Radiotherapy sensitization microneedle and application thereof

Abstract

The invention belongs to the technical field of drug system delivery, and particularly relates to a radiotherapy sensitization microneedle and application thereof. The invention relates to a design of a mitochondrial targeting photosensitive microneedle system and an application of the mitochondrial targeting photosensitive microneedle system in melanoma radiosensitization radiotherapy. According to the invention, an intelligent delivery platform of the microneedles (MNs) is fused with mitochondrial targeted photodynamic therapy, and a new normal form is developed for radiosensitization therapy of melanoma. According to the invention, a soluble hyaluronic acid (HA) microneedle array is adopted, and a mitochondrial targeting photosensitizer is loaded. After penetrating through the cuticle, the microneedle is rapidly dissolved in the corium layer, the photosensitizer is released, and mitochondrial targeted enrichment is achieved. Then, under the condition of 660 nm illumination, the photosensitizer generates active oxygen in a mitochondrial matrix to directly destroy the mitochondrial structure and function, so that the mitochondrial ROS storm is induced, and the tumor radiotherapy sensitivity is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug system delivery, and in particular relates to a radiotherapy sensitization microneedle and an application thereof. Background Art

[0002] Melanoma is a highly invasive skin malignancy. Its treatment system has shifted from traditional cytotoxic drugs to precision therapy based on immune checkpoint blockade (typically represented by CTLA-4 / PD-1 combination therapy).

[0003] In the comprehensive treatment system of melanoma, radiotherapy still has irreplaceable clinical value. Its core mechanism of action relies on ionizing radiation-induced DNA double-strand breaks (DSBs) and reactive oxygen species (ROS)-mediated oxidative stress damage. However, melanoma cells significantly reduce radiosensitivity through a dual defense mechanism: on the one hand, ROS damage is neutralized by overexpression of antioxidant systems such as glutathione (GSH), and on the other hand, efficient DNA repair pathways such as non-homologous end joining (NHEJ) are activated. Although ultra-high-dose radiotherapy is used clinically to overcome radiation resistance, it inevitably causes irreversible damage to adjacent normal tissues, severely limiting its clinical application value. Therefore, the development of new technologies that can enhance tumor radiosensitivity while reducing treatment toxicity has become a key breakthrough in this field.

[0004] Existing delivery systems have significant limitations in the targeted delivery of radiosensitizers: traditional routes of administration (oral / intravenous injection) are limited by the first-pass effect and rapid liver / kidney metabolism, resulting in less than 5%-10% of the conventional dose of drug accumulation in the tumor site, and toxic reactions such as bone marrow suppression caused by systemic exposure have become the main obstacle to dose escalation. Although delivery systems based on liposomes or polymer nanoparticles achieve passive targeting through enhanced permeability and retention effect (EPR), their application is limited by three major technical bottlenecks: ① The blood half-life is generally <4 hours, making it difficult to maintain an effective therapeutic concentration; ② The rapid clearance mechanism of the reticuloendothelial system significantly reduces the delivery efficiency; ③ The immunogenicity of the carrier material may induce anti-carrier antibodies, resulting in accelerated blood clearance during repeated administration.

[0005] Therefore, the development of new sensitization technologies and their targeted delivery systems provides an innovative solution for breaking through the bottleneck of melanoma radiotherapy efficacy, and has important clinical significance for achieving precise dose control and synergistically improving the therapeutic window.

[0006] Photodynamic therapy is an emerging local treatment method for tumors. It generates ROS (such as singlet oxygen, 1 O 2) selectively oxidatively damages tumor tissues, and has the advantages of high temporal and spatial precision, low toxic side effects, and no drug resistance. Photodynamic therapy can be used independently to treat superficial tumors such as skin cancer, and can also be used in conjunction with conventional treatments (such as radiotherapy) to treat tumors such as esophageal cancer and breast cancer. It is worth noting that photodynamic therapy consumes GSH in tumor cells by releasing ROS, inhibits the antioxidant defense system, and enhances radiotherapy sensitivity. In recent years, mitochondrial-targeted photosensitizers have become the research focus in the field of photodynamic therapy. They can effectively alleviate the limitations of the tumor hypoxic microenvironment on photodynamic therapy by inhibiting the oxidative phosphorylation process to reduce oxygen consumption, and provide a new approach for photodynamic therapy of hypoxic tumors. Other studies have shown that mitochondrial-targeted photosensitizers can inhibit protein phosphorylation, block pro-survival signaling pathways, and induce mitochondrial-derived apoptosis, thereby improving tumor radiotherapy sensitivity. Therefore, the development of mitochondrial-targeted photosensitizers and their precise delivery systems, and the regulation of tumor radiotherapy resistance mechanisms through mitochondrial-targeted photodynamic therapy provide new strategies for overcoming tumor radiotherapy resistance.

