BODIPY derivative, nano preparation, corresponding preparation method and application

Abstract

The invention discloses a BODIPY derivative, a nano preparation, a corresponding preparation method and application, and the BODIPY derivative is prepared by taking BODIPY as a mother nucleus and adjusting substituent groups and expanding a conjugated system. According to the BODIPY derivative and the nano preparation, the emission wavelength can extend to an NIR-II region, and the BODIPY derivative and the nano preparation can be used for preparing a medicine for inhibiting tumor cell growth or a near-infrared two-region fluorescence imaging agent.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, relates to a BODIPY derivative, and also relates to a nanoformulation, preparation and application of the BODIPY derivative. Background Art

[0002] Tumor phototherapy guided by the near-infrared region I (NIR-I, 700-900nm) has attracted widespread attention in the biomedical field in recent years. The core advantage of this technology lies in its unique optical properties: compared with visible light, NIR-I has better tissue penetration ability (up to several millimeters), and can effectively reduce the light absorption and scattering effects of biological components such as hemoglobin and water molecules. This spectral characteristic gives it a breakthrough potential in deep tissue treatment, and has promoted the innovative development of the two major technical systems of photothermal therapy (PTT) and photodynamic therapy (PDT). As an emerging research direction in the field of photomedicine, the near-infrared region II (NIR-II, usually referring to 900-1700nm) has shown significant advantages in tissue penetration depth, imaging resolution, photothermal efficiency and biosafety compared to the near-infrared region I (NIR-I, 700-900nm), becoming a key direction to break through the bottleneck of existing technologies. Most existing materials with near-infrared region II emission suffer from poor biocompatibility, low quantum yield, and potential toxicity. Therefore, it is necessary to develop near-infrared region II photosensitizers with relatively simple structures and good biosafety for guided tumor phototherapy. In 2019, Academician Peng Xiaojun et al. reported trifluoromethyl and methyl-substituted fluoroboron dipyrrole (BODIPY) derivatives (Advanced Materials, 2020, 32. DOI: 10.1002 / adma.201907855.) and tested the photothermal conversion ability of the compounds. Among them, the trifluoromethyl-substituted BODIPY derivative had excellent photothermal conversion efficiency, but its maximum absorption wavelength could not be further improved. Summary of the Invention

[0003] Objectives of the invention: The objective of the present invention is to provide a BODIPY derivative to increase the absorption and emission wavelengths of the compound; another objective of the present invention is to provide a nanoformulation prepared from the BODIPY derivative; a further objective of the present invention is to provide an application of the nanoformulation; and the present invention also provides a method for preparing the BODIPY derivative.

[0004] Technical solution: The BODIPY derivative of the present invention has the structure shown in Formula I:

[0005]

[0006] In the structure shown in Formula I, R1 is a thienyl group or a phenyl group, R2 is a hydrogen group or a methyl group, and X is Br or I.

[0007] Preferably, R1 is 2-thienyl, with the structural formula

[0008] Preferably, the BODIPY derivative has the structure shown in Formula I-1, I-2, or I-3;

[0009]

[0010] The nanoformulation of the present invention comprises a BODIPY derivative having a structure shown in Formula I. The BODIPY derivative is coated with a DSPE-PEG layer. The mass ratio of the BODIPY derivative to DSPE-PEG is 1:8-12.

[0011] Preferably, the particle size of the nanoformulation is less than 250 nm.

[0012] The preparation method of the aforementioned nanoformulation is as follows: dissolving the structural compound represented by Formula I and DSPE-PEG in a first organic solvent to obtain a solution, dripping the solution into water at a rotation speed of 300-600 rpm, and stirring until the first organic solvent is completely evaporated to obtain the nanoformulation.

[0013] Preferably, the first organic solvent is one or a mixture of acetone, tetrahydrofuran, methanol and ethanol.

[0014] The aforementioned nanomaterials are directly used to prepare drugs or near-infrared second-zone fluorescent imaging agents that use photodynamic therapy to inhibit tumor cell growth.

[0015] Preferably, the concentration of the nanoformulation is 200-300 μg / mL.

