Photo-response drug-release BODIPY compound as well as preparation method and application thereof

Abstract

The invention discloses a light response drug release BODIPY compound as well as a preparation method and application thereof, and particularly relates to synthesis of the BODIPY compound of a light response drug release immune drug, preparation of a nano-drug and application of the nano-drug in light immune synergistic antitumor. The preparation method comprises the following steps: covalently coupling an immune drug through a sensitive group, further forming a nano-micelle through self-assembly, loading another immune drug, and constructing the photoresponsive self-assembled nano-drug for synergistically delivering multiple immune drugs. Under an illumination condition, the self-assembled nano-drug shows an excellent photodynamic effect and an immune drug controllable release behavior. The dual effects of photodynamic therapy and immunotherapy are verified at the cell and animal level, and light-immune synergistic tumor ablation is realized.

Description

Technical Field

[0001] The invention belongs to biomedical technology, and specifically relates to a fluoroboron dipyrrole compound capable of releasing immune drugs in response to light, and a preparation method and application thereof. Background Art

[0002] Although conventional tumor treatment techniques, such as surgery, chemotherapy and radiotherapy, have made significant progress, clinical tumor treatment still faces challenges such as tumor recurrence, drug resistance and toxic side effects. In recent years, with the rapid development and cross-integration of immunology, molecular biology and oncology, immunotherapy has emerged in the field of tumor treatment and has become a hot topic in the field of tumor treatment. Immunotherapy mainly regulates the activation level of the body's systemic immune system through systemic or local medication, which not only effectively kills primary tumors, but also efficiently eliminates distant metastases; and immunotherapy can inhibit tumor recurrence by producing long-term immune memory effects. Benefiting from the above advantages, immunotherapy has made significant breakthroughs in the clinical practice of various tumors such as hematological tumors, which has greatly inspired research enthusiasm in the field. In immunotherapy, immune drugs often produce anti-tumor effects by activating the body's innate or adaptive immunity, which is a key factor in determining the effectiveness of immunotherapy. However, the effects of immune drugs are usually lacking in selectivity. While producing anti-tumor effects, they also produce multi-level toxic and side effects in normal parts of the body. Therefore, the development of an immune drug delivery system that selectively activates tumor sites is a key issue that needs to be solved to achieve safe and effective immunotherapy.

[0003] The occurrence and development of tumors often form a unique microenvironment, such as hypoxia, low pH, high oxidative stress, and high expression enzyme levels. In response to these unique physical and chemical characteristics, researchers have designed a variety of responsive release or activation of immune drug delivery systems, such as using a carrier that responds to the high expression of tissue proteases in the tumor site to construct a delivery system, which can selectively release STING agonists at the tumor site, activating anti-tumor immune responses while avoiding systemic toxic side effects. In addition to endogenous stimuli, a variety of exogenous stimuli, such as light, radiation, magnetic fields, ultrasound, etc., can also be used to selectively release or activate immune drugs at the tumor site (Nature, 2024, 630, 206). Moreover, compared with endogenous stimuli, exogenous stimuli can more accurately achieve the spatiotemporal selective release or activation of immune drugs. Among them, light-responsive immune drug delivery systems, especially immune drug delivery systems that can respond to near-infrared light with strong tissue penetration ability, have attracted much attention in recent years due to their advantages such as good safety and convenient operation. In the process of anti-tumor treatment with photoresponsive immunodrug delivery system, the photophysical and chemical properties of photoresponsive drugs, such as chemical structure, molecular weight, hydrophilicity, spectral properties and photosensitivity, have a decisive influence on the anti-tumor effect and are issues that must be considered in depth. In addition, the existing immunodrug delivery system that can respond to near-infrared light with strong tissue penetration ability needs to be improved in addition to the performance of inducing selective release of immunodrugs. The problem of immunodrug leakage in non-target sites and toxic side effects also needs to be solved. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a fluoroboron dipyrrole compound that releases immune drugs in response to light. The compound contains fluoroboron dipyrrole fragments, active oxygen sensitive linking groups, immune drugs and hydrophilic polymer chains. The nanomedicine prepared by self-assembly of the compound can respond to active oxygen generated by near-infrared light excitation, break sensitive linking groups, release immune drugs, and has the advantages of good water solubility and stable properties. It shows good photoimmune synergistic anti-tumor effects in tumors, such as various breast cancer and pancreatic cancer models.

[0005] The present invention adopts the following technical solution: A photoresponsive drug-releasing fluoroboron dipyrrole compound having the following chemical structural formula:

[0006] 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.

