Bodipy nanophotosensitizer with mitochondrial targeting and photodynamic activity, and preparation method and application thereof

Abstract

The application discloses a BODIPY nano photosensitizer with mitochondrial targeting and photodynamic activity, and simultaneously provides a preparation method and application thereof, and belongs to the technical field of biological medicines.Several BODIPY photosensitizers with mitochondrial targeting function developed by the application can generate reactive oxygen under light conditions, and effectively act on mitochondria, so as to kill tumor cells. Not only a lower drug concentration can be used, but also a lower light intensity and light irradiation time can be used for treatment. The application cannot cause drug tolerance of tumors, has small side effects, is non-irritating, and has a broad application prospect in the clinic.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically involving several methods for preparing and applying BODIPY nanophotosensitive agents, particularly their application in preparing BODIPY photosensitizers with mitochondrial targeting capabilities. Targeting mitochondria enhances the photodynamic therapy effect on tumor cells. Background Technology

[0002] Photodynamic therapy (PDT) utilizes the interaction between a photosensitizer and light of a specific wavelength to generate reactive oxygen species (ROS), which induce cell damage and death, thereby achieving the therapeutic goals for various diseases such as cancer, skin diseases, and ophthalmic diseases. Due to its advantages such as precise targeting, non-invasiveness, and relatively few side effects, it has been widely used in clinical practice. The three essential elements of PDT are the photosensitizer, the light source, and the ROS, all of which play crucial roles. However, the short lifespan of ROS in PDT is a significant drawback. ROS, or free radicals, have a very short half-life, meaning they quickly lose their activity after formation. This implies a limited duration of action and a limited diffusion distance, potentially failing to effectively kill all target cells and thus affecting treatment efficacy. Furthermore, ROS exhibit a degree of nonspecificity, which may damage surrounding normal tissues, increasing the risk of treatment complications. Therefore, developing photosensitizers capable of targeting specific organelles (cell membrane, nucleus, endoplasmic reticulum, lysosomes, and mitochondria) for anti-tumor applications shows great potential. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the main objective of this invention is to provide several BODIPY photosensitizers with mitochondrial targeting functions, along with their preparation methods and applications. Benefiting from the delocalized lipophilic cationic structure in the molecule, BODIPY derivatives can accumulate on the negatively charged inner mitochondrial membrane, generating a large amount of ROS upon light irradiation, enabling their application in mitochondrial photodynamic therapy. This invention solves the problem of insufficient water solubility of existing BODIPY photosensitizers, while simultaneously possessing mitochondrial targeting capabilities and enhanced photodynamic effects.

[0004] The present invention adopts the following technical solution: A BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity has the following chemical structure: ; In the formula, R1 is a phosphorus-containing aromatic group or heterocyclic group, a nitrogen-containing aromatic group or heterocyclic group, or a nitrogen-containing alkyl group; R2 and R3 are independently selected from hydrogen, phosphorus-containing and oxygen-containing aromatic groups or heterocyclic groups, nitrogen-containing and oxygen-containing aromatic groups or heterocyclic groups, or nitrogen-containing and oxygen-containing alkyl groups; R4 is hydrogen or alkyl.

[0005] Preferably, R1 is one of the following groups: ; Wherein, X is a halogen, such as chlorine, bromine, or iodine, and n is 1 to 20, preferably 1 to 11; R2 and R3 are independently selected from hydrogen or one of the following groups: ; Wherein, X is a halogen, such as chlorine, bromine, or iodine, and m is 1 to 20, preferably 2 to 12; In R4, the alkyl group has 1 to 6 carbon atoms, preferably 1 to 3, such as methyl.

[0006] This invention discloses a method for preparing the above-mentioned BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity, comprising the following steps: (1) After reacting compound 1 and a phosphorus-containing aromatic or heterocyclic compound, an iodination reaction is carried out to obtain the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity. (2) React compound 2 with a nitrogen-containing aromatic or heterocyclic compound to obtain the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity; (3) React compound 2 and nitrogen-containing alkyl compound to obtain the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity; The chemical structural formula of compound 1 is as follows: ; Wherein, X is a halogen, and n is 1 to 20, preferably 2 to 12; R5 and R6 are independently selected from hydrogen and haloalkoxy groups; R4 is hydrogen or alkyl. The chemical structural formula of compound 2 is as follows: ; Wherein, X is a halogen, and n is 1 to 20, preferably 2 to 12; R5 and R6 are independently selected from hydrogen and haloalkoxy groups; R4 is hydrogen or alkyl.