[0016] The preparation method of the aforementioned BODIPY derivative comprises the following steps: subjecting the compound of the structure shown in Formula II to a halogenation reaction to obtain the compound of the structure shown in Formula III; subjecting the compound of the structure shown in Formula III to a condensation reaction with 4-diethylaminobenzaldehyde to obtain the BODIPY derivative of the structure shown in Formula I.

[0017]

[0018] Preferably, the reaction conditions of the halogenation reaction are as follows: the structure represented by Formula II and the halogenation reagent are dissolved in a second organic solvent, and reacted at room temperature for more than 4 hours to obtain a compound represented by Formula III, and the molar ratio of the compound represented by Formula II to the halogenation reagent is 1:2.5 to 3.5, and the second organic solvent is one or a mixture of dichloromethane, chloroform, and 1,2-dichloroethane, and the halogenation reagent is N-bromosuccinimide, N-chlorosuccinimide or N-iodosuccinimide.

[0019] Preferably, the condensation reaction is a Knoevenagel condensation, and the condensation reaction conditions are as follows: the compound represented by formula II and 4-diethylaminobenzaldehyde are dissolved in toluene, acetic acid and piperidine are added as catalysts, and the reaction is carried out at 110-120° C. for not less than 3 hours to obtain the compound represented by formula I, the molar ratio of the compound represented by formula II to 4-diethylaminobenzaldehyde is 1:2.5-3.5; and the amount of catalyst added is 1-3% of the volume of toluene.

[0020] Preferably, the synthesis method of the compound of formula II is:

[0021] (1) dissolving a pyrrole compound and thiophene carboxaldehyde in dichloromethane, adding trifluoroacetic acid as a catalyst, and stirring at room temperature for 12 to 24 hours under a nitrogen atmosphere, wherein the molar ratio of thiophene carboxaldehyde to pyrrole compound is 1:2.1 to 2.5, and the amount of trifluoroacetic acid added is 0.002 to 0.003% by volume of dichloromethane;

[0022] (2) Place in an ice bath, add an amount of 2,3-dichloro-5,6-dicyanobenzoquinone equal to that of thiophene carboxaldehyde, and stir for at least 2 hours;

[0023] (3) adding triethylamine and boron trifluoride etherate, and reacting for not less than 6 hours to obtain a compound of formula II; wherein the amount of triethylamine added is 3 to 3.5 times the molar amount of the pyrrole compound, and the volume ratio of boron trifluoride etherate to triethylamine is 1 to 1.3:1.

[0024] Preferably, the pyrrole compound is 2-methylpyrrole or 2,4-dimethylpyrrole.

[0025] Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: 1. Improving the maximum absorption wavelength and emission wavelength of the compound: The several BODIPY derivatives developed by the present invention extend their emission wavelength to the NIR-II region by adjusting the substituents and expanding the conjugated system, and have broad application prospects in clinical practice; 2. Can be directly used for treatment or guided treatment: The several BODIPY derivatives developed by the present invention, as photosensitizers, can produce near-infrared region II emission under the irradiation of 808nm laser, and can be used as active oxygen promoters for direct treatment, or as near-infrared region II imaging fluorescent agents for tumors or blood vessels to guide treatment; 3. It has photothermal and photodynamic effects and can be directly used to treat tumors; 4. Lower drug concentrations can be used, and lower light intensity and illumination time can be used for treatment. It has good biocompatibility and low toxicity and can be well used in vivo; 5. It will not cause tumor tolerance, has few side effects, and is non-irritating. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1In the figure, A and B are the H NMR spectrum and mass spectrum of Example 1, respectively;

[0027] Figure 2 In the figure, A and B are the H NMR spectrum and mass spectrum of Example 2, respectively;

[0028] Figure 3 In the figure, A and B are respectively the H NMR spectrum and mass spectrum of Example 3;

[0029] Figure 4 In the figure, A and B are the H NMR spectrum and mass spectrum of Comparative Example 1, respectively;

[0030] Figure 5 In the figure, A and B show the particle size distribution and stability of TBDPN2 and TBDPN2 NPs, respectively; C and D show the particle size distribution and stability of TBDPN3 and TBDPN3 NPs, respectively; E and F show the particle size distribution and stability of BBDPN and BBDPNNPs, respectively; G and H show the particle size distribution and stability of TBDPN4 and TBDPN4 NPs, respectively;