[0007] In addition, microneedle (MN) is a new transdermal drug delivery system that has revolutionized drug delivery by enhancing drug transdermal permeability. This technology can efficiently deliver compounds of different molecular weights, such as chemotherapy drugs, peptides, proteins, deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and vaccines. This broad-spectrum delivery capability gives it unique advantages in the treatment of melanoma: (1) Compared with traditional oral or intravenous administration, microneedle administration can accurately deliver drugs to deep melanoma lesions, significantly improving targeting and efficacy; (2) Compared with traditional monotherapy, microneedle administration can achieve multimodal drug combination and responsive drug release, thereby optimizing efficacy and reducing the risk of drug resistance; (3) Compared with traditional chemotherapy or targeted therapy, microneedle administration can minimize the spread of drugs to healthy tissues and organs, thereby reducing side effects and prolonging patient survival; (4) Compared with subcutaneous injection, microneedle administration can achieve better efficacy at a lower dose by concentrating drugs at the tumor site, and its painless nature significantly improves patient compliance. These advantages make it possible for microneedle technology combined with melanoma therapy to be put into clinical application in the future. Summary of the invention

[0008] The shortcomings of the existing technology are as follows: (1) Currently, radiotherapy alone cannot distinguish between normal tissue and tumor tissue, which may cause acute damage or chronic complications to adjacent organs (such as the lungs, heart, and intestines). In addition, the absorption efficiency of tumor tissue for radiation energy is generally low, making it difficult for ROS generation to reach the ideal threshold during treatment.

[0009] (2) The current drug delivery system has shortcomings: on the one hand, since the drug circulates in the body, it is degraded before reaching the lesion, or diffuses into healthy tissues, limiting the therapeutic effect; on the other hand, the dosage is large and it cannot be enriched at the tumor site.

[0010] In order to solve the above-mentioned technical problems, this application provides the following technical solutions: In view of the above-mentioned problems and defects, the present invention relates to the design of a mitochondrial targeted photosensitizer microneedle system and its application in radiosensitization radiotherapy for melanoma. This invention integrates the intelligent delivery platform of microneedles (MNs) with mitochondrial targeted photodynamic therapy, opening up a new paradigm for radiosensitization therapy of melanoma. The present invention adopts a soluble hyaluronic acid microneedle array to load mitochondrial targeted photosensitizers. After the microneedles penetrate the stratum corneum, they quickly dissolve in the dermis, release the photosensitizer and achieve mitochondrial targeted enrichment. Subsequently, under 660 nm light conditions, the photosensitizer produces reactive oxygen in the mitochondrial matrix, directly destroying the mitochondrial structure and function, thereby inducing a mitochondrial ROS storm, thereby improving the sensitivity of tumor radiotherapy.

[0011] The present invention provides a radiotherapy sensitization microneedle, which is obtained by mixing m-BDP and hyaluronic acid and then drying; The structural formula of the m-BDP is as follows: ; wherein n is selected from any integer between 1 and 20, X is a bromine atom or an iodine atom, R 1 and R 2 One selected from the group consisting of methyl, ethyl, propyl and butyl.

[0012] Preferably, the preparation method of the m-BDP is as follows: S11: Under nitrogen protection, triethylene glycol monomethyl ether, p-toluenesulfonyl chloride and triethylamine were added to organic solvent I and reacted in an ice bath for 2 hours to purify the intermediate product 1; the chemical formula of the intermediate product 1 is as follows: ; S12: Under nitrogen protection, the intermediate product 1, 3,4-dihydroxybenzaldehyde and potassium carbonate are added to an organic solvent II and heated at 85-95° C. for reaction for 44-52 hours to purify the intermediate product 2; the chemical formula of the intermediate product 2 is as follows: ; S13: The intermediate product 2, 2,4-dimethylpyrrole, catalyst and 2,3-dichloro-5,6-dicyanobenzoquinone are dissolved in an organic solvent III, and triethylamine and boron trifluoride ether are added after reacting at room temperature (25±5°C) for 10-14 hours, and dilute hydrochloric acid is added after reacting in an ice-water bath for 8-16 hours to purify the intermediate product 3; the chemical formula of the intermediate product 3 is as follows: ; S14: Under nitrogen protection, the intermediate product 3 and N-iodosuccinimide are added to an organic solvent IV for reaction at room temperature for 3-5 hours, and the intermediate product 4 is purified; the chemical formula of the intermediate product 4 is as follows: ; S15: Under nitrogen protection, the intermediate product 4, pyridine-4-carboxaldehyde and piperidine acetate are added to an organic solvent V at 55-65° C. and heated to react for 0.8-1.2 hours, and the intermediate product 5 is purified; the chemical formula of the intermediate product 5 is as follows: ; S16: Under nitrogen protection, the intermediate product 5 and methyl iodide are added to an organic solvent VI and heated at 45-55° C. for reaction for 8-12 hours, and ether is added for purification to obtain the m-BDP.

[0013] Furthermore, the organic solvent I, organic solvent II, organic solvent III, organic solvent IV, organic solvent V and organic solvent VI are independently selected from N,N-dimethylformamide solution (DMF), acetonitrile (CH 3 CN), tetrahydrofuran (THF), dichloromethane (DCM), or ethanol.