[0007] Preferably, the phosphorus-containing aromatic compounds include triphenylphosphine; the nitrogen-containing heterocyclic compounds include 1-methylimidazole; and the nitrogen-containing alkyl compounds include trimethylamine.

[0008] Preferably, the number of carbon atoms in the haloalkoxy group is 1 to 20; for example, the haloalkoxy groups are as follows: ; Where X is a halogen, such as chlorine, bromine, or iodine, and n is 1 to 20, preferably 2 to 12.

[0009] Preferably, the alkyl group has 1 to 6 carbon atoms.

[0010] This invention discloses a nano-formulation comprising the above-mentioned BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity.

[0011] This invention discloses a method for preparing the above-mentioned nano-formulation, comprising the following steps: adding an organic solution of the above-mentioned BODIPY nano-photosensitizer with mitochondrial targeting and photodynamic activity to water to obtain the nano-formulation. Preferably, the organic solvent includes one or more of acetone, tetrahydrofuran, methanol, and ethanol.

[0012] The BODIPY nanophotosensitive agents of this invention, which possess mitochondrial targeting and photodynamic activity, have a delocalized lipophilic cationic structure. Examples of these BODIPY photosensitizers are BDPI-TPP, BDPI-TMA, and BDPI-IMA.

[0013] The preparation method of BDPI-TPP includes the following steps: reacting compound 1 with triphenylphosphine to obtain a positively charged BODIPY photosensitizer, and then reacting it with N-iodosuccinimide to obtain the positively charged iodoBODIPY photosensitizer BDPI-TPP. The preparation methods of BDPI-TMA and BDPI-IMA are as follows: Compound 2 is reacted with trimethylamine and 1-methylimidazole respectively to obtain positively charged iodo-BODIPY photosensitizers BDPI-TMA and BDPI-IMA.

[0014] In the nano-formulation of this invention, the final concentration of the BODIPY nano-photosensitizer with mitochondrial targeting and photodynamic activity is 50–200 µg / mL.

[0015] The preparation method of the above-mentioned nano-formulation includes the following steps: dissolving BODIPY nano-photosensitizer with mitochondrial targeting and photodynamic activity in an organic solvent to obtain a solution, adding the solution dropwise to ultrapure water, and stirring at room temperature until the organic solvent is completely evaporated. The resulting solution is the nano-formulation.

[0016] In this invention, the organic solvent in the organic solution of the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity is acetone, tetrahydrofuran, methanol or ethanol; the concentration of the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity is 0.2 to 2 mg / mL; and the volume ratio of the organic solution to ultrapure water is 1: (2 to 10).

[0017] This invention discloses the application of BODIPY nanophotosensitizers or nanoformulations with mitochondrial targeting and photodynamic activity in the preparation of photosensitizers with mitochondrial targeting function.

[0018] The present invention relates to the application of BODIPY nanophotosensitizers or nanoformulations with mitochondrial targeting and photodynamic activity in the preparation of photosensitizers with mitochondrial targeting function. This allows for targeting of mitochondria in various cell lines, demonstrating universality. Furthermore, it can target mitochondria for anti-tumor therapy via photodynamic therapy, also demonstrating universality. Specifically, the various cell lines include, but are not limited to, HeLa cells, 4T1 cells, and B16-F10 cells.

[0019] This invention discloses a mitochondrial-targeting reagent, including the above-mentioned BODIPY nanophotosensitizer or nanoformulation with mitochondrial targeting and photodynamic activity.

[0020] This invention discloses the application of the above-mentioned BODIPY nanophotosensitizer or nanoformulation with mitochondrial targeting and photodynamic activity in the preparation of mitochondrial targeting reagents.