[0031] Figure 6 In the figure, A and B show the UV absorption and fluorescence emission spectra of TBDPN2 and TBDPN2 NPs, respectively; C and D show the UV absorption and fluorescence emission spectra of TBDPN3 and TBDPN3 NPs, respectively; E and F show the UV absorption and fluorescence emission spectra of TBDPN4 and TBDPN4 NPs, respectively; G and H show the UV absorption and fluorescence emission spectra of BBDPN and BBDPN NPs, respectively;

[0032] Figure 7 In the figure, A and B show the concentration-dependent temperature-increasing curve and power-dependent temperature-increasing curve of TBDPN2 NPs, and C and D show the temperature-increasing and temperature-decreasing curves of TBDPN2 NPs and the calculated curve of photothermal conversion efficiency, respectively;

[0033] Figure 8 In the figure, A and B show the concentration-dependent temperature-increasing curve and power-dependent temperature-increasing curve of TBDPN3 NPs, and C and D show the temperature-increasing and temperature-decreasing curves of TBDPN3 NPs and the calculated curve of photothermal conversion efficiency, respectively;

[0034] Figure 9 In the figure, A and B show the concentration-dependent temperature-increasing curve and power-dependent temperature-increasing curve of BBDPN NPs, and C and D show the temperature-increasing and temperature-decreasing curves of BBDPN NPs and the calculated curve of photothermal conversion efficiency, respectively;

[0035] Figure 10In the figure, A and B show the concentration-dependent temperature rise curve and power-dependent temperature rise curve of TBDPN4 NPs, and C and D show the temperature rise and fall curve of TBDPN4 NPs and the calculated curve of photothermal conversion efficiency, respectively.

[0036] Figure 11 In the figure, the ability of TBDPN2, TBDPN3, BBDPN and TBDPN4 photosensitizers to generate singlet oxygen in solution is shown, as well as the comparison of the ability of different photosensitizers to generate singlet oxygen;

[0037] Figure 12 In the figure, A and B show the ability of TBDPN2 NPs to generate reactive oxygen species in 4T1 cells and the killing effect on 4T1 cells; C and E show the cytotoxic effects of TBDPN2 NPs, TBDPN3 NPs, and BBDPN NPs on 4T1 cells, respectively;

[0038] Figure 13 In the figure, A shows the vascular imaging effects of different nanoformulations, and B shows the imaging effects of TBDPN2 under different wavelength conditions; C, D, and E show the accumulation of TBDPN2 NPs in the tumor site and the accumulation of TBDPN2 NPs in various organs, respectively;

[0039] Figure 14 Figure A shows the treatment plan of the animal experiment, Figure B shows the changes in the temperature of the tumor site during the treatment process, Figure C shows the changes in the weight of mice during the monitoring period, Figure D shows the changes in the monitored tumor size, Figure E shows the final tumor size, and Figure F shows pictures of the tumors between the groups. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below with reference to the accompanying drawings. Unless otherwise specified, all reagents used are commercially available and are used directly without further purification.

[0041] Example 1 Synthesis of TBDPN2 and Preparation of Nanoformulations

[0042] 1.1TBDPN2 synthesis route is as follows:

[0043]

[0044]

[0045] 2-Methylpyrrole (1.78 g, 22 mmol) and 2-thiophenecarboxaldehyde (1.12 g, 10 mmol) were added to a round-bottom flask and dissolved in 500 mL of dichloromethane. Under a nitrogen atmosphere, 10 μL of trifluoroacetic acid was added dropwise and stirred at room temperature overnight. 2,3-Dichloro-5,6-dicyanobenzoquinone (2.27 g, 10 mmol) was added under an ice bath and stirred for two hours. 10 mL of triethylamine was then added. Once the white mist disappeared, 12 mL of boron trifluoride etherate was added and the reaction was allowed to proceed for 6 hours. The organic phase was washed with saturated brine, separated, and the solvent removed. The resulting compound, TBDP, was purified by silica gel column chromatography in a 17% yield.

