A radiotherapy sensitizing microneedle and its application

Abstract

The present invention belongs to the technical field of drug systemic delivery, and specifically relates to a radiosensitizing microneedle and its application. The present invention relates to the design of a mitochondrion-targeted photosensitizing microneedle system and its application in radiosensitizing radiotherapy for melanoma. The present invention combines the intelligent delivery platform of microneedles (MNs) with mitochondrion-targeted photodynamic therapy, opening up a new paradigm for radiosensitizing radiotherapy of melanoma. The present invention uses soluble hyaluronic acid (HA) microneedle arrays loaded with mitochondrion-targeted photosensitizers. After the microneedles penetrate the stratum corneum, they rapidly dissolve in the dermis layer, release the photosensitizers and achieve mitochondrion-targeted enrichment. Subsequently, under the illumination condition of 660 nm, the photosensitizers generate reactive oxygen species in the mitochondrial matrix, directly destroying the mitochondrial structure and function, thereby inducing a mitochondrial ROS storm, and thus enhancing the sensitivity of tumor radiotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug systemic delivery, and particularly relates to a radiosensitizing microneedle and its application. Background Art

[0002] Melanoma, as a highly invasive skin malignancy, its treatment system has shifted from traditional cytotoxic drugs to precision therapy based on immune checkpoint blockade (the typical representative is the combination therapy of CTLA-4 / PD-1).

[0003] In the comprehensive treatment system of melanoma, radiotherapy still has irreplaceable clinical value. Its core mechanism of action depends on DNA double-strand breaks (DSBs) induced by ionizing radiation and oxidative stress damage mediated by reactive oxygen species (ROS). However, melanoma cells significantly reduce radiosensitivity through a dual defense mechanism: on the one hand, they neutralize ROS damage through the overexpression of antioxidant systems such as glutathione (GSH), and on the other hand, they activate highly efficient DNA repair pathways such as non-homologous end joining (NHEJ). Clinically, although ultra-high-dose radiotherapy is used to overcome radioresistance, 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 point in this field.

[0004] Existing delivery systems have significant limitations in the targeted delivery of radiosensitizers: traditional administration routes (oral / intravenous injection) are limited by the first-pass effect and rapid metabolism in the liver / kidney, resulting in drug accumulation at the tumor site being less than 5%-10% of the conventional dose, and toxic reactions such as myelosuppression 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 the enhanced permeability and retention effect (EPR), their applications are 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 innovative solutions for breaking through the efficacy bottleneck of melanoma radiotherapy and has important clinical significance for achieving precise dose regulation and synergistically improving the therapeutic window.

[0006] As an emerging local tumor treatment method, photodynamic therapy generates ROS (such as singlet oxygen) through photosensitizers, 1O2) selectively oxidizes and damages tumor tissues, with advantages such as high spatiotemporal precision, low toxicity and side effects, and no drug resistance. Photodynamic therapy can be independently used for the treatment of superficial tumors such as skin cancer, and can also be used as an adjuvant to conventional treatment methods (such as radiotherapy) for the treatment of 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 the sensitivity to radiotherapy. In recent years, mitochondrion-targeted photosensitizers have become the research focus in the field of photodynamic therapy. By inhibiting the oxidative phosphorylation process and reducing oxygen consumption, they can effectively alleviate the limitation of the tumor hypoxic microenvironment on photodynamic therapy, providing a new approach for the photodynamic therapy of hypoxic tumors. Another study shows that mitochondrion-targeted photosensitizers can induce mitochondrion-derived apoptosis by inhibiting protein phosphorylation and blocking the prosurvival signaling pathway, thereby enhancing the sensitivity of tumor radiotherapy. Therefore, developing mitochondrion-targeted photosensitizers and their precise delivery systems, and regulating the tumor radiotherapy resistance mechanism through mitochondrion-targeted photodynamic therapy, provide a new strategy for overcoming tumor radiotherapy resistance.