[0021] This invention discloses the application of the aforementioned BODIPY nanophotosensitizer or nanoformulation with mitochondrial targeting and photodynamic activity in the preparation of photodynamic therapeutic drugs. These photodynamic therapeutic drugs include medications for treating various diseases such as cancer, skin diseases, and ophthalmic diseases.

[0022] Preferably, the application includes at least one of the following: (1) The application of the BODIPY photosensitizer or nanoformation with a delocalized lipophilic cationic structure in the targeting of mitochondria in various cell lines; (2) The application of the BODIPY photosensitizer or nanoformulation with a delocalized lipophilic cationic structure in the fight against tumors via photodynamic therapy; Preferably, the method can target and photodynamically treat mitochondria in various cell lines, demonstrating universal applicability. Mitochondria, as the "powerhouse" of the cell, are the primary site of aerobic respiration, responsible for producing the vast majority of ATP, providing energy for various intracellular life activities. Simultaneously, mitochondria are also the site of most ROS production, playing a crucial role in regulating intracellular redox homeostasis, apoptosis, and necrosis. When ROS are overexpressed in mitochondria, it causes mitochondrial oxidative stress, leading to mitochondrial dysfunction and cell death. Mitochondria are organelles formed by a double-membrane structure, with distinctive structural features. The electron transport chain occurs on the inner mitochondrial membrane, allowing protons (H+) to pass across the membrane. + Mitochondria, along with other ions, exhibit asymmetry, resulting in a negative membrane potential unlike that of other organelles. Therefore, mitochondria are ideal target organelles for PDT, as they can generate large amounts of ROS, leading to mitochondrial dysfunction and subsequently triggering processes such as apoptosis.

[0023] Preferably, the various cell lines include, but are not limited to, HeLa cells, 4T1 cells, B16-F10 cells, etc.

[0024] Beneficial effects: 1. The BODIPY photosensitizer used in this invention has a simple structure, is easy to synthesize, and has low production cost.

[0025] 2. The several BODIPY photosensitizers with mitochondrial targeting function developed in this invention exhibit low fluorescence quantum yields but high singlet oxygen quantum yields. This is advantageous for their use as photosensitizers in phototherapy (PDT). Treatment can be performed using not only lower drug concentrations but also lower light intensities and durations, resulting in better patient tolerance and compliance.

[0026] 3. The several BODIPY photosensitizers with mitochondrial targeting function developed in this invention can efficiently generate reactive oxygen species under light conditions and effectively act on mitochondria, thereby selectively killing tumor cells. They do not cause drug resistance in tumors, have few side effects, are non-irritating, and have broad clinical application prospects. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 In the figures, A and B are the proton NMR spectra of BDPI-TPP and BDPI-TMA in the examples, respectively.

[0029] Figure 2 In the figures, A and B are the proton NMR spectrum and mass spectrum of BDPI-IMA in the examples, respectively.

[0030] Figure 3 In the figure, AF shows the absorption and fluorescence spectra of BODIPY photosensitizer and its nano-formulation, respectively.

[0031] Figure 4 In the diagram, A and B show the generation of reactive oxygen species in solution of BDPI photosensitizer and its nano-formulation, respectively, while C shows the generation of reactive oxygen species in solution of BDP photosensitizer.

[0032] Figure 5 The image shows the production of reactive oxygen species in BODIPY cells under both darkness and light conditions.

[0033] Figure 6 The image shows CLSM images of HeLa cells co-stained with BODIPY (5µM) and the commercial dye Mito-Tracker.

[0034] Figure 7 The effect of BODIPY on mitochondrial membrane potential under light irradiation is shown in the figure.

[0035] Figure 8 The results show the effect of different BODIPY concentrations on the viability of HeLa cells.

[0036] Figure 9 The results show the effect of different BODIPY concentrations on the viability of 4T1 cells.

[0037] Figure 10 The results show the effect of different BODIPY concentrations on the viability of B16-F10 cells. Detailed Implementation

[0038] To further understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. However, those skilled in the art will understand that the embodiments described below are only some embodiments of the present invention, not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] This invention provides a BODIPY photosensitizer with mitochondrial targeting function, the structural formula of which is as follows: ; In the formula, R1 is a phosphorus-containing aromatic group or heterocyclic group, a nitrogen-containing aromatic group or heterocyclic group, or a nitrogen-containing alkyl group; R2 and R3 are independently selected from hydrogen, phosphorus-containing and oxygen-containing aromatic groups or heterocyclic groups, nitrogen-containing and oxygen-containing aromatic groups or heterocyclic groups, or nitrogen-containing and oxygen-containing alkyl groups; R4 is hydrogen or alkyl.