[0007] The present invention discloses a method for preparing a photoresponsive drug-releasing fluoroboron dipyrrole compound, comprising the following steps: using an alkynyl compound and an azide-containing R compound as raw materials to react and prepare the photoresponsive drug-releasing fluoroboron dipyrrole compound; the chemical structural formula of the alkynyl compound is as follows:

[0008] The azide-containing R compound is preferably an azide-containing hydrophilic polymer.

[0009] In the present invention, the time for preparing the light-responsive drug-releasing fluoroboron dipyrrole compound is 5 to 100 hours, preferably 10 to 75 hours, and more preferably 15 to 50 hours.

[0010] The invention discloses a photoresponsive nanoparticle, the raw materials for preparing the nanoparticle include the photoresponsive drug-releasing fluoroboron dipyrrole compound. Specifically, the photoresponsive nanoparticle is a nanomicelle, and the particle size is 30 to 200 nanometers, preferably 40 to 120 nanometers, and further preferably 50 to 100 nanometers.

[0011] The present invention discloses a method for preparing the above-mentioned light-responsive nanoparticles, which are prepared by using the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds through a thin film dispersion method, a solvent dialysis method or a solvent volatilization method, specifically, the responsive nanoparticles are prepared by self-assembly of the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds.

[0012] The present invention discloses a drug system, including the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compound or light-responsive nanoparticles. Under light conditions, the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compound or light-responsive nanoparticles exhibit excellent photodynamic effect and controlled release behavior of immune drugs. The present invention confirms its dual effects of photodynamic therapy and immunotherapy at the cell and animal levels, and realizes photoimmunosynergistic tumor ablation.

[0013] Furthermore, the drug system also includes other drugs. That is, the light-responsive drug-releasing fluoroboron dipyrrole compounds or light-responsive nanoparticles of the present invention have the dual effects of photodynamic therapy and immunotherapy, and achieve photo-immunotherapy-synergistic tumor ablation; the light-responsive drug-releasing fluoroboron dipyrrole compounds or light-responsive nanoparticles can also be used in combination with other anti-tumor drugs, and other drugs are drugs that have a preventive and therapeutic effect on tumors, including agonists, such as STING agonists, which can be diABZI.

[0014] Furthermore, other drugs are independent of the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds or light-responsive nanoparticles to form a drug system; or other drugs are not independent of the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds or light-responsive nanoparticles to form a drug system. Preferably, the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds are used as carriers of other drugs, that is, the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds and other drugs are used as raw materials, and a drug system is assembled by a thin film dispersion method, a solvent dialysis method or a solvent volatilization method.

[0015] The invention discloses the application of the light-responsive drug-releasing fluoroboron dipyrrole compound as a drug carrier or in the preparation of a drug carrier.

[0016] The invention discloses the use of the light-responsive drug-releasing fluoroboron dipyrrole compounds or light-responsive nanoparticles as photosensitizers or immune stimulants, or the use of the photosensitizers or immune stimulants in the preparation of photosensitizers or immune stimulants.

[0017] The present invention discloses the use of the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds or light-responsive nanoparticles and drug systems in the preparation of drugs. Preferably, the drug is a drug for treating tumors, such as a drug for treating breast cancer and pancreatic cancer.

[0018] The present invention covalently bonds the immune drug molecule R848 to a fluoroborate dipyrrole dye molecule that has near-infrared absorption and generates reactive oxygen through an active oxygen-sensitive thioketal group, and further connects the hydrophilic chain through click chemistry, so that it does not need to be encapsulated by other carriers and can directly self-assemble into a multimodal therapeutic nanomedicine; the reactive oxygen generated by the fluoroborate dipyrrole dye molecule under near-infrared excitation can directly kill tumors on the one hand, and on the other hand, it can break the thioketal group to achieve the local selective release of the immune drug in the tumor.

[0019] The present invention has at least the following advantages: There is no report in the prior art on the construction of a photoresponsive immune drug delivery system based on fluoroboron dipyrrole derivatives by covalent coupling. In the photoresponsive drug-releasing fluoroboron dipyrrole compounds or photoresponsive nanoparticles of the present invention, the immune drug R848 is covalently linked, which solves the problems of low drug loading and drug leakage caused by the general immune drugs relying on macromolecular carriers for encapsulation; near-infrared light precisely regulates drug release, solves the common off-target toxicity problems of immune drugs, and the preliminary safety evaluation at the animal level proves this; the nanomedicine prepared by self-assembly provides a photoresponsive nanodrug delivery platform, which also has obvious long-lasting systemic circulation effects and efficient tumor targeting effects, and this excellent improvement is proved by the results of pharmacokinetic experiments and tissue distribution experiments; at the same time, the concept of multimodal treatment combining photodynamic therapy and immune drugs is introduced to enhance the synergistic anti-tumor effect of drugs, which is also proved in the results of animal experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a route map for synthesizing BTR in Example 1 of the present invention.