[0014] Furthermore, in steps S11, S12, S13 and S15, the purification method is extraction followed by column chromatography.

[0015] Furthermore, in the step S14, the purification method is column chromatography; in the step S16, the ether purification method is to add ether for 8-16 hours and then filter.

[0016] Specifically, the column chromatography uses silica, and the eluent is selected from two of dichloromethane, methanol and petroleum ether.

[0017] Furthermore, the catalyst is selected from trifluoroacetic acid.

[0018] Furthermore, in the step S11, the molar ratio of triethylene glycol monomethyl ether and p-toluenesulfonyl chloride is 1:1-3; the mass of triethylamine is twice the mass of p-toluenesulfonyl chloride; in the step S12, the molar ratio of the intermediate product 1, 3,4-dihydroxybenzaldehyde and potassium carbonate is 1:2-4:6; in the step S13, the molar ratio of the intermediate product 2, 2,4-dimethylpyrrole and 2,3-dichloro-5,6-dicyanobenzoquinone is 1:2:2-6; the mass of boron trifluoride ether is 100 times the mass of 2,4-dimethylpyrrole.

[0019] Furthermore, in the step S15, the molar ratio of the intermediate product 4, pyridine-4-carboxaldehyde and piperidine acetate is 1:10-20:10-20; in the step S16, the molar ratio of the intermediate product 5 and methyl iodide is 1:10; and the mass of the ether is 50 times that of methyl iodide.

[0020] The present invention also provides the use of the radiotherapy sensitization microneedle in local tumor treatment, and after the radiotherapy sensitization microneedle is administered, a light source is used to irradiate the tumor site.

[0021] Preferably, the light source is an LED lamp with a wavelength of 660 nm and a power of 50 mW cm -2 The irradiation time is 20-40 minutes.

[0022] The technical solution of the present invention has the following advantages compared with the prior art: The present invention provides a simple synthesis method of a radiosensitizing compound, and applies the compound to growth inhibition and radiosensitization of mouse melanoma B16F10 cells, which can fully exert the radiosensitization effect while overcoming the limitations of the prior art, and the synthetic preparation route is simple, and has certain transformation prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The synthetic route of the radiosensitizer m-BDP is shown in FIG. 1 , wherein n is 1 to 20; X is a bromine atom or an iodine atom; R 1 and R 2 They can be methyl or ethyl respectively.

[0024] Figure 2 is the radiosensitizer m-BDP in Example 1 1 HNMR spectrum; m-BDP with n = 3; X is iodine atom; R 1 and R 2 Take methyl as an example.

[0025] Figure 3 This is a diagram of the mitochondrial colocalization of the compound m-BDP in Example 1, where the scale bar is 20 μm; a represents the situation of "Hoechst 33342", b represents the situation of "Mito Tracker", c represents the situation of "m-BDP", and d represents the situation of "combining the three pathways of mitochondrial fuel, nuclear dye and m-BDP together".

[0026] Figure 4 This is the γ-H2AX immunofluorescence staining image of the compound m-BDP in Example 1, wherein the scale bar is 10 μm.

[0027] Figure 5These are the single needle morphology characterization diagram and needle arrangement morphology characterization diagram of the MN-m-BDP in Example 6, where the scale of A is 200 μm; the scale of B is 400 μm.

[0028] Figure 6 This is a diagram of the MN-m-BDP tumor inhibition experiment in Example 6. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0030] The specific steps for synthesizing the target molecule compound in this specific embodiment are as follows: The reaction solvents are N,N-dimethylformamide solution (DMF), acetonitrile (CH 3 CN), tetrahydrofuran (THF), dichloromethane (DCM), ethanol (CH 3 CH 2 OH).

[0031] Embodiment 1:

[0032] Triethylene glycol monomethyl ether and p-toluenesulfonyl chloride were added to a reaction vessel in a molar ratio of 1:1, and dichloromethane (10 times the weight of p-toluenesulfonyl chloride) was added as a solvent. Then, triethylamine (2 times the weight of p-toluenesulfonyl chloride) was added, and the mixture was stirred and reacted for 2 hours under an ice bath and nitrogen protection. After the reaction was completed, the mixture was extracted and column chromatography (SiO 2 ; eluent: dichloromethane / petroleum ether), to obtain a white solid product, compound 1, with a yield of 64%.

[0033] Compound 1, 3,4-dihydroxybenzaldehyde and potassium carbonate were added to a reaction vessel in a molar ratio of 1:2:6, and then N,N-dimethylformamide (100 times the weight of 3,4-dihydroxybenzaldehyde) was added as a solvent, and the reaction was carried out at 90°C under the protection of nitrogen for 48 hours. After the reaction was completed, the mixture was extracted and column chromatography (SiO 2 ; eluent: dichloromethane / petroleum ether), to obtain light yellow oily liquid compound 2 with a yield of 70%.

