A photoresponsive drug-releasing fluoroboron dipyrrole compound, its preparation method and application

Abstract

This invention discloses a photoresponsive drug-releasing fluoroboron dipyrrole compound, its preparation method, and its applications. Specifically, it relates to the synthesis of fluoroboron dipyrrole compounds that release immunotherapies in a photoresponsive manner, the preparation of nanomedicines, and their use in photoimmunotherapy for synergistic antitumor treatment. Using fluoroboron dipyrrole dye molecules as the core, immunotherapies are covalently coupled to sensitive groups, and further self-assembled into nanomicelles to load another immunotherapy, constructing a photoresponsive self-assembled nanomedicine that synergistically delivers multiple immunotherapies. Under illumination, the self-assembled nanomedicine exhibits excellent photodynamic effects and controlled release of immunotherapies. This invention has demonstrated its dual effects of photodynamic therapy and immunotherapy at the cellular and animal levels, achieving photoimmunotherapy-synergistic tumor ablation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a fluoroboron dipyrrole compound that releases a photoresponsive immunotherapeutic drug, its preparation method, and its application. Background Technology

[0002] Despite significant advancements in conventional cancer treatments such as surgery, chemotherapy, and radiotherapy, challenges remain in clinical cancer therapy, including tumor recurrence, drug resistance, and toxic side effects. In recent years, with the rapid development and interdisciplinary integration of immunology, molecular biology, and oncology, immunotherapy has emerged as a leading force in cancer treatment, becoming a hot topic. Immunotherapy primarily modulates the activation level of the body's overall immune system through systemic or local drug administration, effectively killing not only the primary tumor but also efficiently eliminating distant metastases. Furthermore, immunotherapy can inhibit tumor recurrence by generating long-term immune memory effects. Benefiting from these advantages, immunotherapy has achieved significant breakthroughs in the clinical practice of various cancers, including hematologic malignancies, greatly encouraging research enthusiasm in the field. In immunotherapy, immunotherapies often exert their anti-tumor effects by activating the body's innate or adaptive immunity, which is a key factor determining the strength of the immunotherapy's efficacy. However, the effects of immunotherapies are often non-selective, producing multi-level toxic side effects in normal parts of the body while generating anti-tumor effects. Therefore, developing tumor-site selectively activated immunotherapeutic drug delivery systems is a crucial issue that urgently needs to be addressed to achieve safe and effective immunotherapy.

[0003] Tumor development often creates unique microenvironments, such as hypoxia, low pH, high oxidative stress, and high levels of expressed enzymes. Targeting these unique physicochemical characteristics, researchers have designed various responsive release or activation immunodrug delivery systems. For example, delivery systems using carriers that respond to the high expression of cathepsins at the tumor site can selectively release STING agonists at the tumor site, activating the anti-tumor immune response while avoiding systemic toxicity. Besides endogenous stimuli, various exogenous stimuli, such as light, radiation, magnetic fields, and ultrasound, can also be used for the selective release or activation of immunodrugs at the tumor site (Nature, 2024, 630, 206). Moreover, compared to endogenous stimuli, exogenous stimuli can more precisely achieve spatiotemporal selective release or activation of immunodrugs. Among these, photoresponsive immunodrug delivery systems, especially those responding to near-infrared light with strong tissue penetration, have attracted considerable attention in recent years due to their advantages such as good safety and ease of operation. In the process of antitumor therapy using photoresponsive immunodrug delivery systems, the photophysicochemical properties of photoresponsive drugs, such as chemical structure, molecular weight, hydrophilicity / hydrophobicity, spectroscopic properties, and photosensitivity, have a decisive influence on the antitumor effect and are issues that must be considered in depth. Furthermore, existing immunodrug delivery systems that respond to near-infrared light with strong tissue penetration capabilities need improvement not only in inducing selective release of immunodrugs, but also in addressing the problems of leakage to non-target sites and the resulting toxic side effects. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a photoresponsive fluoroboron dipyrrole compound that releases an immunodrug. This compound contains a fluoroboron dipyrrole fragment, a reactive oxygen species-sensitive linker, an immunodrug, and a hydrophilic polymer chain. Nanoparticles prepared by self-assembly using this compound can respond to reactive oxygen species generated by near-infrared light excitation, break the sensitive linker, and release the immunodrug. It also has the advantages of good water solubility and stable properties, and exhibits good photoimmunoassay synergistic antitumor effects in tumors, such as various breast cancer and pancreatic cancer models.

[0005] The present invention adopts the following technical solution:

[0006] A photoresponsive drug delivery compound of the fluoroboron dipyrrole class has the following chemical structural formula:

[0007]

[0008] Wherein, R is a hydrophilic group; preferably, R is a hydrophilic polymer group, such as a polyethylene glycol group, a polyethylene glycol monomethyl ether group, a phospholipid-polyethylene glycol group, or a polyvinyl alcohol group. Preferably, the number average molecular weight of R is 1000 to 10000.

