BODIPY dye and application thereof in photo-thermal field

Abstract

The invention discloses a BODIPY dye and application thereof in the photo-thermal field, and belongs to the technical field of functional dyes and biological medicines. A strong electron-withdrawing group trifluoromethyl is modified on the basis of a common fluorine-boron dipyrromethene dye, and after a conjugated structure is prolonged through p-hydroxybenzaldehyde modification, the absorption wavelength of the dye is subjected to significant red shift; and reacting a single-side hydroxyl group with paratoluensulfonyl chloride to obtain the BODIPY dye. According to the BODIPY dye, the BODIPY dye is a BODIPY dye, nanoparticles are formed after the BODIPY dye is wrapped by amphiphilic molecules DSPE-PEG2000, and by combining the characteristic that zeta potential is negative, the BODIPY dye can target tumor cell lysosome, then lysosome escape is achieved, and finally the BODIPY dye targets endoplasmic reticulum. The BODIPY dye can cause endoplasmic reticulum stress and induce tumor cells to generate immunogenic death even if the BODIPY dye is used for photothermal therapy under a relatively mild photothermal condition, so that the tumor cells are effectively killed. The BODIPY dye is simple in synthesis mode, low in dark toxicity and good in cell killing effect, and has the potential of being applied to clinical treatment.

Description

Technical Field

[0001] The present invention relates to the fields of functional dyes and biomedicine, and particularly to the application of BODIPY dyes and nanoparticles prepared therefrom in photothermal therapy. Background Art

[0003] Photothermal therapy refers to the use of the photothermal conversion of photothermal agents (PTAs) to kill cancer cells by raising the local temperature. The photothermal agent needs to be enriched in tumor tissues. Under the irradiation of a laser with a specific absorption wavelength, the absorbed light energy is converted into heat energy, thereby promoting the local temperature rise of the tumor tissue and achieving the purpose of treating tumors. By controlling the laser, the tumor site can be selectively irradiated. At the same time, the photothermal heating effect is positively correlated with the laser power and the concentration of the photothermal agent. Therefore, photothermal therapy can achieve local precision control, kill cancer cells while greatly reducing the damage to normal tissues, and has the characteristics of high selectivity, no drug resistance, and few post-treatment complications. Therefore, photothermal therapy is expected to develop into a new type of tumor treatment method.

[0004] BODIPY dyes have the advantages of high molar extinction coefficient, good photostability, and good modifiability, and are excellent choices for high-performance photothermal agents. However, so far, most of the reported BODIPY dyes aggregate in the lysosomes of tumor cells and cannot achieve lysosomal escape to further target other subcellular organelles. This results in limited cell killing ability of BODIPY dyes during mild photothermal therapy. To enhance the cell killing ability of BODIPY dyes, the tosyl chloride group is modified on one styrene group, which can further target the endoplasmic reticulum on the premise of achieving lysosomal escape, effectively induce endoplasmic reticulum stress, and induce immunogenic cell death, thereby improving the cell killing ability under mild photothermal therapy. Summary of the Invention

[0005] The purpose of the present invention is to provide a BODIPY dye that still has high cell killing ability under mild photothermal therapy conditions, aiming to solve the problem of poor photothermal therapy effect of existing photothermal agents at low temperatures.

[0006] The technical solution of the present invention is as follows:

[0007] A class of BODIPY dye molecules having the structure of formula I:

[0008]

[0009] Wherein, X and Y are each independently an O, N, or S atom. n and m are the number of hydrogen atoms matching X and Y.

[0010] Some specific BODIPY dyes, where X and Y are each independently an O, N, or S atom, and X and Y are generally the same.

[0011] For some specific BODIPY dyes, when X is an N atom, m = 1 and n = 2; when X is an O or S atom, m = 0 and n = 1; when Y is an N atom, m = 1 and n = 2; when Y is an O or S atom, m = 0 and n = 1.

[0012] Some specific BODIPY dyes have a trifluoromethyl substitution at the meso position.

[0013] For some specific BODIPY dyes, only one of X and Y is connected to p-toluenesulfonyl chloride.

[0014] A nanoparticle comprising at least one of the BODIPY dyes of formula I.

[0015] A specific nanoparticle, wherein the BODIPY dye is encapsulated in DSPE-PEG 2000 (distearoyl phosphatidylethanolamine-polyethylene glycol 2000) to form a nanoparticle.

[0016] The mass ratio of the BODIPY dye to DSPE-PEG2000 is 1:5 - 15, and the preferred mass ratio is 1:10 - 15.

[0017] In a specific technical solution, the particle size of the nanoparticle is 50 - 150 nm.

