Aza-BODIPY molecule with NIR-II fluorescence emission and I-type photodynamic activity as well as preparation method, application and photosensitizer of aza-BODIPY molecule

Abstract

The invention provides an aza-BODIPY molecule with NIR-II fluorescence emission and I-type photodynamic activity and a preparation method thereof. The aza-BODIPY molecule has good light absorption in a near-infrared (NIR) region, the emission peak of the aza-BODIPY molecule is prolonged to a near-infrared second region (NIR-II), and the aza-BODIPY molecule shows an ultrahigh NIR-II fluorescence quantum yield of 13%. Meanwhile, the photosensitizer prepared by adopting aza-BODIPY molecules can effectively generate I-type active oxygen species, namely superoxide anion free radicals, under the excitation of 808nm laser. On the basis, high-resolution tumor fluorescence imaging can be realized by utilizing NIR-II fluorescence of the aza-fluorine boron fluorescent sensitizer, and the aza-fluorine boron fluorescent sensitizer is used for accurate and efficient NIR-II fluorescence imaging mediated I-type photodynamic therapy of deep tumor tissues.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and particularly to aaza-boron dipyrromethene molecule with both NIR-II fluorescence emission and type I photodynamic activity, its preparation method, application, and photosensitizer. Background Art

[0002] Traditional cancer therapies, including surgery, chemotherapy, and radiotherapy, have achieved good results clinically, but still have defects such as limited efficacy, severe side effects, and difficulty in radical cure; coupled with factors such as the complex composition and structural characteristics of tumors, the application effect of traditional treatment strategies has been limited to a certain extent. Therefore, it is urgent to develop new and efficient cancer treatment methods to improve the quality of life and overall survival rate of patients.

[0003] Photodynamic therapy (PDT) is a non-invasive treatment method. Compared with traditional chemotherapy, radiotherapy, and surgery, PDT has the advantages of minimally invasive, not affected by drug resistance, and controllable selection of the treatment area. The PDT process involves exposing a photosensitizer to light and generating cytotoxic reactive oxygen species (ROS) through interaction with molecular oxygen under light excitation, and using the generated reactive oxygen species to cause irreversible damage to tumor cells. According to the mechanism and type of reactive oxygen species generation, PDT can be divided into type I PDT and type II PDT. The type II PDT process usually requires a large amount of oxygen to generate singlet oxygen. As the oxygen is consumed, the treatment effect of type II PDT will be severely hindered due to insufficient oxygen levels. Compared with type II PDT with high oxygen dependence and high oxygen consumption, type I PDT generates reactive oxygen species such as superoxide anion radicals and hydroxyl radicals through electron transfer, is not dependent on oxygen, and shows great potential in the treatment of solid tumors with hypoxic tumor microenvironments. Therefore, the development of photosensitizers with type I photodynamic activity has become a current research hotspot.

[0004] Fluorescence imaging (FLI), as a new type of optical imaging technology, has been widely used in basic biomedical research and the diagnosis and treatment of diseases due to its significant advantages such as high sensitivity, high contrast, simple operation, and low radiation. The light that can penetrate biological tissues to the greatest depth is usually in the near-infrared wavelength range, and this specific range is called the "biological near-infrared window". The near-infrared window can be further divided into the first near-infrared window (NIR-I, 700 - 1000 nm) and the second near-infrared window (NIR-II, 1000 - 1700 nm) according to the wavelength. Compared with the NIR-I window, the NIR-II spectral region has lower tissue absorption, scattering, and autofluorescence effects. NIR-II fluorescence shows deeper tissue penetration ability, and real-time imaging of high signal-to-noise ratio and large-depth tissues can be achieved in this window, which gives NIR-II fluorescence imaging significant advantages in intraoperative tumor navigation applications.

[0005] Therefore, constructing materials with both NIR-II fluorescence emission and type I photodynamic behavior can effectively solve the bottleneck problems of insufficient penetration depth, failure in hypoxic environment and separation of diagnosis and treatment in traditional diagnosis and treatment technologies. Compared with inorganic materials and organic semiconductor conjugated polymers, organic small molecules have the characteristics of clear structure, adjustable performance, fast metabolism and high biosafety, and are more suitable for the development of type I photosensitizers and subsequent clinical applications. However, due to the difficulty in accurately regulating the energy dissipation pathway, it is still difficult to construct organic small molecules with both NIR-II fluorescence emission and type I photodynamic behavior. Currently, the reported organic small molecules with type I photodynamic behavior rarely have NIR-II fluorescence at the same time, and the reported organic small molecules with both NIR-II fluorescence emission and type I photodynamic behavior have a generally low NIR-II fluorescence quantum yield (QY), usually less than 1%. The fluorescence quantum yield determines the quality of imaging. Low fluorescence quantum yield will cause poor resolution and signal-to-noise ratio of NIR-II fluorescence imaging, which in turn leads to low imaging penetration depth, which limits its application in fields such as bioimaging and photodynamic therapy. Summary of the invention

[0006] The purpose of the present invention is to synthesize an aza-fluoro-boron fluorescent sensitizer molecule with ultra-high NIR-II fluorescence quantum yield and type I photodynamic activity, to solve the problems of scarcity and insufficient diversity of current type I PDT photosensitizers, as well as low NIR-II fluorescence quantum yield, and to improve the problem of insufficient tissue penetration in traditional tumor photodiagnosis and treatment, and the inability to diagnose and treat deep tumors.

