Nir-ii fluorescent and type i photodynamic active aza-fluorophore molecule, preparation method, application and photosensitizer thereof

Abstract

The present application provides a kind of NIR-II fluorescent emission and I type photodynamic activity of nitrogen hetero fluorine boron fluorescent molecule and its preparation method, nitrogen hetero fluorine boron fluorescent molecule has good light absorption in near infrared (NIR) region, and its emission peak is extended to near infrared two (NIR-II) and exhibits 13% of super high NIR-II fluorescence quantum yield.Meanwhile, the photosensitizer prepared by using nitrogen hetero fluorine boron fluorescent molecule can also effectively produce I type active oxygen species-super oxide anion radical under 808nm laser excitation.Based on this, the NIR-II fluorescence of nitrogen hetero fluorine boron fluorescent photosensitizer can realize high-resolution tumor fluorescence imaging, for deep tumor tissue precise, efficient NIR-II fluorescence imaging mediated I type photodynamic therapy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a nitrogen-containing fluorine boron fluorescent molecule with NIR-II fluorescence emission and type I photodynamic activity, a preparation method, application and photosensitizer thereof. BACKGROUND

[0002] Traditional cancer therapies including surgery, chemotherapy and radiotherapy have achieved good results in clinical practice, but still have defects such as limited efficacy, serious side effects and difficulty in radical treatment. In addition, the application effect of traditional treatment strategies is limited due to the complexity of tumor components and structural characteristics. 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, which has the advantages of minimally invasive, not affected by drug resistance, controllable treatment area selection compared with traditional chemotherapy, radiotherapy and surgery. The PDT process involves exposing photosensitizers to a light source and generating cytotoxic reactive oxygen species (ROS) through interaction with molecular oxygen under light excitation, which uses 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 produce singlet oxygen, and with the consumption of oxygen, the treatment effect of type II PDT will be severely hindered due to insufficient oxygen level. Compared with type II PDT with high oxygen dependence and high oxygen consumption, type I PDT generates reactive oxygen species such as superoxide anion free radicals and hydroxyl radicals through electron transfer, which has no dependence on oxygen and shows great potential in treating solid tumors with hypoxic tumor microenvironment. Therefore, developing photosensitizers with type I photodynamic activity has become a research hotspot.

[0004] Fluorescence imaging (FLI) as a new optical imaging technology has a wide range of applications in biomedical basic research and disease diagnosis and treatment due to its high sensitivity, high contrast, simple operation and low radiation. The light that can penetrate biological tissues with the maximum depth is usually located in the near-infrared wavelength range, which 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 effect, and NIR-II fluorescence shows deeper tissue penetration capability. In this window, real-time imaging of high signal-to-noise ratio and large depth of tissue can be achieved, which makes NIR-II fluorescence imaging have significant advantages in tumor intraoperative navigation applications.

[0005] Therefore, constructing a material with NIR-II fluorescence emission and type I photodynamic behavior can effectively solve the problems of insufficient penetration depth, invalidity in hypoxic environment and separation of diagnosis and treatment in traditional diagnosis and treatment technology. Compared with inorganic materials, organic semiconductor conjugated polymers and organic small molecules, organic small molecules have the characteristics of clear structure, adjustable performance, fast metabolism and high biological safety, and are more suitable for the development of type I photosensitizer and subsequent clinical application. However, due to the difficulty in accurately regulating the energy dissipation path, it is still difficult to construct an organic small molecule with NIR-II fluorescence emission and type I photodynamic behavior. At present, few organic small molecules with type I photodynamic behavior can also have NIR-II fluorescence. The NIR-II fluorescence quantum yield (QY) of the organic small molecules reported to have NIR-II fluorescence emission and type I photodynamic behavior is generally low, usually less than 1%. The fluorescence quantum yield determines the quality of imaging. Low fluorescence quantum yield will cause low resolution and signal-to-noise ratio of NIR-II fluorescence imaging, and further cause low imaging penetration depth, which limits its application in the fields of biological imaging and photodynamic therapy. SUMMARY

[0006] The present application aims to synthesize a nitrogen-fluorine boron fluorescent sensitizer molecule with ultrahigh NIR-II fluorescence quantum yield and type I photodynamic activity, to solve the problems of the current type I PDT photosensitizer, such as the lack of types, insufficient diversity and low NIR-II fluorescence quantum yield, and to improve the problem of insufficient tissue penetration in traditional tumor photodiagnosis and treatment, which cannot diagnose and treat deep tumors.

