in a tube furnace at 350 ℃ with a temperature of 5 ℃ / min under N2 atmosphere for 2 hours. Add the obtained product to 80 mL of anhydrous methanol and stir for 20 min. Centrifuge at 11000 r / min for 20 min. Filter the supernatant through a 0.22 μm oil film to remove carbon flakes. Then remove the methanol solvent by rotation. Add 30 mL of DI water to convert to an aqueous phase. Filter through a 0.22 μm oil film to obtain purified Fe-CDs. 3. Dissolve 30 mg Zr-MOF and 3 mg DOX in 60 mL of deionized water, stir for 24 h, centrifuge, wash three times with deionized water to remove excess DOX, suspend in 40 mL of deionized water, add 20 mL of 1.5 mg / mL Fe-CDs solution, stir for 24 h, centrifuge the solution, wash three times with deionized water to remove excess Fe-CDs, and obtain DOX / Zr-MOF / Fe-CDs.

[0017] like Figure 1 The images show TEM and HRTEM images of the prepared DOX / Zr-MOF / Fe-CDs. It can be seen from the images that the particle size distribution of DOX / Zr-MOF / Fe-CDs is 20 nm, and under high magnification electron microscopy, it has obvious lattice stripes.

[0018] Example 2 Acoustodynamic, chemodynamic, and GSH consumption performance tests of DOX / Zr-MOF / Fe-CDs sonosensitive agents: (a) The DOX / Zr-MOF / Fe-CDs acoustic sensor prepared in this invention can generate a large amount of singlet oxygen under low-intensity ultrasound. 1 O2), by using 1,3-diphenylisobenzofuran (DPBF) as 1 O2 probe to detect DOX / Zr-MOF / Fe-CDs sonosensitive agent under ultrasonic irradiation 1 O2 generation efficiency was used to evaluate its acoustic and dynamic performance.

[0019] (b) The DOX / Zr-MOF / Fe-CDs sonosensitive agent prepared in this invention can undergo a Fenton reaction under acidic conditions to generate a large number of hydroxyl radicals (•OH). The chemical kinetic properties of the DOX / Zr-MOF / Fe-CDs sonosensitive agent are evaluated by using 3,3',5,5'-tetramethylbenzidine (TMB) as a •OH probe.

[0020] (c) The DOX / Zr-MOF / Fe-CDs acoustic sensor prepared in this invention can consume GSH. The ability of the DOX / Zr-MOF / Fe-CDs acoustic sensor to consume GSH was evaluated by using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a GSH probe.

[0021] Test results are as follows Figure 2 , Figure 3 As shown, Figure 2 a) is DOX / Zr-MOF / Fe-CDs 1 O2 generation rate test; b) Comparison of generation rates between Zr-MOF and DOX / Zr-MOF / Fe-CDs; c) Comparison of ESR spectra of Zr-MOF and DOX / Zr-MOF / Fe-CDs. The figures show that the acoustic-dynamic performance of DOX / Zr-MOF / Fe-CDs is significantly better than that of Zr-MOF nanoparticles.

[0022] Figure 3 Figure a) shows the •OH generation rate test of DOX / Zr-MOF / Fe-CDs; b) compares the •OH generation rates of Zr-MOF and DOX / Zr-MOF / Fe-CDs; c) compares the ESR spectra of Zr-MOF and DOX / Zr-MOF / Fe-CDs; d) tests the GSH consumption performance of DOX / Zr-MOF / Fe-CDs. The figures show that the chemical kinetic performance of DOX / Zr-MOF / Fe-CDs is significantly better than that of Zr-MOF nanoparticles; furthermore, DOX / Zr-MOF / Fe-CDs can efficiently consume GSH.

[0023] Example 3 In vitro sonodynamic and chemodynamic therapy with DOX / Zr-MOF / Fe-CDs sonosensitive agents: This embodiment uses the MTT assay to detect cell viability after DOX / Zr-MOF / Fe-CDs US irradiation treatment. Mouse colorectal cancer cells (CT26) were seeded into 96-well plates at a density of 5000 cells per well and cultured for 24 hours. Then, different concentrations (0, 40, 80, 120, 160, 200 μg / mL) of DOX / Zr-MOF / Fe-CDs were added and cultured for 4 hours. The cells were then irradiated with US (50 kHz, 3.0 W cm⁻¹). -2 Irradiation was performed for 5 minutes, and then the efficacy of extracorporeal sonodynamic therapy with DOX / Zr-MOF / Fe-CDs was tested using a standard MTT assay.

[0024] Test results are as follows Figure 4 As shown, Figure 4Image a) shows a confocal image of reactive oxygen species generated by Zr-MOF and DOX / Zr-MOF / Fe-CDs; images b and c) show DOX / Zr-MOF / Fe-CDs irradiated with US (50 kHz, 3.0 W cm⁻¹). -2 The figure shows a comparison of cell survival rates under US irradiation (5 min) or without irradiation. It can be seen from the figure that DOX / Zr-MOF / Fe-CDs can generate a large amount of reactive oxygen species under US irradiation; under US irradiation, DOX / Zr-MOF / Fe-CDs can completely kill tumor cells.