[0007] According to the first aspect of the present invention, there is provided an aza-fluoro-boron fluorescent molecule having both NIR-II fluorescence emission and type I photodynamic activity, the aza-fluoro-boron fluorescent molecule is denoted as BDPPZO, and the chemical structure is shown in Formula I:

[0008]

[0009] According to a second aspect of the present invention, there is provided a method for preparing the aforementioned nitrogen-fluorine-boron fluorescent molecule having both NIR-II fluorescence emission and type I photodynamic activity, comprising the following steps:

[0010] Under a nitrogen atmosphere, 1-(10-hexyl-10H-phenothiazin-3-yl)ethanone, 4-(diphenylamino)benzaldehyde and potassium hydroxide are dissolved in ethanol, and reacted under the desired reaction conditions to obtain a first product;

[0011] dissolving the first product, nitromethane and triethylamine in methanol, and reacting under desired reaction conditions to obtain a second product;

[0012] dissolving the second product and ammonium acetate in n-butanol, and reacting under desired reaction conditions to obtain a third product;

[0013] Dissolve the third product in 1,2-dichloroethane, add N,N-diisopropylethylamine and boron trifluoride diethyl ether, and carry out the reaction under the required reaction conditions to obtain the fourth product;

[0014] Under atmospheric pressure conditions, dissolve the fourth product in dichloromethane, use m-chloroperoxybenzoic acid as an oxidant, and carry out the reaction under the required reaction conditions to obtain the azaboron dipyrromethene molecule BDPPZO.

[0015] As an alternative embodiment, the molar ratio of 1-(10-hexyl-10H-phenothiazin-3-yl)ethanone, 4-(diphenylamino)benzaldehyde and potassium hydroxide is 1:1:(1-2), and the required reaction conditions include reacting at a temperature of 30°C - 35°C for 18h - 24h.

[0016] As an alternative embodiment, the molar ratio of the first product, nitromethane and triethylamine is 1:(2-3):(4-5), and the required reaction conditions include reacting at a temperature of 70°C - 75°C for 24h - 30h.

[0017] As an alternative embodiment, the molar ratio of the second product and ammonium acetate is 1:(50-60), and the required reaction conditions include reacting at a temperature of 120°C - 130°C for 30h - 36h.

[0018] As an alternative embodiment, the molar ratio of the third product, N,N-diisopropylethylamine and boron trifluoride diethyl ether is 1:(12-16):(10-12), and the required reaction conditions include reacting at a temperature of 50°C - 60°C for 20h - 24h.

[0019] As an alternative embodiment, the molar ratio of the fourth product and m-chloroperoxybenzoic acid is 1:(3-5), and the required reaction conditions include reacting at room temperature for 6h - 10h.

[0020] According to the third aspect of the object of the present invention, there is provided an application of the aforementioned azaboron dipyrromethene molecule with both NIR-II fluorescence emission and type I photodynamic activity in the preparation of a drug for type I photodynamic therapy mediated by NIR-II fluorescence imaging.

[0021] According to the third aspect of the object of the present invention, there is provided a photosensitizer containing the aforementioned azaboron dipyrromethene molecule with both NIR-II fluorescence emission and type I photodynamic activity.

[0022] As an alternative embodiment, the photosensitizer includes nanoparticles prepared by self-assembly of BDPPZO and the amphiphilic polymer F127.

[0023] As can be seen from the above-mentioned BODIPY molecule of the present invention with both NIR-II fluorescence emission and type-I photodynamic activity, the BODIPY molecule has good absorption in the NIR region, has an ultra-high NIR-II fluorescence quantum yield of 13%, can achieve high-resolution imaging of deep tissues, has a high imaging signal-to-noise ratio, and provides an effective tool for diagnosis and guiding treatment;

[0024] Meanwhile, the nanoparticles prepared by self-assembly of the BODIPY molecule and the amphiphilic polymer F127 exhibit excellent superoxide anion radical generation performance under 808 nm laser excitation, and can be used for precise and efficient NIR-II fluorescence imaging-mediated type-I photodynamic therapy of tumor tissues, reducing tumor treatment damage and side effects, improving treatment efficiency and accuracy, and having good application prospects as a novel fluorescent sensitizer in the precise diagnosis and treatment of cancer. Brief Description of the Drawings

[0025] Figure 1 is the synthetic reaction roadmap of BDPPZO of the present invention.