[0034] Compound 2 and 2,4-dimethylpyrrole were added to a reaction vessel in a molar ratio of 1:2, and then tetrahydrofuran (100 times the weight of 2,4-dimethylpyrrole) as a solvent and trifluoroacetic acid (10 times the weight of 2,4-dimethylpyrrole) as a catalyst were added.

[0035] Subsequently, 2,3-dichloro-5,6-dicyanobenzoquinone in a molar ratio of 1:2.2 to 2,4-dimethylpyrrole and dissolved in tetrahydrofuran (10 times the weight of 2,4-dimethylpyrrole) was added to the reaction solution, and the mixture was stirred at room temperature for 12 hours.

[0036] Finally, triethylamine in an amount 100 times the weight of 2,4-dimethylpyrrole was added, and boron trifluoride ether in an amount 100 times the weight of 2,4-dimethylpyrrole was added dropwise in an ice-water bath to react overnight.

[0037] After the reaction was completed, a small amount of dilute hydrochloric acid was added and stirred for 3 hours. After the reaction was completed, the mixture was extracted and column chromatography (SiO 2 ; eluent: petroleum ether / dichloromethane) to obtain compound 3 with a yield of 50%.

[0038] Compound 3 and N-iodosuccinimide were added to a reaction vessel in a molar ratio of 1:3, and then dichloromethane (10 times the weight of N-iodosuccinimide) was added as a solvent. The mixture was stirred at room temperature for 4 hours under nitrogen protection. After the reaction was completed, the mixture was purified by column chromatography (SiO 2 ; eluent: petroleum ether / dichloromethane) to obtain compound 4 with a yield of 90%.

[0039] Compound 4, pyridine-4-carboxaldehyde and piperidine acetate were added to the reaction vessel in a molar ratio of 1:10:10, and then acetonitrile (30 times the weight of pyridine-4-carboxaldehyde) was added as a solvent, and the mixture was reacted at 60°C under nitrogen protection for 1 hour. After the reaction, the mixture was extracted and purified by column chromatography (SiO 2 ; eluent: dichloromethane / methanol), to obtain blue product compound 5 with a yield of 70%.

[0040] Compound 5 and methyl iodide were added to the reaction vessel at a molar ratio of 1:10, and then N,N-dimethylformamide was added as a solvent at 2 times the amount of methyl iodide, and the reaction was carried out at 50°C under the protection of nitrogen for 10 hours. After the reaction was completed, 50 times the amount of ether was added to methyl iodide overnight, and the reaction was filtered through a sand plate Buchner funnel to obtain a green product m-BDP with a yield of 95%.

[0041] Embodiment 2:

[0042] Triethylene glycol monomethyl ether and p-toluenesulfonyl chloride were added to a reaction vessel in a molar ratio of 1:1, tetrahydrofuran (10 times the weight of p-toluenesulfonyl chloride) was added as a solvent, and then triethylamine (2 times the weight of p-toluenesulfonyl chloride) was added, and the reaction was stirred for 2 hours under ice bath and nitrogen protection. After the reaction was completed, the column chromatography was purified by extraction (SiO 2 ; eluent: dichloromethane / petroleum ether), to obtain a white solid product, compound 1, with a yield of 63%.

[0043] Compound 1, 3,4-dihydroxybenzaldehyde and sodium carbonate were added to the reaction vessel in a molar ratio of 1:3:6, and then N,N-dimethylformamide (100 times the weight of 3,4-dihydroxybenzaldehyde) was added as a solvent, and the reaction was carried out at 90°C under the protection of nitrogen for 48 hours. After the reaction was completed, the column chromatography was purified by extraction (SiO 2 ; eluent: dichloromethane / petroleum ether), to obtain light yellow oily liquid compound 2 with a yield of 70%.

[0044] Compound 2 and 2,4-dimethylpyrrole were added to the reaction vessel in a molar ratio of 1:2, and then 100 times the weight of tetrahydrofuran of 2,4-dimethylpyrrole was added as a solvent and 10 times the weight of trifluoroacetic acid of 2,4-dimethylpyrrole was added as a catalyst. Subsequently, 2,3-dichloro-5,6-dicyanobenzoquinone was dissolved in 10 times the weight of 2,4-dimethylpyrrole in a molar ratio of 1:3 to 2,4-dimethylpyrrole in tetrahydrofuran, and the reaction was stirred at room temperature for 12 hours. Finally, 50 times the weight of 2,4-dimethylpyrrole of triethylamine was added, and 100 times the weight of 2,4-dimethylpyrrole of boron trifluoride ether was added dropwise in an ice water bath to react overnight. After the reaction was completed, a small amount of dilute hydrochloric acid was added and stirred for 3 hours. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; eluent: petroleum ether / dichloromethane) to obtain compound 3 with a yield of 45%.