[0021] Figure 2 It is the hydrogen spectrum nuclear magnetic resonance image of the product BTR in Example 1 of the present invention.

[0022] Figure 3 This is an electron microscope image of the BTR-NPs prepared in Example 2 of the present invention.

[0023] Figure 4 This is an electron microscope image of dBTR-NPs prepared in Example 5 of the present invention.

[0024] Figure 5 These are the UV (a) and fluorescence spectra (b) of the BTR obtained in Example 1 and the BTR-NPs obtained in Example 2 of the present invention.

[0025] Figure 6 This is the result of the BTR-NPs prepared in Example 2 of the present invention generating active oxygen under light.

[0026] Figure 7 This is the result of the BTR-NPs prepared in Example 2 of the present invention releasing R848 under light.

[0027] Figure 8 This is the effect of BTR-NPs prepared in Example 2 of the present invention in killing tumor cells under light.

[0028] Fig. 9 This is the evaluation result of the tumor targeting of dBTR-NPs prepared in Example 5 of the present invention.

[0029] Fig.10 The therapeutic effect of dBTR-NPs prepared in Example 5 of the present invention on breast cancer 4T1 orthotopic tumor: (a) dosing regimen, (b) experimental grouping, (c) average tumor volume of each group, and (d) survival rate of mice in each group.

[0030] Fig.11 The therapeutic effect of dBTR-NPs prepared in Example 5 of the present invention on pancreatic cancer PANC02 subcutaneous tumor: (a) dosing regimen, (b) experimental grouping, (c) average tumor volume of each group, and (d) survival rate of mice in each group.

[0031] Fig.12 This is the safety evaluation result of dBTR-NPs prepared in Example 5 of the present invention. DETAILED DESCRIPTION

[0032] In the process of anti-tumor treatment with photoresponsive immune drug delivery system, the photophysical and chemical properties of photoresponsive drugs, such as chemical structure, molecular weight, hydrophilicity, spectral properties and photosensitivity, have a decisive influence on the anti-tumor effect and are issues that must be considered in depth. In addition, the existing immune drug delivery system that can respond to near-infrared light with strong tissue penetration ability needs to improve the performance of inducing selective release of immune drugs, and also needs to solve the problem of toxic side effects caused by leakage of immune drugs in non-target sites.

[0033] The present invention discloses a fluoroboron dipyrrole compound capable of releasing an immune drug in response to light, and the chemical structure thereof is as follows:

[0034] 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; further, the molecular weight of R is 1000 to 10000.

[0035] The present invention discloses a method for preparing the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compound, comprising the following steps: (1) reacting the compound represented by formula (II) with the immune agonist R848 to obtain a R848 derivative having a terminal thioketone chain group represented by formula (III); (2) The R848 derivative having a thioketone chain group at the end of formula (III) is reacted with a fluoroborane dipyrrole derivative of formula (IV) to obtain a compound of formula (V); (3) reacting the alkynyl group on the compound represented by formula (V) with a hydrophilic polymer containing azide to obtain a photoresponsive drug-releasing fluoroboron dipyrrole compound; Wherein, the chemical structural formula of the above compound is as follows:

[0036]

[0037] 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.

[0038] Specifically, the present invention discloses the synthesis of a fluoroboron dipyrrole compound that releases an immune drug in response to light and its new use in pharmaceutical manufacturing, specifically the synthesis of a fluoroboron dipyrrole compound that releases an immune drug in response to light, the preparation of nanomedicines, and the use in photoimmunosynergistic anti-tumor. The fluoroboron dipyrrole dye molecule is used as the parent core, and the immune drug is covalently coupled through a sensitive group, and further formed into nanomicelles through self-assembly, and another immune drug is loaded to construct a photoresponsive self-assembled nanomedicine that synergistically delivers multiple immune drugs. Under illumination conditions, the self-assembled nanomedicine exhibits excellent photodynamic effects and controlled release behavior of immune drugs. The present invention confirms its dual effects of photodynamic therapy and immunotherapy at the cell and animal levels, and achieves photoimmunosynergistic tumor ablation.

[0039] The above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds or light-responsive nanoparticles disclosed for the first time in the present invention have the dual effects of photodynamic therapy and immunotherapy, and realize photoimmunosynergistic tumor ablation; the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds or light-responsive nanoparticles can also be used in combination with other anti-tumor drugs, such as STING agonists, which can be diABZI.