[0007] According to the first aspect of the object of the present application, a nitrogen-fluorine boron fluorescent molecule with NIR-II fluorescence emission and type I photodynamic activity is provided, which is denoted as BDPPZO, and the chemical structure is shown as formula I:

[0008]

[0009] According to the second aspect of the object of the present application, a preparation method of the aforementioned nitrogen-fluorine boron fluorescent molecule with NIR-II fluorescence emission and type I photodynamic activity is provided, which comprises the following steps:

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

[0011] The first product, nitromethane and triethylamine are dissolved in methanol, and the reaction is carried out under the required reaction conditions to obtain a second product;

[0012] The second product and ammonium acetate are dissolved in n-butanol, and the reaction is carried out under the required reaction conditions to obtain a third product;

[0013] The third product is dissolved in 1,2-dichloroethane, N,N-diisopropylethylamine and boron trifluoride ether are added, and the reaction is carried out under the required reaction conditions to obtain the fourth product;

[0014] The fourth product is dissolved in dichloromethane under normal pressure, and m-chloroperoxybenzoic acid is used as an oxidant, and the reaction is carried out under the required reaction conditions to obtain the nitrogen hetero fluorine boron fluorescent molecule BDPPZO.

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

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

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

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

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

[0020] According to the third aspect of the object of the present application, a kind of nitrogen hetero fluorine boron fluorescent molecule with the application of the foregoing NIR-II fluorescent emission and I type photodynamic activity in preparation for NIR-II fluorescent imaging mediated I type photodynamic therapy drug.

[0021] According to the third aspect of the object of the present application, a kind of photosensitizer contains the nitrogen hetero fluorine boron fluorescent molecule with the foregoing NIR-II fluorescent emission and I type photodynamic activity.

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

[0023] It can be known from the above aza-fluorine boron fluorescent molecule of the present application that has NIR-II fluorescence emission and type I photodynamic activity that the aza-fluorine boron fluorescent molecule has good absorption in the NIR region, has a super-high NIR-II fluorescence quantum yield of 13%, can realize high-resolution imaging of deep tissue, and has a high imaging signal-to-noise ratio, thereby providing an effective tool for diagnosis and treatment guidance;

[0024] Meanwhile, the nanoparticles prepared by self-assembly of the aza-fluorine boron fluorescent molecule and the amphiphilic polymer F127 exhibit excellent superoxide anion radical production performance under 808 nm laser excitation, can be used for precise and efficient NIR-II fluorescence imaging mediated type I photodynamic therapy of tumor tissue, reduces tumor treatment damage and side effects, improves treatment efficiency and accuracy, and has a good application prospect in precise diagnosis and treatment of cancer as a new fluorescent sensitizer. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a synthesis reaction line diagram of the BDPPZO of the present application.

[0026] Figure 2 is a BDPPZO in deuterium with tetrachloroethane in the present application 1 H-NMR spectrum.

[0027] Figure 3 is a BDPPZO in deuterium with chloroform in the present application 13 C-NMR spectrum.

[0028] Figure 4 is a mass spectrum of the BDPPZO in the present application.

[0029] Figure 5 is a NIR-II fluorescence quantum yield test diagram of the BDPPZO in the present application.

[0030] Figure 6 is a dynamic light scattering particle size distribution test diagram of the BDPPZO nanoparticles in the present application.

[0031] Figure 7 is a UV absorption and fluorescence emission spectrum diagram of the BDPPZO nanoparticles in water in the present application.

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

[0033] Figure 9 is a 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 is a staining diagram of the BDPPZO nanoparticles of the present application example after incubation with 4T1 cells using superoxide anion fluorescent probe dihydroethidium (DHE).

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

[0036] Figure 12 is a tumor volume change diagram of a breast cancer model mouse during a 14-day treatment process. DETAILED DESCRIPTION

[0037] In order to better understand the technical content of the present application, specific embodiments are described below with reference to the accompanying drawings.

[0038] Aspects of the present application are described in this disclosure with reference to the accompanying drawings, in which a number of illustrative embodiments are shown. The embodiments of this disclosure need not necessarily include all aspects of the present application. It should be understood that the various concepts and embodiments introduced above, and those described in more detail below, can be implemented in any of numerous ways.