[0018] The preparation method of the nanoparticle: Dissolve the BODIPY dye and DSPE-PEG 2000 in an organic solvent miscible with water, and slowly add it dropwise to ultrapure water under ultrasonic action, and then continue ultrasonic treatment for 5 min until the solution is clear. The solution is filtered through a 0.45 μm and a 0.22 μm aqueous filter membrane in sequence and then dialyzed to remove the organic solvent to obtain the aqueous solution of the nanoparticle.

[0019] Or,

[0020] The nanoparticle dispersion is concentrated by an ultrafiltration tube to obtain a high-concentration nanoparticle dispersion; the organic solvent miscible with water is one or more of THF, DMSO, and DMF. A specific compound has the following structure:

[0021]

[0022] The present invention provides a synthesis method of a specific BODIPY dye, and the steps are as follows:

[0023] (1) Synthesis of Compound 1

[0024] Under nitrogen protection, PhSiCl3 was added to a CH2Cl2 solution containing CF3COOH and 2,4-dimethylpyrrole at room temperature. The mixture was stirred for 15 min. Then Et3N was added and the reaction was stirred for another 15 min. After that, BF3·Et2O was added and the reaction was further stirred for 3 h. After the reaction was completed, the reaction was quenched with water. The reaction mixture was washed with water and extracted with CH2Cl2. The combined organic layers were dried over anhydrous sodium sulfate, filtered and the solvent was evaporated. The crude product was purified by silica gel column chromatography to obtain a dark red solid compound 1.

[0025] (2) Synthesis of compound 2

[0026] Under nitrogen protection, p-hydroxybenzaldehyde, compound 1, acetic acid, and piperidine were added to dry toluene, and a little activated 4Å molecular sieve was added. The mixture was refluxed and stirred at 100 °C for 3 h. After the product was cooled to room temperature, it was extracted with CH2Cl2. The combined organic layers were washed with water, dried over anhydrous sodium sulfate and then rotary evaporated under reduced pressure. The crude product was separated and purified by silica gel column chromatography to obtain compound 2.

[0027] (3) Synthesis of compound 3

[0028] Under nitrogen protection, compound 2 and p-toluenesulfonyl chloride were added to dry N,N-dimethylformamide and reacted for 1 h, during which TLC was used for monitoring. After the reaction was completed, the solution was rotary evaporated under reduced pressure. The crude product was separated and purified by silica gel column chromatography to obtain compound 3.

[0029] A preparation method of a specific nanoparticle

[0030] Compound 3 and DSPE-PEG 2000 were dissolved in an organic solvent to obtain a mixed solution. Then the mixed solution was added dropwise to ultrapure water under ultrasonic treatment, and ultrasonic treatment was continued for 5 min until the system became clear. The solution was first filtered through 0.45 μm and 0.22 μm aqueous filters, and then dialyzed through a dialysis bag overnight to finally obtain an aqueous solution of nanoparticles.

[0031] The above BODPY dye and nanoparticles can be applied in the field of photothermal.

[0032] Furthermore, the BODIPY dye is applied to prepare a drug or reagent for tumor photothermal therapy.

[0033] Furthermore, the nanoparticles are used to prepare a drug or reagent for tumor photothermal therapy.

[0034] A preparation method of a specific reagent, the nanoparticles are concentrated by an ultrafiltration tube or dispersed in ultrapure water, PBS and common pharmaceutical solutions.

[0035] Compared with the prior art, the present invention has the following prominent advantages:

[0036] By connecting two functional modules together, such BODIPY dyes greatly improve the immune killing effect on tumor cells while ensuring the photothermal therapy effect. The first functional module, trifluoromethyl BODIPY parent body, ensures the photothermal conversion efficiency of the dye molecule, endowing it with good photothermal conversion ability, which provides a basis for subsequent damage to tumor cell sub-organelles and induction of immune response. The second functional module, p-toluenesulfonic acid group, ensures that the dye efficiently targets the endoplasmic reticulum after lysosomal escape, which is a prerequisite for the endoplasmic reticulum stress-induced immune response under photothermal therapy. The two are connected through the reaction of hydroxyl (amino) group with p-toluenesulfonyl chloride, complementing each other, combining photothermal therapy and immunotherapy together, and having high tumor cell killing ability.

[0037] (1) The functional modules of the dye are clear and the preparation is simple.