[0040] Preferably, R1 is one of the following groups: ; Wherein, X is a halogen, such as chlorine, bromine, or iodine, and n is 1 to 20, preferably 1 to 11; R2 and R3 are independently selected from hydrogen or one of the following groups: ; Wherein, X is a halogen, such as chlorine, bromine, or iodine, and m is 1 to 20, preferably 2 to 12; In R4, the alkyl group has 1 to 6 carbon atoms, preferably 1 to 3.

[0041] Preferably, the structural formula of the BODIPY photosensitizer with mitochondrial targeting function is BDPI-TPP, BDPI-TMA, or BDPI-IMA:

[0042] In this invention, compound 1 is reacted with triphenylphosphine to obtain a positively charged BODIPY photosensitizer, which is then reacted with N-iodosuccinimide to obtain a positively charged iodoBODIPY photosensitizer BDPI-TPP; preferably, the reaction solvent is acetonitrile.

[0043] In this invention, compound 2 is reacted with trimethylamine and 1-methylimidazole respectively to obtain positively charged BODIPY photosensitizers BDPI-TMA and BDPI-IMA; preferably, the solvent for the preparation reaction of BDPI-TMA is ethanol, and the solvent for the preparation reaction of BDPI-IMA is acetonitrile.

[0044] The structural formula of compound 1 is as follows: ; The structural formula of compound 2 is as follows: ; Wherein, X is a halogen, and n is 1 to 20, preferably 2 to 12; R5 and R6 are independently selected from hydrogen and haloalkoxy groups; R4 is hydrogen or alkyl.

[0045] Preferably, the phosphorus-containing aromatic compounds include triphenylphosphine; the nitrogen-containing heterocyclic compounds include 1-methylimidazole; and the nitrogen-containing alkyl compounds include trimethylamine.

[0046] Preferably, the number of carbon atoms in the haloalkoxy group is 1 to 20; for example, the haloalkoxy groups are as follows:

[0047] Where X is a halogen, such as chlorine, bromine, or iodine, and n is 1 to 20, preferably 2 to 12.

[0048] Preferably, the alkyl group has 1 to 6 carbon atoms.

[0049] The present invention also provides nanoformulations containing the above-mentioned BODIPY photosensitizer with mitochondrial targeting function.

[0050] Preferably, the final concentration of the BODIPY photosensitizer with mitochondrial targeting function in the nano-formulation is 50-200 µg / mL.

[0051] The present invention also provides a method for preparing the above-mentioned nano-formulation, the steps of which are as follows: dissolving the BODIPY photosensitizer with mitochondrial targeting function in an organic solvent to obtain a solution, adding the solution dropwise to ultrapure water, and stirring at room temperature until the organic solvent is completely evaporated, the resulting solution is the nano-formulation.

[0052] Preferably, the organic solvent is acetone, tetrahydrofuran, methanol or ethanol; the concentration of BODIPY photosensitizer with mitochondrial targeting function in the organic solution is 0.2~2 mg / mL; the volume ratio of organic solution to ultrapure water is 1:(2~10).

[0053] The present invention also provides the application of the above-mentioned BODIPY photosensitizer with mitochondrial targeting function in the preparation of photosensitizer with mitochondrial targeting function.

[0054] The BODIPY photosensitizer of the present invention, which has a delocalized lipophilic cationic structure, is prepared by a simple chemical reaction. By introducing a delocalized lipophilic cationic structure into the BODIPY structure, it is endowed with mitochondrial targeting ability.

[0055] Specifically, the preparation method of BODIPY photosensitizer with delocalized lipophilic cationic structure having the structural formula BDPI-TPP is as follows: Compound 1 is reacted with triphenylphosphine to obtain a positively charged BODIPY photosensitizer, and then reacted with N-iodosuccinimide to obtain a positively charged iodoBODIPY photosensitizer.