[0045] Compound 3 and N-iodosuccinimide were added to a reaction vessel in a molar ratio of 1:3, and then dichloromethane (10 times the weight of N-iodosuccinimide) was added as a solvent. The mixture was stirred at room temperature for 6 hours under nitrogen protection, and then vacuum distilled and purified by column chromatography (SiO 2 ; eluent: petroleum ether / dichloromethane) to obtain compound 4 with a yield of 82%.

[0046] Compound 4, pyridine-4-carboxaldehyde and piperidine acetate were added to the reaction vessel in a molar ratio of 1:20:20, and then acetonitrile (30 times the weight of pyridine-4-carboxaldehyde) was added as a solvent, and the reaction was carried out at 50°C under nitrogen protection for 1 hour. After the reaction was completed, the mixture was extracted and column chromatography (SiO 2 ; eluent: dichloromethane / methanol), to obtain blue product compound 5 with a yield of 72%.

[0047] Compound 5 and methyl iodide were added to the reaction vessel at a molar ratio of 1:9, and then 3 times the amount of N,N-dimethylformamide was added as a solvent for methyl iodide, and the reaction was carried out at 40°C under the protection of nitrogen for 10 hours. After the reaction was completed, 50 times the amount of ether for methyl iodide was added overnight, and the reaction was filtered through a sand plate Buchner funnel to obtain a green product m-BDP with a yield of 96%.

[0048] Embodiment 3:

[0049] Triethylene glycol monomethyl ether and p-toluenesulfonyl chloride were added to the reaction vessel in a molar ratio of 1:1, and N,N-dimethylformamide (10 times the weight of p-toluenesulfonyl chloride) was added as a solvent. Then, triethylamine (2 times the weight of p-toluenesulfonyl chloride) was added, and the reaction was stirred for 2 hours under ice bath and nitrogen protection. After the reaction was completed, the column chromatography was purified by extraction (SiO 2 ; eluent: dichloromethane / petroleum ether), to obtain a white solid product, compound 1, with a yield of 64%.

[0050] Compound 1, 3,4-dihydroxybenzaldehyde and potassium carbonate were added to the reaction vessel in a molar ratio of 1:4:6, and then N,N-dimethylformamide (100 times the weight of 3,4-dihydroxybenzaldehyde) was added as a solvent, and the reaction was carried out at 80°C under the protection of nitrogen for 48 hours. After the reaction was completed, the column chromatography was purified by extraction (SiO 2 ; eluent: dichloromethane / petroleum ether), to obtain light yellow oily liquid compound 2 with a yield of 70%.

[0051] Compound 2 and 2,4-dimethylpyrrole were added to the reaction vessel in a molar ratio of 1:2, and then tetrahydrofuran 100 times the weight of 2,4-dimethylpyrrole was added as a solvent and trifluoroacetic acid 10 times the weight of 2,4-dimethylpyrrole was added as a catalyst. Subsequently, 2,3-dichloro-5,6-dicyanobenzoquinone was dissolved in tetrahydrofuran 10 times the weight of 2,4-dimethylpyrrole in a molar ratio of 1:2 to 2,4-dimethylpyrrole, and the reaction was stirred at room temperature for 12 hours. Finally, triethylamine 100 times the weight of 2,4-dimethylpyrrole was added, and boron trifluoride ether 50 times the weight of 2,4-dimethylpyrrole was added dropwise in an ice water bath to react overnight. After the reaction was completed, a small amount of dilute hydrochloric acid was added and stirred for 3 hours. After the reaction was completed, it was extracted and purified by column chromatography (SiO 2 ; eluent: petroleum ether / dichloromethane) to obtain compound 3 with a yield of 42%.

[0052] Compound 3 and N-iodosuccinimide were added to a reaction vessel in a molar ratio of 1:3, and then dichloromethane (10 times the weight of N-iodosuccinimide) was added as a solvent. The mixture was stirred at room temperature for 5 hours under nitrogen protection, and then vacuum distilled and purified by column chromatography (SiO 2 ; eluent: petroleum ether / dichloromethane) to obtain compound 4 with a yield of 87%.

[0053] Compound 4, pyridine-4-carboxaldehyde and piperidine acetate were added to the reaction vessel in a molar ratio of 1:15:15, and then acetonitrile (30 times the weight of pyridine-4-carboxaldehyde) was added as a solvent, and the reaction was carried out at 60°C under nitrogen protection for 2 hours. After the reaction was completed, the mixture was extracted and purified by column chromatography (SiO 2; eluent: dichloromethane / methanol), to obtain blue product compound 5 with a yield of 65%.

[0054] Compound 5 and methyl iodide were added to the reaction vessel at a molar ratio of 1:10, and then N,N-dimethylformamide was added as a solvent at 3 times the amount of methyl iodide, and the reaction was carried out at 50°C under the protection of nitrogen for 8 hours. After the reaction was completed, ether was added at 50 times the amount of methyl iodide overnight, and the reaction was filtered through a sand plate Buchner funnel to obtain a green product m-BDP with a yield of 93%.

[0055] The synthetic routes of Examples 1 to 3 are shown in the figure below: .