[0040] Furthermore, other drugs are independent of the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds or light-responsive nanoparticles to form a drug system; or other drugs are not independent of the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds or light-responsive nanoparticles to form a drug system. Preferably, the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds are used as carriers of other drugs, that is, the above-mentioned light-responsive drug-releasing fluoroboron dipyrrole compounds and other drugs are used as raw materials, and a drug system is assembled by a thin film dispersion method, a solvent dialysis method or a solvent volatilization method.

[0041] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail with the accompanying drawings as follows. The specific implementation methods of the present invention are further described in detail below in conjunction with the drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present 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 Soochow University. The difference between the two groups was tested using a T test or a one-sided ANOVA test, *P<0.05, **P<0.01, and ***P<0.001. Embodiment 1

[0042] See also Figure 1 , preparing a fluoroborane dipyrrole compound for releasing immunodrugs in response to light, wherein R is a polyethylene glycol group (molecular weight of 5000), the specific steps are as follows: 4-Hydroxybenzaldehyde, potassium carbonate and potassium iodide were dissolved in acetonitrile (50 times the mass of 4-hydroxybenzaldehyde) in a molar ratio of 1:1.5:0.1, stirred at room temperature, and then tert-butyl 2-bromoacetate was added dropwise in 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 with a sample 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%.

[0043] Compound 1 was dissolved in dichloromethane, and an equal volume of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 24 hours. The mixture was filtered, and the filter cake was washed with n-hexane to obtain a white solid, namely compound 2, with a yield of 97%.

[0044] R848 and carbonyldiimidazole in a molar ratio of 1:1.5 were dissolved in dichloromethane (30 times the mass of R848), stirred at room temperature for 8 hours, and then compound 3 (the molar ratio of compound 3 to R848 was 1:2) was added to continue the reaction for 24 hours. The solvent was pressurized and dried, and purified by column chromatography (ethyl acetate) to obtain a yellow solid, which was compound 4, with a yield of 80%.

[0045] 3,5-diiodo-4-hydroxybenzaldehyde and 2,4-dimethylpyrrole were placed in a flask in a molar ratio of 1:2, a dichloromethane solution of 50 times the mass of 3,5-diiodo-4-hydroxybenzaldehyde was added to dissolve it, trifluoroacetic acid (4 drops) was added dropwise, and stirred at room temperature for 12 hours in a dark place, and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (the molar ratio of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to 3,5-diiodo-4-hydroxybenzaldehyde was 1:1) was added, and stirring at room temperature was continued for 12 hours; then the reaction solution was moved to an ice bath, triethylamine (the molar ratio of 2,3-diiodo-4-hydroxybenzaldehyde to 3,5-diiodo-4-hydroxybenzaldehyde was 1:40) was added, and boron trifluoride ether (the molar ratio of 2,3-diiodo-4-hydroxybenzaldehyde to 3,5-diiodo-4-hydroxybenzaldehyde was 1:40) was added dropwise, and the reaction was allowed to react at room temperature for 12 hours after the addition was completed, and the mixture was dried under reduced pressure, mixed with silica gel, and eluted by column chromatography to obtain a yellow-brown solid, which was compound 5, with a yield of 51%.

[0046] Compound 5, bromoacetonitrile and potassium carbonate were dissolved in acetone (10 times the mass of compound 6) in a molar ratio of 1:1.5:2, refluxed for 2 hours, cooled to room temperature, filtered through diatomaceous earth, the filter cake was washed with dichloromethane, the filtrate was spin-dried and the sample was separated by column chromatography to obtain a yellow solid, which was compound 6, with a yield of 96%.

[0047] Compound 6 was dissolved in anhydrous ethanol with a mass of 10 times that of compound 6, and iodine was added (the molar ratio of iodine to compound 6 was 1:2.5, and it was ultrasonically dissolved in anhydrous ethanol with a mass of 2 times that of iodine) to obtain a reaction solution; iodic acid (the molar ratio of iodine to compound 6 was 1:2) was weighed and ultrasonically dissolved in water (0.5 times the mass of iodic acid), and then added dropwise to the reaction solution; then stirred at room temperature for 15 hours in a dark place, and then a saturated solution of sodium thiosulfate (20 times the mass of compound 6) was added, and stirred at room temperature for 30 minutes; then extracted with dichloromethane three times, the organic phases were combined and dried over anhydrous sodium sulfate for 1 hour, dried by rotary evaporation under reduced pressure, mixed, and separated by column chromatography to obtain a red solid, namely compound 7, with a yield of 59.6%.