[0007] In addition, as a novel transdermal drug delivery system, microneedles (MN) have revolutionized the drug delivery method by enhancing the transdermal permeability of drugs. This technology can efficiently deliver compounds with different molecular weights, such as chemotherapeutic drugs, polypeptides, proteins, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and vaccines. This broad-spectrum delivery ability makes it show unique advantages in the treatment of melanoma: (1) Compared with traditional oral or intravenous administration, microneedle administration can precisely deliver drugs to the deep lesions of melanoma, significantly improving the targeting and efficacy; (2) Compared with traditional single therapies, microneedle administration can not only achieve multimodal combination of drugs but also release drugs in a responsive manner, optimizing the efficacy while reducing the risk of drug resistance; (3) Compared with traditional chemotherapy or targeted therapy, microneedle administration can avoid the diffusion of drugs to healthy tissues and organs to the greatest extent, thereby reducing side effects and prolonging the survival period of patients; (4) Compared with subcutaneous injection, microneedle administration can achieve better efficacy with a lower dose by enriching drugs in the tumor site, and its painless feature significantly improves patient compliance. These advantages make it possible for the microneedle technology combined with melanoma therapy to be clinically applied in the future. Summary of the Invention

[0008] The disadvantages of the current existing technologies are as follows:

[0009] (1) Currently, radiotherapy alone cannot distinguish between normal tissues and tumor tissues, which may lead to acute injuries or chronic complications in adjacent organs (such as the lungs, heart, and intestines). In addition, the absorption efficiency of tumor tissues for radiation energy is generally low, making it difficult to reach the ideal threshold for the generation of ROS during the treatment process.

[0010] (2) Disadvantages of current drug systemic delivery: On the one hand, since the drug circulates in the body, it degrades prematurely before reaching the lesion or diffuses into healthy tissues, limiting the therapeutic effect; on the other hand, the dosage used is relatively large and it cannot accumulate at the tumor site.

[0011] To solve the above-mentioned technical problems, the present application provides the following technical solutions:

[0012] In view of the above problems and defects, the present invention relates to the design of a mitochondrial-targeted photosensitive microneedle system and its application in radiosensitizing radiotherapy for melanoma. This invention combines the intelligent delivery platform of microneedles (MNs) with mitochondrial-targeted photodynamic therapy, opening up a new paradigm for radiosensitizing treatment of melanoma. The present invention uses a soluble hyaluronic acid microneedle array loaded with a mitochondrial-targeted photosensitizer. After the microneedles penetrate the stratum corneum, they rapidly dissolve in the dermis layer, release the photosensitizer and achieve mitochondrial-targeted enrichment. Subsequently, under the illumination condition of 660 nm, the photosensitizer generates reactive oxygen species in the mitochondrial matrix, directly destroying the mitochondrial structure and function, thereby inducing a mitochondrial ROS storm, thus enhancing the sensitivity of tumor radiotherapy.

[0013] The present invention provides a radiosensitizing microneedle, which is obtained by drying the mixture of m-BDP and hyaluronic acid;

[0014] The structural formula of the m-BDP is as follows:

[0015] ; wherein, n is selected from any integer in 1-20, X is a bromine atom or an iodine atom, and R1 and R2 are selected from one of methyl, ethyl, propyl and butyl.

[0016] Preferably, the preparation method of the m-BDP is as follows:

[0017] S11: Under nitrogen protection, add triethylene glycol monomethyl ether, p-toluenesulfonyl chloride and triethylamine to organic solvent I and react in an ice bath for 2 hours, and purify to obtain intermediate product 1; the chemical formula of the intermediate product 1 is as follows:

[0018] ;

[0019] S12: Under nitrogen protection, add the intermediate product 1, 3,4-dihydroxybenzaldehyde and potassium carbonate to organic solvent II and heat and react at 85-95 °C for 44-52 hours, and purify to obtain intermediate product 2; the chemical formula of the intermediate product 2 is as follows:

[0020] ;

[0021] S13: Dissolve the intermediate 2, 2,4 - dimethylpyrrole, the catalyst, and 2,3 - dichloro - 5,6 - dicyanobenzoquinone in organic solvent III, react at room temperature (25 ± 5 °C) for 10 - 14 hours, then add triethylamine and boron trifluoride diethyl etherate, react in an ice - water bath for 8 - 16 hours, and then add dilute hydrochloric acid. After purification, intermediate 3 is obtained. The chemical formula of intermediate 3 is as follows:

[0022] ;

[0023] S14: Under nitrogen protection, add the intermediate 3 and N - iodosuccinimide to organic solvent IV and react at room temperature for 3 - 5 hours. After purification, intermediate 4 is obtained. The chemical formula of intermediate 4 is as follows:

[0024] ;

[0025] S15: Under nitrogen protection, add the intermediate 4, pyridine - 4 - carbaldehyde, and piperidine acetate to organic solvent V and heat - react at 55 - 65 °C for 0.8 - 1.2 hours. After purification, intermediate 5 is obtained. The chemical formula of intermediate 5 is as follows:

[0026] ;

[0027] S16: Under nitrogen protection, add the intermediate 5 and iodomethane to organic solvent VI and heat - react at 45 - 55 °C for 8 - 12 hours, then add ether for purification to obtain the m - BDP.