[0026] Figure 2 is the 1 1H-NMR spectrum of BDPPZO of the present invention in deuterated tetrachloroethane.

[0027] Figure 3 is the 13 13C-NMR spectrum of BDPPZO of the present invention in deuterated chloroform.

[0028] Figure 4 is the mass spectrum of BDPPZO of the present invention in the example.

[0029] Figure 5 is the test chart of the NIR-II fluorescence quantum yield of BDPPZO of the present invention in the example.

[0030] Figure 6 is the test chart of the dynamic light scattering particle size distribution of BDPPZO nanoparticles of the present invention in the example.

[0031] Figure 7 is the ultraviolet absorption and fluorescence emission spectrum of BDPPZO nanoparticles of the present invention in water in the example.

[0032] Figure 8 is the generation diagram of superoxide anion radicals detected by using DHR123 as a probe for BDPPZO nanoparticles.

[0033] Figure 9 is the relative survival rate of different concentrations of BDPPZO nanoparticles incubated with mouse breast cancer cells (4T1) for 24 hours under light or dark conditions.

[0034] Figure 10 It is a diagram of the BDPPZO nanoparticles in an example of the present invention after incubation with 4T1 cells and stained with the superoxide anion fluorescent probe dihydroethidium (DHE).

[0035] Figure 11 It is the NIR-II fluorescence imaging diagram of the BDPPZO nanoparticles in an example of the present invention in a breast cancer model mouse; among them, a is the fluorescence imaging diagram at each time point after injecting the BDPPZO nanoparticles, and b is the change in the fluorescence intensity of the corresponding mouse tumor.

[0036] Figure 12 It is a diagram of the change in tumor volume during the 14-day treatment of a breast cancer model mouse. Specific Embodiments

[0037] To better understand the technical content of the present invention, specific embodiments are hereby given and described in conjunction with the accompanying drawings as follows.

[0038] In the present disclosure, aspects of the present invention are described with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to cover all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those concepts and embodiments described in more detail below, can be implemented in any of many ways.

[0039] Aza-BODIPY molecule

[0040] The exemplary azaboron dipyrromethene molecule of the present invention has both NIR-II fluorescence emission and type I photodynamic activity. This azaboron dipyrromethene molecule is denoted as BDPPZO, and its chemical structure is shown in Formula I:

[0041]

[0042] The aforementioned exemplary azaboron dipyrromethene molecule has good absorption in the NIR region and its fluorescence emission extends to the NIR-II region, and has an ultra-high NIR-II fluorescence quantum yield of 13%.

[0043] Preparation method

[0044] Combined Figure 1 As shown, in an exemplary embodiment of the present invention, a preparation method of the aforementioned azaboron dipyrromethene molecule with both NIR-II fluorescence emission and type I photodynamic activity uses 1-(10-hexyl-10H-phenothiazin-3-yl)ethanone and 4-(diphenylamino)benzaldehyde as starting materials and reacts according to Figure 1 the reaction route for preparation.

[0045] In an alternative example, the method for preparing aaza-BODIPY molecules with both NIR-II fluorescence emission and type I photodynamic activity includes the following steps:

[0046] Under a nitrogen atmosphere, dissolve 1-(10-hexyl-10H-phenothiazin-3-yl)ethanone (Compound 1), 4-(diphenylamino)benzaldehyde (Compound 2) and potassium hydroxide in ethanol, react under the required reaction conditions, and then extract the reaction solution with dichloromethane and water to obtain a first crude product, which is purified to obtain a first product (Compound 3);

[0047] Dissolve the first product, nitromethane and triethylamine in methanol, react under the required reaction conditions, cool, acidify and extract with dichloromethane after the reaction is completed to obtain a second crude product, which is purified to obtain a second product (Compound 4);

[0048] Dissolve the second product and ammonium acetate in n-butanol, react under the required reaction conditions, then cool to room temperature, and filter and wash with cold ethanol to obtain a third product (Compound 5);

[0049] Dissolve the third product in 1,2-dichloroethane, add N,N-diisopropylethylamine and boron trifluoride diethyl etherate, react under the required reaction conditions, then extract with dichloromethane and water, collect the organic layer, dry and evaporate to obtain a third crude product. After purification, a fourth product (Compound 6) is obtained;

[0050] Under normal pressure conditions, dissolve the fourth product in dichloromethane, slowly add m-chloroperbenzoic acid under an ice bath condition, react under the required reaction conditions, treat the reaction mixture with an aqueous sodium hydroxide solution until the pH is neutral after the reaction is completed, extract with dichloromethane, and distill off the solvent under reduced pressure to obtain a third crude product, which is purified to obtain the aaza-BODIPY molecule BDPPZO.

[0051] In an alternative example, the molar ratio of 1-(10-hexyl-10H-phenothiazin-3-yl)ethanone, 4-(diphenylamino)benzaldehyde and potassium hydroxide is 1:1:(1 - 2), and the required reaction conditions include reacting at a temperature of 30°C - 35°C for 18h - 24h.

