BODIPY photosensitizer, nano preparation and application and preparation method of BODIPY photosensitizer and nano preparation

Abstract

The invention discloses a BODIPY photosensitizer, a nano preparation and application and a preparation method thereof, the molecular structure of the BODIPY photosensitizer is adjusted, and dithiodipropionic acid is used for bonding PEG (Polyethylene Glycol), so that the BODIPY photosensitizer has GSH (Glutathione) response and BBB (Boron Boron Boron) crossing capabilities at the same time. The photosensitizer disclosed by the invention has good photothermal conversion and photodynamic capacity, a nano preparation formed by self-assembly can efficiently enrich brain glioma in a targeted manner, a new preparation and a nano drug delivery system are provided for phototherapy of the brain glioma, and the photosensitizer has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a BODIPY photosensitizer, a nano-formulation of BODIPY photosensitizer self-assembly, and also to its corresponding uses and preparation methods. Background Technology

[0002] Glioblastoma (GBM) is one of the most common primary brain tumors of the central nervous system, accounting for approximately 40% of intracranial malignancies. It is highly invasive and progresses rapidly, and one of the major challenges in its treatment is the presence of the blood-brain barrier (BBB), which makes it difficult for most drugs to be effectively delivered to the brain tumor. Traditional phototherapy methods typically require the use of targeting ligands to enhance the tumor targeting of drugs and also require packaging materials to fabricate nanoparticles. Common brain-targeting strategies include antibody targeting and peptide targeting. However, antibody targeting suffers from problems such as immunogenicity, high cost, poor stability, and size limitations. Peptide targeting suffers from non-specific binding, poor stability, and complex delivery systems. Nanoparticles fabricated using packaging materials also suffer from low hydrophilic drug encapsulation efficiency and low brain-targeting penetration due to size limitations. Therefore, developing a photosensitizer that can self-assemble, requires no packaging materials, and does not require targeting ligand modification to achieve efficient brain tumor targeting has significant clinical implications. Summary of the Invention

[0003] Purpose of the invention: The purpose of this invention is to provide a BODIPY photosensitizer that has the ability to cross the blood-brain barrier. Another purpose of this invention is to provide a nano-formulation formed by the self-assembly of the BODIPY photosensitizer. Yet another purpose of this invention is to provide a method for preparing the BODIPY photosensitizer and the nano-formulation, as well as their uses.

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

[0005]

[0006] Where R is a fatty amine or aromatic amine, R′ is a hydrophilic polymer, and X is a halogen.

[0007] Preferably, R is selected from dimethylamino, diethylamino, or diphenylamino, and X is hydrogen or iodine.

[0008] Preferably, in order to enhance the blood-brain barrier crossing ability of the compound, the BODIPY photosensitizer has the structure shown in Formula II or Formula III:

[0009] .

[0010] Preferably, R′ is selected from at least one of polyethylene glycol, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, poloxamer, poloxamer, hydroxymethylcellulose, carboxyethylcellulose, polysarcosine-lipid conjugate, and conjugates of polysarcosine and lipid substances.

[0011] Preferably, R′ is selected from polyethylene glycol, wherein the weight-average molecular weight of polyethylene glycol is 1900~2200.

[0012] Preferably, the BODIPY photosensitizer has the structure of Formula IV or the formula shown:

[0013] n=42~50.

[0014] The preparation method of the BODIPY photosensitizer with the structures shown in Formulas I to V includes the following steps:

[0015] The compound with the structure shown in formula BDP a undergoes a first esterification reaction with 1 to 5 equivalents of dithiodipropionic acid at room temperature to obtain the compound with the structure shown in formula BDP b; the compound with the structure shown in formula BDP b undergoes a second esterification reaction with 1 to 2 equivalents of a hydrophilic polymer at room temperature to obtain the compound with the structure shown in formula I.

[0016] .

[0017] Preferably, the conditions for the first esterification reaction and the second esterification reaction are as follows: the solvent used is DCM, the catalyst is EDC·HCl and DMAP, the reaction temperature is room temperature, the duration of the first esterification reaction is 24~48 hours, and the duration of the second esterification reaction is 72~96 hours.

[0018] Preferably, the method for synthesizing the compound with the structure shown in formula BDP a is as follows:

[0019] The compound with the structure shown in formula BDP1 was subjected to a halogenation reaction with 2 to 4.5 equivalents of a halogenating reagent at room temperature to obtain the compound with the structure shown in formula BDP2, wherein the halogenating reagent was N-iodosuccinimide, and the reaction time was 6 hours.