[0048] Compound 7, compound 2, acetic acid and piperidine were placed in a reactor in a molar ratio of 1:4:50:25, and acetonitrile (20 times the mass of compound 7) was added. The mixture was stirred at room temperature for 1 hour, refluxed for 3 hours, and naturally cooled to room temperature. The mixture was extracted three times with dichloromethane, and the organic phases were combined, dried over anhydrous sodium sulfate, dried under reduced pressure, mixed, and separated by column chromatography to obtain a green solid, namely compound 8, with a yield of 70%.

[0049] Compound 8, compound 4, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine were placed in a reactor in a molar ratio of 1:2.2:2.5:4, and tetrahydrofuran (100 times the mass of compound 8) was added, and the mixture was stirred at room temperature for 18 hours; then water was added to quench the reaction, and the mixture was extracted three times with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, and the sample was dried under reduced pressure and separated by column chromatography to obtain a green solid, namely compound 9, with a yield of 80%.

[0050] Compound 9 (80 mg, 0.06 mmol), monomethylazidePEG 5000 (317 mg, 0.06 mmol), copper sulfate pentahydrate (19 mg, 0.076 mmol), sodium ascorbate (25 mg, 0.126 mmol) were dissolved in 5 mL of dimethyl sulfoxide, and stirred at room temperature for 24 h under nitrogen protection; then the dimethyl sulfoxide was removed by freeze drying, and 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 image: 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). Embodiment 2

[0051] Photoresponsive nanomicelles were prepared using a solvent dialysis method. The specific steps are as follows: weigh 10.0 mg of the BTR compound, dissolve it in 500 μL of dimethyl sulfoxide, and sonicate for 2 s at an ultrasonic power of 400 W, with an interval of 2 s, for a total of 2 min; then add 15 ml of ultrapure water for 2 minutes, continue sonicating for another 10 minutes, and then put it into a dialysis bag with a cutoff molecular weight of 13,000 Daltons, and place it in 2 liters of ultrapure water for dialysis. The liquid was changed at 2, 4, 6, 8, and 24 hours. At the end of the dialysis, the green liquid in the ultrafiltration bag was filtered through a 0.45-micron filter membrane to obtain the photoresponsive nanomicelles BTR-NPs. The size distribution and morphology of the obtained nanomicelles were characterized by electron microscopy. The average particle size was about 54.4 nanometers. The results are as follows Figure 3 shown. Embodiment 3

[0052] The steps of this embodiment are basically the same as those of the second embodiment, except that: this embodiment adopts a thin film dispersion method to prepare light-responsive nano-micelles. The specific steps are: weigh 2 mg of the sample, dissolve it in 5 ml of tetrahydrofuran, remove the tetrahydrofuran by rotary evaporation, so that the substance forms a thin film at the bottom of the flask, and add 10 ml of water under ultrasonic conditions to dissolve it to obtain nano-micelles. Embodiment 4

[0053] The steps of this embodiment are basically the same as those of the second embodiment, except that this embodiment adopts the solvent evaporation method to prepare the photoresponsive nanomicelles. The specific steps are as follows: weigh 2 mg of the sample, dissolve it in 1 ml of tetrahydrofuran, drip it dropwise into 10 ml of water under magnetic stirring, and stir at 40°C until the acetone evaporates completely. Embodiment 5

[0054] The photoresponsive nanomicelles loaded with the STING agonist diABZI were prepared by solvent dialysis. The specific steps are as follows: weigh 10.0 mg of BTR solid and dissolve it in 500 μL of dimethyl sulfoxide; weigh 1.50 mg of STING agonist diABZI and dissolve it in 500 μL of dimethyl sulfoxide, then mix the two solutions evenly, ultrasonicate at an ultrasonic power of 400 W, ultrasonicate for 2 s, with an interval of 2 s, for a total of 2 min; then add 15 ml of ultrapure water for 2 minutes, and continue ultrasonicating for 10 minutes, put it into a dialysis bag with a molecular weight cutoff of 13,000 Daltons, and dialyze it in 2 liters of ultrapure water. The liquid was changed at 2, 4, 6, 8, and 24 hours. At the end of dialysis, the green liquid in the ultrafiltration bag was filtered through a 0.45-micron filter membrane to obtain the photoresponsive nanomicelles dBTR-NPs loaded with the STING agonist diABZI. The particle size of dBTR-NPs is about 95.8 nanometers, and the transmission electron microscopy image of dBTR-NPs is shown as follows Figure 4 As shown; the diABZI drug loading in dBTR-NPs was 9.4%.