[0039] Azafluoroboradiazaindacene

[0040] The exemplary azaborafly molecule of the present application has both NIR-II fluorescence emission and type I photodynamic activity, and is denoted as BDPPZO, and the chemical structure is shown in formula I:

[0041]

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

[0043] Preparation method

[0044] Combination Figure 1 As shown in the exemplary embodiments of the present application, a preparation method of the aforementioned azaborafly molecule having both NIR-II fluorescence emission and type I photodynamic activity is provided, which is prepared by reacting 1-(10-hexyl-10H-phenothiazine-3-yl) ethanone and 4-(diphenylamino) benzaldehyde as initial raw materials according to the reaction route of Figure 1 .

[0045] In an alternative example, the method of preparing aza-fluoroboradiazaindacene molecule having NIR-II fluorescence emission and type I photodynamic activity comprises the steps of:

[0046] dissolving 1-(10-hexyl-10H-phenothiazin-3-yl)ethanone (compound 1), 4- (diphenylamino)benzaldehyde (compound 2) and potassium hydroxide in ethanol under nitrogen atmosphere, carrying out the reaction under the desired reaction conditions, followed by extracting the reaction solution with dichloromethane and water to obtain a first crude product, purifying to obtain a first product (compound 3);

[0047] dissolving the first product, nitromethane and triethylamine in methanol, carrying out the reaction under the desired reaction conditions, cooling, acidifying and extracting with dichloromethane after the completion of the reaction to obtain a second crude product, purifying to obtain a second product (compound 4);

[0048] dissolving the second product and ammonium acetate in n-butanol, carrying out the reaction under the desired reaction conditions, followed by cooling to room temperature and extracting with cold ethanol to obtain a third product (compound 5);

[0049] dissolving the third product in 1,2-dichloroethane, adding N,N- diisopropylethylamine and boron trifluoride etherate, carrying out the reaction under the desired reaction conditions, followed by extracting with dichloromethane and water, collecting the organic layer, drying and evaporating to obtain a third crude product. Purifying to obtain a fourth product (compound 6);

[0050] dissolving the fourth product in dichloromethane under normal pressure, slowly adding m-chloroperoxybenzoic acid under ice bath conditions, carrying out the reaction under the desired reaction conditions, treating the reaction mixture with aqueous sodium hydroxide until the pH is neutral after the completion of the reaction, extracting with dichloromethane, removing the solvent under reduced pressure to obtain a third crude product, purifying to obtain aza-fluoroboradiazaindacene 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 desired reaction conditions include carrying out the reaction at a temperature of 30-35°C for 18-24h.

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

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

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

[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 6-10h.

[0056] Application

[0057] In another exemplary embodiment of the present application, there is provided a use of the aforementioned nitrogen-containing fluorine-containing fluorescent molecule with both NIR-II fluorescence emission and type I photodynamic activity in the preparation of a drug for NIR-II fluorescence imaging mediated type I photodynamic therapy.

[0058] In another exemplary embodiment of the present application, there is provided a photosensitizer containing the aforementioned nitrogen-containing fluorine-containing fluorescent 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 808nm laser excitation, and can be applied to precise and efficient NIR-II fluorescence imaging mediated type I photodynamic therapy of tumor tissue, reducing tumor treatment damage and side effects, and improving treatment efficiency and accuracy.

[0061] For better understanding, the present application is further described below in conjunction with several specific examples, but the preparation process is not limited thereto, and the content of the present application 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] Compound 3 (1.219 g, 52%) was obtained as a red oil by dissolving 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) in ethanol (30 mL) and stirring at 30 °C overnight; the reaction solution was extracted with dichloromethane and water (1:1 by volume) to obtain the crude product; finally, the product was purified by column chromatography on silica gel using dichloromethane / petroleum ether (1:2) as eluent to obtain compound 3 (1.219 g, 52%) as a red oil.