[0020] The compound with the structure shown in formula BDP 2 was reacted with 2-4.5 equivalents of dimethylaminobenzaldehyde, diethylaminobenzaldehyde, and diphenylaminobenzaldehyde under inert gas protection at 120-130°C for 1-2 hours to obtain the compound with the structure shown in formula BDP a. The inert gas was N2.

[0021]

[0022] The compound with the structure shown in formula BDP 1 is synthesized as follows:

[0023] (1) Terephthalaldehyde was dissolved in tetrahydrofuran, anhydrous ethanol was added, sodium borohydride was added under ice bath, and the mixture was stirred for 6 h. After the reaction was completed, hydrochloric acid solution was added dropwise to adjust the pH to 5-6, and the mixture was purified to obtain 4-hydroxymethylbenzaldehyde;

[0024] (2) 4-Hydroxymethylbenzaldehyde and 2,4-dimethylpyrrole were dissolved in anhydrous dichloromethane. Trifluoroacetic acid was added dropwise, and the mixture was stirred overnight under N2 conditions. 2,3-Dichloro-5,6-dicyanobenzoquinone was added, and the mixture was stirred for 2 h. Triethylamine was added, and boron trifluoride diethyl ether was added dropwise under constant pressure in an ice bath. The mixture was stirred for 6 h, and BDP 1 was purified.

[0025] The present invention describes a nano-formulation formed by the self-assembly of at least one of the compounds with structures shown in formulas I to V.

[0026] The aforementioned method for preparing nano-formulations involves dissolving at least one of the compounds with structures shown in formulas I to V in an organic solvent to form a solution. Under stirring conditions, the solution is slowly added dropwise to water, and the mixture is stirred until all the organic solvent has evaporated to obtain the nano-formulation.

[0027] Preferably, the organic solvent is one or more of acetone, tetrahydrofuran, methanol, and ethanol.

[0028] Preferably, the concentration of the solution is 0.2~2 mg / mL; the volume ratio of the organic solution to ultrapure water is 1:2~10.

[0029] Any one of the compounds with the structure shown in Formulas I to V, the aforementioned nanoformulations have photothermal conversion, reactive oxygen species generation, and fluorescence imaging capabilities, and can be used to prepare tumor therapeutic drugs, photoacoustic imaging agents, near-infrared fluorescent imaging agents, or for drug tracing imaging.

[0030] Preferably, the tumor is a glioma.

[0031] Preferably, in the tumor treatment drug, the dosage of the nano-formulation or compound is 7.0~8.5 μg / g. More preferably, the dosage is 7.4~8.4 μg / g.

[0032] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. Near-infrared fluoroboron dipyrrole is prepared by introducing different amino donors through molecular engineering strategies, and a photosensitizer is constructed by bridging hydrophilic polymers with disulfide bonds. This photosensitizer can effectively cross the blood-brain barrier; 2. The compounds prepared by this invention do not require a carrier and can be self-assembled into nanoparticles, reducing the complexity and cost of drug preparation. After being formulated into nanoparticles, they possess self-targeting capabilities, can efficiently cross the blood-brain barrier, and can accumulate in situ in gliomas, enhancing the therapeutic effect; 3. PEG is a non-toxic, water-soluble polymer that can reduce phagocytosis and renal clearance, thereby prolonging the circulation time of the drug in the body; 4. The introduction of dimethylaminophenyl, diethylaminophenyl, and diphenylaminophenyl increases... Intramolecular charge transfer (ICT) allows BODIPY to exhibit absorption and emission spectra in the near-infrared region, enabling its use in infrared fluorescence imaging; 5. The introduction of iodine atoms enhances intersystem crossing (ISC), improving photodynamic performance; 6. The introduction of reduction-responsive SS bonds can consume excess GSH (glutathione) in tumor cells, disrupting the tumor's antioxidant system, increasing reactive oxygen species production, and enhancing photodynamic therapy efficacy. Simultaneously, the breaking of disulfide bonds leads to the dissociation of the nano-formulation, restoring fluorescence; 7. The BODIPY photosensitizer of this invention has a simple structure, is easy to synthesize, and has low production costs; 8. The BODIPY photosensitizer nano-formulation exhibits highly efficient photodynamic and photothermal properties under near-infrared light irradiation, enabling precise photodynamic and / or photothermal therapy, and has broad application prospects in clinical practice. Attached Figure Description

[0033] Figure 1 The 1H NMR spectrum and mass spectrometry of MBDP-PEG prepared in Example 1;

[0034] Figure 2 The 1H NMR spectrum and mass spectrometry of EBDP-PEG prepared in Example 2 are shown.