[0028] Further, 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 (CH3CN), tetrahydrofuran (THF), dichloromethane (DCM), or ethanol.

[0029] Further, in steps S11, S12, S13, and S15, the purification method is extraction followed by column chromatography.

[0030] Further, in step S14, the purification method is column chromatography; in step S16, the method of ether purification is to filter after adding ether for 8 - 16 hours.

[0031] Specifically, the column chromatography uses silica gel, and the eluent is selected from two of dichloromethane, methanol, and petroleum ether.

[0032] Further, the catalyst is selected from trifluoroacetic acid.

[0033] Further, in the step S11, the molar ratio of triethylene glycol monomethyl ether to p-toluenesulfonyl chloride is 1:1 - 3; the mass of triethylamine is 2 times the mass of p-toluenesulfonyl chloride; in the step S12, the molar ratio of intermediate 1, 3,4-dihydroxybenzaldehyde to potassium carbonate is 1:2 - 4:6; in the step S13, the molar ratio of intermediate 2, 2,4-dimethylpyrrole to 2,3-dichloro-5,6-dicyano-1,4-benzoquinone is 1:2:2 - 6; the mass of boron trifluoride diethyl etherate is 100 times the mass of 2,4-dimethylpyrrole.

[0034] Further, in the step S15, the molar ratio of intermediate 4, pyridine-4-carboxaldehyde to piperidine acetate is 1:10 - 20:10 - 20; in the step S16, the molar ratio of intermediate 5 to iodomethane is 1:10; the mass of diethyl ether is 50 times the mass of iodomethane.

[0035] The present invention also provides the application of the above radiotherapy sensitizing microneedles in local tumor treatment, and after administering the radiotherapy sensitizing microneedles, the tumor site is irradiated with a light source.

[0036] Preferably, the light source is an LED lamp with a wavelength of 660 nm and a power of 50 mW / cm -2 , and the irradiation time is 20 - 40 minutes.

[0037] The technical solution of the present invention has the following advantages compared with the prior art:

[0038] The present invention provides a simple synthesis method of a radiotherapy sensitizing compound, and applies it to the growth inhibition and radiotherapy sensitization of murine melanoma cell B16F10 cells, which can fully exert the radiotherapy sensitization effect while overcoming the limitations of the prior art, and the synthetic preparation route is simple, having a certain transformation prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is a synthetic route diagram of the radiotherapy sensitizer m-BDP, where n is 1 - 20; X is a bromine atom or an iodine atom; R1 and R2 can be methyl or ethyl respectively.

[0040] Figure 2 For the radiotherapy sensitizer m-BDP in Example 1 1 HNMR spectrum; m-BDP takes n = 3; X is an iodine atom; R1 and R2 are methyl as an example.

[0041] Figure 3It is a diagram of the mitochondrial co-localization of compound m-BDP in Example 1, where the scale bar is 20 μm; a shows the case of "Hoechst 33342", b shows the case of "Mito Tracker", c shows the case of "m-BDP", and d shows the case of combining the three channels of mitochondrial fuel, nuclear dye, and m-BDP.

[0042] Figure 4 It is an immunofluorescence staining diagram of γ-H2AX of compound m-BDP in Example 1, where the scale bar is 10 μm.

[0043] Figure 5 It is a single-needle morphology characterization diagram and a needle-type arrangement morphology characterization diagram of MN-m-BDP in Example 6, where the scale bar of A is 200 μm; the scale bar of B is 400 μm.

[0044] Figure 6 It is an anti-tumor experiment diagram of MN-m-BDP in Example 6. Detailed implementation manners

[0045] The present invention will be 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 be able to implement it, but the examples given are not intended to limit the present invention.

[0046] The specific synthesis steps of the target molecular compound in this specific embodiment are as follows:

[0047] Among them, the reaction solvents are N,N-dimethylformamide solution (DMF), acetonitrile (CH3CN), tetrahydrofuran (THF), dichloromethane (DCM), ethanol (CH3CH2OH).

[0048] Example 1:

[0049] Triglycol monomethyl ether and p-toluenesulfonyl chloride were added to a reaction vessel in a molar ratio of 1:1, 10 times the weight of p-toluenesulfonyl chloride of dichloromethane was added as a solvent, and then 2 times the weight of triethylamine 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, it was extracted and column chromatographed (SiO2; the eluent was 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 a reaction vessel in a molar ratio of 1:2:6, and then 100 times the weight of 3,4-dihydroxybenzaldehyde of N,N-dimethylformamide was added as a solvent, and the reaction was carried out at 90 °C under nitrogen protection for 48 hours. After the reaction was completed, it was extracted and column chromatographed (SiO2; the eluent was dichloromethane / petroleum ether) to obtain a pale yellow oily liquid, compound 2, with a yield of 70%.