[0052] In an alternative example, the molar ratio of the first product, nitromethane and triethylamine is 1:(2 - 3):(4 - 5), and the required reaction conditions include reacting at a temperature of 70°C - 75°C for 24h - 30h.

[0053] In an alternative example, the molar ratio of the second product and ammonium acetate is 1:(50 - 60), and the required reaction conditions include reacting at a temperature of 120°C - 130°C for 30h - 36h.

[0054] In an alternative example, the molar ratio of the third product, N,N-diisopropylethylamine, and boron trifluoride diethyl etherate is 1:(12 - 16):(10 - 12), and the required reaction conditions include reacting at a temperature of 50°C - 60°C for 20 h - 24 h.

[0055] In an alternative example, the molar ratio of the fourth product and meta-chloroperoxybenzoic acid is 1:(3 - 5), and the required reaction conditions include reacting at room temperature for 6 h - 10 h.

[0056] Application

[0057] In another exemplary embodiment of the present invention, there is provided an application of the aforementioned aza-boron dipyrromethene molecule with both NIR-II fluorescence emission and type-I photodynamic activity in the preparation of a type-I photodynamic therapy drug mediated by NIR-II fluorescence imaging.

[0058] In another exemplary embodiment of the present invention, there is provided a photosensitizer containing the aforementioned aza-boron dipyrromethene molecule with both NIR-II fluorescence emission and type-I photodynamic activity.

[0059] In an alternative example, the aforementioned photosensitizer includes nanoparticles prepared by self-assembly of BDPPZO and amphiphilic polymer F127.

[0060] The aforementioned photosensitizer exhibits bright NIR-II fluorescence and excellent reactive oxygen species generation performance under 808 nm laser excitation, and can be applied to precise and efficient type-I photodynamic therapy mediated by NIR-II fluorescence imaging of tumor tissues, reducing tumor treatment damage and side effects, and improving treatment efficiency and accuracy.

[0061] For better understanding, the present invention will be further described below in conjunction with several specific examples, but the preparation process is not limited thereto, and the content of the present invention is not limited thereto.

[0062] Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0063] Example 1

[0064] [Synthesis of Compound 3]

[0065] 1-(10-Hexyl-10H-phenothiazin-3-yl)ethanone (1.302 g, 4 mmol), 4-(diphenylamino)benzaldehyde (1.093 g, 4 mmol) and potassium hydroxide (0.449 g, 8 mmol) were dissolved in ethanol (30 mL) and stirred overnight at 30 °C; the reaction solution was extracted with dichloromethane and water (volume ratio 1:1) to obtain the crude product; finally, column chromatography purification was performed on a silica gel column using dichloromethane / petroleum ether (1:2) as the eluent to obtain the red oily product compound 3 (1.219 g, 52%).

[0066] 1 H NMR (400 MHz, CDCl3): δ ppm 7.87 (dd, J = 8.4, 2.0 Hz, 1H, Ar-H), 7.83 - 7.76 (m, 2H, Ar-H), 7.50 (d, J = 8.8 Hz, 2H, Ar-H), 7.40 (d, J = 15.2 Hz, 1H, CH-C=O), 7.34 - 7.29 (m, 4H, Ar-H), 7.18 - 7.10 (m, 8H, Ar-H), 7.05 (d, J = 8.4 Hz, 2H, Ar-H), 6.94 (t, J = 7.2 Hz, 1H), 6.87 (d, J = 8.4 Hz, 2H), 3.87 (t, J = 7.2 Hz, 2H, N-CH2), 1.87 - 1.79 (m, 2H, CH2), 1.47 - 1.32 (m, 6H, CH2), 0.92 - 0.89 (m, 3H, CH3).

[0067] 13 C NMR (100 MHz, CDCl3): δ ppm 187.79, 150.12, 149.24, 147.00, 143.97, 143.91, 132.77, 129.84, 129.66, 128.64, 128.22, 127.81, 127.63, 127.59, 125.55, 124.39, 124.21, 124.03, 123.35, 121.81, 119.01, 115.85, 114.67, 47.92, 31.56, 26.84, 26.69, 22.75, 14.19.

[0068] It can be determined that the structure of compound 3 is formula II;

[0069]

[0070] [Synthesis of Compound 4]

[0071] Compound 3 (1.162 g, 2 mmol), nitromethane (0.610 g, 10 mmol) and triethylamine (1.012 g, 10 mmol) were dissolved in methanol (40 mL) and heated at 80 °C for 24 h; after cooling, it was neutralized with dilute hydrochloric acid (2 M), and then extracted with dichloromethane to obtain a dark orange oily crude product; the crude product was eluted with dichloromethane / petroleum ether (1:2), and further purified by silica gel column chromatography to obtain an orange oily product, compound 4 (0.882 g, 69%).