[0055] The performance test of the nano micelles prepared in the present invention is as follows: 1. Test the absorption and fluorescence spectra of the polymer (BTR) and nanomicelles (BTR-NPs) synthesized in the present invention. The specific steps are: accurately weigh 2.0 mg of the polymer and dissolve it in 1 ml of N,N-dimethylformamide, dilute to a final concentration of 15 micromoles per liter, scan the full wavelength (wavelength range 250-900 nanometers) with a UV-visible spectrophotometer, and then scan the fluorescence emission spectrum with a fluorescence spectrophotometer (excitation wavelength 660 nanometers, emission wavelength 680-900 nanometers). Prepare nanomicelles of the same concentration, dilute to 15 micromoles per liter with ultrapure water, and scan the absorption and emission spectra of each nanoparticle under the same conditions of UV and fluorescence spectrophotometers. The results are as follows. Figure 5 As shown in the UV (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.

[0056] 2. Test the active oxygen yield of the nanomicelles (BTR-NPs) prepared in the present invention. The specific steps are: measure 2.97 ml of a certain concentration of nanomicelles, mix with 1,3-diphenylisobenzofuran (DPBF) solution (0.3 mmol, 30 μl), place on a magnetic stirrer, illuminate with 660 nm, 0.5 W / cm2, and scan the ultraviolet absorption of each sample at 415 nm at 0, 5, 10, 15, 20, 25, and 30 seconds. Repeat the above experiment with zinc phthalocyanine as the control compound to calculate the active oxygen quantum yield of the nanomicelles. The results are as follows: Figure 6As shown in the figure, the absorbance of DPBF decreased significantly with the extension of illumination time, indicating that reactive oxygen species were produced. Further calculations showed that the quantum yield of reactive oxygen species in BTR-NPs was 0.28.

[0057] 3. Test the release of R848 in response to light by the nanomicelles (BTR-NPs) prepared in the present invention. The specific steps are: take a certain concentration of nanomicelles, dilute it to 100 micromoles per liter with pH 5.0 buffer (50 mmol per liter sodium acetate, pH 5.0), take 500 microliters to a 1.5 ml centrifuge tube, irradiate with 660 nanometers of different intensities (0, 0.15, 0.3, 0.5 watts per square centimeter) for 20 minutes, take 20 microliters of sample at 0, 5, 10, 15, and 20 minutes respectively and place it in a 1.5 ml centrifuge tube, add 180 microliters of dimethyl sulfoxide to mix, centrifuge (12000 rpm, 5 minutes), take 150 microliters of supernatant, place it in an inner liner tube, and use high performance liquid chromatography detection. The amount of R848 released in response was calculated according to the standard curve. The results are as follows: Figure 7 The results showed that R848 was not released without light, but was released rapidly under light conditions, and the cumulative release of R848 was positively correlated with light intensity.

[0058] 4. Test the effect of the nano-micelles (BTR-NPs) prepared in the present invention on killing tumor cells in response to light. The specific steps are: collect 4T1 breast cancer cells in the logarithmic growth phase and culture them in a 96-well plate with a seeding density of 1×10 per well. 4 The cells were placed in a carbon dioxide cell culture incubator and cultured for 12 hours to allow them to adhere to the wall. Then the culture medium in the 96-well plate was removed, and 100 microliters of culture medium containing nanomicelles were added to each well to set up an illumination group and a non-illumination group. After 12 hours of drug administration in the illumination group, the drug-containing culture medium was discarded and replaced with a basal culture medium. The light was irradiated at 660 nanometers and 0.3 watts / square centimeter for 4 minutes. After continuing to culture for 24 hours, the culture medium was discarded, and MTT solution (5.0 mg / ml, 20.0 microliters) was added to each well and cultured for 4 hours. After 24 hours of drug administration in the non-illumination group, MTT was incubated for 4 hours in the same way, the solution was discarded, dimethyl sulfoxide (100.0 microliters) was added to each well, and the plate was shaken for 10 minutes. The absorbance value of each well at 490 nanometers was measured with an enzyme reader, and the cell survival rate was calculated. The results are as follows Figure 8 As shown. The results show that in the absence of light, BTR-NPs have almost no cytotoxicity; under light conditions, the cytotoxicity is significantly enhanced with the increase of BTR-NPs concentration, and the calculated half inhibition concentration is 0.32 micromol / L. The tumor cell killing effect of BTR-NPs disclosed in the present invention is significantly improved compared with the fluoroboron dipyrrole derivative nanomicelles (half inhibition concentration of 2.0 to 5.1 micromol / L) previously disclosed by the applicant.