[0056] The preparation method of BODIPY photosensitizers with delocalized lipophilic cationic structures having the structural formulas BDPI-TMA and BDPI-IMA is as follows: Compound 2 is reacted with trimethylamine and 1-methylimidazole, respectively, to obtain positively charged BODIPY photosensitizers.

[0057] The present invention relates to a nanoformulation of BODIPY photosensitizer with a delocalized lipophilic cationic structure; preferably, the final concentration of BODIPY photosensitizer with a delocalized lipophilic cationic structure in the nanoformulation is 50-200 µg / mL.

[0058] The preparation method of the nano-formulation of the present invention comprises the following steps: dissolving a BODIPY photosensitizer having a delocalized lipophilic cationic structure in an organic solvent to obtain a solution; adding the solution dropwise to ultrapure water; and stirring at room temperature until the organic solvent is completely evaporated. The resulting solution is the nano-formulation. Preferably, the organic solvent is acetone, tetrahydrofuran, methanol, or ethanol; the concentration of the BODIPY photosensitizer having a delocalized lipophilic cationic structure in the organic solution is 0.2~2 mg / mL; and the volume ratio of the organic solution to ultrapure water is 1:(2~10).

[0059] The present invention relates to the application of the BODIPY photosensitizer with a delocalized lipophilic cationic structure and its nano-formulation in the preparation of photosensitizers with mitochondrial targeting function. Applications include, but are not limited to: (1) the application of the BODIPY photosensitizer or nano-formulation with the delocalized lipophilic cationic structure in targeting mitochondria in various cell lines; and (2) the application of the BODIPY photosensitizer or nano-formulation with the delocalized lipophilic cationic structure in anti-tumor therapy via photodynamic therapy.

[0060] The BODIPY photosensitizer and its nanoformation with a delocalized lipophilic cationic structure of the present invention can target and photodynamically treat mitochondria in various cell lines, and have universal applicability. Specific cell lines include, but are not limited to, HeLa cells, 4T1 cells, and B16-F10 cells.

[0061] The terminology used in this invention generally has the meanings commonly understood by those skilled in the art, unless otherwise stated. To enable those skilled in the art to better understand the technical solutions of this invention, the invention will be further described in detail below with reference to embodiments.

[0062] In the following embodiments, various processes and tests not described in detail are conventional methods known in the art. Unless otherwise specified, the materials, reagents, apparatus, instruments, equipment, etc., used in the following embodiments are commercially available.

[0063] The present invention will be further illustrated below with reference to the embodiments.

[0064] Example 1: Synthesis of BDPI-TPP BDP-Br (151 mg, 0.3 mmol) and triphenylphosphine (105 mg, 0.4 mmol) were added to a round-bottom flask, dissolved in 15 mL of acetonitrile, and refluxed at 85 °C for 24 h under a nitrogen atmosphere. After removing the solvent, the compound was purified by silica gel column chromatography, and the obtained compound was BDP-TPP.

[0065] BDP-TPP (38 mg, 0.05 mmol) and N-iodosuccinimide (NIS, 45 mg, 0.2 mmol) were added to a round-bottom flask, followed by 15 mL of dichloromethane. The mixture was reacted at room temperature for 6 hours. After the reaction was complete, the product BDPI-TPP was purified by silica gel column chromatography using dichloromethane and ethanol as eluents, yielding 36%.

[0066] The specific synthesis route is shown below:

[0067] Example 2 Preparation of BDPI-TPP nanoformulation The BDPI-TPP (1 mg) prepared in Example 1 was dissolved in 5 mL of acetone. 10 mL of ultrapure water was added dropwise to the solution under stirring. The solution was stirred at room temperature for 8 h until the organic solvent was completely evaporated. The resulting solution was the BDPI-TPP nanoformulation BDPI-TPPNPs.

[0068] Comparative Example 1 The BDP-TPP (1 mg) prepared in Example 1 was dissolved in 5 mL of acetone. 10 mL of ultrapure water was added dropwise to the solution under stirring. The solution was stirred at room temperature for 8 h until the organic solvent was completely evaporated. The resulting solution was the BDP nano-formulation BDP-TPP NPs.