[0072] 1 H NMR (400 MHz, CDCl3): δ ppm 7.73 (d, J = 8.4 Hz, 1H, Ar-H), 7.64 (s, 1H, Ar-H), 7.23 (d, J = 7.2 Hz, 3H, Ar-H), 7.18–7.10 (m, 4H, Ar-H), 7.07 - 6.92 (m, 10H, Ar-H), 6.85 (dd, J = 17.4, 8.6 Hz, 2H, Ar-H), 4.80 (dd, J = 12.4, 6.4 Hz, 1H, CH2-NO2), 4.65 (dd, J = 12.4, 8.0 Hz, 1H, CH2-NO2), 4.14 (m, 1H, CH), 3.87 (t, J = 7.2 Hz, 2H, N-CH2), 3.39 - 3.24 (m, 2H, O=C-CH2), 1.84 - 1.76 (m, 2H, CH2), 1.44 - 1.29 (m, 6H, CH2), 0.88 (t, J = 5.6 Hz, 3H, CH3).

[0073] The structure of compound 4 can be determined as formula Ⅲ;

[0074]

[0075] [Synthesis of Compound 5]

[0076] Compound 4 (0.642 g, 1 mmol), ammonium acetate (4.625 g, 60 mmol) and n-butanol (10 mL) were added to a 100 mL round-bottom flask and heated under reflux at 130 °C for 36 h; after cooling to room temperature, it was filtered with cold ethanol and washed three times to obtain a dark blue solid product, compound 5 (0.480 g, 80%).

[0077] 11H NMR (400 MHz, CDCl3): δ ppm 7.93 (d, J = 8.8 Hz, 4H, Ar-H), 7.70 (dd, J = 6.8, 2 Hz, 4H, Ar-H), 7.19 - 7.15 (m, 10H, Ar-H), 7.13 - 7.10 (m, 8H, Ar-H), 7.05 (s, 2H, Ar-H), 7.03 (s, 2H, pyrrole-H), 7.01 - 6.92 (m, 12H, Ar-H), 6.89 (d, J = 8 Hz, 2H), 3.91 (t, J = 7.2 Hz, 4H, N-CH2), 1.91 - 1.84 (m, 4H, CH2), 1.49 - 1.32 (m, 12H, CH2), 0.88 (t, J = 6.8 Hz, 6H, CH3).

[0078] 13 13C NMR (100 MHz, CDCl3): δ ppm 153.08, 149.93, 147.54, 146.58, 144.35, 141.72, 129.82, 129.32, 128.30, 127.54, 127.50, 126.55, 126.12, 125.22, 124.55, 124.04, 123.25, 123.16, 122.95, 115.65, 115.49, 112.82, 47.93, 31.57, 26.91, 26.78, 22.74, 14.12.

[0079] The structure of compound 5 can be determined as formula Ⅳ;

[0080]

[0081] [Synthesis of Compound 6]

[0082] Dissolve compound 5 (0.418 g, 0.35 mmol) in dry 1,2-dichloroethane (25 mL), treat it with N,N-diisopropylethylamine (0.498 g, 3.85 mmol) and boron trifluoride diethyl etherate (0.795 g, 5.6 mmol), stir at 50 °C for 24 h under a nitrogen atmosphere; extract with dichloromethane and water, collect the organic layer, dry it, and evaporate to obtain the crude product; purify by column chromatography eluting with dichloromethane / petroleum ether (1:1) to obtain the dark blue product compound 6 (0.200 g, 46%).

[0083] 11H NMR (400 MHz, CDCl3): δ ppm 8.06 (dd, J = 8.4, 1.6 Hz, 2H, Ar-H), 7.93 (d, J = 8.8 Hz, 4H, Ar-H), 7.69 (d, J = 2.0 Hz, 2H, Ar-H), 7.22 (t, J = 8.4 Hz, 8H, Ar-H), 7.15 (d, J = 7.2 Hz, 9H, Ar-H), 7.12 (dd, J = 4.0, 1.6 Hz, 2H, Ar-H), 7.10 (s, 1H, pyrrole-H), 7.07 (s, 1H, pyrrole-H), 7.05 - 7.02 (m, 7H, Ar-H), 6.94 - 6.84 (m, 8H, Ar-H), 3.88 (t, J = 7.2 Hz, 4H, N-CH2), 1.88 - 1.80 (m, 4H, CH2), 1.46 - 1.32 (m, 12H, CH2), 0.90 (t, J = 6.8 Hz, 6H, CH3).

[0084] 13 13C NMR (100 MHz, CDCl3): δ ppm 156.12, 148.85, 147.33, 147.12, 145.64, 143.90, 142.10, 130.26, 129.62, 129.48, 128.23, 127.54, 127.42, 126.35, 126.01, 125.22, 123.95, 123.88, 123.04, 122.20, 116.65, 115.59, 115.04, 47.95, 31.57, 26.90, 26.78, 22.74, 14.13.