[0009] This invention discloses a method for preparing a photoresponsive drug-releasing fluoroboron dipyrrole compound, comprising the following steps: using an alkyne compound and an azide-containing R compound as raw materials, the photoresponsive drug-releasing fluoroboron dipyrrole compound is prepared by reaction; the chemical structural formula of the alkyne compound is as follows:

[0010]

[0011] The R compound containing azide is preferably a hydrophilic polymer containing azide.

[0012] In this invention, the preparation time of the photoresponsive drug-releasing fluoroboron dipyrrole compound is 5 to 100 hours, preferably 10 to 75 hours, and more preferably 15 to 50 hours.

[0013] This invention discloses a photoresponsive nanoparticle, the raw materials for which include the above-mentioned photoresponsive drug-releasing fluoroboron dipyrrole compound. Specifically, the photoresponsive nanoparticle is a nanomicelle with a particle size of 30-200 nanometers, preferably 40-120 nanometers, and more preferably 50-100 nanometers.

[0014] This invention discloses a method for preparing the above-mentioned photoresponsive nanoparticles, which are prepared by using the above-mentioned photoresponsive drug-releasing fluoroboron dipyrrole compound via thin film dispersion, solvent dialysis, or solvent evaporation. Specifically, the photoresponsive nanoparticles are prepared by self-assembly of the above-mentioned photoresponsive drug-releasing fluoroboron dipyrrole compound.

[0015] This invention discloses a drug system comprising the aforementioned photoresponsive drug-releasing fluoroboron dipyrrole compound or photoresponsive nanoparticles. Under illumination, the aforementioned photoresponsive drug-releasing fluoroboron dipyrrole compound or photoresponsive nanoparticles exhibit excellent photodynamic effects and controlled release of immunotherapeutic drugs. This invention has demonstrated its dual effects of photodynamic therapy and immunotherapy at the cellular and animal levels, achieving synergistic photoimmunotherapy for tumor ablation.

[0016] Furthermore, the aforementioned drug system also includes other drugs. Specifically, the photoresponsive drug-releasing fluoroboron dipyrrole compounds or photoresponsive nanoparticles of this invention possess dual effects of photodynamic therapy and immunotherapy, achieving synergistic photoimmunotherapy for tumor ablation. Furthermore, the aforementioned photoresponsive drug-releasing fluoroboron dipyrrole compounds or photoresponsive nanoparticles can be used in combination with other anti-tumor drugs, which are drugs with anti-tumor effects, including agonists such as STING agonists, which can be diABZI.

[0017] Furthermore, other drugs are independent of the aforementioned photoresponsive drug-releasing fluoroboron dipyrrole compounds or photoresponsive nanoparticles, forming a drug system; or other drugs are not independent of the aforementioned photoresponsive drug-releasing fluoroboron dipyrrole compounds or photoresponsive nanoparticles, forming a drug system. Preferably, the aforementioned photoresponsive drug-releasing fluoroboron dipyrrole compounds serve as carriers for other drugs, that is, a drug system is prepared by assembling the aforementioned photoresponsive drug-releasing fluoroboron dipyrrole compounds and other drugs as raw materials using thin-film dispersion, solvent dialysis, or solvent evaporation methods.

[0018] This invention discloses the use of the above-mentioned photoresponsive drug-releasing fluoroboron dipyrrole compounds as drug carriers or in the preparation of drug carriers.

[0019] This invention discloses the application of the above-mentioned photoresponsive drug-releasing fluoroboron dipyrrole compounds or photoresponsive nanoparticles as photosensitizers or immune agonists, or their application in the preparation of photosensitizing reagents or immune agonist reagents.

[0020] This invention discloses the application of the above-mentioned photoresponsive drug-releasing fluoroboron dipyrrole compounds or photoresponsive nanoparticles and drug systems in the preparation of drugs. Preferably, the drug is a drug for treating tumors, such as drugs for treating breast cancer or pancreatic cancer.

[0021] This invention covalently binds the immunodrug molecule R848 with a fluoroboron dipyrrole dye molecule that has near-infrared absorption and generates reactive oxygen species through a reactive oxygen species-sensitive thioclase group. Furthermore, it connects the hydrophilic chain through click chemistry, allowing it to directly self-assemble into a multimodal therapeutic nanodrug without the need for encapsulation by other carriers. The reactive oxygen species generated by the near-infrared excitation of the fluoroboron dipyrrole dye molecule can directly kill tumors on the one hand, and break the thioclase group on the other hand, achieving selective release of immunodrugs at the tumor site.

[0022] The present invention has at least the following advantages:

[0023] No existing technologies have reported the construction of photoresponsive immunodrug delivery systems based on fluoroboron dipyrrole derivatives via covalent coupling. This invention covalently links the immunodrug R848 to photoresponsive fluoroboron dipyrrole compounds or photoresponsive nanoparticles, solving the problems of low drug loading and drug leakage associated with conventional immunodrug encapsulation by macromolecular carriers. Near-infrared light precisely regulates drug release, addressing the common off-target toxicity issues of immunodrugs, as demonstrated by preliminary animal safety evaluations. The self-assembled nanodrug provides a photoresponsive nanodrug delivery platform, exhibiting significant long-lasting systemic circulation and highly efficient tumor targeting effects, as evidenced by pharmacokinetic and tissue distribution experiments. Furthermore, the invention introduces a multimodal treatment concept combining photodynamic therapy and immunodrugs, enhancing the synergistic anti-tumor effect, as also demonstrated in animal experiments. Attached Figure Description

[0024] Figure 1 This is a roadmap for synthesizing BTR in Embodiment 1 of this invention.

