A perylene monoimide-based NIR-II fluorescent dye, nanoparticles and preparation method and application thereof

Abstract

This invention discloses a perylene-imide-based NIR-II fluorescent dye, nanoparticles, its preparation method, and its applications. The perylene-imide-based NIR-II fluorescent dye consists of a perylene-imide backbone and a polymethylbenzene backbone. Nanoparticles are obtained by ultrasonically mixing the dye with an anticancer drug and an amphiphilic polymer. The synthesis method of this invention is simple, the fluorescent dye has a well-defined structure, and exhibits good water solubility, biocompatibility, and stability. Both its absorption and fluorescence emission spectra are located in the near-infrared II region. The perylene-imide-based NIR-II fluorescent dye nanoparticles prepared by this invention have a fluorescence quantum yield of 1.7% under 980 nm laser irradiation, demonstrating excellent NIR-IIa fluorescence imaging capabilities, enabling high-resolution imaging diagnosis of in situ lung cancer. Under 1064 nm laser irradiation, the photothermal conversion efficiency is 40.3%, exhibiting excellent photothermal conversion performance, allowing for precise NIR-II phototherapy for in situ lung cancer.

Description

Technical Field

[0001] This invention belongs to the field of photothermal therapy preparation technology, specifically relating to a perylene monoimide-based NIR-II fluorescent dye, nanoparticles, their preparation method and application. Background Technology

[0002] Cancer is one of the major diseases that seriously threaten human health. Traditional cancer treatments, including radiotherapy, chemotherapy, and surgery, are often accompanied by a wide range of side effects. Therefore, research on non-invasive and non-surgical treatments has always been a hot topic in the medical field. Phototherapy, due to its advantages of high safety and high selectivity, has been widely used in cancer treatment in recent years. During phototherapy, non-toxic phototherapy agents can be activated under light irradiation, selectively killing cancer cells without causing serious side effects. Through careful design of phototherapy agents and good control of light irradiation at the lesion site (such as tumor tissue), phototherapy can reduce the side effects of traditional chemotherapy and radiotherapy. Photothermal therapy (PTT), as a type of phototherapy, relies on nanomaterials with photothermal effects to convert light into heat to kill cancer cells, effectively avoiding side effects on normal cells.

[0003] Precise imaging diagnosis of cancer sites is crucial for cancer treatment. Traditional near-infrared I (NIR-I, 650-900 nm) fluorescence imaging techniques suffer from limitations such as low tissue penetration depth, low spatial resolution, and low signal-to-noise ratio. To address this challenge, in the past decade, researchers have developed a new imaging method utilizing near-infrared II (NIR-II, 1000-1700 nm) fluorescence imaging technology to achieve deeper tissue penetration, higher resolution, and lower background noise. The NIR-II region can be divided into NIR-IIa' (1000-1300 nm), NIR-IIa (1300-1400 nm), and NIR-IIb (1500-1700 nm), with increasing wavelength leading to improved imaging resolution and quality. Currently reported photothermal therapy drugs still suffer from problems such as complex synthesis, absorption wavelengths located in the NIR-I region, imaging wavelengths limited to the NIR-IIa' region, and unsatisfactory therapeutic effects. Summary of the Invention

[0004] The purpose of this invention is to provide a perylene monoimide-based NIR-II fluorescent dye, nanoparticles, their preparation method, and applications.

[0005] The perylene-monimide-based NIR-II fluorescent dye is composed of two parts: perylene-monimide and a polymethyl ether backbone, and its structural formula is shown below: .

[0006] The preparation method of the perylene monoimide-based NIR-II fluorescent dye is as follows: (1) Dissolve perylene monoimide compounds in liquid bromine at a molar ratio of 1:1-2 in chloroform and react under sealed conditions in an ice bath for 2-5 hours; extract with NaOH aqueous solution, and dry the organic phase to obtain bromoperylene monoimide Py-1; (2) Py-1, tert-butyl hydrazinoate, sodium tert-butoxide, tris(dibenzylacetone)dipalladium and 2,2'-bis(diphenylphosphine)-1,1'-binaphthylene in a molar ratio of 1:1-1.5:4-4.5:0.1-0.2:0.2-0.4 were dissolved in anhydrous tetrahydrofuran and reacted at 70-90 °C for 10-20 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, and the organic solvent was removed by vacuum distillation. The crude product was separated and purified by chromatographic column chromatography to obtain the Boc protected perylene monoimide derivative Py-2. (3) Dissolve Py-2 and trifluoroacetic acid in anhydrous dichloromethane at a molar ratio of 1:6-10 and react at room temperature for 1-3 hours; add saturated sodium bicarbonate aqueous solution to the reaction solution for extraction, and after drying the organic phase, separate by column chromatography to obtain hydrazone-substituted perylene monoimide Py-3. (4) Py-3 and 3-methyl-2-butanone in a molar ratio of 1:2-3 were dissolved in glacial acetic acid and refluxed at 100-140°C for 10-15 hours; the reaction solution was precipitated with saturated saline, filtered, the precipitate was washed with deionized water, and purified by column chromatography to obtain perylene monoimide and indoline Py-4. (5) Py-4 and 1,3-propanesulfonyl lactone in a molar ratio of 1:4-6 were dissolved in 1,2-dichloroethane. The oxygen was removed by freezing-vacuuming-thawing three times with liquid nitrogen. The reaction was carried out at 160-200°C for 20-30 hours under nitrogen protection. The product was precipitated with methyl tert-butyl ether and washed. The product was purified by column chromatography to obtain quaternary ammonium perylene monoimide and indoline derivative Py-5. (6) Dissolve 1-cyclohexene-1-carboxaldehyde and Py-5 in a mixed solvent of acetic acid and acetic anhydride at a molar ratio of 1:2-2.5, then add sodium acetate, and react at 100-140°C for 12-24 hours under nitrogen protection. Precipitate with methyl tert-butyl ether and wash, then separate and purify by column chromatography to obtain perylene monoimide NIR-II fluorescent dye.