[0051] Compound 2 and 2,4-dimethylpyrrole were added to a reaction vessel in a molar ratio of 1:2, and then 100 times the weight of 2,4-dimethylpyrrole of tetrahydrofuran was added as a solvent and 10 times the weight of 2,4-dimethylpyrrole of trifluoroacetic acid was added as a catalyst.

[0052] Subsequently, 2,3-dichloro-5,6-dicyanobenzoquinone with a molar ratio of 1:2.2 to 2,4-dimethylpyrrole was dissolved in 10 times the weight of 2,4-dimethylpyrrole of tetrahydrofuran and added to the above reaction solution, and the reaction was stirred at room temperature for 12 hours.

[0053] Finally, 100 times the weight of 2,4-dimethylpyrrole of triethylamine was added, and 100 times the weight of 2,4-dimethylpyrrole of boron trifluoride diethyl ether was added dropwise under an ice-water bath and reacted overnight.

[0054] 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 (SiO2; eluent: petroleum ether / dichloromethane) to obtain Compound 3 with a yield of 50%.

[0055] Compound 3 and N-iodosuccinimide were added to a reaction vessel in a molar ratio of 1:3, and then 10 times the weight of N-iodosuccinimide of dichloromethane was added as a solvent, and the reaction was stirred at room temperature for 4 hours under nitrogen protection. After the reaction was completed, it was purified by column chromatography (SiO2; eluent: petroleum ether / dichloromethane) to obtain Compound 4 with a yield of 90%.

[0056] Compound 4, pyridine-4-carbaldehyde and piperidine acetate were added to a reaction vessel in a molar ratio of 1:10:10, and then 30 times the weight of pyridine-4-carbaldehyde of acetonitrile was added as a solvent, and the reaction was carried out at 60 °C under nitrogen protection for 1 hour. After the reaction was completed, it was extracted and purified by column chromatography (SiO2; eluent: dichloromethane / methanol) to obtain the blue product Compound 5 with a yield of 70%.

[0057] Compound 5 and iodomethane were added to a reaction vessel in a molar ratio of 1:10, and then 2 times the weight of iodomethane of N,N-dimethylformamide was added as a solvent, and the reaction was carried out at 50 °C under nitrogen protection for 10 hours. After the reaction was completed, 50 times the weight of iodomethane of diethyl ether was added overnight, and it was filtered with a sintered glass Buchner funnel to obtain the green product m-BDP with a yield of 95%.

[0058] Example 2:

[0059] Triethylene glycol monomethyl ether and p-toluenesulfonyl chloride were added to a reaction vessel in a molar ratio of 1:1. Tetrahydrofuran, which is 10 times the weight of p-toluenesulfonyl chloride, was added as a solvent. Then, triethylamine, which is 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, it was extracted and purified by column chromatography (SiO2; eluent: dichloromethane / petroleum ether) to obtain a white solid product, compound 1, with a yield of 63%.

[0060] Compound 1, 3,4-dihydroxybenzaldehyde, and sodium carbonate were added to a reaction vessel in a molar ratio of 1:3:6. Then, N,N-dimethylformamide, which is 100 times the weight of 3,4-dihydroxybenzaldehyde, was added as a solvent, and the mixture was reacted at 90 °C under nitrogen protection for 48 hours. After the reaction, it was extracted and purified by column chromatography (SiO2; eluent: dichloromethane / petroleum ether) to obtain a pale yellow oily liquid, compound 2, with a yield of 70%.

[0061] Compound 2 and 2,4-dimethylpyrrole were added to a reaction vessel in a molar ratio of 1:2. Then, tetrahydrofuran, which is 100 times the weight of 2,4-dimethylpyrrole, was added as a solvent, and trifluoroacetic acid, which is 10 times the weight of 2,4-dimethylpyrrole, was added as a catalyst. Subsequently, 2,3-dichloro-5,6-dicyanobenzoquinone, which has a molar ratio of 1:3 with 2,4-dimethylpyrrole, was dissolved in tetrahydrofuran, which is 10 times the weight of 2,4-dimethylpyrrole, and added to the above reaction solution, and the mixture was stirred at room temperature for 12 hours. Finally, triethylamine, which is 50 times the weight of 2,4-dimethylpyrrole, was added, and boron trifluoride diethyl etherate, which is 100 times the weight of 2,4-dimethylpyrrole, was added dropwise under an ice-water bath and reacted overnight. After the reaction, a small amount of dilute hydrochloric acid was added and stirred for 3 hours. After the reaction, it was extracted and purified by column chromatography (SiO2; eluent: petroleum ether / dichloromethane) to obtain compound 3 with a yield of 45%.