[0035] Figure 3 The 1H NMR spectrum and mass spectrometry of TBDP-PEG prepared in Example 3 are shown.

[0036] Figure 4 The spectra of MBDP-PEG, EBDP-PEG, TBDP-PEG and their corresponding nano-formulations are shown: (A) absorption spectrum, (B) fluorescence spectrum;

[0037] Figure 5 For using DPBF as a probe 1O2 detection curves: (A) Absorption spectrum of DPBF, (B) Absorption spectrum of DPBF + MBDP-PEG, (C) Absorption spectrum of DPBF + EBDP-PEG, (D) Absorption spectrum of DPBF + TBDP-PEG, (E) Absorbance decay rate.

[0038] Figure 6 O2 using DHR123 as a probe •− Detection curves: (A) Fluorescence spectrum of DHR123, (B) Fluorescence spectrum of MBDP-PEG+DHR123, (C) Fluorescence spectrum of EBDP-PEG+DHR123, (D) Fluorescence spectrum of TBDP-PEG+DHR123, (E) Enhancement rate of fluorescence intensity.

[0039] Figure 7 In the figure, A shows the change in the relative absorbance of DHR123 at 418 nm in the presence of MBDP-PEG NPs, EBDP-PEG NPs and TBDP-PEG NPs, and B shows the relative fluorescence intensity of DHR123 at 525 nm in the presence of MBDP-PEG NPs, EBDP-PEG NPs and TBDP-PEG NPs.

[0040] Figure 8 In the figure, A shows the particle size distribution of MBDP-PEG NPs, EBDP-PEG NPs and TBDP-PEG NPs, while B, C and D show the changes in particle size and PDI of MBDP-PEG NPs, EBDP-PEG NPs and TBDP-PEG NPs after 8 days of storage, respectively.

[0041] Figure 9 In the diagram, A, B, and C show different concentrations of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs under laser conditions (808 nm, 0.8 W / cm²). 2 The heating curves under irradiation are shown in Figures D, E, and F, respectively, which show the linear relationship between -Ln(θ) and time obtained by MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs during the cooling period after laser irradiation.

[0042] Figure 10In the diagram, A shows a CLSM image of GL261-luc cells after co-incubation with MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs for 4 h; B shows a CLSM image of U87-MG cells after co-incubation with MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs for 4 h; C shows a flow cytometry graph of ROS generation in GL261-luc cells co-incubated with MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs; and D shows a flow cytometry bar graph of ROS generation in GL261-luc cells co-incubated with MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs.

[0043] Figure 11 Table A shows the effects of different concentrations of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs on the viability of GL261-luc cells; Table B shows the effects of different concentrations of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs on the viability of U87-MG cells; Table C shows the effects of different concentrations of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs on the viability of U87-MG cells under 808 nm laser light (1 W / cm²). 2 The effects of different concentrations of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs on the viability of GL261-luc cells under 808 nm laser (1 W / cm²) irradiation (5 min) are shown in Figure D. 2 Results of the effect of irradiation (5 min) on the viability of U87-MG cells;

[0044] Figure 12 In the figure, A shows the blood-brain barrier (BBB) ​​model established in vitro using bEnd.3 cells; B shows the survival rate of bEnd.3 cells after co-incubation with MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs; C shows the flow cytometry analysis of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs after incubation with GL261-luc cells for 2, 4, and 12 h after simulating crossing the BBB in vitro.

[0045] Figure 13Table A shows fluorescence images of mice bearing GL261-Luc tumors after intravenous injection of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs. Table B shows fluorescence images of mice bearing U87-MG tumors after intravenous injection of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs. Tables C and D show the in vitro fluorescence distribution of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs in the heart, liver, spleen, lung, and kidney of mice bearing GL261-Luc and U87-MG tumors, respectively, 6 h after intravenous injection. Table E shows the fluorescence imaging and bioluminescence maps of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs in the brain.

[0046] Figure 14 In Figure A, the results show mice with orthotopic GL261-luc tumors after intravenous injection of MBDP-PEG NPs and EBDP-PEG NPs followed by irradiation with an 808 nm laser (1 W / cm²). 2 A) Infrared thermographic images of mice; B) Changes in body weight of mice after treatment with PBS, PBS+Light, MBDP-PEG NPS, EBDP-PEG NPs, MBDP-PEG NPs + Light, and EBDP-PEG NPs + Light; C) Survival curves of mice after treatment with PBS, PBS+Light, MBDP-PEG NPS, EBDP-PEG NPs, MBDP-PEG NPs + Light, and EBDP-PEG NPs + Light.