[0025] Figure 2 This is the proton NMR spectrum of the product BTR in Example 1 of this invention.

[0026] Figure 3 This is an electron microscope image of the BTR-NPs prepared in Example 2 of this invention.

[0027] Figure 4 This is an electron microscope image of dBTR-NPs prepared in Example 5 of this invention.

[0028] Figure 5 The ultraviolet (a) and fluorescence spectra (b) of the BTR obtained in Example 1 and the BTR-NPs obtained in Example 2 of this invention are shown.

[0029] Figure 6 This is the result of BTR-NPs prepared in Example 2 of this invention generating reactive oxygen species under light.

[0030] Figure 7 This is the result of the BTR-NPs prepared in Example 2 of this invention releasing R848 under light irradiation.

[0031] Figure 8 This describes the effect of BTR-NPs prepared in Example 2 of this invention on killing tumor cells under light irradiation.

[0032] Figure 9 This is the tumor targeting evaluation result of dBTR-NPs prepared in Example 5 of this invention.

[0033] Figure 10 The therapeutic effects of dBTR-NPs prepared in Example 5 of this invention on 4T1 in situ breast cancer tumors are as follows: (a) administration regimen, (b) experimental groups, (c) average tumor volume of each group, and (d) survival rate of mice in each group.

[0034] Figure 11 The therapeutic effects of dBTR-NPs prepared in Example 5 of this invention on subcutaneous pancreatic cancer PANC02 tumors are as follows: (a) administration regimen, (b) experimental groups, (c) average tumor volume of each group, and (d) survival rate of mice in each group.

[0035] Figure 12 This is the safety evaluation result of the dBTR-NPs prepared in Example 5 of this invention. Detailed Implementation

[0036] In the process of antitumor therapy using photoresponsive immunodrug delivery systems, the photophysicochemical properties of photoresponsive drugs, such as chemical structure, molecular weight, hydrophilicity / hydrophobicity, spectral properties, and photosensitivity, have a decisive influence on the antitumor efficacy and are issues that must be thoroughly considered. Furthermore, existing immunodrug delivery systems that respond to near-infrared light with strong tissue penetration capabilities require not only improvements in inducing selective release of immunodrugs but also solutions to address the toxic side effects caused by leakage of immunodrugs to non-target sites.

[0037] This invention discloses a fluoroboron dipyrrole compound that releases a photoresponsive immunodrug, the chemical structural formula of which is as follows:

[0038]

[0039] In this process, the hydrophilic polymer chain R is a polyethylene glycol group, a polyethylene glycol monomethyl ether group, a phospholipid-polyethylene glycol group, or a polyvinyl alcohol group; furthermore, the molecular weight of R is 1000 to 10000.

[0040] This invention discloses a method for preparing the above-mentioned photoresponsive drug-releasing fluoroboron dipyrrole compound, comprising the following steps:

[0041] (1) The compound shown in formula (II) was reacted with the immune agonist R848 to obtain the R848 derivative with a thioketone chain group at the end shown in formula (III);

[0042] (2) The R848 derivative with a thioketone chain group at the end shown in formula (III) reacts with the fluoroboron dipyrrole derivative shown in formula (IV) to obtain the compound shown in formula (V);

[0043] (3) By reacting the alkynyl group on the compound shown in formula (V) with a hydrophilic polymer containing azide, a photoresponsive drug-releasing fluoroboron dipyrrole compound is obtained;

[0044] The chemical structural formulas of the above compounds are as follows:

[0045]

[0046]

[0047] Wherein, the hydrophilic polymer chain R is a polyethylene glycol group, a polyethylene glycol monomethyl ether group, a phospholipid-polyethylene glycol group, or a polyvinyl alcohol group. Correspondingly, the hydrophilic polymer containing azide is polyethylene glycol containing azide, polyethylene glycol monomethyl ether containing azide, phospholipid-polyethylene glycol containing azide, or polyvinyl alcohol containing azide.

[0048] Specifically, this invention discloses the synthesis of a fluoroboron dipyrrole compound for photoresponsive release of immunotherapies and its novel applications in pharmaceuticals. Specifically, it relates to the synthesis of the fluoroboron dipyrrole compound for photoresponsive release of immunotherapies, the preparation of nanomedicines, and their application in photoimmunotherapy for synergistic antitumor treatment. Using a fluoroboron dipyrrole dye molecule as the core, immunotherapies are covalently coupled to sensitive groups, and further self-assembled into nanomicelles to load another immunotherapy, constructing a photoresponsive self-assembled nanomedicine that synergistically delivers multiple immunotherapies. Under illumination, the self-assembled nanomedicine exhibits excellent photodynamic effects and controlled release of immunotherapies. This invention has demonstrated its dual effects of photodynamic therapy and immunotherapy at the cellular and animal levels, achieving photoimmunotherapy-synergistic tumor ablation.