[0062] Compound 3 and N-iodosuccinimide were added to a reaction vessel in a molar ratio of 1:3. Then, dichloromethane, which is 10 times the weight of N-iodosuccinimide, was added as a solvent, and the mixture was stirred at room temperature for 6 hours under nitrogen protection. After vacuum distillation, it was purified by column chromatography (SiO2; eluent: petroleum ether / dichloromethane) to obtain compound 4 with a yield of 82%.

[0063] Compound 4, pyridine-4-carboxaldehyde, and piperidine acetate were added to a reaction vessel in a molar ratio of 1:20:20. Then, acetonitrile, which is 30 times the weight of pyridine-4-carboxaldehyde, was added as a solvent, and the mixture was reacted at 50 °C under nitrogen protection for 1 hour. After the reaction, it was extracted and purified by column chromatography (SiO2; eluent: dichloromethane / methanol) to obtain a blue product, compound 5, with a yield of 72%.

[0064] Compound 5 and iodomethane were added to the reaction vessel in a molar ratio of 1:9, and then N,N-dimethylformamide three times the amount of iodomethane was added as a solvent. The reaction was carried out at 40 °C under nitrogen protection for 10 hours. After the reaction, ether 50 times the amount of iodomethane was added overnight, and the mixture was filtered through a sintered Buchner funnel to obtain the green product m-BDP with a yield of 96%.

[0065] Example 3:

[0066] Triethylene glycol monomethyl ether and p-toluenesulfonyl chloride were added to the reaction vessel in a molar ratio of 1:1. N,N-dimethylformamide 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. The reaction was stirred at ice bath temperature under nitrogen protection for 2 hours. After the reaction, the product was extracted and purified by column chromatography (SiO2; eluent: dichloromethane / petroleum ether) to obtain the white solid product Compound 1 with a yield of 64%.

[0067] Compound 1, 3,4-dihydroxybenzaldehyde and potassium carbonate were added to the reaction vessel in a molar ratio of 1:4:6. 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 nitrogen protection for 48 hours. After the reaction, the product was extracted and purified by column chromatography (SiO2; eluent: dichloromethane / petroleum ether) to obtain the pale yellow oily liquid Compound 2 with a yield of 70%.

[0068] Compound 2 and 2,4-dimethylpyrrole were added to the reaction vessel in a molar ratio of 1:2. 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-dicyano-1,4-benzoquinone with a molar ratio of 1:2 to 2,4-dimethylpyrrole was dissolved in tetrahydrofuran 10 times the weight of 2,4-dimethylpyrrole and added to the above reaction solution. 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 diethyl etherate 50 times the weight of 2,4-dimethylpyrrole was added dropwise under ice bath conditions and the reaction was carried out overnight. After the reaction, a small amount of dilute hydrochloric acid was added and stirred for 3 hours. After the reaction, the product was extracted and purified by column chromatography (SiO2; eluent: petroleum ether / dichloromethane) to obtain Compound 3 with a yield of 42%.

[0069] Compound 3 and N-iodosuccinimide were added to the reaction vessel in a molar ratio of 1:3. Dichloromethane 10 times the weight of N-iodosuccinimide was added as a solvent, and the reaction was stirred at room temperature under nitrogen protection for 5 hours. After distillation under reduced pressure, the product was purified by column chromatography (SiO2; eluent: petroleum ether / dichloromethane) to obtain Compound 4 with a yield of 87%.

[0070] Compound 4, pyridine-4-carboxaldehyde, and piperidine acetate were added to a reaction vessel in a molar ratio of 1:15:15. Then, acetonitrile, which was 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, the product was extracted and purified by column chromatography (SiO2; eluent: dichloromethane / methanol) to obtain blue product Compound 5 with a yield of 65%.

[0071] Compound 5 and iodomethane were added to a reaction vessel in a molar ratio of 1:10. Then, N,N-dimethylformamide, which was 3 times the amount of iodomethane, was added as a solvent, and the reaction was carried out at 50 °C under nitrogen protection for 8 hours. After the reaction, diethyl ether, which was 50 times the amount of iodomethane, was added and left overnight, and then filtered through a sintered Buchner funnel to obtain green product m-BDP with a yield of 93%.