[0056] Embodiment 4:

[0057] The mitochondrial co-localization of the m-BDP prepared in Example 1 at the cellular level was tested, and the specific operation was as follows: B16F10 cells in the logarithmic growth phase were cultured at 2 × 10 4 Cells were inoculated at a density of 100 cells / well in a laser confocal culture dish, and 1 mL of high-glucose RPMI1640 medium containing 10% fetal bovine serum (FBS) was added to each well. The cells were placed in a cell culture incubator and incubated for 12 hours at a constant temperature. After the cells adhered to the wall, the culture medium was discarded, and the cells were washed twice with phosphate buffered saline (PBS). The m-BDP solution (10 μg mL -1 , 1 mL), the cells were placed in the incubator and incubated again for 24 hours, the drug-containing medium was discarded, and the cells were rinsed three times with PBS. After rinsing, the mitochondrial stain MitoTracker GreenFM (0.2 μM, 1 mL) was added to stain the cells for 20 minutes. After staining, the cells were rinsed three times with PBS, and then the cells were stained with the nuclear stain Hoechst 33342 (10 μM, 1 mL) for 10 minutes, washed with PBS three times, and finally the co-localization of m-BDP with mitochondria after entering the cells was observed with a laser confocal microscope. (Mitochondrial stain: excitation wavelength is 488nm, detection wavelength is 510-570nm; nuclear probe: excitation wavelength is 405 nm, detection wavelength is 425-475nm; m-BDP: excitation wavelength is 633nm, detection wavelength is 650-750nm).

[0058] Embodiment 5:

[0059] The nuclear damage of the compound m-BDP prepared in Example 1 was tested under different conditions. The specific operation was as follows: B16F10 cells in the logarithmic growth phase were inoculated on the slides in a 12-well plate at a density of 2 × 105 / mL, and cultured in a cell culture incubator for 12 hours. After confirming that the cells were attached to the wall, the culture medium was discarded, and the cells were washed twice with PBS. The m-BDP solution prepared with the culture medium was added, 1 mL per well, and the concentration of each group was 2.00 μg mL -1 After 24 hours of incubation, the culture medium was replaced and the light group ( hv ) were respectively exposed to 660 nm LED light (50 mW cm -2 ) condition, illuminate for 10 minutes, return to the incubator and continue to culture for 2 hours. The dose of the X-ray irradiation group (+ X-ray) was 6 Gy, and the non-X-ray irradiation group (-X-ray) did not receive any treatment. After 6 hours, the culture medium was removed, washed 3 times with PBS, and the fixative (4% paraformaldehyde) was added. 1 ml of fixative was added to each well and fixed for 15 minutes. The fixative was removed and washed 3 times with PBS. Immunostaining permeabilization solution (Triton x 100) containing 5% bovine serum albumin was added and blocked overnight at 4°C. The immunostaining blocking solution was removed, and γ-H2AX rabbit monoclonal antibody (1:500) was added and incubated at 4°C for 12 hours. Washed 3 times with PBS, 5-10 minutes each time. Added rabbit monoclonal antibody secondary antibody fluorescein isothiocyanate (FITC) (1:200) and incubated at room temperature for 1 hour. Washed 3 times with PBS, 5-10 minutes each time, added nuclear staining solution (DAPI), and stained at room temperature for about 5 minutes. The sections were mounted on glass slides and the cell nuclear damage was observed using a laser confocal microscope.

[0060] Embodiment 6:

[0061] HA of 5 kDa, HA of 50 kDa and the m-BDP aqueous solution in Example 1 were mixed and stirred at a mass fraction of 1.5:1.5:7, and ultrasonically mixed to ensure uniform mixing in a sample bottle, followed by centrifugation to remove bubbles (2500 rpm, 10 minutes). At the same time, the mold was pre-vacuumed for 10 minutes, and then the above mixed sample was dripped on the mold to form a micro-convex shape, and vacuumed again for 5 minutes. Then, the bubbles were removed with a gun tip, placed in a dryer and dried for 24 hours, and the MN-m-BDP soluble microneedles were removed from the mold and stored in a drying oven.

[0062] Embodiment 7:

[0063] HA of 5 kDa, HA of 50 kDa and the m-BDP aqueous solution in Example 1 were mixed and stirred at a mass fraction of 2:1:7, and ultrasonically mixed to ensure uniform mixing in a sample bottle, followed by centrifugation to remove bubbles (2500 rpm, 10 minutes). At the same time, the mold was pre-vacuumed for 5 minutes, and then the above-mentioned mixed sample was dripped on the mold to form a micro-convex shape, and vacuumed again for 10 minutes. Then, the bubbles were removed with a gun tip, placed in a dryer and dried for 24 hours, and the MN-m-BDP soluble microneedles were removed from the mold and stored in a drying oven.