[0066] 1 H NMR (400 MHz, CDC13): δ ppm 7.87 (dd, J = 8.4, 2.0 Hz, 1 H, Ar-H), 7.83-7.76 (m, 2 H, Ar-H), 7.50 (d, J = 8.8 Hz, 2 H, Ar-H), 7.40 (d, J = 15.2 Hz, 1 H, CH-C=0), 7.34-7.29 (m, 4 H, Ar-H), 7.18-7.10 (m, 8 H, Ar-H), 7.05 (d, J = 8.4 Hz, 2 H, Ar-H), 6.94 (t, J = 7.2 Hz, 1 H), 6.87 (d, J = 8.4 Hz, 2 H), 3.87 (t, J = 7.2 Hz, 2 H, N-CH2), 1.87-1.79 (m, 2 H, CH2), 1.47-1.32 (m, 6 H, CH2), 0.92-0.89 (m, 3 H, CH3).

[0067] 13 C NMR (100 MHz, CDC13): 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] The structure of compound 3 can be determined as 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, neutralized with dilute hydrochloric acid (2 M) and then extracted with dichloromethane to obtain the crude product as a dark orange oil; the crude product was further purified by column chromatography on silica gel eluting with dichloromethane / petroleum ether (1 :2) to obtain the product compound 4 as an orange oil (0.882 g, 69%).

[0072] 1 H NMR (400 MHz, CDC13): δ ppm 7.73 (d, J = 8.4 Hz, 1 H, Ar-H), 7.64 (s, 1 H, 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, 1 H, CH2-NO2), 4.65 (dd, J = 12.4, 8.0 Hz, 1 H, CH2-NO2), 4.14 (m, 1 H, 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 was determined to be 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 to reflux at 130 °C for 36 h; cooled to room temperature, extracted with cold ethanol and washed three times to obtain the product compound 5 as a dark blue solid (0.480 g, 80%).

[0077] 1H NMR (400 MHz, CDC13): 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 C NMR (100 MHz, CDC13): 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] Compound 5 (0.418 g, 0.35 mmol) was dissolved in dry 1,2-dichloroethane (25 mL) and treated with N,N-diisopropylethylamine (0.498 g, 3.85 mmol) and boron trifluoride etherate (0.795 g, 5.6 mmol) and stirred at 50 °C under nitrogen atmosphere for 24 h; extracted with dichloromethane and water, collected the organic layer, dried, evaporated to get crude product; purified by column chromatography eluting with dichloromethane / pet ether (1:1) to get dark blue product compound 6 (0.200 g, 46%).

[0083] 1H NMR (400 MHz, CDC13): 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), 7.07-7.02 (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 C NMR (100 MHz, CDC13): 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] Compound 6 (186 mg, 0.15 mmol) was dissolved in 20 mL of dichloromethane, and m-chloroperbenzoic acid (104 mg, 0.6 mmol) was slowly added under ice bath conditions. After stirring at room temperature and normal pressure for 6 h, it was extracted with aqueous sodium hydroxide solution (2M) and dichloromethane, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (silica, ethyl acetate:dichloromethane = 1:1) to obtain a dark blue solid BDPPZO (70 mg, 37%).

[0089] As shown in Figure 2 , the structure of compound 6 can be determined as Formula V. 1H 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 shown in Figure 3 , the molecular structure of BDPPZO can be determined as Formula I. 13 C 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 shown in Figure 4 , the MALDI-TOF mass spectrum (m / z): calcd for C 80 H 70 BF2N7O2S2, [M+Na] + : 1296.5094; found: 1296.3481.

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

[0093]

[0094] Example 2

[0095] [BDPPZO NIR-II fluorescent quantum yield test]

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

[0097] IR1061 was dissolved in dichloromethane, and five solutions with different concentrations were prepared, and the absorption at 808 nm of each solution was not higher than 0.10 (the absorption values were 0.02, 0.04, 0.06, 0.08, and 0.1, respectively). BDPPZO was also subjected to the same operation. The ultraviolet spectrum and fluorescence spectrum of each solution were tested respectively, the integral area of each fluorescence peak was calculated, and the linear fitting curve of absorbance-fluorescence peak area was drawn to further calculate the fluorescence quantum yield of BDPPZO.

[0098] As shown in Figure 5 , the fluorescence quantum yield of BDPPZO in dichloromethane was 13%, which was 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 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 ultrasonicated for 5 min.

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

[0104] As shown in Figure 6 , the dynamic light scattering particle size distribution test results of BDPPZO nanoparticles showed that the hydrated particle size was 58 nm, which met the requirements of the enhanced permeability and retention (EPR) effect.

[0105] As shown in Figure 7 , the maximum absorption peak of BDPPZO nanoparticles in water was 830 nm, and the fluorescence emission was centered at 1020 nm, which indicated that the BDPPZO nanoparticles of the present application had good NIR-II fluorescence emission behavior.