[0038] (2) The dye can perform photothermal therapy and combine with the immune response induced by endoplasmic reticulum stress to effectively kill tumor cells under mild light irradiation and low concentration conditions. Description of the Drawings

[0039] Figure 1 is the 1H NMR spectrum of Compound 3;

[0040] Figure 2 is the high-resolution mass spectrum of Compound 3;

[0041] Figure 3 is the UV absorption spectrum of the nanoparticles;

[0042] Figure 4 is the transmission electron microscopy image of the nanoparticles;

[0043] Figure 5 is the in vitro photothermal temperature rise (a, b) and temperature rise-cooling cycle (c) images of the nanoparticles under laser irradiation;

[0044] Figure 6 is the test result image of the light-dark toxicity of the nanoparticles to cells. Detailed Embodiments

[0045] The present invention provides a preparation method and application of a BODIPY dye and its nanoparticles. To make the invention purpose and technical solution of the present invention clearer and more definite, the following further explains the present invention in detail. It should be clear that the examples of the specific embodiments described herein are only for further explaining the present invention, rather than limiting the present invention.

[0046] In the examples of the present invention, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000 Purchased from Bidepharm, PBS buffer was purchased from Beijing Solarbio Science & Technology Co., Ltd. The names and units in the examples are common names and units in this field. For example, μM is the concentration unit micromole per milliliter.

[0047] Example 1

[0048] Preparation of a BODIPY dye and its nanoparticles (X = O)

[0049]

[0050] (1) Synthesis of Compound 1

[0051] Under nitrogen protection, at room temperature, PhSiCl3 (0.75 mL, 4.68 mM) was added to a CH2Cl2 solution containing CF3COOH (0.35 mL, 4.71 mM) and 2,4 - dimethylpyrrole (1.0 mL, 9.71 mM). The reaction was stirred for 10 min. Then Et3N (1.5 mL, 10 mM) was added, and the reaction was stirred for another 15 min. After that, 2.0 mL of 16 mM BF3·Et2O was added, and the reaction was further stirred for 3 h and then quenched with water. The reaction mixture was washed with water (2×100 mL) and extracted with CH2Cl2. The combined organic layers were dried over magnesium sulfate, filtered, and evaporated. The crude product was purified by silica gel column chromatography (CH2Cl2 / n - hexane = 1:1) to obtain a dark red solid compound 1 (300 mg).

[0052] (2) Synthesis of Compound 2

[0053] Under nitrogen protection, p - hydroxybenzaldehyde (309 mg, 2.5 mM), compound 1 (200 mg, 0.63 mM), acetic acid (0.1 mL), and piperidine (0.1 mL) were added to dry toluene (10 mL). A little activated 4Å molecular sieve was added, and the mixture was refluxed and stirred at 100 °C for 3 h. After the product was cooled to room temperature, it was extracted with CH2Cl2. The combined organic layers were washed with water, dried over anhydrous sodium sulfate, and then rotary evaporated under reduced pressure. The crude product was separated and purified by silica gel column chromatography (CH2Cl2 / CH3OH = 100:1) to obtain compound 2 (150 mg).

[0054] (3) Synthesis of Compound 3

[0055] Under nitrogen protection, compound 2 (100 mg, 0.19 mM) and p-toluenesulfonyl chloride (38 mg, 0.2 mM) were added to dry N,N-dimethylformamide (5 ml), and the reaction was carried out for 1 h. The resulting solution was rotary evaporated under reduced pressure, and the crude product obtained was separated and purified by silica gel column chromatography (CH2Cl2 / CH3OH = 80:1) to obtain compound 3 (50 mg). 1 H NMR (400 MHz, DMSO-d6) δ10.31 (s, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.64 (d, J = 8.4 Hz, 2H), 7.58 (d, J= 7.9 Hz, 2H), 7.51 (d, J = 8.2 Hz, 2H), 7.40 – 7.32 (m, 1H), 7.21 – 7.10 (m,2H), 6.93 (dd, J = 15.2, 8.5 Hz, 2H), 3.19 (d, J = 5.1 Hz, 1H), 2.46 (s, 3H),2.34 (s, 5H), 1.25 (s, 5H), 0.85 (s, 2H). HRMS: found: 677.1700, calculated: C 35 H 27 BF5N2O4S + , 677.1783. The results are as Figure 1 and Figure 2 shown.

[0056] (4) A method for preparing nanoparticles

[0057] Weigh 1 mg of compound 3 and 10 mg of DSPE-PPEG 2000 Dissolve them in 1 mL of THF. The resulting solution was added dropwise to 9 ml of ultrapure water under the action of ultrasonic waves at 300 W, and ultrasonic treatment was continued for 5 min until the nanoparticle solution system became clear. The initially obtained nanoparticle solution was filtered through 0.45 μm and 0.22 μm aqueous filters, and dialyzed overnight in a dialysis bag with a molecular weight cut-off of 3500 to remove the organic solvent, obtaining the final aqueous nanoparticle solution.