[0064] Embodiment 8:

[0065] HA of 5 kDa, HA of 50 kDa and the aqueous solution of m-BDP in Example 1 were mixed and stirred at a mass fraction of 2:2:6, and ultrasonically mixed to ensure uniform mixing in a sample bottle, followed by centrifugation to remove bubbles (2500 rpm, 10 minutes). At the same time, the mold was pre-vacuumed for 10 minutes, and then the mixed sample was dripped on the mold to form a micro-convex shape, and vacuumed again for 15 minutes. Then, the bubbles were removed with a gun tip, placed in a dryer and dried for 24 hours, and the MN-m-BDP soluble microneedles were removed from the mold and stored in a drying oven.

[0066] Embodiment 9:

[0067] HA of 5 kDa, HA of 50 kDa and the m-BDP aqueous solution in Example 1 were mixed and stirred at a mass fraction of 1:3:6, and ultrasonically mixed to ensure uniform mixing in a sample bottle, followed by centrifugation to remove bubbles (2500 rpm, 10 minutes). At the same time, the mold was pre-vacuumed for 10 minutes, and then the above-mentioned mixed sample was dripped on the mold to form a micro-convex shape, and vacuumed again for 5 minutes. Then, the bubbles were removed with a gun tip, placed in a dryer and dried for 36 hours, and the MN-m-BDP soluble microneedles were removed from the mold and stored in a drying oven.

[0068] Embodiment 10:

[0069] HA of 5 kDa, HA of 50 kDa and the aqueous solution of m-BDP in Example 1 were mixed and stirred at a mass fraction of 3:1:6, and ultrasonically mixed to ensure uniform mixing in a sample bottle, followed by centrifugation to remove bubbles (2500 rpm, 10 minutes). At the same time, the mold was pre-vacuumed for 10 minutes, and then the mixed sample was dripped on the mold to form a micro-convex shape, and vacuumed again for 5 minutes. Then, the bubbles were removed with a gun tip, and the mold was placed in a dryer for 48 hours. The MN-m-BDP soluble microneedles were removed from the mold and stored in a drying oven.

[0070] Embodiment 11:

[0071] HA of 5 kDa, HA of 50 kDa and the m-BDP aqueous solution in Example 1 were mixed and stirred at a mass fraction of 1.5:2.5:6, and ultrasonically mixed to ensure uniform mixing in a sample bottle, followed by centrifugation to remove bubbles (2500 rpm, 10 minutes). At the same time, the mold was pre-vacuumed for 15 minutes, and then the above mixed sample was dripped on the mold to form a micro-convex shape, and vacuumed again for 10 minutes. Then, the bubbles were removed with a gun tip, placed in a dryer and dried for 24 hours, and the MN-m-BDP soluble microneedles were removed from the mold and stored in a drying oven.

[0072] Embodiment 12:

[0073] HA of 5 kDa, HA of 50 kDa and the m-BDP aqueous solution in Example 1 were mixed and stirred at a mass fraction of 2.5:1.5:6, and ultrasonically mixed to ensure uniform mixing in a sample bottle, followed by centrifugation to remove bubbles (2500 rpm, 10 minutes). At the same time, the mold was pre-vacuumed for 10 minutes, and then the above-mentioned mixed sample was dripped on the mold to form a micro-convex shape, and then vacuumed again for 20 minutes. Then, the bubbles were removed with a gun tip, placed in a dryer and dried for 24 hours, and the MN-m-BDP soluble microneedles were removed from the mold and stored in a drying oven.

[0074] Application Example 1: The tumor volume was 80 mm 3 The animal model was made by using female C57BL / 6 mice bearing melanoma (B16F10) subcutaneously. The mice were randomly divided into six groups, PBS (blank control group), X-ray (X-ray irradiation group alone), MN-c-BDP (microneedle patch group alone), MN-c-BDP / hv (microneedle patch plus light irradiation group), MN-c-BDP / X-ray (microneedle patch plus X-ray group), and MN-c-BDP / hv / X-ray (microneedle patch plus light irradiation group plus X-ray group), with 5 mice in each group. Four hours after microneedle administration, the light irradiation group was illuminated with an LED light (wavelength: 660 nm, power: 50 mW cm -2 ) The tumor site of the mice was illuminated for 30 minutes, while the non-illumination group was not subjected to this operation. After 12 hours of illumination, the X-ray irradiation group was anesthetized and then irradiated with X-rays, with an irradiation dose of 6 Gy. Lead sheets were used to shield the mice except for the tumor site. During the treatment, the weight and tumor volume of the mice were recorded every 2 days, and the weight change curve and tumor growth curve of the mice were plotted. When the tumor volume exceeded 1500 mm 3 At about 2 d, the mice were considered dead and euthanized.