[0106] Example 4

[0107] [BDPPZO nanoparticle photodynamic performance test]

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

[0109] As shown in Figure 8 , the fluorescence intensity at 525 nm increased with time, indicating that the BDPPZO nanoparticles of the present application have superior superoxide anion radical production capacity.

[0110] Example 5

[0111] [Cell toxicity experiment of BDPPZO nanoparticles]

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

[0113] After 12 h of incubation, one of the plates was irradiated with 808 nm laser (1 W / cm 2 ) at room temperature for 5 min per well, and the other plate was incubated in the dark to test the dark toxicity of the BDPPZO nanoparticles.

[0114] After the irradiation was completed, the plates were further incubated in the dark for 12 h, 200 μL of MTT solution was added to each well and incubated in the dark for 4 h. The culture medium was removed, 200 μL of dimethyl sulfoxide was added to dissolve the blue-purple formazan crystals, and the absorbance value at 490 nm was read by an enzyme marker instrument. The survival rate of 4T1 cells under different concentrations of BDPPZO nanoparticles was calculated. The cell survival rate calculation formula is: average absorbance value of treatment group / average absorbance value of control group × 100%.

[0115] As shown in Figure 9 , under dark conditions, the survival rate of 4T1 cells could remain above 80% in the concentration range of 0-15 μg / mL; however, under irradiation conditions, 10 μg / mL of BDPPZO nanoparticles could kill 60% of 4T1 cells, and 15 μg / mL of BDPPZO nanoparticles could kill up to 95% of 4T1 cells; this indicates that the BDPPZO nanoparticles of the present application have good biocompatibility and phototoxicity.

[0116] Example 6

[0117] [Intracellular reactive oxygen species production experiment of BDPPZO nanoparticles]

[0118] 4T1 cells were seeded into 6-well cell culture plates at a density of 5 x 10 5 cells per well and incubated at 37°C in a dark light environment with 5% CO2 for 24 h. Then, the culture medium was removed and washed with PBS for 3 times, and fresh culture medium containing 30 μg / mL of BDPPZO nanoparticles was added and incubated for 12 h. Then, the culture medium was replaced with fresh culture medium containing 10 μM DHE and incubated for 0.5 h. After that, the culture medium was removed and washed with PBS for 3 times. One well was irradiated with an 808 nm laser (1 W / cm 2 ) for 10 min, and the other well was not irradiated. After the irradiation, the fluorescent images were rapidly captured with a fluorescence inverted microscope.

[0119] As shown in Figure 10 , compared with no irradiation, there was obvious superoxide anion radical production in 4T1 tumor cells incubated with BDPPZO nanoparticles under irradiation, which indicated that the BDPPZO nanoparticles of the present application could effectively generate superoxide anion radicals in tumor cells.

[0120] Example 7

[0121] [Fluorescence imaging of BDPPZO nanoparticles in mice in vivo]

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

[0123] As shown in Figure 11 , after the mice were injected with BDPPZO nanoparticles intravenously, the fluorescence signal of the tumor site gradually increased with time, reached a maximum at 12 h, and then decreased, which indicated that the distribution of BDPPZO could be detected by fluorescence imaging, and the BDPPZO nanoparticles of the present application could effectively enrich in the tumor site, reached a maximum enrichment at 12 h, which was the best treatment time window, thereby further guiding the type I photodynamic therapy.

[0124] It can be seen that the BDPPZO nanoparticles of the present application can achieve high-resolution imaging of deep tissues and accurately locate tumors on the one hand due to their high fluorescence quantum yield, and can improve the treatment effect on hypoxic tumors on the other hand.

[0125] Example 8

[0126] [Experiment of tumor treatment of BDPPZO nanoparticles]

[0127] Subcutaneous injection of 4T1 cancer cells to construct 4T1 tumor-bearing mouse model, when the tumor volume reaches 50mm 3 Afterwards, the subsequent experiment can be carried out.

[0128] The 4T1 tumor-bearing mice were randomly divided into 3 groups (PBS+L, NPs, NPs+L, 3 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 ) via the tail vein. After 12 hours of injection, the mice in the PBS+L group and the NPs+L group were irradiated with 808 nm laser (1.0 W cm -2 ) for 5 min. The second treatment was carried out on the 5th day of the experiment, and the tumor size was measured every 2 days, lasting until the 14th day.