[0059] 5. Test the tumor targeting efficiency of the diABZI-loaded light-responsive nanomicelles (dBTR-NPs) prepared in the present invention in mice. The specific steps are: collect 4T1 cells in good condition and in the logarithmic growth phase, with a cell concentration of 2×10 7 / mL, 50 μL of cell suspension was injected into the third and fourth mammary pads of 6-8 week-old Balb / c mice, and the tumors grew to 80-100 mm in 6-7 days. 3 At the same time, 200 μL of nanomicelles with a concentration of 400 μmol / L were injected into tumor-bearing mice through the tail vein. After 24 hours, the mice were killed by dislocation, and the heart, liver, spleen, lung, kidney, tumor, and lymphoid organs were removed, weighed and recorded, and then placed in a 10-ml centrifuge tube, chopped with scissors, and homogenized with 1 ml of ultrapure water for 3 minutes. 1 ml of organic phase (methanol: dichloromethane = 10:1) was added and vortexed for 3 minutes, centrifuged (6000 rpm, 3 minutes), the lower organic phase was aspirated, extracted 3 times to combine the organic phases, evaporated in a fume hood, 1 ml of dimethyl sulfoxide was added for ultrasonic dissolution, vortexed for 3 minutes, centrifuged (12000 rpm, 3 minutes), the supernatant was aspirated to test the fluorescence spectrum, the drug concentration was calculated by annotating the curve, and the tumor targeting efficiency of the nanomicelles in mice was further calculated. The results are shown in Fig. 9 The results showed that dBTR-NPs were mainly distributed in the liver, spleen and tumor in tumor-bearing mice, and the tumor enrichment efficiency could reach 10.6% injected dose / g (ID% g -1 ). The present invention uses covalent bonds to couple the fluoroborane dipyrrole core and the immune drug for the first time, which can efficiently load the drug while reducing the leakage of the drug in the blood; at the same time, at the tumor site, light irradiation generates active oxygen, breaks the covalent bonds, and releases the drug.

[0060] 6. Test the effect of the diABZI-loaded light-responsive nanomicelles (dBTR-NPs) prepared in the present invention on the treatment of breast cancer 4T1 in situ tumors. The specific steps are: collect 4T1-Luc cells in good condition and in the logarithmic growth phase, with a cell concentration of 2×10 7 / ml, 50 μl of cell suspension was injected into the third and fourth pairs of mammary pads of 6-8 week-old Balb / c mice. After the tumors grew to 80-100 mm3, 200 μl of drug or control was injected through the tail vein on days 1, 4, and 7. Light was applied (0.3 W / cm2, 8 min) on days 2, 5, and 8. The dosing schedule was as follows Fig.10a. In order to demonstrate the superiority of nanomicelles, a mixture of free diABZI, free R848 and a fluoroborane dipyrrole compound represented by formula (VI) (abbreviated as d / R / B mixture, the mixing molar ratio is 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 illumination group, BTR-NPs illumination group, dBTR-NPs illumination group, as shown in Fig.10 As shown in b, the dosage of fluoroboron dipyrrole derivatives in each group was 4.0 μmol / kg. The tumor volume of mice was measured with a vernier caliper (tumor volume = (length × width 2 ) / 2, such as Fig.10 c), and draw the survival curve of each group of mice (as shown in Fig.10 d). The results showed that compared with free drugs, dBTR-NPs can significantly inhibit the growth of breast cancer 4T1 orthotopic tumors and significantly prolong the survival of mice. More importantly, the fluoroboron dipyrrole derivative nano-micelles of the present invention can almost completely eliminate the 4T1 orthotopic tumor model at a dosage of 4.0 micromoles per kilogram, and the effect is significantly better than the fluoroboron dipyrrole derivative nano-micelles previously disclosed by the applicant.

[0061]

[0062] 7. Test the effect of the diABZI-loaded light-responsive nanomicelles (dBTR-NPs) prepared in the present invention on the treatment of pancreatic cancer PANC02 subcutaneous tumors. The specific steps are: collect Panc02 cells in good condition and in the logarithmic growth phase, with a cell concentration of 6×10 7 / ml, 50 μl of cells were injected into the dorsal thigh of 6-8 week old C57 mice, and after the tumor grew to 80 cubic millimeters, 200 μl of drug or control was injected through the tail vein on days 1, 4, and 7, and light was applied (0.3 W / cm2, 8 min) on days 2, 5, and 8. The dosing schedule was as follows Fig.11 The experimental mice were divided into the following 4 groups: PBS control group, d / R / B mixture group (same as above), dBTR-NPs group, dBTR-NPs illumination group, as shown in Fig.11 As shown in b, the dosage of fluoroboron dipyrrole derivatives in each group was 4.0 μmol / kg. The tumor volume of mice was measured with a vernier caliper (tumor volume = (length × width 2 ) / 2, such as Fig.11 c), and draw the survival curve of each group of mice, as shown in Fig.11 d. The results showed that compared with free drugs, dBTR-NPs could significantly inhibit the growth of subcutaneous pancreatic cancer Panc02 tumors and significantly prolong the survival of mice.