[0075] Effect evaluation 1: The present invention will obtain the photosensitizer m-BDP through multi-step synthetic retrosynthesis ( Figure 1 ), and its structure was characterized by 1 HNMR nuclear magnetic resonance ( Figure 2 ); Next, the mitochondrial targeting ability of compound m-BDP against murine melanoma cells (B16F10) was tested through cell experiments ( Figure 3 ) (the scale bar is 20 μm) (Merged represents the merging of the three channels of mitochondrial dye, nuclear dye, and m-BDP), and radiosensitization tests were carried out ( Figure 4 ) (the scale bar is 10 μm) (Merged represents the merging of the two channels of γ-H2AX immunofluorescence dye and DAPI dye). Subsequently, the morphology of the microneedles was characterized ( Figure 5 ) (the scale bar of A is 200 μm, and the scale bar of B is 400 μm). Finally, the antitumor experiment of microneedle MN-m-BDP was carried out by constructing a subcutaneous female C57BL / 6 tumor-bearing mouse model of melanoma (B16F10) ( Figure 6 ).

[0076] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A radiotherapy sensitization microneedle, characterized in that: It is obtained by mixing m-BDP and hyaluronic acid and then drying; The structural formula of the m-BDP is as follows: ; wherein n is selected from any integer between 1 and 20, X is a bromine atom or an iodine atom, and R1 and R2 are selected from methyl, ethyl, propyl or butyl.

2. The radiotherapy sensitization microneedle according to claim 1, characterized in that: The preparation method of the m-BDP is as follows: S11: Under nitrogen protection, triethylene glycol monomethyl ether, p-toluenesulfonyl chloride and triethylamine are added to organic solvent I for reaction in an ice bath, and purified to obtain intermediate product 1; the chemical formula of intermediate product 1 is as follows: ； S12: Under nitrogen protection, the intermediate product 1, 3,4-dihydroxybenzaldehyde and potassium carbonate are added to an organic solvent II and heated at 85-95° C. for reaction for 44-52 hours to purify the intermediate product 2; the chemical formula of the intermediate product 2 is as follows: ； S13: The intermediate product 2, 2,4-dimethylpyrrole, a catalyst and 2,3-dichloro-5,6-dicyanobenzoquinone are dissolved in an organic solvent III, and triethylamine and boron trifluoride ether are added after reacting at room temperature for 10-14 hours, and hydrochloric acid is added after reacting in an ice-water bath for 8-16 hours to purify to obtain an intermediate product 3; the chemical formula of the intermediate product 3 is as follows: ； S14: Under nitrogen protection, the intermediate product 3 and N-iodosuccinimide are added to an organic solvent IV for reaction at room temperature for 3-5 hours, and the intermediate product 4 is purified; the chemical formula of the intermediate product 4 is as follows: ； S15: Under nitrogen protection, the intermediate product 4, pyridine-4-carboxaldehyde and piperidine acetate are added to an organic solvent V at 55-65° C. and heated to react for 0.8-1.2 hours, and the intermediate product 5 is purified; the chemical formula of the intermediate product 5 is as follows: ； S16: Under nitrogen protection, the intermediate product 5 and methyl iodide are added to an organic solvent VI and heated at 45-55° C. for reaction for 8-12 hours, and ether is added for purification to obtain the m-BDP.

3. The radiotherapy sensitization microneedle according to claim 2, characterized in that: The organic solvent I, organic solvent II, organic solvent III, organic solvent IV, organic solvent V and organic solvent VI are independently selected from N,N-dimethylformamide solution, acetonitrile, tetrahydrofuran, dichloromethane or ethanol.

4. The radiotherapy sensitization microneedle according to claim 2, characterized in that: In the steps S11, S12, S13 and S15, the purification method is extraction followed by column chromatography.

5. The radiotherapy sensitization microneedle according to claim 2, characterized in that: In the step S14, the purification method is column chromatography; in the step S16, the ether purification method is to add ether for 8-16 hours and then filter.

6. The radiotherapy sensitization microneedle according to claim 4 or 5, characterized in that: The column chromatography uses silicon dioxide, and the eluent is selected from two of dichloromethane, methanol and petroleum ether.

7. The radiotherapy sensitization microneedle according to claim 2, characterized in that: The catalyst is selected from trifluoroacetic acid.

8. The radiotherapy sensitization microneedle according to claim 2, characterized in that: In the step S11, the molar ratio of triethylene glycol monomethyl ether and p-toluenesulfonyl chloride is 1:1-3; the mass of triethylamine is twice the mass of p-toluenesulfonyl chloride; in the step S12, the molar ratio of the intermediate product 1, 3,4-dihydroxybenzaldehyde and potassium carbonate is 1:2-4:6; in the step S13, the molar ratio of the intermediate product 2, 2,4-dimethylpyrrole and 2,3-dichloro-5,6-dicyanobenzoquinone is 1:2:2-6.

9. The radiotherapy sensitization microneedle according to claim 2, characterized in that: In the step S15, the molar ratio of the intermediate product 4, pyridine-4-carboxaldehyde and piperidine acetate is 1:10-20:10-20; in the step S16, the molar ratio of the intermediate product 5 and methyl iodide is 1:

10.

10. Use of the radiosensitizing microneedle according to any one of claims 1 to 9 in local tumor treatment, characterized in that: After administering the radiotherapy sensitizing microneedles, a light source is used to irradiate the tumor site.

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