[0047] Figure 15 In Figure A, the image shows mice with orthotopic U87-MG tumors subjected to intravenous injection of EBDP-PEG NPs followed by irradiation with an 808 nm laser (1 W / cm²). 2 A) Infrared thermal imaging of mice; B) Body weight changes of mice after treatment with PBS, PBS+Light, EBDP-PEG NPs and EBDP-PEG NPs + Light; C) Survival curves of mice after treatment with PBS, PBS+Light, EBDP-PEG NPs and EBDP-PEG NPs + Light. Detailed Implementation

[0048] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0049] Example 1: Synthesis of MBDP-PEG. The specific synthetic route is as follows:

[0050]

[0051] 4-Hydroxymethylbenzaldehyde (1.36 g, 10 mmol) and 2,4-dimethylpyrrole (2.09 g, 22 mmol) were dissolved in 400 mL of anhydrous DCM (dichloromethane). After adding 5 drops of trifluoroacetic acid (TFA), the mixture was stirred overnight under N2 conditions. Then, 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ 2.27 g, 10 mmol) was added, and the mixture was stirred for 2 h. 10 mL of triethylamine was added, and 12 mL of boron trifluoride diethyl ether (BF3·Et2O) was added dropwise under constant pressure in an ice bath. The mixture was stirred for 6 h, and the resulting solution was purified to give BDP 1 (0.50 g, 15% yield).

[0052] BDP 1 (0.35 g, 1 mmol) and N-iodosuccinimide (NIS 0.90 g, 4 mmol) were dissolved in 40 mL of anhydrous dichloromethane. The mixture was stirred at room temperature for 6 h to purify BDP 2 (0.55 g, 91% yield).

[0053] BDP 2 (0.35 g, 0.58 mmol) and 4-dimethylaminobenzaldehyde (0.60 g, 4 mmol) were dissolved in 30 mL of toluene. 2.7 mL of piperidine and 0.7 mL of acetic acid were added under N2 conditions. The mixture was stirred at 120 °C for 1 h. After removing the solvent, MBDP (0.20 g, 33% yield) was eluted by column chromatography.

[0054] MBDP (87 mg, 0.1 mmol) and dithiodipropionic acid (105 mg, 0.5 mmol) were dissolved in anhydrous DCM, and EDC·HCl (56 mg, 0.29 mmol) and DMAP (4 mg, 0.03 mmol) were added. The mixture was stirred at room temperature for 24 hours. After the reaction was complete, DCM was removed under reduced pressure, and the product BDP 5 was purified by silica gel column chromatography. Yield: 73.7%.

[0055] BDP 5 (53 mg, 0.05 mmol) and polyethylene glycol 2000 (150 mg, 0.075 mmol) were dissolved in anhydrous DCM, and EDC·HCl (19 mg, 0.1 mmol) and DMAP (1 mg, 0.008 mmol) were added. The mixture was stirred at room temperature for 72 hours. After the reaction was complete, DCM was removed under reduced pressure, and the product MBDP-PEG was purified by silica gel column chromatography. Yield: 66.2%.

[0056] Example 2: Synthesis of EBDP-PEG, the specific synthetic route is as follows:

[0057]

[0058] BDP 2 (0.35 g, 0.58 mmol) and 4-diethylaminobenzaldehyde (0.60 g, 4 mmol) were dissolved in 30 mL of toluene. 2.7 mL of piperidine and 0.7 mL of acetic acid were added under N2 conditions. The mixture was stirred at 120 °C for 1 h. After removing the solvent, EBDP (0.20 g, 27% yield) was eluted by column chromatography.

[0059] EBDP (92 mg, 0.1 mmol) and dithiodipropionic acid (105 mg, 0.5 mmol) were dissolved in anhydrous DCM, and EDC·HCl (56 mg, 0.29 mmol) and DMAP (4 mg, 0.03 mmol) were added. The mixture was stirred at room temperature for 24 hours. After the reaction was complete, DCM was removed under reduced pressure, and the product BDP 6 was purified by silica gel column chromatography. Yield: 66.8%

[0060] BDP 6 (56 mg, 0.05 mmol) and polyethylene glycol (150 mg, 0.075 mmol) were dissolved in anhydrous DCM, and EDC·HCl (19 mg, 0.1 mmol) and DMAP (1 mg, 0.008 mmol) were added. The mixture was stirred at room temperature for 72 hours. After the reaction was complete, DCM was removed under reduced pressure, and the product EBDP-PEG was purified by silica gel column chromatography. Yield: 55.6%.