[0063] 8. Safety test of the diABZI-loaded photoresponsive nanomicelles (dBTR-NPs) prepared in the present invention. The specific steps are: collecting 4T1 cells in good condition and in the logarithmic growth phase, with a cell concentration of 2×10 7 / ml, 50 μl of cell suspension was injected into the third and fourth mammary pads of 6-8 week old Balb / c mice. When the tumor grew to 80-100 mm3 on day 6-7, the mice were treated according to the following five groups: PBS control group, dBTR-NPs group, d / R / B mixture illumination group (same as above), BTR-NPs illumination group, dBTR-NPs illumination group. On days 1, 4, and 7, 200 μl of drug or control was injected into the tail vein. On days 2, 5, and 8, light was applied (0.3 W / cm2, 8 minutes). The drug dosages were diABZI (3 mg / kg), R848 (2.5 mg / kg), and fluoroboron dipyrrole derivatives (4 μmol / kg). The day after the injection, the animals were killed and their hearts, livers, spleens, lungs, kidneys and tumors were removed and placed in 4% paraformaldehyde. The tissues were embedded in paraffin and sliced, which were then stained with hematoxylin-eosin. The pathological morphological changes of the stained tissue slices were observed under a fluorescent upright microscope. Fig.12 The results showed that under light conditions, dBTR-NPs could produce significant cell apoptosis at the tumor site, but had no effect on normal tissues such as the heart, liver, spleen, lung, and kidney.

[0064] The present invention actively conducts research and innovation, creates a fluoroboron dipyrrole compound that releases immune drugs in response to near-infrared light, and self-assembles into drug-loaded nano-micelles, so that it can efficiently deliver and controllably release immune drugs, and is applied to the photoimmunotherapy of malignant tumors such as breast cancer and pancreatic cancer. In the present invention, the photoresponsive drug-releasing fluoroboron dipyrrole compound exhibits excellent photophysical properties such as high extinction coefficient and adjustable absorption spectrum, and can also be activated by light to produce highly reactive oxygen species, which can directly kill tumors, attack reactive oxygen sensitive groups, and regulate drug responsive release, and is very suitable for constructing a photoresponsive immune drug delivery system; and the method of the present invention solves the problems of non-target site leakage and toxic side effects of immune drugs that are inevitable in the prior art of using photoresponsive carriers to load immune drugs through physical encapsulation methods.

Claims

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

2. The light-responsive drug-releasing fluoroboranedipyrrole compound according to claim 1, characterized in that: R is a hydrophilic polymer group.

3. The method for preparing the light-responsive drug-releasing fluoroboranedipyrrole compound according to claim 1, characterized in that: The photoresponsive drug-releasing fluoroboron dipyrrole compound is prepared by reaction using an alkynyl compound and an azide-containing R compound as raw materials; the chemical structural formula of the alkynyl compound is as follows: 。 4. A photoresponsive nanoparticle, characterized in that: The raw materials for preparing the photoresponsive nanoparticles include the photoresponsive drug-releasing fluoroboron dipyrrole compound according to claim 1.

5. The method for preparing the light-responsive nanoparticles according to claim 4, characterized in that: The light-responsive drug-releasing fluoroboron dipyrrole compound of claim 1 is prepared by a thin film dispersion method, a solvent dialysis method or a solvent volatilization method.

6. A pharmaceutical system, characterized in that It comprises the light-responsive drug-releasing fluoroboron dipyrrole compound according to claim 1 or the light-responsive nanoparticles according to claim 4.

7. The pharmaceutical system according to claim 6, characterized in that: Other medicines are also included.

8. Use of the light-responsive drug-releasing fluoroborane dipyrrole compound of claim 1 or the light-responsive nanoparticle of claim 4 as a photosensitizer or an immunostimulant, or in the preparation of a photosensitizer or an immunostimulant.

9. Use of the light-responsive drug-releasing fluoroborane dipyrrole compound of claim 1, the light-responsive nanoparticles of claim 4, or the drug system of claim 6 in the preparation of drugs.

10. Use of the light-responsive drug-releasing fluoroboron dipyrrole compound of claim 1 as a drug carrier or in the preparation of a drug carrier.

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