[0072] The synthetic routes of Examples 1 to 3 are shown in the following figure:

[0073] 。

[0074] Example 4:

[0075] The mitochondrial co-localization of m-BDP prepared in Test Example 1 was tested at the cellular level, and the specific operation was as follows:

[0076] B16F10 cells in the logarithmic growth phase were seeded in a confocal dish at a density of 2 × 10 4 cells / well and cultured. 1 mL of high-glucose RPMI1640 medium containing 10% fetal bovine serum (FBS) was added to each well, and the cells were incubated in a cell culture incubator at a constant temperature for 12 hours. After the cells adhered to the wall, the culture medium was discarded, and the cells were washed twice with phosphate buffer (PBS). Then, an m-BDP solution (10 μg mL -1 , 1 mL) prepared with the medium was added, and the cells were incubated in the incubator for another 24 hours. The medium containing the drug was discarded, and the cells were rinsed three times with PBS. After rinsing, the mitochondrial stain MitoTracker Green FM (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 nuclear stain Hoechst 33342 (10 μM, 1 mL) was added to stain the cells for 10 minutes. The cells were washed 3 times with PBS, and finally, the co-localization of m-BDP with mitochondria after entering the cells was observed using a confocal microscope. (Mitochondrial stain: excitation wavelength 488 nm, detection wavelength 510 - 570 nm; nuclear probe: excitation wavelength 405 nm, detection wavelength 425 - 475 nm; m-BDP: excitation wavelength 633 nm, detection wavelength 650 - 750 nm).

[0077] Example 5:

[0078] Test the nuclear damage of the compound m-BDP prepared in Example 1 under different conditions. The specific operation is as follows:

[0079] Inoculate B16F10 cells in the logarithmic growth phase on the coverslips in a 12-well plate at an inoculation density of 2 × 10 5 / mL, and place them in a cell incubator for constant temperature culture for 12 hours. After determining that the cells have adhered, pour out the culture medium, wash twice with PBS, and add the m-BDP solution prepared with the culture medium, 1 mL per well. The concentration of each group is 2.00 μg mL -1 . After culturing in the incubator for 24 hours, change the culture medium. The light irradiation group ( hv ) is irradiated with a 660 nm LED lamp (50 mW cm -2 ) for 10 minutes under the condition, and then put back into the incubator for continuous culture for 2 hours. The dose of the X-ray irradiation group (+ X-ray) is 6 Gy, and the non-X-ray irradiation group (-X-ray) is not treated. After 6 hours, aspirate the culture medium, wash 3 times with PBS, add the fixing solution (4% paraformaldehyde), add 1 ml of the fixing solution to each well, and fix for 15 minutes. Aspirate the fixing solution and wash 3 times with PBS. Add the immunostaining permeabilization solution (Triton x 100) containing 5% bovine serum albumin and block overnight at 4°C. Aspirate the immunostaining blocking solution, add γ-H2AX rabbit monoclonal antibody (1:500), and incubate at 4°C for 12 hours. Wash 3 times with PBS, 5 - 10 minutes each time. Add the secondary antibody fluorescein isothiocyanate (FITC) of rabbit monoclonal antibody (1:200) and incubate at room temperature for 1 hour. Wash 3 times with PBS, 5 - 10 minutes each time, add the nuclear staining solution (DAPI), and stain at room temperature for about 5 minutes. Mount on a glass slide and observe the nuclear damage through a laser confocal microscope.

[0080] Example 6:

[0081] Mix 5 kDa HA, 50 kDa HA and the m-BDP aqueous solution in Example 1 according to a mass fraction of 1.5:1.5:7, stir and ultrasonically ensure uniform mixing in a sample bottle, and then centrifuge to remove air bubbles (2500 rpm, 10 minutes). At the same time, first evacuate the mold for 10 minutes, then drop the above mixed sample on the mold to form a micro-protrusion, and evacuate again for 5 minutes. Then use a pipette tip to remove air bubbles, place it in a desiccator and dry for 24 hours, and remove it from the mold to obtain MN-m-BDP soluble microneedles, which are placed in a drying oven for storage.

[0082] Example 7:

[0083] Mix 5 kDa HA, 50 kDa HA, and the m-BDP aqueous solution from Example 1 at a mass fraction of 2:1:7, stir and sonicate to ensure uniform mixing in a sample bottle, and then centrifuge to remove air bubbles (2500 rpm, 10 minutes). Meanwhile, first evacuate the mold for 5 minutes, then drop the above mixed sample onto the mold to form a slightly convex shape, and evacuate again for 10 minutes. Then use a pipette tip to remove air bubbles, place it in a desiccator and dry for 24 hours, and remove it from the mold to obtain MN-m-BDP soluble microneedles, which are stored in a drying oven.