[0061] Example 3: Synthesis of TBDP-PEG, the specific synthetic route is as follows:

[0062]

[0063] BDP 2 (0.35 g, 0.5 mmol) and 4-diphenylaminobenzaldehyde (0.46 g, 1.7 mmol) were dissolved in 30 mL of toluene. 2.7 mL of piperidine and 0.7 mL of acetic acid were added under N2 atmosphere. The mixture was stirred at 120 °C for 1 h. After removing the solvent, TBDP (0.20 g, 31.3% yield) was eluted by column chromatography.

[0064] TBDP (117 mg, 0.1 mmol) and dithiodipropionic acid (105 mg, 0.5 mmol) were dissolved in anhydrous DCM, and EDC·HCl (56 mg, 0.29 mmol) and DMAP (4 mg, 0.03 mmol) were added. The mixture was stirred at room temperature for 24 hours. After the reaction was complete, DCM was removed under reduced pressure, and the product BDP 7 was purified by silica gel column chromatography. Yield: 82.1%.

[0065] BDP 7 (65 mg, 0.05 mmol) and polyethylene glycol (150 mg, 0.075 mmol) were dissolved in anhydrous DCM, and EDC·HCl (19 mg, 0.1 mmol) and DMAP (1 mg, 0.008 mmol) were added. The mixture was stirred at room temperature for 72 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the product TBDP-PEG was purified by silica gel column chromatography. Yield: 51.1%.

[0066] Example 4 Preparation of BDP nanoformulation

[0067] Take 10 mg of MBDP-PEG, EBDP-PEG, and TBDP-PEG prepared in Examples 1, 2, and 3, respectively, dissolve them in 5 mL of tetrahydrofuran, and then slowly add them dropwise to 10 mL of deionized water, stirring until the organic solvent evaporates. After dialysis for 24 h, nano-formulations MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs are obtained.

[0068] The MBDP-PEG, EBDP-PEG, and TBDP-PEG prepared in Examples 1-3 were characterized by NMR and mass spectrometry analysis. Figures 1 to 3 As shown, the structure and purity of MBDP-PEG, EBDP-PEG and TBDP-PEG were confirmed by proton nuclear magnetic resonance spectroscopy and mass spectrometry.

[0069] Example 5: Detection of photophysical properties

[0070] The absorption and emission spectra of the BODIPY photosensitizers MBDP-PEG, EBDP-PEG, and TBDP-PEG prepared in this invention in DMF were detected, as well as the absorption and emission spectra of the corresponding nano-formulations of BODIPY photosensitizers in water. The results are shown in Table 1. Figure 4 As shown in Table 1, the superscript [a] represents the maximum absorption wavelength in the DMF; [b] represents the maximum emission wavelength in the DMF; [c] represents the fluorescence quantum yield in the DMF; [d] represents the singlet oxygen quantum yield in the DMF; and [e] represents the photothermal conversion efficiency.

[0071] Table 1. Photophysical properties of M / E / TBDP-PEG.

[0072]

[0073] Example 6 Reactive Oxygen Detection

[0074] To evaluate the type I and type II ROS generation capabilities of free MBDP-PEG, EBDP-PEG, TBDP-PEG, and their corresponding nanoformulations, DPBF was used as a probe molecule. Fluorescence quenching of DPBF was detected to assess the performance of MBDP-PEG, EBDP-PEG, and TBDP-PEG. 1 O2 generation capability, the operation steps are as follows:

[0075] A DMF solution of DPBF was added to the test solution, with the DMF solution of DPBF used as a blank control. An 808nm laser (20 mW / cm²) was used. 2 Irradiate the test solution and blank control for 80 seconds, and measure the absorption spectrum of the mixed solution every 10 seconds.

[0076] like Figure 5 As shown in sections A to E, in solutions containing only DPBF, the absorbance at 418 nm remained unchanged. In solutions containing MBDP-PEG, EBDP-PEG, and TBDP-PEG, the absorbance gradually decreased with illumination time. Among these solutions, the absorbance decreased most rapidly in the EBDP-PEG solution, followed by MBDP-PEG, with TBDP-PEG showing the slowest decrease.

[0077] The in vitro O2 generation of BODIPY photosensitizers with different structures and their corresponding nano-formulations was detected by measuring the increase in fluorescence intensity of dihydrorhodamine 123 (DHR123). •− The capability and operation steps are as follows:

[0078] A DMF solution of DHR123 was added to the test solution, with the DMF solution of DHR123 used as a blank control. An 808nm laser (20 mW / cm²) was used. 2 Irradiate the test solution and blank control for 80 seconds, and measure the absorption spectrum of the mixed solution every 10 seconds.