[0046] Add TBDP (100 mg, 0.33 mmol) and N-bromosuccinimide (NBS, 178 mg, 1 mmol) to a round-bottom flask and dissolve in 200 mL of dichloromethane. Stir and react at room temperature for 4-6 hours. Remove the solvent and purify by silica gel column chromatography to obtain TBDP-Br. Yield: 90%.

[0047] TBDP-Br (101 mg, 0.22 mmol) and p-diethylaminobenzaldehyde (117 mg, 0.66 mmol) were added to a flask and dissolved in 30 mL of toluene. The mixture was refluxed at 110-120°C for 3-6 hours under a nitrogen atmosphere in the presence of acetic acid (0.7 mL) and piperidine (2.7 mL). The solvent was removed and the product was purified by silica gel column chromatography to obtain TBDPN2 in an 18% yield.

[0048] 1.2 TBDPN2 Nanoformulation: 1 mg of synthesized TBDPN2 and 10 mg of distearoylphosphatidylethanolamine-polyethylene glycol (DSPE)-PEG2000 were dissolved in 5 mL of tetrahydrofuran and thoroughly mixed under ultrasound. The solution was then added dropwise to 10 mL of ultrapure water under stirring (300-600 rpm). The mixture was stirred at room temperature for 8 hours. After the organic solvent was completely evaporated, the resulting solution was the TBDPN2 nanoformulation (TBDPN2 NPs).

[0049] Example 2 Synthesis of TBDPN3 and Preparation of Nanoformulations

[0050] 2.1 Synthesis route of TBDPN3:

[0051]

[0052] TBDP (100 mg, 0.33 mmol) and N-iodosuccinimide (NIS, 225 mg, 1 mmol) were added to a round-bottom flask and dissolved in 200 mL of dichloromethane. The mixture was stirred at room temperature for 4-6 hours. The solvent was removed and the mixture was purified by silica gel column chromatography to obtain TBDP-I.

[0053] TBDP-I (100 mg, 0.18 mmol) and p-diethylaminobenzaldehyde (96 mg, 0.54 mmol) were added to a flask and dissolved in 30 mL of toluene. The mixture was refluxed at 110-120°C for 3-6 hours under a nitrogen atmosphere in the presence of acetic acid (0.7 mL) and piperidine (2.7 mL). The solvent was removed and the mixture was purified by silica gel column chromatography to obtain TBDPN3 in a 20% yield.

[0054] 2.2TBDPN3 Nanoformulation: Dissolve TBDPN3 (1 mg) and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE)-PEG2000 (10 mg) in 5 mL of tetrahydrofuran and thoroughly mix under ultrasound. Add the solution dropwise to 10 mL of ultrapure water while stirring (300-600 rpm). Stir at room temperature for 8 hours. After the organic solvent has completely evaporated, the resulting solution is the TBDPN3 nanoformulation.

[0055] Example 3 Synthesis of BBDPN and Preparation of Nanoformulations

[0056] 3.1 Synthesis route of BBDPN:

[0057]

[0058] 2-Methylpyrrole (1.78 g, 22 mmol) and benzaldehyde (1.06 g, 10 mmol) were added to a round-bottom flask and dissolved in 500 mL of dichloromethane. Under a nitrogen atmosphere, 10 μL of trifluoroacetic acid was added dropwise, and the mixture was stirred at room temperature overnight. 2,3-Dichloro-5,6-dicyanobenzoquinone (2.27 g, 10 mmol) was added under an ice bath and stirred for two hours. 10 mL of triethylamine was then added, and after the white mist disappeared, 12 mL of boron trifluoride etherate was added and the reaction was allowed to proceed for 6 hours. The organic phase was washed with saturated brine, separated, and the solvent removed. Purification was performed by silica gel column chromatography to obtain the compound obtained, BBDP, in a 15% yield.

[0059] BBDP (101 mg, 0.34 mmol) and N-bromosuccinimide (NBS, 178 mg, 1 mmol) were added to a round-bottom flask and dissolved in 200 mL of dichloromethane. The mixture was stirred at room temperature for 4-6 hours. The solvent was removed and the mixture was purified by silica gel column chromatography to obtain BBDP-Br. The yield was 95%.