[0007] The preparation method of the perylene-imide-based NIR-II fluorescent dye nanoparticles is as follows: the perylene-imide-based NIR-II fluorescent dye and the anticancer drug are dissolved in DMF to obtain a mixed solution; the amphiphilic polymer is dissolved in deionized water, and the mixed solution is added under ultrasonic conditions, and ultrasonication is continued for 3-10 minutes; the organic solvent is removed by dialysis, and the nanoparticles are obtained by ultrafiltration concentration.

[0008] The anticancer drug in question is osimertinib.

[0009] The amphiphilic polymer is a phospholipid-polyethylene glycol-epidermal growth factor peptide.

[0010] The nanoparticles of the perylene imide-based NIR-II fluorescent dye prepared above have a maximum absorption peak at 1053 nm and a maximum fluorescence emission peak at 1129 nm in aqueous solution, which can extend to the NIR-IIa (1300-1400 nm) region.

[0011] The perylene monoimide-based NIR-II fluorescent dye nanoparticles prepared above are used as contrast agents for near-infrared II fluorescence imaging. The excitation wavelength is 980 nm.

[0012] The application of the perylene monoimide-based NIR-II fluorescent dye nanoparticles prepared above in the preparation of cancer photothermal therapy agents. The laser irradiation wavelength was 1064 nm.

[0013] The perylene monoimide-based NIR-II fluorescent dye synthesized in this invention is simple to synthesize, has a well-defined structure, and exhibits good water solubility, biocompatibility, and stability. Its absorption and fluorescence emission spectra are both located in the near-infrared II region, and its fluorescence emission spectrum extends to the NIR-IIa (1300-1400 nm) region. Under 980 nm laser irradiation, it exhibits a fluorescence quantum yield of 1.7%, demonstrating excellent NIR-IIa fluorescence imaging capabilities, enabling high-resolution imaging diagnosis of in situ lung cancer. Under 1064 nm laser irradiation, its photothermal conversion efficiency is 40.3%, demonstrating excellent photothermal conversion performance, which can efficiently kill cancer cells and enable precise NIR-II phototherapy for in situ lung cancer. Attached Figure Description

[0014] Figure 1 This is a flowchart illustrating the synthesis of the Py1053 molecule in Example 1 of this invention.

[0015] Figure 2 This is the proton NMR spectrum of the Py1053 compound molecule in Example 1 of the present invention.

[0016] Figure 3 This is the mass spectrum of the Py1053 compound molecule in Example 1 of the present invention.

[0017] Figure 4 This is an absorption and emission diagram of the Py1053 molecule in dichloromethane in Application Example 1 of the present invention.

[0018] Figure 5 The Py1053 / Osi@DSPE-PEG in Application Example 2 of this invention 2000 - Hydrated particle size and transmission electron microscopy image of GE11 nanoparticles.

[0019] Figure 6 The Py1053 / Osi@DSPE-PEG in Application Example 2 of this invention 2000 - Absorption and emission spectra of GE11 nanoparticles in water.

[0020] Figure 7 The Py1053 / Osi@DSPE-PEG in Application Example 2 of this invention 2000 - Graphs showing the photothermal heating effect of GE11 at different concentrations.

[0021] Figure 8 The Py1053 / Osi@DSPE-PEG in Application Example 2 of this invention 2000 -Graphic stability effect of GE11.

[0022] Figure 9 The Py1053 / Osi@DSPE-PEG in Application Example 3 of this invention 2000 -Graphs showing the (A) dark toxicity of GE11 and its (B) cell-killing effect under light conditions.