[0025] Real-time Example 4 In vivo sonodynamic and chemodynamic therapy with DOX / Zr-MOF / Fe-CDs sonosensitizers: In this embodiment, 100 μL (2 million cells) of mouse colorectal cancer cells (CT26) were subcutaneously implanted in the left axilla of 3-5 week old female mice. The tumors were allowed to grow to 100 mm². 3 The mice were divided into 6 groups (n=5 per group): (1) saline, (2) US irradiation alone (50 kHz, 3.0 W cm⁻¹). -2 (3) Zr-MOF (200 μg / mL) + US, (4) Fe-CDs (200 μg / mL) + US irradiation (50 kHz, 3.0 W cm-2, 5 min), (5) DOX / Zr-MOF / Fe-CDs (200 μg / mL), (6) DOX / Zr-MOF / Fe-CDs (200 μg / mL) + US irradiation (50 kHz, 3.0 W cm-2, 5 min). The tumor size was measured every other day and the weight of the mice was recorded daily to evaluate the in vivo sonodynamic therapeutic efficiency of DOX / Zr-MOF / Fe-CDs sonosensitive agents.

[0026] Test results are as follows Figure 5 As shown, Figure 5 In Figure 1, a) shows the change in tumor volume after intravenous injection of DOX / Zr-MOF / Fe-CDs; b) shows the survival time of mice after treatment; and c) shows the change in body weight of mice during tumor treatment. As can be seen from the figures, compared to Zr-MOF alone, DOX / Zr-MOF / Fe-CDs exhibit the best tumor treatment effect under US irradiation, completely inhibiting tumor growth; DOX / Zr-MOF / Fe-CDs can prolong the survival time of mice; and DOX / Zr-MOF / Fe-CDs did not show significant long-term in vivo toxicity.

[0027] The above descriptions are merely embodiments of the present invention, and common knowledge regarding specific technical solutions or characteristics is not elaborated upon here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing a Zr-based heterojunction acoustic sensor, characterized in that, Includes the following steps: S1. At room temperature, H2BDC and ZrOCl2·8H2O were simultaneously dissolved in DMF, and then glacial acetic acid was added. The solution was then stored in an 80°C oven for 4 hours, centrifuged at 3000 r / min for 5 min, washed with DMF and ethanol, and finally dried under vacuum to obtain solid Zr-MOF powder. S2. Dissolve EDTA-FeNa powder in deionized water and mix uniformly under US for 10 min. Grind the dried product into a fine powder and load the powder into a quartz boat. Anneal the powder in a tube furnace at 350℃ and 5℃ / min under N2 atmosphere for 2 hours. Add the obtained product to anhydrous methanol and stir for 20 min. Centrifuge at 11000 r / min for 20 min. Filter the supernatant through a 0.22 μm oil film to remove carbon flakes. Then remove the methanol solvent by rotation. Add DI water to convert to an aqueous phase. Filter through a 0.22 μm oil film to obtain purified Fe-CDs. S3. Dissolve Zr-MOF and DOX in deionized water, stir for 24 h, centrifuge, wash with deionized water to remove excess DOX, suspend in deionized water, add Fe-CDs solution and stir for 24 h, finally centrifuge the solution, wash with deionized water to remove excess Fe-CDs, and obtain DOX / Zr-MOF / Fe-CDs.

2. The method for preparing a Zr-based heterojunction acoustic sensor according to claim 1, characterized in that: In step S1, 498.4 mg H2BDC and 147.78 mg ZrOCl2·8H2O are simultaneously dissolved in 40 mL DMF, and then 18 mL of glacial acetic acid is added.

3. The method for preparing a Zr-based heterojunction acoustic sensor according to claim 1, characterized in that: In step S2, 2 g of EDTA-FeNa powder is dissolved in 10 mL of deionized water and mixed. 1 g of powder is loaded into a quartz boat. The annealed product is added to 80 mL of anhydrous methanol. 30 mL of DI water is added to convert it into an aqueous phase.

4. The method for preparing a Zr-based heterojunction acoustic sensor according to claim 1, characterized in that: In step S3, 30 mg Zr-MOF and 3 mg DOX are dissolved in 60 mL of deionized water. After removing excess DOX, the solution is suspended in 40 mL of deionized water and then 20 mL of Fe-CDs solution at 1.5 mg / mL is added.

5. The method for preparing a Zr-based heterojunction acoustic sensor according to claim 1, characterized in that: The washing process in steps S1-S3 needs to be performed three times.

6. The DOX / Zr-MOF / Fe-CDs product prepared by the method for preparing a Zr-based heterojunction acoustic sensor according to any one of claims 1-5, characterized in that, This product is used in sonodynamic therapy and chemodynamic therapy to improve anti-tumor efficacy.

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