[0085] The structure of compound 6 can be determined as formula V,

[0086]

[0087] [Synthesis of BDPPZO]

[0088] Dissolve compound 6 (186 mg, 0.15 mmol) in 20 mL of dichloromethane. Slowly add meta-chloroperoxybenzoic acid (104 mg, 0.6 mmol) under ice bath conditions. Stir for 6 h at room temperature and atmospheric pressure. Then extract with aqueous sodium hydroxide solution (2 M) and dichloromethane. Remove the solvent by rotary evaporation under reduced pressure. The crude product is purified by column chromatography (silica gel, ethyl acetate:dichloromethane = 1:1) to obtain dark blue solid BDPPZO (70 mg, 37%).

[0089] As Figure 2 shown 11H NMR (400 MHz, C2D2Cl4): δ ppm 8.59 (s, 2H, Ar-H), 8.46 (d, J = 8.8 Hz, 2H, Ar-H), 8.00 - 7.94 (m, 6H, Ar-H), 7.71 (t, J = 8.0 Hz, 2H, Ar-H), 7.52 (d, J = 9.2 Hz, 2H, Ar-H), 7.46 (d, J = 8.4 Hz, 2H, Ar-H), 7.33 (t, J = 7.2 Hz, 2H, Ar-H), 7.25 (t, J = 6.8 Hz, 8H, Ar-H), 7.19 - 7.16 (m, 8H, Ar-H), 7.11 - 7.04 (m, 10H, Ar-H), 4.24 (t, J = 8.0 Hz, 4H, N-CH2), 2.03 - 1.94 (m, 4H, CH2), 1.58 - 1.51 (m, 4H, CH2), 1.44 - 1.36 (m, 8H, CH2), 0.94 (t, J = 7.2 Hz, 6H, CH3).

[0090] As Figure 3 shown, 13 13C NMR (100 MHz, CDCl3) δ ppm 155.66, 149.12, 146.83, 145.75, 142.96, 139.23, 137.68, 133.77, 133.53, 133.08, 131.88, 130.32, 129.40, 125.80, 125.27, 124.27, 123.99, 122.43, 121.81, 116.48, 115.91, 115.87, 48.50, 31.39, 26.51, 26.29, 22.65, 13.99.

[0091] As Figure 4 shown, MALDI-TOF mass spectrum (m / z): calcd for C 80 H 70 BF2N7O2S2, [M+Na] + : 1296.5094; found: 1296.3481.

[0092] Combined with Figures 2-4 shown, the molecular structure of BDPPZO can be determined as Formula I.

[0093]

[0094] Example 2

[0095] [BDPPZO NIR-II Fluorescence Quantum Yield Test]

[0096] IR1061 was selected as the reference substance (fluorescence quantum yield in dichloromethane is 1.7%), and the fluorescence quantum yield of BDPPZO was determined by the comparison method.

[0097] IR1061 was dissolved in dichloromethane to prepare five solutions with different concentrations. The absorbance of each solution at 808 nm was not higher than 0.10 (the absorbance values were 0.02, 0.04, 0.06, 0.08, 0.1), and the same operation was performed on BDPPZO. The ultraviolet spectra and fluorescence spectra of each solution were measured respectively, the integral areas of each fluorescence peak were calculated, the linear fitting curve of absorbance - fluorescence peak area was plotted, and the fluorescence quantum yield of BDPPZO was further calculated.

[0098] As Figure 5 shown, the fluorescence quantum yield of BDPPZO in dichloromethane is 13%, which is the highest fluorescence quantum yield among the existing aza - BODIPY - based NIR - II organic fluorescent small molecules.

[0099] Example 3

[0100] [Preparation of BDPPZO nanoparticles]

[0101] BDPPZO nanoparticles were prepared by the nanoprecipitation method as follows:

[0102] BDPPZO molecules (1 mg) (prepared according to the method of Example 1) and Pluronic F - 127 (10 mg) were dissolved in tetrahydrofuran (1 mL) and sonicated for 5 min.

[0103] Then, the solution was quickly injected into deionized water (10 mL) and sonicated for 1 h. After completely removing tetrahydrofuran by stirring and volatilization, it was filtered through a 0.22 - μm filter head to obtain a clear and transparent blue - green BDPPZO nanoparticle solution, which was stored at 4 °C for later use.

[0104] As Figure 6 shown, the test results of the dynamic light scattering particle size distribution of BDPPZO nanoparticles show that its hydrodynamic diameter is 58 nm, and this size meets the requirements of the enhanced permeability and retention (EPR) effect.

[0105] As Figure 7 shown, BDPPZO nanoparticles have a maximum absorption peak at 830 nm in water and show fluorescence emission centered at 1020 nm, indicating that the BDPPZO nanoparticles of the present invention have good NIR - II fluorescence emission behavior.

[0106] Example 4

[0107] [Photodynamic performance test of BDPPZO nanoparticles]

[0108] Add DHR123 fluorescent probe (50 μL, 200 μg / mL) to the aqueous solution of BDPPZO nanoparticles (1 mL, 20 μg / mL), and irradiate with an 808 nm laser (50 mW / cm 2 ), and observe the change in fluorescence intensity at 525 nm.