[0049] The above-mentioned photoresponsive drug-releasing fluoroboron dipyrrole compounds or photoresponsive nanoparticles disclosed for the first time in this invention have dual effects of photodynamic therapy and immunotherapy, realizing photoimmunotherapy synergistic tumor ablation; the above-mentioned photoresponsive drug-releasing fluoroboron dipyrrole compounds or photoresponsive nanoparticles can also be used in combination with other drugs that can fight tumors, such as STING agonists, which can be diABZI.

[0050] Furthermore, other drugs are independent of the aforementioned photoresponsive drug-releasing fluoroboron dipyrrole compounds or photoresponsive nanoparticles, forming a drug system; or other drugs are not independent of the aforementioned photoresponsive drug-releasing fluoroboron dipyrrole compounds or photoresponsive nanoparticles, forming a drug system. Preferably, the aforementioned photoresponsive drug-releasing fluoroboron dipyrrole compounds serve as carriers for other drugs, that is, a drug system is prepared by assembling the aforementioned photoresponsive drug-releasing fluoroboron dipyrrole compounds and other drugs as raw materials using thin-film dispersion, solvent dialysis, or solvent evaporation methods.

[0051] The above description is merely an overview of the technical solution of the present invention. To better understand the technical means of the present invention and to facilitate its implementation according to the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. The specific implementation methods of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are used to illustrate the present invention but are not intended to limit the scope of the invention. The raw materials involved in the present invention are all existing products, and the specific preparation operations and performance tests are all conventional techniques. Animal experiments meet the relevant requirements of Suzhou University. Differences between two groups were analyzed using a t-test or a one-sided ANOVA test, with *P<0.05, **P<0.01, and ***P<0.001. Example 1

[0052] See Figure 1 The specific steps for preparing a fluoroboron dipyrrole compound for a photoresponsive release immunodrug with R being a polyethylene glycol group (molecular weight 5000) are as follows:

[0053] 4-Hydroxybenzaldehyde, potassium carbonate, and potassium iodide were dissolved in acetonitrile at a molar ratio of 1:1.5:0.1 in 50 times the mass of 4-hydroxybenzaldehyde. The mixture was stirred at room temperature, and then tert-butyl 2-bromoacetate was added dropwise at a molar ratio of 1:1.5 to 4-hydroxybenzaldehyde. The mixture was refluxed for 8 hours, then cooled to room temperature, filtered, and the filter cake was washed with ethyl acetate. The filtrate was mixed and separated by column chromatography (petroleum ether:ethyl acetate (V:V) = 10:1-5:1) to obtain a brown solid, which was compound 1, with a yield of 89%.

[0054] Compound 1 was dissolved in dichloromethane, and an equal volume of trifluoroacetic acid was added. The mixture was stirred at room temperature for 24 hours, filtered, and the filter cake was washed with n-hexane to obtain a white solid, which was compound 2, with a yield of 97%.

[0055] R848 and carbonyl diimidazole in a molar ratio of 1:1.5 were dissolved in dichloromethane at a mass of 30 times that of R848. The mixture was stirred at room temperature for 8 hours. Then, compound 3 (in a molar ratio of 1:2 to R848) was added and the reaction was continued for 24 hours. The solvent was evaporated under pressure and purified by column chromatography (ethyl acetate) to obtain a yellow solid, which was compound 4, with a yield of 80%.

[0056] 3,5-Diiodo-4-hydroxybenzaldehyde and 2,4-dimethylpyrrole were placed in a flask at a molar ratio of 1:2. A solution of dichloromethane, 50 times the mass of 3,5-diiodo-4-hydroxybenzaldehyde, was added to dissolve them. Four drops of trifluoroacetic acid were added dropwise. After stirring at room temperature in the dark for 12 hours, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (molar ratio of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to 3,5-diiodo-4-hydroxybenzaldehyde was added, and stirring continued at room temperature for another 12 hours. The reaction mixture was then transferred to an ice bath, and triethylamine (molar ratio of 2,3-diiodo-4-hydroxybenzaldehyde to 3,5-diiodo-4-hydroxybenzaldehyde to 1:40) was added. Boron trifluoride diethyl ether (molar ratio of 2,3-diiodo-4-hydroxybenzaldehyde to 3,5-diiodo-4-hydroxybenzaldehyde to 1:40) was added dropwise. After the addition was complete, the mixture was reacted at room temperature for 12 hours. The mixture was then evaporated to dryness under reduced pressure, mixed with silica gel, and eluted by column chromatography to obtain a yellowish-brown solid, which was compound 5, with a yield of 51%.

[0057] Compound 5, bromoacetonitrile, and potassium carbonate were dissolved in acetone (10 times the mass of compound 6) at a molar ratio of 1:1.5:2. The mixture was refluxed for 2 hours, cooled to room temperature, filtered through diatomaceous earth, the filter cake was washed with dichloromethane, and the filtrate was evaporated, stirred, and eluted by column chromatography to obtain a yellow solid, which was compound 6, with a yield of 96%.