[0069] Example 3 Synthesis of BDPI-TMA BDP-Br (503.2 mg, 1 mmol) and N-iodosuccinimide (NIS, 89.9 mg, 4 mmol) were added to a round-bottom flask, followed by 15 mL of dichloromethane. The mixture was reacted at room temperature for 6 hours. After the reaction was complete, the mixture was washed with saturated brine and dried over anhydrous NaSO4. The filtrate was then filtered to obtain the product BDPI-Br. The solvent was removed by vacuum distillation. Finally, the product was purified by silica gel column chromatography to obtain BDPI-Br.

[0070] BDPI-Br (113 mg, 0.15 mmol) was added to a round-bottom flask and dissolved in 20 mL of ethanol. Trimethylamine (0.1 mL, 0.3 mmol) was added with stirring at room temperature, and the mixture was refluxed at 60 °C for 24 h. After the reaction was complete, the solvent was removed, and the product, BDPI-TMA, was precipitated by diethyl ether. The yield was 39%.

[0071] The specific synthesis route is shown below:

[0072] Example 4 Preparation of BDPI-TMA Nanoformulation The BDPI-TMA (1 mg) prepared in Example 3 was dissolved in 5 mL of acetone. 10 mL of ultrapure water was added dropwise to the solution under stirring. The solution was stirred at room temperature for 8 h until the organic solvent was completely evaporated. The resulting solution was the BDPI-TMA nano-formulation BDPI-TMA NPs.

[0073] Comparative Example 2 BDP-Br (76 mg, 0.15 mmol) was added to a round-bottom flask and dissolved in 20 mL of ethanol. Trimethylamine (0.1 mL, 0.3 mmol) was added with stirring at room temperature, and the mixture was refluxed at 80 °C for 12 h. After the reaction was completed, the solvent was removed, and the product, BDP-TMA, was obtained by precipitating with diethyl ether.

[0074] The prepared BDP-TMA (1 mg) was dissolved in 5 mL of acetone. 10 mL of ultrapure water was added dropwise to the solution under stirring. The solution was stirred at room temperature for 8 h until the organic solvent was completely evaporated. The resulting solution is the nano-formulation of BDP-TMA, BDP-TMA NPs.

[0075] Example 5 Synthesis of BDPI-IMA BDPI-Br (226.5 mg, 0.3 mmol) and 1-methylimidazole (0.24 mL, 3 mmol) were added to a round-bottom flask, dissolved in 45 mL of acetonitrile, and refluxed at 85 °C for 12 h under a nitrogen atmosphere. After removing the solvent, the compound BDPI-IMA was obtained by column chromatography. The yield was 65%.

[0076] The specific synthesis route is shown below:

[0077] Example 6 Preparation of BDPI-IMA Nanoformulation The BDPI-IMA (1 mg) prepared in Example 3 was dissolved in 5 mL of acetone. 10 mL of ultrapure water was added dropwise to the solution under stirring. The solution was stirred at room temperature for 8 h until the organic solvent was completely evaporated. The resulting solution was the BDPI-IMA nano-formulation BDPI-IMA NPs.

[0078] Comparative Example 3 BDP-Br (151 mg, 0.3 mmol) and 1-methylimidazole (0.24 mL, 3 mmol) were added to a round-bottom flask, dissolved in 45 mL of acetonitrile, and refluxed at 85 °C for 24 h under a nitrogen atmosphere. After removing the solvent, the compound BDP-IMA was obtained by column chromatography.

[0079] The prepared BDP-IMA (1 mg) was dissolved in 5 mL of acetone. 10 mL of ultrapure water was added dropwise to the solution under stirring. The solution was stirred at room temperature for 8 h until the organic solvent was completely evaporated. The resulting solution is the nano-formulation of BDP-IMA, BDP-IMA NPs.

[0080] Test characterization Nuclear magnetic resonance (NMR) analysis was performed on BDPI-TPP prepared in Example 1, BDPI-TMA prepared in Example 3, and BDPI-IMA prepared in Example 5. Mass spectrometry (MS) analysis was performed on BDPI-IMA. The NMR and MS measurements confirmed that BDPI-TPP (… Figure 1 Medium A), BDPI-TMA ( Figure 1 (B) and BDPI-IMA ( Figure 2 The structure and purity of A and B).