[0109] As Figure 8 shown, the fluorescence intensity at 525 nm increases with time, indicating that the BDPPZO nanoparticles of the present invention have excellent superoxide anion radical generation ability.

[0110] Example 5

[0111] [Cytotoxicity experiment of BDPPZO nanoparticles]

[0112] Inoculate 4T1 cells at a density of 5.0×10 3 per well into two 96-well cell culture plates, and incubate at 37 °C in a dark environment with 5% CO2 for 24 h; add different concentrations of BDPPZO nanoparticles (0, 5, 7.5, 10, 12.5, 15 μg / mL) to the plates and incubate for 12 h.

[0113] After incubating for 12 h, irradiate one of the plates with an 808 nm laser (1 W / cm 2 ) at room temperature for 5 min per well, and incubate the other plate in the dark to test the dark cytotoxicity of BDPPZO nanoparticles on cells.

[0114] After the light irradiation is completed, continue to incubate the plates in the dark for 12 h, then add 200 μL of MTT solution to each well and incubate in the dark for 4 h. Remove the culture medium from the plates, add 200 μL of dimethyl sulfoxide to dissolve the purple formazan crystals, and read the absorbance value at 490 nm with an enzyme-linked immunosorbent assay reader to calculate the survival rate of 4T1 cells under different concentrations of BDPPZO nanoparticles. The formula for calculating the cell survival rate is: average absorbance of the treatment group / average absorbance of the control group × 100%.

[0115] As Figure 9 shown, in the dark, within the concentration range of 0 - 15 μg / mL, the survival rate of 4T1 cells can remain above 80%; however, under light irradiation conditions, only 10 μg / mL of BDPPZO nanoparticles can kill 60% of 4T1 cells, and 15 μg / mL of BDPPZO nanoparticles can even cause the mortality rate of 4T1 cells to be as high as 95%; this shows that the BDPPZO nanoparticles of the present invention have good biocompatibility and phototoxicity.

[0116] Example 6

[0117] [Experiment on Intracellular Reactive Oxygen Species Generation of BDPPZO Nanoparticles]

[0118] Seed 4T1 cells into a 6-well cell culture plate at a density of 5×10 5 cells per well, incubate in the dark at 37 °C and 5% CO2 for 24 h. Then, remove the medium and wash 3 times with PBS. Add fresh medium containing 30 μg / mL BDPPZO nanoparticles and incubate for 12 h. Then, replace the medium with fresh medium containing 10 μM DHE and incubate for 0.5 h. After that, remove the medium and wash three times with PBS. One well is irradiated with an 808 nm laser (1 W / cm 2 ) for 10 min, and the other well is not irradiated. After the irradiation is completed, quickly capture the fluorescence images with a fluorescence inverted microscope.

[0119] As Figure 10 shown, compared with the non-irradiated group, under the light irradiation condition, obvious superoxide anion radicals are generated in the 4T1 tumor cells incubated with BDPPZO nanoparticles, indicating that the BDPPZO nanoparticles of the present invention can effectively generate superoxide anion radicals in tumor cells.

[0120] Example 7

[0121] [Fluorescence Imaging of BDPPZO Nanoparticles in Mice]

[0122] Inject BDPPZO nanoparticles (200 μL, 500 μg / mL) into mice via the tail vein, and record the fluorescence images of the tumor site at different time points (pre, 3, 6, 9, 12, 24 h).

[0123] As Figure 11 shown, after intravenous injection of BDPPZO nanoparticles in mice, the fluorescence signal at the tumor site gradually increases with time, reaches the maximum value at 12 h, and then weakens, indicating that the distribution of BDPPZO can be detected by fluorescence imaging. The BDPPZO nanoparticles of the present invention can effectively accumulate at the tumor site, reach the maximum accumulation at 12 h, which is the optimal treatment time window, and thus can further guide type I photodynamic therapy.

[0124] It can be proved from this that on the one hand, due to the high fluorescence quantum yield of the BDPPZO nanoparticles of the present invention, high-resolution imaging of deep tissues can be achieved to accurately locate tumors, and on the other hand, the therapeutic effect on hypoxic tumors can be improved.

[0125] Example 8

[0126] [Tumor Therapy Experiment of BDPPZO Nanoparticles]

[0127] A 4T1 tumor-bearing mouse model was constructed by subcutaneous injection of 4T1 cancer cells. When the tumor volume reached about 50 mm 3 or so, subsequent experiments could be carried out.