[0084] Example 8:

[0085] Mix 5 kDa HA, 50 kDa HA, and the m-BDP aqueous solution from Example 1 at a mass fraction of 2:2:6, stir and sonicate to ensure uniform mixing in a sample bottle, and then centrifuge to remove air bubbles (2500 rpm, 10 minutes). Meanwhile, first evacuate the mold for 10 minutes, then drop the above mixed sample onto the mold to form a slightly convex shape, and evacuate again for 15 minutes. Then use a pipette tip to remove air bubbles, place it in a desiccator and dry for 24 hours, and remove it from the mold to obtain MN-m-BDP soluble microneedles, which are stored in a drying oven.

[0086] Example 9:

[0087] Mix 5 kDa HA, 50 kDa HA, and the m-BDP aqueous solution from Example 1 at a mass fraction of 1:3:6, stir and sonicate to ensure uniform mixing in a sample bottle, and then centrifuge to remove air bubbles (2500 rpm, 10 minutes). Meanwhile, first evacuate the mold for 10 minutes, then drop the above mixed sample onto the mold to form a slightly convex shape, and evacuate again for 5 minutes. Then use a pipette tip to remove air bubbles, place it in a desiccator and dry for 36 hours, and remove it from the mold to obtain MN-m-BDP soluble microneedles, which are stored in a drying oven.

[0088] Example 10:

[0089] Mix 5 kDa HA, 50 kDa HA, and the m-BDP aqueous solution from Example 1 at a mass fraction of 3:1:6, stir and sonicate to ensure uniform mixing in a sample bottle, and then centrifuge to remove air bubbles (2500 rpm, 10 minutes). Meanwhile, first evacuate the mold for 10 minutes, then drop the above mixed sample onto the mold to form a slightly convex shape, and evacuate again for 5 minutes. Then use a pipette tip to remove air bubbles, place it in a desiccator and dry for 48 hours, and remove it from the mold to obtain MN-m-BDP soluble microneedles, which are stored in a drying oven.

[0090] Example 11:

[0091] Mix 5 kDa HA, 50 kDa HA and the m-BDP aqueous solution in Example 1 at a mass fraction of 1.5:2.5:6, stir and ultrasonically ensure uniform mixing in a sample bottle, and then centrifuge to remove air bubbles (2500 rpm, 10 minutes). At the same time, first evacuate the mold for 15 minutes, then drop the above mixed sample on the mold to form a micro-protrusion, and evacuate again for 10 minutes. Then use a pipette tip to remove air bubbles, place it in a dryer and dry for 24 hours, remove it from the mold to obtain MN-m-BDP soluble microneedles, and store them in a drying oven.

[0092] Example 12:

[0093] Mix 5 kDa HA, 50 kDa HA and the m-BDP aqueous solution in Example 1 at a mass fraction of 2.5:1.5:6, stir and ultrasonically ensure uniform mixing in a sample bottle, and then centrifuge to remove air bubbles (2500 rpm, 10 minutes). At the same time, first evacuate the mold for 10 minutes, then drop the above mixed sample on the mold to form a micro-protrusion, and evacuate again for 20 minutes. Then use a pipette tip to remove air bubbles, place it in a dryer and dry for 24 hours, remove it from the mold to obtain MN-m-BDP soluble microneedles, and store them in a drying oven.

[0094] Application Example 1:

[0095] Select female C57BL / 6 tumor-bearing mice with subcutaneous melanoma (B16F10) with a tumor volume of about 80 mm 3 as an animal model. Randomly divide the mice into six groups: PBS (blank control group), X-ray (single X-ray irradiation group), MN-c-BDP (single microneedle patch group), MN-c-BDP / hv (microneedle patch plus light group), MN-c-BDP / X-ray (microneedle patch plus X-ray group), MN-c-BDP / hv / X-ray (microneedle patch plus light group plus X-ray group), with 5 mice in each group. The light group was irradiated with an LED lamp (wavelength: 660 nm, power: 50 mW cm -2 ) on the tumor site of the mice for 30 minutes 4 hours after microneedle administration, while the non-light group did not perform this operation. 12 hours after light irradiation, the X-ray irradiation group was anesthetized and irradiated with X-rays at a dose of 6 Gy. The mice were shielded with lead sheets except for the tumor site. During the treatment process, the body weight and tumor volume of the mice were recorded every 2 days, and the body weight change curve and tumor growth curve of the mice were plotted. When the tumor volume exceeded about 1500 mm 3 , the mice were considered dead and euthanized.