[0079] like Figure 6 As shown in sections A to E, almost no fluorescence was produced in the solution containing only DHR123, while enhanced fluorescence was observed in the DMF solution containing MBDP-PEG, EBDP-PEG, and TBDP-PEG, indicating that O2 •− Effective generation. Among them, the fluorescence intensity of the solution containing MBDP-PEG increased by 40.5 times, the fluorescence intensity of the solution containing TBDP-PEG increased by 66.5 times, and the fluorescence intensity of the solution containing EBDP-PEG increased by 77.5 times, exhibiting the strongest O2... •− Generative capability. For example... Figure 7 As shown in sections A to B, EBDP-PEG NPs exhibit the strongest photodynamic activity.

[0080] like Figure 8 In Part A, DLS measurements showed that the particle sizes of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs were 90.2 nm, 95.1 nm, and 104.3 nm, respectively, with PDI values ​​of 0.19, 0.17, and 0.22, respectively. Figure 8 From part B to part D, the prepared nanoparticles exhibit good colloidal stability. All nanoparticles can remain stable in water for more than a week, during which time the particle size and PDI do not change.

[0081] Example 7 Photothermal Performance Testing

[0082] Keeping other conditions constant (808 nm laser, 0.8 W / cm²), 2 The photothermal conversion capacity of the nano-formulation was investigated under different concentrations (0, 5, 10, 15, 20, 30 μM) in water after irradiation for 5 minutes. The resulting heating curves are shown in the figure. Figure 9 As shown in sections A, B, and C, the temperature change of the nanoparticles gradually increases with increasing concentration. Within the same time frame, at the same NP concentration, EBDP-PEGNPs exhibit the greatest temperature increase. Figure 9 The D to F sections show the photothermal heating and cooling curves of three nano-photosensitizers. The MBDP-PEG was calculated using standard methods. The photothermal conversion efficiencies of NPs, EBDP-PEG NPs, and TBDP-PEG NPs were 20.49%, 24.3%, and 14.3%, respectively (Table 1). These results indicate that EBDP-PEG... NPs exhibit the best photothermal heating capability.

[0083] Example 8: Cell endocytosis and detection of intracellular reactive oxygen species

[0084] The uptake of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs by co-incubating with two glioblastoma cell lines (GL261-luc and U87-MG) for 4 h was detected using CLSM. The experimental conclusions are as follows: Figure 10 As shown in sections A and B, bright red fluorescence was observed in GL261-luc and U87-MG cells, indicating that the nanoparticles can be rapidly taken up and dissociated by tumor cells, emitting NIR fluorescence.

[0085] Based on the above, a DCFH-DA probe was added for ROS measurement. For example... Figure 10 In parts A and B, tumor cells treated with MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs all showed significant green fluorescence under laser irradiation.

[0086] To further quantify intracellular ROS production, we used flow cytometry to quantitatively analyze changes in ROS levels within GL261-luc cells. The experimental conclusions are as follows: Figure 10 As shown in sections C and D, compared with the PBS control group, the mean fluorescence intensity of the drug-only group and the light-only group did not change significantly. However, the ROS levels of the MBDP-PEG NPs + Laser, EBDP-PEG NPs + Laser, and TBDP-PEG NPs + Laser groups increased by 4.46, 6.38, and 2.74 times, respectively. These results indicate that the three nanoformulations can generate a large amount of ROS under 808 nm laser irradiation and can be used for tumor phototherapy (PDT). Among them, EBDP-PEG NPs exhibited the strongest ROS generation capacity under laser irradiation.

[0087] Example 9 Cytotoxicity test

[0088] The biocompatibility and phototoxicity of nanoparticles at different concentrations were determined using a CCK8 kit. Figure 11 Parts A and B show the effects of different concentrations of nano-formulations on the viability of GL261-luc and U87-MG cells, respectively. After co-incubation with the three nano-formulations for 48 h, the cell viability remained above 90%, indicating that MBDP-PEGNPs, EBDP-PEG NPs, and TBDP-PEG NPs have good cell compatibility; Figure 11 The C and D parts are irradiated with an 808 nm laser (1 W / cm²). 2 At 5 min, the cell viability of both GL261-luc and U87-MG cells gradually decreased with increasing nanoparticle concentration. Under the same treatment conditions, EBDP-PEG NPs exhibited superior phototoxicity under laser irradiation. These results indicate that EBDP-PEG NPs possess the best ROS generation and photothermal properties, giving them the best antitumor potential.