[0060] BBDP-Br (100 mg, 0.22 mmol) and p-diethylaminobenzaldehyde (117 mg, 0.66 mmol) were added to a flask and dissolved in 30 mL of toluene. The mixture was refluxed at 90°C under a nitrogen atmosphere for 4-6 hours in the presence of acetic acid (0.7 mL) and piperidine (2.7 mL). The solvent was removed and the mixture was purified by silica gel column chromatography to obtain BBDPN in a 23% yield.

[0061] 3.2TBDPN3 Nanoformulation: Dissolve BBDPN (1 mg) and DSPE-PEG2000 (10 mg) in 5 mL of tetrahydrofuran and thoroughly mix under ultrasound. Add the solution dropwise to 10 mL of ultrapure water while stirring (300-600 rpm). Stir at room temperature for 8 hours. After the organic solvent has completely evaporated, the resulting solution is the TBDPN3 nanoformulation, BBDPN NPs.

[0062] Comparative Example 1: Synthesis of TBDPN4 and Preparation of Nanoformulations

[0063]

[0064] 2,4-Methylpyrrole (2.09 g, 22 mmol) and 2-thiophenecarboxaldehyde (1.12 g, 10 mmol) were added to a round-bottom flask and dissolved in 500 mL of dichloromethane. 10 μL of trifluoroacetic acid was added dropwise under a nitrogen atmosphere and stirred at room temperature overnight. 2,3-Dichloro-5,6-dicyanobenzoquinone (2.27 g, 10 mmol) was added under an ice bath and stirred for two hours. 10 mL of triethylamine was then added. Once the white mist disappeared, 12 mL of boron trifluoride etherate was added and the reaction was allowed to react for 6 hours. The organic phase was washed with saturated brine, separated, and the solvent removed. The resulting compound, BDP2, was purified by silica gel column chromatography in a 23% yield.

[0065] BDP2 (109 mg, 0.33 mmol) and N-bromosuccinimide (NBS, 178 mg, 1 mmol) were added to a round-bottom flask and dissolved in 200 mL of dichloromethane. The mixture was stirred at room temperature for 4-6 hours. The solvent was removed and the mixture was purified by silica gel column chromatography to obtain BDP2-Br in a 92% yield.

[0066] BDP2-Br (107 mg, 0.22 mmol) and p-diethylaminobenzaldehyde (117 mg, 0.66 mmol) were added to a flask and dissolved in 30 mL of toluene. The mixture was refluxed at 110-120°C for 3-6 hours under a nitrogen atmosphere in the presence of acetic acid (0.7 mL) and piperidine (2.7 mL). The solvent was removed and the product was purified by silica gel column chromatography to obtain TBDPN4, a 24% yield.

[0067] 1.2 TBDPN4 Nanoformulation: Dissolve the synthesized TBDPN2 (1 mg) and distearoylphosphatidylethanolamine-polyethylene glycol (DSPE)-PEG2000 (10 mg) in 5 mL of tetrahydrofuran and thoroughly mix under ultrasound. Add the solution dropwise to 10 mL of ultrapure water while stirring (300-600 rpm). Stir at room temperature for 8 hours. After the organic solvent has completely evaporated, the resulting solution is the TBDPN4 nanoformulation (TBDPN4 NPs).

[0068] Example 4 Molecular Structure and Nanoformulation Characterization

[0069] The TBDPN2, TBDPN3, BBDPN and TBDPN4 prepared in Examples 1 to 3 and Comparative Example 1 were subjected to nuclear magnetic resonance and mass spectrometry analysis. The test results are as follows: Figures 1 to 4 As shown in Figure 2, the structures and purities of TBDPN2, TBDPN3, BBDPN and TBDPN4 were confirmed by H NMR and mass spectrometry. Figure 5 As shown, the prepared nanoformulation has a size of less than 250 nm, is uniform in size, has a dispersion index (PDI) of less than 0.2, and maintains its original size after being stored at room temperature for 7 days, with high stability.