[0058] Example 2

[0059] Preparation of a BODIPY dye and its nanoparticles (X = N)

[0060]

[0061] (1) Synthesis of compound 1

[0062] Under nitrogen protection, PhSiCl3 (4.68 mM) was added to a CH2Cl2 solution containing CF3COOH (4.71 mM) and 2,4-dimethylpyrrole (9.71 mM) at room temperature. The reaction mixture was stirred for 15 min. Then, Et3N (10 mM) was added, and the reaction was stirred for another 15 min. After that, BF3·Et2O was added, and the reaction was further stirred for 3 h. After the reaction was completed, the reaction was quenched with water. The reaction mixture was washed with water and extracted with CH2Cl2. The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The crude product was purified by silica gel column chromatography to obtain a dark red solid compound 1.

[0063] (2) Synthesis of Compound 4

[0064] Under nitrogen protection, p-aminobenzaldehyde (155 mg, 1.28 mmol), compound 1 (100 mg, 0.32 mmol), acetic acid (0.1 mL), and piperidine (0.1 mL) were added to dry toluene. A little activated 4Å molecular sieve was added, and the mixture was refluxed and stirred at 100 °C for 3 h. After the product was cooled to room temperature, it was extracted with CH2Cl2. The combined organic layers were washed with water, dried over anhydrous sodium sulfate, and then rotary evaporated under reduced pressure. The crude product was separated and purified by silica gel column chromatography to obtain compound 4.

[0065] (3) Synthesis of Compound 5

[0066] Under nitrogen protection, compound 4 (100 mg, 0.19 mmol) and p-toluenesulfonyl chloride (19.1 mg, 0.1 mmol) were added to dry N,N-dimethylformamide (3 mL), and the reaction was carried out for 1 h while monitoring by TLC. After the reaction was completed, the solution was rotary evaporated under reduced pressure, and the crude product was separated and purified by silica gel column chromatography to obtain compound 5.

[0067] The preparation of the nanoparticles was the same as in Example 1.

[0068] Example 3

[0069] Calibration of the Concentration of Aqueous Nanoparticle Solution

[0070] A certain mass of compound 3 was dissolved in the organic solvent DMSO to prepare a stock solution with a concentration of 3 mM. 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μL of the stock solution were respectively taken into 3 mL of DMSO solvent, and their absorbance values were measured using a UV-visible absorption spectrometer, and a standard concentration-absorbance standard curve was plotted, as Figure 3As shown. Take 0.15 ml, 0.3 ml, and 0.6 ml of the nanoparticle solution, dilute it to 3 ml with the organic solvent DMSO, measure its absorbance, substitute it into the standard curve, and calculate and average to obtain the concentration of the nanoparticle solution as 40 μM. If a higher concentration of the nanoparticle solution is needed, use an ultrafiltration tube to concentrate the nanoparticle solution. If a lower concentration of the nanoparticle solution is needed, add PBS for dilution.

[0071] Example 4

[0072] Nanoparticle DLS and TEM experiments

[0073] Perform dynamic light scattering (DLS) experiments on the nanoparticles using a Zetasizer Nano-ZS90 ( Figure 4 In a), the hydrodynamic diameter of the nano-photothermal reagent is measured to be 100 nm, and the PDI is 0.038. The lower PDI indicates that the nanoparticles have good dispersibility. Immediately afterwards, the morphology of the nano-photothermal reagent was also tested using a transmission electron microscope (TEM), as Figure 4 shown in b).

[0074] Example 5

[0075] In vitro photothermal heating and heating-cooling cycle experiments of nanoparticles

[0076] Tested the in vitro heating conditions of nanoparticles with different concentrations (0 μM, 20 μM, 40 μM, 60 μM) under 760 nm laser irradiation with a light power density of 500 mW / cm 2 (shown in b). The specific operation is to place the nanoparticle solutions with different concentrations under a 760 nm laser for irradiation, and use an infrared camera FLIR-1910582 to monitor the solution temperature every 30 s for a total of 5 min to obtain the heating situation of the nanoparticle solutions. Using the same method as above, the heating situations of nanoparticle solutions with a concentration of 20 μM under different powers ( Figure 5 (shown in a) and the heating-cooling cycle situation of the nanoparticle solution under the conditions of a concentration of 20 μM and a power of 500 mW / cm Figure 5 (shown in c). 2 Figure 5

[0077] Example 6:

[0078] In vitro photothermal therapy experiments of nanoparticles

[0079] Test the cytotoxicity of the nanoparticles through MTT experiments. Culture A549 cells, MCF-7 cells, and 4T1 cells in 96-well plates (about 1×10 4 ​​cells, and 100 μL of DMEM medium was added thereto). When the cells adhered and reached a density of 80%, media containing 2, 4, 6, 8, 10, 12 μM nanoparticles were added to the MCF-7 cell plates, media containing 2.5, 5, 7.5, 10, 12.5, 15 μM nanoparticles were added to the 4T1 cell plates, and 2.5, 5, 7.5, 10, 12.5, 15 μM nanoparticles were added to the A549 cell plates; and they were incubated for 5 h, and then irradiated with a 760 nm laser (500 mW / cm 2 ) for 15 min. At the same time, another control group with the same experimental conditions but without laser irradiation was also prepared for the study of cell dark toxicity. After incubating again at 37 °C for 24 h, 100 μL of DMEM solution with 0.5 mg / mL MTT was added to each well and incubated for another 4 h. Subsequently, the media in the 96-well plates were removed, and 100 μL of DMSO was added to each well to dissolve the formed formazan crystals. A Bio-Rad microplate reader was used to measure the absorbance of the solution at 490 nm and calculate the cell viability:

[0080]

[0081] Among them, the experimental group represents the cell culture groups treated with different concentrations of nanoparticles; the blank group represents the cell culture group with only medium added; the control group represents the cell culture group without nanoparticles added; OD is the absorbance value measured at 490 nm of the DMSO solution dissolving the formazan crystals. Each experiment was performed in parallel 4 times. The test results of the phototoxicity and dark toxicity of nanoparticles to cells are as Figure 6 shown. It can be seen from the figure that the nanoparticles have strong phototoxicity to A549 cells, MCF-7 cells and 4T1 cells, low dark toxicity and good cell killing effect; for MCF-7 cells, the cells are basically inactivated when 10 μM nanoparticles are added; for 4T1 cells, the cells are basically inactivated when 15 μM nanoparticles are added; for A549 cells, the cells are basically inactivated when 12.5 μM nanoparticles are added.

[0082] The above is only the preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered within the protection scope of the present invention. Those skilled in the art should understand that any improvement to the present invention, the equivalent substitution of the raw materials selected for the present invention, and the substitution and selection of auxiliary components, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A BODIPY dye, characterized in that: The BODIPY dye has the structure of Formula I: ； Wherein, X and Y are independently N, O, or S atoms, and n and m are the numbers of hydrogen atoms matching X and Y.

2. A BODIPY dye according to claim 1, characterized in that: When X is an N atom, m=1, n=2; when X is an O or S atom, m=0, n=1; when Y is an N atom, m=1, n=2; when Y is an O or S atom, m=0, n=1.

3. A BODIPY dye according to claim 1, characterized in that: X and Y are the same atom.

4. A nanoparticle, characterized in that: The nanoparticles include at least one of the BODIPY dyes described in claim 1.

5. A nanoparticle according to claim 4, characterized in that: The nanoparticles further include distearoylphosphatidylethanolamine-polyethylene glycol DSPE-PEG for encapsulating the BODIPY dye 2000 .

6. A nanoparticle according to claim 5, characterized in that: The BODIPY dye is mixed with distearoylphosphatidylethanolamine-polyethylene glycol DSPE-PEG 2000 The mass ratio is 1:5-15.

7. A nanoparticle according to claim 6, characterized in that: The diameter of the nanoparticles is 50-150 nm.

8. A nanoparticle according to any one of claims 4 to 7, characterized in that: Prepared by the following method: (1) The BODIPY dye, DSPE-PEG 2000 After being dissolved in an organic solvent, the mixture is added dropwise to the dispersion medium under ultrasonic action; or, The BODIPY dye is dissolved in an organic solvent and then added dropwise to the DSPE-PEG under ultrasound. 2000 In a solution with a dispersion medium; (2) The mixture was sonicated until no turbidity was observed, filtered through 0.45 μm and 0.22 μm water filter membranes, and dialyzed to remove the organic solvent to obtain a nanoparticle dispersion; or, The nanoparticle dispersion is concentrated through an ultrafiltration tube to obtain a high-concentration nanoparticle dispersion; The organic solvent is a solvent miscible with water, and the dispersion medium is ultrapure water or PBS buffer; The organic solvent is one or more of DMSO, THF and DMF.

9. The use of a BODIPY dye according to any one of claims 1 to 3, characterized in that: The BODIPY dye is used to prepare drugs for photothermal therapy of tumors.

10. The use of the nanoparticles according to any one of claims 4 to 7, characterized in that: The nanoparticles are used to prepare drugs for photothermal therapy of tumors.

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