[0081] Example 7: Detection of photophysical properties The absorption and emission spectra of BODIPY photosensitizer in DMSO and the corresponding nano-formulation in water were detected. Results are as follows: Figure 3 As shown, the absorption wavelength of BODIPY photosensitizer in DMSO is around 501 nm, and the emission wavelength is around 560 nm. Table 1 collects the maximum absorption wavelength and emission wavelength of BODIPY photosensitizer, Stokes shift, and corresponding fluorescence quantum yield (φ) and singlet oxygen quantum yield (Φ) of nano-formulations.

[0082] Table 1. Photophysical properties of different BODIPY photosensitizers

[0083] Where, λ abs = Absorption spectral peak; λ em = Fluorescence spectrum peak value; Stokes shift; φ = Fluorescence quantum yield, with Rhodamine 6G as a reference; Φ = Singlet oxygen quantum yield, with methylene blue (MB) in dichloromethane as a reference.

[0084] Example 8 Reactive Oxygen Detection The ability of BODIPY photosensitizer and its nanoparticles to generate reactive oxygen species (ROS) in vitro was detected by measuring changes in the absorbance of 1,3-diphenylisobenzofuran (DPBF). Using DPBF as a probe molecule, the ROS generation ability of BODIPY photosensitizer was determined by detecting the fluorescence quenching of DPBF. A solution of DPBF in dimethyl sulfoxide (DMSO) was added to a BODIPY solution, with DPBF in DMSO serving as a control. The mixed solution was subjected to green light (10 mW / cm²). 2Irradiation was performed for 70 seconds, and the absorption spectrum of the mixed solution was measured every 10 seconds. The ability of the BODIPY nanoparticle formulation to generate reactive oxygen species in vitro was tested using the same method. Figure 4 As shown, the results indicate that all BODIPY photosensitizers efficiently generate reactive oxygen species (ROS), with BDPI-IMA exhibiting the best performance, generating more than twice the amount of ROS compared to other photosensitizers within the same timeframe. Figure 4 As can be seen from C, under the same concentration and test conditions, compounds without iodine only showed a negligible reactive oxygen species generation capacity.

[0085] Example 9 Intracellular Reactive Oxygen Species Detection To determine the production of intracellular reactive oxygen species (ROS) and their inhibitory effect on cancer cells, the DCFH-DA probe was first used for ROS measurement. HeLa cells were seeded at 100,000 cells / mL and incubated overnight at 37°C. Afterward, the probe was administered (1 µg / mL) and incubated for 30 minutes. Then, freshly prepared DCFH-DA probe solution was added under light-protected conditions, and the cells were incubated at 37°C for another 30 minutes. Following this, the cells were irradiated in darkness or under green light for 3 minutes. Finally, imaging was performed using a laser confocal microscope. The experimental results are shown below. Figure 5 As shown, different BODIPY photosensitizers can all generate reactive oxygen species, and the fluorescence intensity of the BDPI-IMA group is significantly higher than that of other groups, indicating that BDPI-IMA has a stronger ability to generate reactive oxygen species, which is consistent with the detection results in solution.

[0086] Example 10 Mitochondrial Targeting Experiment HeLa cells were seeded at 100,000 cells / mL and incubated overnight at 37°C. Afterward, the drug (5 µg / mL) was administered and incubated for 30 minutes. The culture medium was then removed, and freshly prepared medium containing the Mito-Tracker (a mitochondrial fluorescent probe) was added. After another 25 minutes of incubation, imaging was performed using a laser confocal microscope. Figure 6 As shown, mitochondrial structures are clearly visible in the green fluorescent region of BODIPY, indicating that BODIPY is located in the mitochondria of living cells. The Pearson correlation coefficient represents the degree of linear dependence between two variables and is used to quantify the overlap of staining regions between BODIPY and the commercially available Mito-Tracker. It can be seen that the fluorescence signals from the two different channels almost completely overlap, with Pearson correlation coefficients exceeding 90% for both.