[0128] The 4T1 tumor-bearing mice were randomly divided into 3 groups (PBS+L, NPs, NPs+L, 3 mice in each group, NPs represents BDPPZO nanoparticles, and L represents laser irradiation). The mice in the PBS+L group were injected with PBS (200 μL) via the tail vein, and the mice in the NPs group and the NPs+L group were injected with BDPPZO nanoparticles (200 μL, 200 μg mL -1 ). 12 h after injection, the tumor sites of the mice in the PBS+L group and the NPs+L group were irradiated with an 808 nm laser (1.0 W cm -2 ) for 5 min. The second treatment was carried out on the 5th day after the start of the experiment, and the tumor size was measured every 2 days until the 14th day.

[0129] As Figure 12 shown, after 2 treatments, the tumors of the mice in the NPs+L group could be completely eliminated on the 10th day after treatment and there was no recurrence within 14 days, while the tumor volumes of the PBS+L group and the NPs group increased by 5-6 times on the 14th day after treatment. This indicates that the BDPPZO nanoparticles of the present invention can effectively kill tumor cells and cure tumors under light irradiation.

[0130] Combined with the above tests, it can be proved that the photosensitizer prepared from the azafluoroboron fluorescence molecule of the present invention can be used for precise and efficient type I photodynamic therapy mediated by NIR-II fluorescence imaging of tumor tissues, reducing tumor treatment damage and side effects, and improving treatment efficiency and accuracy.

[0131] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Those with ordinary knowledge in the technical field to which the present invention pertains can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be subject to what is defined by the claims.

Claims

1. An aza-BODIPY molecule with both NIR-II fluorescence emission and type-I photodynamic activity, characterized in that, The azaboron dipyrromethene molecule is denoted as BDPPZO, and its chemical structure is shown in Formula I:

2. A method for preparing aaza-boron dipyrromethene molecule with both NIR-II fluorescence emission and type I photodynamic activity as described in claim 1, characterized in that, The method includes the following steps: Under a nitrogen atmosphere, 1-(10-hexyl-10H-phenothiazin-3-yl)ethanone, 4-(diphenylamino)benzaldehyde, and potassium hydroxide are dissolved in ethanol and reacted under the required reaction conditions to obtain a first product; The first product, nitromethane, and triethylamine are dissolved in methanol and reacted under the required reaction conditions to obtain a second product; The second product and ammonium acetate are dissolved in n-butanol and reacted under the required reaction conditions to obtain a third product; The third product is dissolved in 1,2-dichloroethane, N,N-diisopropylethylamine and boron trifluoride diethyl etherate are added, and the reaction is carried out under the required reaction conditions to obtain a fourth product; Under normal pressure conditions, the fourth product is dissolved in dichloromethane, and m-chloroperoxybenzoic acid is used as an oxidant, and the reaction is carried out under the required reaction conditions to obtain the azaboron dipyrromethene molecule BDPPZO.

3. The preparation method of theaza-BODIPY molecule with both NIR-II fluorescence emission and type I photodynamic activity according to claim 2, characterized in that, The molar ratio of 1-(10-hexyl-10H-phenothiazin-3-yl)ethanone, 4-(diphenylamino)benzaldehyde, and potassium hydroxide is 1:1:(1 - 2), and the required reaction conditions include reacting at a temperature of 30°C - 35°C for 18 h - 24 h.

4. The preparation method of theaza-BODIPY molecule with both NIR-II fluorescence emission and type I photodynamic activity according to claim 2, characterized in that, The molar ratio of the first product, nitromethane, and triethylamine is 1:(2 - 3):(4 - 5), and the required reaction conditions include reacting at a temperature of 70°C - 75°C for 24 h - 30 h.

5. The preparation method of the BODIPY molecule with both NIR-II fluorescence emission and type-I photodynamic activity according to claim 2, characterized in that, The molar ratio of the second product and ammonium acetate is 1:(50 - 60), and the required reaction conditions include reacting at a temperature of 120°C - 130°C for 30 h - 36 h.

6. The preparation method of theaza-BODIPY molecule with both NIR-II fluorescence emission and type-I photodynamic activity according to claim 2, characterized in that, The molar ratio of the third product, N,N-diisopropylethylamine, and boron trifluoride diethyl etherate is 1:(12 - 16):(10 - 12), and the required reaction conditions include reacting at a temperature of 50°C - 60°C for 20 h - 24 h.

7. The preparation method of theaza-BODIPY molecule with both NIR-II fluorescence emission and type I photodynamic activity according to claim 2, characterized in that, The molar ratio of the fourth product and m-chloroperoxybenzoic acid is 1:(3 - 5), and the required reaction conditions include reacting at room temperature for 6 h - 10 h.

8. Use of the azaboron dipyrromethene molecule with both NIR-II fluorescence emission and type I photodynamic activity as claimed in claim 1 in the preparation of a type I photodynamic therapy drug mediated by NIR-II fluorescence imaging.

9. A photosensitizer, characterized in that, Contains the azaboron dipyrromethene molecule with both NIR-II fluorescence emission and type I photodynamic activity as claimed in claim 1.

10. The photosensitizer according to claim 9, wherein The photosensitizer includes nanoparticles prepared by self-assembly of BDPPZO and the amphiphilic polymer F127.

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