[0089] Example 10: In vitro simulated BBB crossing experiment of M / E / TBDP-PEG NPs

[0090] Effective penetration of the BBB by nanoformulations is essential for drug delivery to the GBM to exert a therapeutic effect. To investigate the BBB penetration capabilities of three nanoformulations, such as... Figure 12 As shown in Part A, a BBB model was established in vitro using bEnd.3 cells. Figure 12As shown in Part B, after incubating the three nano-formulations with bEnd.3 cells for 48 h, the cell viability remained above 97%, indicating that the three nano-formulations have good cell compatibility. When the monolayer transmembrane resistance is greater than 200 Ω·cm... 2 Nanoparticles were added to the upper chamber of a transwell and co-incubated with GL261-luc cells for 2, 4, and 12 h. Flow cytometry was used to analyze the uptake of the nanoparticles by the GL261-luc cells. Figure 12 As shown in section C, the endocytosis of MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs increased significantly with increasing incubation time. At the same incubation time, GL261-luc cells showed the highest uptake of EBDP-PEG NPs, followed by MBDP-PEG NPs, with TBDP-PEG NPs showing the lowest uptake. These results confirm that EBDP-PEG NPs possess the best in vitro BBB-crossing ability and are enriched in GL261-Luc cells.

[0091] Example 11: In vivo fluorescence imaging experiment of M / E / TBDP-PEG NPs in an in situ GBM-loaded model

[0092] To gain a deeper understanding of the ability of nanoparticles to cross the blood-brain barrier and aggregate at brain tumor sites, near-infrared fluorescence imaging was performed on mouse models bearing orthotopic GL261-luc and U87-MG tumors using an in vivo imaging system. After intravenous injection of 5 mg / kg MBDP-PEG NPs, EBDP-PEG NPs, and TBDP-PEG NPs into tumor-bearing mice, the NIR fluorescence of the nanoparticle formulations was monitored using IVIS at 2, 4, 6, 8, 12, 24, and 36 h after injection. The results are as follows: Figure 13 As shown in sections A and B, MBDP-PEG NPs and EBDP-PEG NPs rapidly accumulated NIR fluorescence in the brain after intravenous injection, reaching their peak fluorescence intensity 6 hours after injection. However, after injection of TBDP-PEG NPs, the fluorescence in the brain was negligible. Figure 13 In the C and D portions, the in vitro distribution showed that the fluorescence signals of MBDP-PEG NPs and EBDP-PEG NPs were mainly enriched in the liver, lungs, and kidneys, indicating that they undergo in vivo metabolism through these organs. Furthermore, as... Figure 13In the E portion, after injection of MBDP-PEG NPs and EBDP-PEG NPs, we found that their NIR fluorescence could achieve excellent co-localization with tumor bioluminescence. This is because the disulfide bonds introduced in the nano-formulation break in response to the redox tumor microenvironment, leading to the disappearance of the aggregation fluorescence quenching (ACQ) effect, thereby enabling fluorescence activation and tumor localization at the tumor site. These results demonstrate that the nano-formulation prepared in this invention can achieve BBB crossing and GBM targeting.

[0093] Example 12: Therapeutic effect of M / E / TBDP-PEG NPs

[0094] An orthotopic GL261-luc tumor-bearing mouse model was established, with mice weighing 18-20g. The therapeutic effects of MBDP-PEG NPs and EBDP-PEG NPs were evaluated: MBDP-PEG NPs and EBDP-PEG NPs (1 mg / mL, 150 μL / mouse) were intravenously injected on days 5 and 10 after tumor inoculation. Six hours after administration, the brain tumor site was irradiated with an 808 nm laser for 5 min (1 W / cm²). 2 ).like Figure 14 In Part A, thermal imaging was used to record temperature changes at the tumor site during light irradiation during treatment. Infrared thermal imaging results showed that EBDP-PEG NPs had superior temperature-raising ability at the tumor site. Figure 14 In Part C, the median survival of mice in the MBDP-PEG NPs + Laser group was prolonged to 32 days, while the median survival of mice in the EBDP-PEG NPs + Laser group was prolonged to 33 days. EBDP NPs showed the best therapeutic effect under light irradiation; compared to PBS, mice receiving EBDP NPs phototherapy had a median survival increased by nearly 70%. Figure 14 In Part B, during the treatment period, the body weight of all mice did not change significantly.