[0023] Figure 10 PCy / Osi@DSPE-PEG in Application Example 4 of this invention 2000 - GE11 targeting effect on EGFR in cells.

[0024] Figure 11 PCy / Osi@DSPE-PEG in Application Example 5 of this invention 2000 - In vivo NIR-II fluorescence imaging of GE11.

[0025] Figure 12 PCy / Osi@DSPE-PEG in Application Example 6 of this invention 2000 - In vivo photoacoustic imaging of GE11. Detailed Implementation Example 1

[0026] (1) A perylene monoimide compound (1.0 g, 2.08 mmol) was dissolved in 200 mL of chloroform, followed by the addition of 1.5 mL of liquid bromine. The reaction system was sealed to prevent leakage of the liquid bromine, and the reaction flask was placed in an ice-water bath for 3 hours. The mixture was extracted with NaOH aqueous solution, and the organic and aqueous phases were separated. After evaporating the organic phase, an orange-red solid bromoperylene monoimide compound, Py-1, was obtained. The reaction yield was 99%.

[0027] (2) Py-1 (0.56 g, 1 mmol), tert-butyl hydrazine carboxylate (0.158 g, 1.2 mmol), sodium tert-butoxide (0.81 g, 4.1 mmol), tris(dibenzylacetone)palladium (0.10 g, 0.11 mmol), and 2,2'-bis(diphenylphosphine)-1,1'-binaphthyl (0.15 g, 0.24 mmol) were dissolved in 10 mL of anhydrous tetrahydrofuran and reacted at 80 °C for 16 hours under nitrogen protection. After the reaction was complete, the mixture was cooled to room temperature and the organic solvent was removed by vacuum distillation to obtain a crude purple solid. The crude product was purified by chromatographic column chromatography to obtain the Boc-protected perylene monoimide derivative Py-2 (0.36 g, 50%).

[0028] (3) Py-2 (0.59 g, 1 mmol) was dissolved in anhydrous dichloromethane, and 8 mmol of trifluoroacetic acid was slowly added dropwise under ice bath conditions. After the addition was completed, the reaction was carried out at room temperature for 2 hours. Saturated sodium bicarbonate aqueous solution was added to the reaction solution for extraction, and the organic phase was dried by rotary evaporation. The crude product was separated by column chromatography to obtain hydrazone-substituted perylene monoimide Py-3 (0.31 g, 50%).

[0029] (4) Py-3 (0.876 g, 1.76 mmol) was dissolved in 8 mL of glacial acetic acid, followed by the addition of 4.4 mmol of 3-methyl-2-butanone. The mixture was then slowly heated to 120°C and reacted for 12 hours. After the reaction was complete, the reaction solution was precipitated with saturated brine, filtered, and the precipitate was washed with deionized water, filtered again, and dried under vacuum to obtain a red solid. The crude product was purified by chromatographic column chromatography to obtain perylene monoimide-indoline Py-4 (0.45 g, 51%).

[0030] (5) Py-4 (0.843 g, 1.5 mmol) was placed in a 10 mL Shrek tube, and 0.5 mL of 1,2-dichloroethane and 1,3-propanesulfonyl lactone (0.913 g, 7.5 mmol) were added. The mixture was deoxygenated by freezing, evacuating and thawing three times with liquid nitrogen. The reaction was carried out at 180 °C for 24 hours under nitrogen protection. The reaction mixture was precipitated with methyl tert-butyl ether and the precipitate was washed. The crude product was separated and purified by chromatographic column chromatography to obtain the quaternary ammonium perylene monoimide and indoline derivative Py-5 (0.47 g, 56%).

[0031] (6) Py-5 (0.133 g, 2 mmol) and 1-cyclohexene were dissolved in 10 mL of a mixed solvent of acetic acid and acetic anhydride (containing 4 mL of acetic acid and 6 mL of acetic anhydride). Sodium acetate (0.082 g, 1 mmol) was then added, and the mixture was reacted at 120 °C for 12 hours under nitrogen protection. The mixture was cooled to room temperature, precipitated with methyl tert-butyl ether, washed several times, and dried. The crude product was purified by silica gel chromatography to obtain Py1053 (0.54 g, 37%). Example 2

[0032] Py1053 (1 mg) and osimertinib (0.5 mg) were dissolved in DMF to prepare a mixed solution, and the amphiphilic polymer DSPE-PEG was used. 2000 -GE11 (5 mg) was dissolved in deionized water to prepare DSPE-PEG. 2000 -GE11 aqueous solution; then, while sonicating the DSPE-PEG2000-GE11 aqueous solution, a DMF solution of Py1053 and osimertinib was rapidly injected, and the mixed solution was sonicated for 5 minutes, the DMF organic solvent was removed by dialysis, and the solution was concentrated by ultrafiltration to obtain an aqueous solution of Py1053 / Osi@DSPE-PEG2000-GE11 nanoparticles.