[0070] Example 5 Photophysical property detection

[0071] The compounds prepared in Examples 1 to 3 and Comparative Example 1 were dissolved in dichloromethane, and the nanoformulations prepared in Examples 1 to 3 and Comparative Example 1 were dissolved in water. The absorption and emission spectra were tested. The results were as follows: Figure 6 As shown in Table 1, the maximum absorption and emission wavelengths, as well as the Stokes shifts, of the compounds and nanoformulations prepared in Examples 1-3 are summarized. Compared to the comparative examples, Examples 1-3 have redder emission wavelengths and larger Stokes shifts. The maximum emission peak of TBDPN2 NPs is at 1032 nm, located in the second near-infrared region, making it most suitable for tumor imaging. The maximum emission peaks in Comparative Example 1 are all smaller than those in Examples 1-3, and the Stokes shift is also the smallest, making it least suitable for fluorescence imaging.

[0072] Table 1:

[0073] Reagents <![CDATA[λ abs / nm]]> <![CDATA[λ em / nm]]> Stokes shift TBDPN2 818 939 121 TBDPN2 NPs 892 1032 140 TBDPN3 777 918 141 TBDPN3 NPs 863 968 105 BBDPN 794 906 112 BBDPN NPs 846 975 129 TBDPN4 787 884 97 TBDPN4 NPs 841 942 101

[0074] In Table 1, λ abs = absorption spectrum peak; λ em = fluorescence spectrum peak; Stokes shift = Stokes shift.

[0075] Example 6 Photothermal Capacity Detection

[0076] Using an 808nm laser, the in vitro photothermal capacity of TBDPN2 NPs, TBDPN3 NPs, BBDPN NPs, and TBDPN4 NPs was tested. Nanoformulations of different concentrations were added to a 96-well plate in a volume of 200μL. After irradiation with an 808nm laser for five minutes, the temperature change was recorded using a thermocouple thermometer. The photothermal properties of each nanoformulation were obtained in concentration dependence and power dependence, and the heating and cooling curves were obtained through hot and cold cycles. The test results are shown in Figure 2. Figures 7 to 10 As shown in Figure 2, the photothermal conversion efficiency (PCE) of each nanoformulation was calculated and its excellent thermal stability was verified, as shown in Figure 2. Figure 7 , TBDPN2 NPs has the highest PCE, reaching 23.1%. Figure 10 As shown, the PCE of Comparative Example 1 is the lowest, which is only 5%.

[0077] Example 7 Detection of Active Oxygen Species

[0078] The ability of different BODIPY derivatives and their nanoparticles to generate reactive oxygen species in vitro was detected by measuring the change in the absorbance of 1,3-diphenylisobenzofuran (DPBF). Using DPBF as a probe molecule, the ability of different BODIPY derivatives to generate reactive oxygen species was determined by detecting the fluorescence quenching of DPBF. A dichloromethane (DCM) solution of DPBF was added to different BODIPY derivatives, and DPBF in DCM was used as a control. The mixed solution was irradiated with an 808nm laser (20mW / cm 2 ) irradiate for 50 seconds, and measure the absorption spectrum of the mixed solution every 10 seconds. Figure 11 As shown, the results showed that both can effectively generate reactive oxygen species.

[0079] Example 8 Detection of Intracellular Reactive Oxygen Species

[0080] In order to measure the production of intracellular reactive oxygen species (ROS) and the inhibition of cancer cells. First, the DCFH-DA probe was used for ROS determination. 4T1 cells were selected to be plated at 400,000 cells / mL, incubated at 37°C overnight, and then the drug (10μg / mL) was administered. They were incubated together for 30 minutes, and freshly prepared DCFH-DA probe solution was added in a light-proof environment. The cells were placed in an incubator at 37°C and incubated together for 30 minutes. Then, they were placed in the dark or irradiated with an 808nm laser for 10 minutes. Finally, imaging was performed using a laser confocal microscope. The experimental results are shown in the figure below. Figure 12 As shown in A, TBDPN2 NPs can produce reactive oxygen species. Compared with the PBS group and the dark group, the drug-treated and light-exposed group can produce more reactive oxygen species.