[0096] Effect Evaluation 1:

[0097] The photosensitizer m-BDP ( Figure 1 ) will be obtained by multi-step synthetic retrosynthesis in the present invention, and its structure was characterized by 1 HNMR nuclear magnetic resonance ( Figure 2 ). Next, the mitochondrial targeting ability ( Figure 3 ) (scale bar is 20 μm) (Merged represents the combination of the mitochondrial dye, nuclear dye, and m-BDP three channels) and radiosensitization test ( Figure 4 ) (scale bar is 10 μm) (Merged represents the combination of the γ-H2AX immunofluorescence dye and DAPI dye two channels) of the compound m-BDP on murine melanoma cells (B16F10) were carried out through cell experiments. Subsequently, the morphology of the microneedles was characterized ( Figure 5 ) (scale bar of A is 200 μm, scale bar of B is 400 μm). Finally, the antitumor experiment of the microneedle MN-m-BDP was carried out by constructing a subcutaneous female C57BL / 6 tumor-bearing mouse model of melanoma (B16F10) ( Figure 6 ).

[0098] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation modes. 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 modes here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A radiotherapy-sensitizing microneedle, characterized in that, It is obtained by drying the mixture of m-BDP and hyaluronic acid; The structural formula of the m-BDP is as follows: 。 2. The radiosensitizing microneedle according to claim 1, wherein The preparation method of the m-BDP is as follows: S11: Under nitrogen protection, add triethylene glycol monomethyl ether, p-toluenesulfonyl chloride and triethylamine into organic solvent I and react under ice bath, and then purify to obtain intermediate 1; The chemical formula of the intermediate 1 is as follows: ； S12: Under nitrogen protection, add the intermediate 1, 3,4-dihydroxybenzaldehyde and potassium carbonate into organic solvent II and heat and react at 85-95 °C for 44-52 hours, and then purify to obtain intermediate 2; The chemical formula of the intermediate 2 is as follows: ； S13: Dissolve the intermediate 2, 2,4-dimethylpyrrole, catalyst and 2,3-dichloro-5,6-dicyanobenzoquinone in organic solvent III, react at room temperature for 10-14 hours, then add triethylamine and boron trifluoride diethyl etherate, react under ice bath for 8-16 hours, and then add hydrochloric acid, and purify to obtain intermediate 3; The chemical formula of the intermediate 3 is as follows: ； S14: Under nitrogen protection, add the intermediate 3 and N-iodosuccinimide into organic solvent IV and react at room temperature for 3-5 hours, and then purify to obtain intermediate 4; The chemical formula of the intermediate 4 is as follows: ； S15: Under nitrogen protection, add the intermediate 4, pyridine-4-carboxaldehyde and piperidine acetate into organic solvent V and heat and react at 55-65 °C for 0.8-1.2 hours, and then purify to obtain intermediate 5; The chemical formula of the intermediate 5 is as follows: ； S16: Under nitrogen protection, add the intermediate 5 and iodomethane into organic solvent VI and heat and react at 45-55 °C for 8-12 hours, add ether for purification, and obtain the m-BDP.

3. The radiosensitizing microneedle according to claim 2, wherein 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 radiosensitizing microneedle according to claim 2, wherein In the steps S11, S12, S13 and S15, the purification method is extraction followed by column chromatography.

5. The radiosensitizing microneedle according to claim 2, wherein, In the step S14, the purification method is column chromatography; In the step S16, the method of ether purification is to filter after adding ether for 8-16 hours.

6. The radiosensitizing microneedle according to claim 4 or 5, wherein The column chromatography uses silica gel, and the eluent is selected from two of dichloromethane, methanol and petroleum ether.

7. The radiosensitizing microneedle according to claim 2, wherein The catalyst is selected from trifluoroacetic acid.

8. The radiosensitizing microneedle according to claim 2, wherein In the step S11, the molar ratio of triethylene glycol monomethyl ether to p-toluenesulfonyl chloride is 1:1-3; The mass of triethylamine is 2 times the mass of p-toluenesulfonyl chloride; In the step S12, the molar ratio of intermediate 1, 3,4-dihydroxybenzaldehyde to potassium carbonate is 1:2-4:6; In the step S13, the molar ratio of intermediate 2, 2,4-dimethylpyrrole to 2,3-dichloro-5,6-dicyanobenzoquinone is 1:2:2-6.

9. The radiosensitizing microneedle according to claim 2, wherein In the step S15, the molar ratio of intermediate 4, pyridine-4-carboxaldehyde to piperidine acetate is 1:10-20:10-20; In the step S16, the molar ratio of intermediate 5 to iodomethane is 1:

10.

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

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