[0129] As shown in Figure 12 , after 2 treatments, the tumors of the mice in the NPs+L group were completely eliminated on the 10th day after treatment and did not recur within 14 days, while the tumor volumes of the mice in the PBS+L group and the NPs group increased by 5-6 times on the 14th day after treatment, which indicates that the BDPPZO nanoparticles of the present application can effectively kill tumor cells and cure tumors under irradiation.

[0130] In combination with the above tests, it can be proved that the photosensitizer prepared from the nitrogen-containing fluorine boron fluorescent molecule of the present application can be used for precise and efficient NIR-II fluorescence imaging mediated type I photodynamic therapy of tumor tissue, reduces the damage and side effects of tumor treatment, and improves the treatment efficiency and accuracy.

[0131] Although the present application has been disclosed as above with preferred embodiments, it is not intended to limit the present application. Those skilled in the art without departing from the spirit and scope of the present application can make various modifications and decorations. Therefore, the protection scope of the present application shall be subject to the definition of the claims.

Claims

1. A nitrogen-containing fluorinated boron fluorescent molecule possessing both NIR-II fluorescence emission and type I photodynamic activity, characterized in that, This nitrogen-containing fluoride boron fluorescent molecule is designated BDPPZO, and its chemical structure is shown in Formula I: Formula I.

2. A method for preparing a nitrogen-containing fluorinated boron fluorescent molecule possessing both NIR-II fluorescence emission and type I photodynamic activity as described in claim 1, characterized in that, The preparation method follows the reaction route below: ； Includes the following steps: Under a nitrogen atmosphere, 1-(10-hexyl-10H-phenthiazin-3-yl) ethyl ketone, 4-(diphenylamino)benzaldehyde and potassium hydroxide were dissolved in ethanol and reacted under the desired reaction conditions to give the first product compound 3. The first product, nitromethane, and triethylamine were dissolved in methanol and reacted under the desired reaction conditions to obtain the second product, compound 4. The second product and ammonium acetate were dissolved in n-butanol and reacted under the desired reaction conditions to obtain the third product, compound 5. The third product was dissolved in 1,2-dichloroethane, and N,N-diisopropylethylamine and boron trifluoride diethyl ether were added. The reaction was carried out under the desired reaction conditions to give the fourth product, compound 6. Under normal pressure, the fourth product is dissolved in dichloromethane, and meta-chloroperoxybenzoic acid is used as an oxidant. The reaction is carried out under the required reaction conditions to obtain the nitrogen-containing fluorinated boron fluorescent molecule BDPPZO.

3. The method for preparing the nitrogen-containing fluorinated boron fluorescent 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-phenthiazin-3-yl) ethyl ketone, 4-(diphenylamino)benzaldehyde and potassium hydroxide is 1:1:(1-2), and the required reaction conditions include reacting at 30°C-35°C for 18-24 h.

4. The method for preparing the nitrogen-containing fluorinated boron fluorescent 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 24-30 h.

5. The method for preparing the nitrogen-containing fluorinated boron fluorescent 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 to ammonium acetate is 1:(50-60), and the required reaction conditions include reacting at a temperature of 120°C-130°C for 30-36 h.

6. The method for preparing the nitrogen-containing fluorinated boron fluorescent 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 ether is 1:(12-16):(10-12), and the required reaction conditions include reacting at a temperature of 50°C-60°C for 20-24 h.

7. The method for preparing the nitrogen-containing fluorinated boron fluorescent 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 to m-chloroperoxybenzoic acid is 1:(3-5), and the required reaction conditions include reacting at room temperature for 6-10 h.

8. The use of the nitrogen-fluorinated boron fluorescent molecule of claim 1, which possesses both NIR-II fluorescence emission and type I photodynamic activity, in the preparation of a type I photodynamic therapeutic drug mediated by NIR-II fluorescence imaging.

9. A photosensitizer, characterized in that, The molecule contains the nitrogen-containing fluorine boron fluorescent molecule as described in claim 1, which possesses both NIR-II fluorescence emission and type I photodynamic activity.

10. The photosensitizer according to claim 9, characterized in that, The photosensitizer comprises nanoparticles prepared by self-assembly of BDPPZO and the amphiphilic polymer F127.

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