[0081] Example 9 4T1 cell live-dead staining experiment

[0082] In order to visually see the killing effect of TBDPN2 nanoformulation on 4T1 cells, Calcein-AM / PropidiumIodide (PI) was used for cell live and dead staining. 4T1 cells were seeded on a confocal dish at a density of 500,000 / mL. They were grown in a 37°C constant temperature cell culture incubator for 24 hours. TBDPN2 NPs (10 μg / mL) were then added and incubated for 30 minutes. An 808 nm laser was then used (illumination for 10 minutes per well). The cells were stained with Calcein-AM and PI for 15 minutes and then observed using a confocal microscope. Figure 12 B, A large area of ​​red fluorescence appeared in the nanoformulation plus illumination group, indicating that the TBDPN2 nanoformulation had a killing effect on 4T1 cells under these conditions.

[0083] Example 10 Cytotoxicity Experiment

[0084] The cytotoxicity of the four nanoformulations on 4T1 cells was evaluated using the MTT assay. 3000 cells / well were plated in a 96-well plate and incubated at 37°C overnight. Different concentrations of nanoformulations were added to the culture medium. The dark group was incubated in the dark for 24 hours and the cell viability was measured. The light group was incubated for 18 hours and then illuminated with an 808 nm laser (0.8 W / cm 2 ) for 5 min, and then incubate for 4 h before detecting cell viability. Figure 12 C. Figure 12 D. Figure 12 E and Figure 12 Figure F shows the effects of different concentrations of TBDPN2 NPs, TBDPN3 NPs, TBDPN4 NPs, and BBDPN NPs on 4T1 cell viability. The results show that cell viability remained unchanged in the dark group, with cell survival rates exceeding 80%. The light-exposed group exhibited a significant phototherapeutic effect, with cell viability decreasing gradually with increasing concentrations of the BODIPY derivatives.

[0085] Example 11 In vivo near-infrared second-zone imaging

[0086] like Figure 13 The near-infrared imaging effect of different nanoformulations was evaluated by whole-body vascular imaging of mice. Different nanoformulations (3*10 -7 mol / L), and then perform near-infrared zone II whole-body vascular imaging. Figure 13As shown in A, it can be seen that the three nanoformulations prepared in Examples 1 to 3 all show a certain vascular imaging effect, and the blood vessels in the hind limbs of mice are clearly visible. The nanoformulation prepared in Comparative Example 1 has extremely poor vascular imaging ability, and the blood vessels in the hind limbs are blurred. At the same time, the cross-sections of the hind limb blood vessels were taken, and the signal-to-noise ratios of the imaging signals of different nanoformulations were calculated. The half-peak width of the signal was calculated by Gaussian fitting, as shown in Table 2. As shown in the figure, the TBDPN2 nanoformulation has the highest signal-to-noise ratio and the lowest half-peak width, indicating that it has the best near-infrared zone II imaging ability. However, the nanoformulation in Comparative Example 1 has the lowest signal-to-noise ratio and the largest half-peak width, and the near-infrared zone II imaging ability is the worst. Based on this, the TBDPN2 nanoformulation will be used to test its signal under different long-pass filters. As shown in Figure 13 B. As the wavelength of the long-pass filter increases, the resolution of the signal increases, as shown in Table 3. This is consistent with the theory. The tumor accumulation effect of the BODIPY derivative nanoformulation was then tested on 4T1 tumor-bearing mice. The BODIPY derivative nanoformulation (3*10 -7 mol / L), and then infrared zone II imaging of the tumor site was performed at different time points. Figure 13 C and Figure 13 D, The fluorescence intensity of the tumor site of mice injected with TBDPN2 NPs increased over time and reached the highest at 72 hours, indicating that TNDPN2 NPs continued to accumulate in the tumor site and were slowly metabolized over time. The mice were then killed and their organs were removed for near-infrared zone II imaging. Figure 14 F shows that the fluorescence intensity of the liver, spleen, and tumor is significantly higher than that of other organs.