[0087] Example 11 Mitochondrial membrane potential detection To determine the effect of BODIPY photosensitizer on mitochondrial function, the JC-1 fluorescent probe was used to detect mitochondrial membrane potential. HeLa cells were seeded at 100,000 cells / mL and incubated overnight at 37°C. The cells were then incubated with the JC-1 probe (1 µg / mL) for 30 minutes, followed by 7 minutes of green light irradiation. The cells were then stained with JC-1 and imaged using a laser confocal microscope. The experimental results are as follows: Figure 7 As shown, JC-1 cells treated with BODIPY photosensitizer all changed from red fluorescence to green fluorescence, indicating a decrease in mitochondrial membrane potential. Therefore, BODIPY photosensitizer can effectively induce mitochondrial dysfunction and lead to early apoptosis.

[0088] Example 12 Cytotoxicity Experiment The cytotoxicity of BODIPY to different cell lines was evaluated using the MTT assay. (See attached table.) Figure 8 , Figure 9 as well as Figure 10 The effects of different BODIPY concentrations on the viability of HeLa cells, 4T1 cells, and B16-F10 cells were shown. Cells were seeded at 3000 cells / well in 96-well plates and incubated overnight at 37°C. Different concentrations of BODIPY nanoparticles were then added to the culture medium. Cell viability was assessed in the dark group after 24 hours of incubation; in the light group, cell viability was assessed after 18 hours of incubation under green light (10 mW / cm²). 2 Cell viability was assessed after irradiation for 25 min and incubation for another 4 hours. Results showed no significant change in cell viability in the dark group, with cell survival rates exceeding 80%. The light group exhibited a significant PDT effect, with cell viability decreasing progressively with increasing BODIPY photosensitizer concentration. Table 2 lists the half-maximal inhibitory concentrations (IC50) of BODIPY photosensitizer for different cell lines. 50 The iodine-free compounds BDP NPs did not exhibit significant cytotoxicity or photodynamic activity, even at high concentrations.

[0089] Table 2. Half-inhibitory concentrations (IC50) of BODIPY photosensitizer for different cell lines. 50 ).

[0090]

[0091] Among them, BDP NPs refer to BDP-TPP NPs, BDP-TMA NPs and BDP-IMA NPs. The nano-formulation BDP NPs without iodine compounds did not show obvious cytotoxicity even at high concentrations and did not have photodynamic activity.

[0092] To date, mitochondrial targeting reagents based on organic fluorophores have been developed, such as cyanine, rhodamine, and coumarin. However, cyanine dyes suffer from poor photostability, significantly hindering their application; rhodamine dyes are highly hydrophobic and difficult to modulate spectrally; and coumarin derivatives typically have short absorption and emission wavelengths, resulting in poor tissue penetration and potential biotoxicity. In contrast, the BODIPY derivative provided in this invention not only solves the above problems but also possesses superior mitochondrial targeting ability and photodynamic activity compared to similar mitochondrial-specific probes. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity, characterized in that, The chemical structure of the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity is shown below: 。 2. The method for preparing the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity as described in claim 1, characterized in that, Includes the following steps: The compounds 2 and 1-methylimidazole were reacted to obtain the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity. The chemical structural formula of compound 2 is as follows: 。 3. A nano-formulation, characterized in that, Includes the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity as described in claim 1.

4. The method for preparing the nano-formulation according to claim 3, characterized in that, The process includes the following steps: adding an organic solution of the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity as described in claim 1 to water to obtain a nanoformulation.

5. The method for preparing the nano-formulation according to claim 4, characterized in that, Organic solvents include one or more of acetone, tetrahydrofuran, methanol, and ethanol.

6. A mitochondrial-targeting reagent, comprising the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity as described in claim 1, or the nanoformulation as described in claim 3.

7. The application of the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity as described in claim 1 or the nanoformulation as described in claim 3 in the preparation of mitochondrial targeting reagents.

8. The application of the BODIPY nanophotosensitizer with mitochondrial targeting and photodynamic activity as described in claim 1, or the nanoformulation as described in claim 3, in the preparation of photodynamic therapeutic drugs.

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