[0058] Compound 6 was dissolved in 10 times its mass of anhydrous ethanol. Iodine (in a molar ratio of 1:2.5 to compound 6, dissolved by sonication in 2 times its mass of anhydrous ethanol) was added to obtain a reaction solution. Iodic acid (in a molar ratio of 1:2 to compound 6) was weighed and dissolved by sonication in water (0.5 times its mass of iodic acid), and added dropwise to the reaction solution. The mixture was then stirred at room temperature in the dark for 15 hours. A saturated sodium thiosulfate solution (20 times its mass of compound 6) was added, and the mixture was stirred at room temperature for 30 minutes. The mixture was then extracted three times with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate for 1 hour, evaporated under reduced pressure, mixed, and separated by column chromatography to obtain a red solid, which was compound 7, with a yield of 59.6%.

[0059] Compound 7, compound 2, acetic acid, and piperidine were placed in a reactor in a molar ratio of 1:4:50:25. Acetonitrile of 20 times the mass of compound 7 was added. The mixture was stirred at room temperature for 1 hour, refluxed for 3 hours, and allowed to cool naturally to room temperature. The mixture was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The mixture was stirred and eluted by column chromatography to obtain a green solid, which was compound 8, with a yield of 70%.

[0060] Compound 8, compound 4, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, and N,N-diisopropylethylamine were placed in a reactor at a molar ratio of 1:2.2:2.5:4. Tetrahydrofuran, at a mass ratio of 100 times that of compound 8, was added, and the mixture was stirred at room temperature for 18 hours. The reaction was then quenched with water, and the mixture was extracted three times with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, and the mixture was stirred under reduced pressure. The mixture was then separated by column chromatography to obtain a green solid, which was compound 9, with a yield of 80%.

[0061] Compound 9 (80 mg, 0.06 mmol), monomethyl PEG 5000 Copper sulfate pentahydrate (317 mg, 0.06 mmol), copper sulfate pentahydrate (19 mg, 0.076 mmol), and sodium antiscorbutate (25 mg, 0.126 mmol) were dissolved in 5 mL of dimethyl sulfoxide (DMSO) under nitrogen protection and stirred at room temperature for 24 h. The DMSO was then removed by freeze-drying. The solid was purified by column chromatography (dichloromethane:methanol = 50:1-10:1) and dried under reduced pressure to obtain 330 mg (69%) of a green solid, named BTR. Figure 2 Its NMR spectrum: 1H NMR (400MHz, CDCl3) δ8.10(d, J =18.3 Hz, 4H), 7.72(d, J = 30.5 Hz, 2H), 7.65– 7.40(m,10H), 7.02 – 6.78(m, 5H), 4.68(d, J = 7.9 Hz, 18H), 4.39(d, J = 6.1 Hz, 9H), 3.99(s, 5H), 2.96(dd, J = 15.6, 6.6 Hz, 8H), 1.79 – 1.41(m, 24H), 1.38 – 1.01(m, 39H). Example 2

[0062] Photoresponsive nanomicelles were prepared using a solvent dialysis method. The specific steps were as follows: 10.0 mg of BTR compound was weighed and dissolved in 500 μL of dimethyl sulfoxide. The solution was sonicated at 400 W for 2 seconds, with a 2-second interval, for a total of 2 minutes. Then, 15 mL of ultrapure water was added after 2 minutes, and sonication continued for another 10 minutes. The solution was then placed in a dialysis bag with a molecular weight cutoff of 13,000 Daltons and dialyzed in 2 L of ultrapure water. The solution was changed at 2, 4, 6, 8, and 24 hours. After dialysis, the green liquid in the ultrafiltration bag was filtered through a 0.45 μm filter membrane to obtain the photoresponsive nanomicelles BTR-NPs. The size distribution and morphology of the obtained nanomicelles were characterized using electron microscopy. The average particle size was approximately 54.4 nm. The results are shown below. Figure 3 As shown. Example 3

[0063] The steps in this embodiment are basically the same as those in Embodiment 2, except that this embodiment uses a thin-film dispersion method to prepare photoresponsive nanomicelles. The specific steps are as follows: 2 mg of sample is weighed and dissolved in 5 mL of tetrahydrofuran. The tetrahydrofuran is removed by rotary evaporation, allowing the material to form a thin film at the bottom of the flask. Then, 10 mL of water is added under ultrasonic conditions to dissolve the nanomicelles. Example 4

[0064] The steps in this embodiment are basically the same as those in Embodiment 2, except that this embodiment uses a solvent evaporation method to prepare photoresponsive nanomicelles. The specific steps are as follows: Weigh 2 mg of the sample, dissolve it in 1 mL of tetrahydrofuran, and add it dropwise to 10 mL of water under magnetic stirring, while stirring at 40°C until the acetone evaporates completely. Example 5

[0065] Photoresponsive nanomicelles loaded with the STING agonist diABZI were prepared using a solvent dialysis method. The specific steps were as follows: 10.0 mg of solid BTR was weighed and dissolved in 500 μL of dimethyl sulfoxide (DMSO); 1.50 mg of the STING agonist diABZI was weighed and dissolved in 500 μL of DMSO. The two solutions were then mixed thoroughly and sonicated at 400 W for 2 seconds, with a 2-second interval, for a total of 2 minutes. After 2 minutes, 15 mL of ultrapure water was added, and sonication continued for another 10 minutes. The mixture was then placed in a dialysis bag with a molecular weight cutoff of 13,000 Daltons and immersed in 2 L of ultrapure water for dialysis. The solution was changed at 2, 4, 6, 8, and 24 hours. After dialysis, the green liquid in the ultrafiltration bag was filtered through a 0.45 μm filter membrane to obtain photoresponsive nanomicelles dBTR-NPs loaded with the STING agonist diABZI. The particle size of dBTR-NPs was approximately 95.8 nm. The transmission electron microscopy (TEM) images of dBTR-NPs are shown below. Figure 4 As shown, the diABZI loading in dBTR-NPs was 9.4%.