[0087] Table 2:

[0088] TBDPN2 NPs TBDPN3 NPs BBDPN NPs TBDPN4 NPs Signal-to-noise ratio 1.56 1.39 1.42 1.46 Half-peak width 0.33 0.43 0.42 0.50

[0089] Table 3:

[0090] 1000nm LP 1100nm LP 1200nm LP Signal-to-noise ratio 1.50 1.64 1.99 Half-peak width 0.66 0.64 0.56

[0091] Example 12 Treatment of 4T1 tumor-bearing mice

[0092] like Figure 14 A, The inhibitory effect of TBDPN2 nanoformulation on tumors was verified by 4T1 tumor-bearing mice. 4T1 tumor-bearing mice were divided into 4 groups (n=5). The treatment plan is shown in the figure. Tumors were inoculated on day -7, and TBDPN2 nanoformulation (300μg / mL) was injected through the tail vein on day 0. After the nanoformulation accumulated in the tumor site for about 8 hours, the tumor site was phototreated. An 808nm laser (0.8W / cm 2 ) Expose the sample to light for approximately five minutes and record the temperature rise within five minutes. Figure 14B, we can see that the temperature of the mouse tumor site increased significantly. After the end of the light exposure, we continued to monitor for 22 days and found that there was no significant change in the weight of the mice in each group ( Figure 14 D), and the tumor volume of mice in the treatment group was significantly suppressed (e.g. Figure 14 C, 13E), the tumor was completely ablated. Then, on the 22nd day, all mice were killed and the tumors were removed. It can be seen that the tumors in the treatment group were completely suppressed (as shown in Figure 1). Figure 14 F), while the tumors in the control group grew normally.

Claims

1. A BODIPY derivative having the structure shown in Formula I: In the structure shown in Formula I, R1 is a thienyl group or a phenyl group, R2 is a hydrogen group or a methyl group, and X is Br or I.

2. The BODIPY derivative according to claim 1, characterized in that R1 is 2-thienyl.

3. A nano preparation, characterized in that: The invention relates to a BODIPY derivative having a structure shown in formula I. The BODIPY derivative is coated with a DSPE-PEG layer. The mass ratio of the BODIPY derivative to DSPE-PEG is 1:8-12.

4. The nanoformulation according to claim 3, characterized in that The particle size of the nanoparticles is less than 250 nm.

5. The method for preparing the nanoformulation according to claim 3, characterized in that: The following steps are involved: The structural compound represented by formula I and DSPE-PEG are dissolved in a first organic solvent to obtain a solution. The solution is dropped into water at a rotation speed of 300-600 rpm and stirred until the first organic solvent is completely evaporated to obtain a nano preparation.

6. The method for preparing the nanoformulation according to claim 5, characterized in that: The first organic solvent is one of acetone, tetrahydrofuran, methanol, and ethanol, or a mixture of several of them.

7. Use of the nanoformulation according to claim 3 in the preparation of a drug or near-infrared fluorescent imaging agent for inhibiting tumor cell growth using photodynamic therapy.

8. The method for preparing the BODIPY derivative according to claim 1, characterized in that: The following steps are involved: The compound of formula II undergoes a halogenation reaction to obtain a compound of formula III, and the compound of formula III undergoes a condensation reaction with 4-diethylaminobenzaldehyde to obtain a BODIPY derivative of formula I:

9. The preparation method according to claim 8, characterized in that The reaction conditions of the halogenation reaction are as follows: the structural compound represented by Formula II and the halogenation reagent are dissolved in a second organic solvent, reacted at room temperature for more than 4 hours to obtain the structural compound represented by Formula III, the molar ratio of the structural compound represented by Formula II to the halogenation reagent is 1:2.5 to 3.5, the second organic solvent is one or a mixture of dichloromethane, chloroform, and 1,2-dichloroethane, and the halogenation reagent is N-bromosuccinimide, N-chlorosuccinimide, or N-iodosuccinimide.

10. The preparation method according to claim 8, characterized in that The reaction conditions of the condensation reaction are as follows: the compound with the structure shown in formula II and 4-diethylaminobenzaldehyde are dissolved in toluene, acetic acid and piperidine are added as catalysts, and the reaction is carried out at 80-120° C. for 3-6 hours under inert gas protection to obtain a BODIPY derivative with the structure shown in formula I, wherein the molar ratio of the compound with the structure shown in formula II to 4-diethylaminobenzaldehyde is 1:2.5-3.5; and the amount of the catalyst added is 10-13% of the volume of the toluene.

Images