[0095] Encouraged by the high BBB penetration and excellent antitumor effects of EBDP-PEG NPs in the GL261-luc model, we further evaluated its therapeutic effect in mice bearing U87-MG tumors in situ. EBDP-PEG NPs (150 μL / mouse) were administered intravenously on days 4 and 8 post-tumor inoculation. Six hours after administration, the tumor site in the brain was irradiated with 808 nm laser light for 5 minutes (1 W / cm²). 2 ),like Figure 15 Part A involved using thermal imaging to record temperature changes at the tumor site during illumination. In the EBDP-PEGNPs + Laser group, the highest temperature at the tumor site in mice reached 50.2 ℃. Figure 15In Part B of the drug, no significant decrease in mouse body weight was observed after 12 days of administration, indicating that EBDP-PEG NPs and laser irradiation have good in vivo safety. Figure 15 In Part C, mice in the PBS group began to lose weight and rapidly develop the disease 12 days after vaccination, with a median survival of only 17 days. Mice in the EBDP-PEG NPs + Laser group had a median survival extended to 25 days, improving the survival rate by approximately 52.9%. This indicates that EBDP-PEG NPs possess excellent biocompatibility and superior anti-tumor effects.

[0096] In summary, this invention provides a BODIPY photosensitizer, a nano-formulation, and corresponding preparation methods and applications. NIR-absorbed BODIPY is chemically bonded to PEG via GSH-responsive disulfide bonds, thereby constructing EBDP-PEG NPs, a nano-formulation capable of effectively crossing the blood-brain barrier. These nanoparticles exhibit excellent photothermal and photodynamic properties under 808 nm laser irradiation, with a photothermal conversion efficiency as high as 24.3%. Experimental results show that EBDP-PEG NPs can efficiently cross the blood-brain barrier and accumulate at brain tumor sites for extended periods. This invention not only provides a novel formulation and nano-drug delivery system for phototherapy of gliomas but also offers new ideas and methods for designing and developing novel phototherapy drugs capable of crossing the blood-brain barrier, potentially promoting the development and advancement of phototherapy technology for brain tumors.

Claims

1. A BODIPY photosensitizer, characterized in that, It has the structure shown in Equation I: Where R is a fatty amine or aromatic amine, R′ is a hydrophilic polymer, and X is a halogen.

2. The BODIPY photosensitizer according to claim 1, characterized in that, R is selected from dimethylamino, diethylamino, or diphenylamino, and X is hydrogen or iodine.

3. The BODIPY photosensitizer according to claim 2, characterized in that, R′ is selected from at least one of polyethylene glycol, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, poloxamer, poloxamer, hydroxymethylcellulose, carboxyethylcellulose, polysarcosine-lipid conjugate, and polysarcosine-lipid conjugate.

4. The BODIPY photosensitizer according to claim 3, characterized in that, The BODIPY photosensitizer has the structure shown in Formula IV or Formula V: ，n=42~50。 5. The method for preparing the BODIPY photosensitizer according to any one of claims 1 to 4, characterized in that, Includes the following steps: The compound with the structure shown in formula BDP a undergoes a first esterification reaction with 1 to 5 equivalents of dithiodipropionic acid at room temperature to obtain the compound with the structure shown in formula BDP b; the compound with the structure shown in formula BDP b undergoes a second esterification reaction with 1 to 2 equivalents of a hydrophilic polymer at room temperature to obtain the compound with the structure shown in formula I.

6. The method for preparing the BODIPY photosensitizer according to claim 4, characterized in that, The conditions for the first and second esterification reactions are as follows: the solvent used is DCM, the catalysts are EDC·HCl and DMAP, the reaction temperature is room temperature, the duration of the first esterification reaction is 24~48 hours, and the duration of the second esterification reaction is 72~96 hours.

7. A nano-formulation, characterized in that, It is formed by the self-assembly of at least one of the compounds with the structures shown in Formula I, Formula IV, and Formula V.

8. The method for preparing the nano-formulation according to claim 7, characterized in that, At least one of the compounds with structures shown in Formula I, Formula IV, and Formula V is dissolved in an organic solvent to form a solution. Under stirring conditions, the solution is slowly added dropwise to water and stirred until the organic solvent is completely evaporated to obtain a nano-formulation. The organic solvent is one or more of acetone, tetrahydrofuran, methanol, and ethanol.

9. The use of the BODIPY photosensitizer according to any one of claims 1 to 4 or the nanoformulation according to claim 7 in the preparation of tumor therapeutic drugs, the preparation of photoacoustic imaging agents, infrared fluorescent imaging agents, or in drug tracing imaging.

10. The application according to claim 9, wherein the tumor is a glioma.

Images