[0066] The performance of the nanomicelles prepared in this invention was tested as follows:

[0067] 1. The absorption and fluorescence spectra of the polymer (BTR) and nanomicelles (BTR-NPs) synthesized in this invention were tested. The specific steps were as follows: 2.0 mg of the polymer was accurately weighed and dissolved in 1 mL of N,N-dimethylformamide, diluted to a final concentration of 15 μmol / L, and the entire wavelength range (250-900 nm) was scanned using a UV-Vis spectrophotometer. Then, the fluorescence emission spectrum (excitation wavelength 660 nm, emission wavelength 680-900 nm) was scanned using a fluorescence spectrophotometer. Nanomicelles of the same concentration were prepared, diluted to 15 μmol / L with ultrapure water, and the absorption and emission spectra of each nanoparticle were scanned under the same UV and fluorescence spectrophotometric conditions. The results are as follows: Figure 5 As shown, in the ultraviolet (a) and fluorescence spectra (b), the absorption and fluorescence peaks of BTR are at 666 nm and 715 nm, respectively; while the absorption peak of BTR-NPs is significantly red-shifted and the fluorescence peak is almost completely quenched, indicating that BTR has successfully self-assembled to form a nanostructure.

[0068] 2. Testing of the reactive oxygen species (ROS) yield of the nanomicelles (BTR-NPs) prepared in this invention. The specific steps were as follows: 2.97 mL of nanomicelles of a certain concentration were measured and mixed with a 0.3 mmol (30 μL) solution of 1,3-diphenylisobenzofuran (DPBF). The mixture was placed on a magnetic stirrer and irradiated with 660 nm light at 0.5 W / cm². The UV absorption at 415 nm for each sample was scanned at 0, 5, 10, 15, 20, 25, and 30 seconds. The above experiment was repeated using zinc phthalocyanine as a control compound, and the ROS quantum yield of the nanomicelles was calculated. The results are as follows: Figure 6 As shown, the absorbance of DPBF decreased significantly with prolonged illumination, indicating the generation of reactive oxygen species. Further calculations revealed that the reactive oxygen quantum yield of BTR-NPs was 0.28.

[0069] 3. Testing the light-responsive release of R848 from the nanomicelles (BTR-NPs) prepared in this invention. The specific steps are as follows: A certain concentration of nanomicelles was diluted to 100 μmol / L with pH 5.0 buffer (50 mmol / L sodium acetate, pH 5.0). 500 μL of this solution was transferred to a 1.5 mL centrifuge tube and irradiated for 20 minutes with lasers of different intensities (0, 0.15, 0.3, 0.5 W / cm²) at 660 nm. At 0, 5, 10, 15, and 20 minutes, 20 μL of the sample was collected and placed in a 1.5 mL centrifuge tube. 180 μL of dimethyl sulfoxide was added and mixed. After centrifugation (12000 rpm, 5 minutes), 150 μL of the supernatant was collected and placed in an inner liner tube for detection using high-performance liquid chromatography (HPLC). The R848 release amount was calculated based on the standard curve. The results are as follows. Figure 7 As shown in the figure. The results show that R848 is not released in the absence of light; however, under light conditions, R848 is released rapidly, and the cumulative amount of R848 released is positively correlated with the light intensity.

[0070] 4. Testing the tumor cell killing effect of the nanomicelles (BTR-NPs) prepared in this invention under light irradiation. The specific steps are as follows: 4T1 breast cancer cells in the logarithmic growth phase are collected and seeded in 96-well plates at a seeding density of 1 × 10⁶ cells per well. 4 Cells were cultured in a CO2 incubator for 12 hours to allow them to adhere. Then, the culture medium in the 96-well plates was removed, and 100 μL of medium containing nanomicelles was added to each well, creating light-controlled and non-light-controlled groups. In the light-controlled group, after 12 hours of drug administration, the drug-containing medium was discarded, replaced with basal medium, and the cells were exposed to 660 nm light at 0.3 W / cm² for 4 minutes. After 24 hours of culture, the medium was discarded, and MTT solution (5.0 mg / mL, 20.0 μL) was added to each well for 4 hours. In the non-light-controlled group, after 24 hours of drug administration, the cells were incubated with MTT for 4 hours, the solution was discarded, and 100.0 μL of dimethyl sulfoxide was added to each well. The cells were shaken for 10 minutes, and the absorbance at 490 nm was measured using a microplate reader. Cell viability was calculated. Results are as follows: Figure 8 As shown in the figure. The results showed that BTR-NPs had almost no cytotoxicity in the absence of light; however, under light conditions, cytotoxicity increased significantly with increasing BTR-NPs concentration, with a calculated half-maximal inhibitory concentration (IC50) of 0.32 μmol / L. The tumor cell killing effect of the BTR-NPs disclosed in this invention is significantly improved compared to the fluoroboron dipyrrole derivative nanomicelles (IC50 of 2.0–5.1 μmol / L) previously disclosed by the applicant.

[0071] 5. The tumor-targeting efficiency of the diABZI-loaded photoresponsive nanomicelles (dBTR-NPs) prepared in this invention was tested in mice. The specific steps were as follows: 4T1 cells in good condition and in the logarithmic growth phase were collected, with a cell concentration of 2 × 10⁻⁶ cells / cells. 7 50 μL of cell suspension was injected into the third and fourth mammary pads of 6-8 week old Balb / c mice. Tumors grew to 80-100 mm in 6-7 days. 3 200 μL of 400 μmol / L nanomicelles were injected into tumor-bearing mice via the tail vein. Twenty-four hours later, the mice were euthanized by dislocation, and their heart, liver, spleen, lungs, kidneys, tumor, and lymph nodes were harvested, weighed, and recorded. These organs were then placed in 10 mL centrifuge tubes, shredded, and homogenized with 1 mL of ultrapure water for 3 minutes. 1 mL of organic phase (methanol:dichloromethane = 10:1) was added, vortexed for 3 minutes, and centrifuged (6000 rpm, 3 minutes). The lower organic phase was collected, extracted three times, and the combined organic phases were evaporated in a fume hood. 1 mL of dimethyl sulfoxide was added, dissolved by sonication, vortexed for 3 minutes, and centrifuged (12000 rpm, 3 minutes). The supernatant was collected, and the fluorescence spectrum was tested. The drug concentration was calculated using a labeled curve, and the tumor-targeting efficiency of the nanomicelles in mice was further calculated. Results are as follows: Figure 9 As shown in the figure. The results showed that dBTR-NPs were mainly distributed in the liver, spleen, and tumor in tumor-bearing mice, with a tumor enrichment efficiency of up to 10.6% of the injection dose / g (ID% g). -1 This invention is the first to covalently couple a fluoroboron dipyrrole core with an immunomodulatory drug, achieving efficient drug loading while reducing drug leakage in the bloodstream; simultaneously, at the tumor site, light exposure generates reactive oxygen species, breaking the covalent bonds and releasing the drug.

[0072] 6. The efficacy of the diABZI-loaded photoresponsive nanomicelles (dBTR-NPs) prepared in this invention for treating 4T1 orthotopic breast cancer was tested. The specific steps were as follows: 4T1-Luc cells in good condition and in the logarithmic growth phase were collected at a cell concentration of 2 × 10⁻⁶. 7 / ml, inject 50 μL of cell suspension into the third and fourth mammary pads of 6-8 week old Balb / c mice. After the tumor grows to 80-100 cubic millimeters, inject 200 μL of drug or control via tail vein on days 1, 4, and 7. Expose to light (0.3 W / cm², 8 minutes) on days 2, 5, and 8. The dosing regimen is as follows. Figure 10As shown in Figure a. To demonstrate the superiority of nanomicelles, a mixture of free diABZI, free R848, and the fluoroboron dipyrrole compound shown in formula (VI) (abbreviated as d / R / B mixture, with a molar ratio of 1.1:1:2) was used as one of the controls. The experimental mice were divided into the following 5 groups: PBS control group, dBTR-NPs group, d / R / B mixture light irradiation group, dBTR-NPs light irradiation group, and dBTR-NPs light irradiation group, as shown in Figure a. Figure 10 As shown in b, the dosage of the fluoroboron dipyrrole derivative in each group was 4.0 μmol per kilogram. The tumor volume in mice was measured using vernier calipers (tumor volume = (length × width)). 2 ) / 2, such as Figure 10 As shown in c), and plot the survival curves for each group of mice (as shown in c). Figure 10 (As shown in d). The results showed that, compared with free drug, dBTR-NPs significantly inhibited the growth of 4T1 in situ breast cancer tumors and significantly prolonged the survival of mice. More importantly, the fluoroboron dipyrrole derivative nanomicelles of the present invention, at a dosage of 4.0 μmol / kg, can almost completely eliminate the 4T1 in situ tumor model, and the effect is significantly better than the fluoroboron dipyrrole derivative nanomicelles previously disclosed by the applicant.

[0073]

[0074] 7. The efficacy of the diABZI-loaded photoresponsive nanomicelles (dBTR-NPs) prepared in this invention in treating pancreatic cancer PANC02 subcutaneous tumors was tested. The specific steps were as follows: Panc02 cells in good condition and in the logarithmic growth phase were collected at a cell concentration of 6 × 10⁻⁶. 7 / ml, inject 50 μL of cells into the back thigh of 6-8 week old C57 mice. After the tumor grows to 80 cubic millimeters, inject 200 μL of the drug or control via the tail vein on days 1, 4, and 7. Expose to light (0.3 W / cm², 8 minutes) on days 2, 5, and 8. The dosing regimen is as follows. Figure 11 As shown in a. The experimental mice were divided into the following 4 groups: PBS control group, d / R / B mixture group (same as above), dBTR-NPs group, and dBTR-NPs light irradiation group, as shown in a. Figure 11 As shown in b, the dosage of the fluoroboron dipyrrole derivative in each group was 4.0 μmol per kilogram. The tumor volume in mice was measured using vernier calipers (tumor volume = (length × width)). 2 ) / 2, such as Figure 11 (as shown in c), and plot the survival curves for each group of mice, as shown. Figure 11 As shown in d. The results showed that, compared with the free drug, dBTR-NPs significantly inhibited the growth of pancreatic cancer Panc02 subcutaneous tumors and significantly prolonged the survival of mice.

[0075] 8. Safety testing of the diABZI-loaded photoresponsive nanomicelles (dBTR-NPs) prepared in this invention. The specific steps are as follows: collect healthy 4T1 cells in the logarithmic growth phase, with a cell concentration of 2 × 10⁻⁶. 7 / ml, inject 50 μL of cell suspension into the third and fourth mammary pads of 6-8 week old Balb / c mice. When the tumors grow to 80-100 cubic millimeters in 6-7 days, they are treated in the following 5 groups: PBS control group, dBTR-NPs group, d / R / B mixture light irradiation group (same as above), BTR-NPs light irradiation group, and dBTR-NPs light irradiation group. 200 μL of drug or control is injected into the tail vein on days 1, 4, and 7. Light irradiation (0.3 W / cm², 8 min) is performed on days 2, 5, and 8. The drug dosage is diABZI (3 mg / kg), R848 (2.5 mg / kg), and fluoroboron dipyrrole derivative (4 μmol / kg). The day after the injection, the patient was euthanized, and the heart, liver, spleen, lungs, kidneys, and tumor were removed. The tissues were embedded in 4% paraformaldehyde and prepared into sections. The sections were then stained with hematoxylin and eosin, and the pathological morphological changes of the stained tissue sections were observed under a fluorescence upright microscope. Figure 12 The results showed that under light irradiation, dBTR-NPs could induce significant apoptosis at the tumor site, but had no effect on normal tissues such as the heart, liver, spleen, lungs, and kidneys.

[0076] This invention actively researches and innovates, creating a near-infrared light-responsive fluoroboron dipyrrole compound that releases immunotherapies. This compound self-assembles into drug-loaded nanomicelles, enabling efficient delivery and controlled release of immunotherapies for use in photoimmunotherapy of malignant tumors such as breast cancer and pancreatic cancer. In this invention, the photoresponsive fluoroboron dipyrrole compound exhibits excellent photophysical properties such as a high extinction coefficient and tunable absorption spectrum. Furthermore, it can be photoactivated to generate highly reactive reactive oxygen species (ROS), which can directly kill tumor cells and attack ROS-sensitive groups, regulating the responsive release of drugs. This makes it highly suitable for constructing photoresponsive immunotherapies. Moreover, this invention solves the problems of leakage of immunotherapies from non-target sites and toxic side effects that inevitably occur with existing technologies using photoresponsive carriers to physically encapsulate immunotherapies.

Claims

1. A photoresponsive drug-releasing fluoroboron dipyrrole compound, characterized in that, The chemical structural formula of the photoresponsive drug-releasing fluoroboron dipyrrole compound is as follows: ; Where R is a hydrophilic polymer group.

2. The method for preparing the photoresponsive drug-releasing fluoroboron dipyrrole compound according to claim 1, characterized in that, The photoresponsive drug-releasing fluoroboron dipyrrole compound was prepared by reacting alkyne compounds and compounds containing azide and hydrophilic polymer groups R as raw materials; the chemical structural formula of the alkyne compound is as follows: 。 3. A photoresponsive nanoparticle, characterized in that, The raw materials for preparing the photoresponsive nanoparticles include the photoresponsive drug-releasing fluoroboron dipyrrole compound as described in claim 1.

4. The method for preparing the photoresponsive nanoparticles according to claim 3, characterized in that, The photoresponsive drug-releasing fluoroboron dipyrrole compound of claim 1 is prepared by thin-film dispersion, solvent dialysis or solvent evaporation.

5. A pharmaceutical system, characterized in that, It includes the photoresponsive drug-releasing fluoroboron dipyrrole compound of claim 1 or the photoresponsive nanoparticle of claim 3.

6. The pharmaceutical system according to claim 5, characterized in that, It also includes other medications.

7. The use of the photoresponsive drug-releasing fluoroboron dipyrrole compound of claim 1 or the photoresponsive nanoparticle of claim 3 in the preparation of photosensitizing reagents or immunostimulatory reagents.

8. The use of the photoresponsive drug-releasing fluoroboron dipyrrole compound of claim 1, the photoresponsive nanoparticle of claim 3, or the drug system of claim 5 in the preparation of a drug.

9. The use of the photoresponsive drug-releasing fluoroboron dipyrrole compound of claim 1 in the preparation of drug carriers.

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