[0033] Application Example 1 A dichloroethane solution of Py1053 was prepared using NIR-II dye IR26 (with a quantum efficiency of 0.05% in dichloroethane) as a standard. Based on IR26, the Py1053 / Osi@DSPE-PEG ratio was calculated using a formula. 2000 -GE11 has a fluorescence quantum yield of 1.7%.

[0034] Application Example 2 Preparation of different concentrations of Py1053 / Osi@DSPE-PEG 2000 -GE11 aqueous solution (1 mL), using a 1064 nm laser (1.0 W / cm²) 2 The Py1053 / Osi@DSPE-PEG was irradiated, and its temperature was recorded at different time points using an infrared camera. The photothermal calculation formula was then used to calculate the Py1053 / Osi@DSPE-PEG temperature. 2000 - The photothermal conversion efficiency of GE11 is 40.3%.

[0035] Application Example 3 Contains Py1053 / Osi@DSPE-PEG 2000- GE11 medium was co-cultured with NCl-H1975 cells for 24 hours. The medium was then replaced with medium containing 10% CCK-8 and incubated for another 0.5 to 2 hours. Subsequently, relative cell viability was detected using a microplate reader to test Py1053 / Osi@DSPE-PEG. 2000 - GE11's cytotoxicity. Further, cells were co-cultured with Pcy / Osi@GE11-containing medium in 96-well plates for 12 hours, followed by exposure to a 1064 nm laser (1.0 W / cm²). 2 Irradiation for 10 minutes. Cell viability was assessed using the same CCK-8 assay for Py1053 / Osi@DSPE-PEG. 2000 The cytotoxic effect of GE11 was demonstrated. Simultaneously, cell viability and mortality of Py1053 / Osi@DSPE-PEG were further verified using calcein-propidium iodide staining. 2000 -The cell-killing effect of GE11.

[0036] Application Example 4 NCl-H1975 cells were seeded into glass-bottomed culture dishes and Py1053 / Osi@DSPE-PEG was added. 2000 Cells were co-incubated with GE11 nanoparticles for 12 hours. After culture, cells were fixed with 4% paraformaldehyde and blocked with 3% bovine serum albumin. Cells were then incubated with anti-EGFR monoclonal antibody and imaged under a fluorescence microscope.

[0037] Application Example 5 Balb / c mice with orthotopic lung cancer were selected, and Py1053 / Osi@DSPE-PEG was injected into the tail vein of the mice. 2000 -GE11 nanoparticles, at a dose of 100 μL / mouse (100 μM), were used to observe the brightness of the lung tumor location in mice at different time points using a near-infrared II in vivo imaging system.

[0038] Application Example 6 Select NCl-H1975 cell-bearing Balb / c mice and inject them via tail vein with Py1053 / Osi@DSPE-PEG. 2000 -GE11 nanoparticles, at a dose of 100 μL / mouse (100 μM), were used to observe the brightness of the tumor location in mice at different time points using in vivo photoacoustic imaging.

Claims

1. A perylenemonoimide-based NIR-II fluorescent dye, characterized in that, The perylene-imide-based NIR-II fluorescent dye consists of two parts: perylene-imide and a polymethylene backbone, and its structural formula is shown below: 。 2. A method for preparing a perylene monoimide-based NIR-II fluorescent dye, characterized in that, The specific steps of the preparation method are as follows: (1) Dissolve perylene monoimide compounds in liquid bromine at a molar ratio of 1:1-2 in chloroform and react under sealed conditions in an ice bath for 2-5 hours; extract with NaOH aqueous solution, and dry the organic phase to obtain bromoperylene monoimide Py-1; (2) Py-1, tert-butyl hydrazinoate, sodium tert-butoxide, tris(dibenzylacetone)palladium and 2,2'-bis(diphenylphosphine)-1,1'-binaphthylene in a molar ratio of 1:1-1.5:4-4.5:0.1-0.2:0.2-0.4 were dissolved in anhydrous tetrahydrofuran and reacted at 70-90 °C for 10-20 hours under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, the organic solvent was removed by vacuum distillation, and the crude product was separated and purified by chromatographic column to obtain the Boc protected perylene monoimide derivative Py-2. (3) Dissolve Py-2 and trifluoroacetic acid in anhydrous dichloromethane at a molar ratio of 1:6-10 and react at room temperature for 1-3 hours; add saturated sodium bicarbonate aqueous solution to the reaction solution for extraction, and after drying the organic phase, separate by column chromatography to obtain hydrazone-substituted perylene monoimide Py-3. (4) Py-3 and 3-methyl-2-butanone in a molar ratio of 1:2-3 were dissolved in glacial acetic acid and refluxed at 100-140°C for 10-15 hours; the reaction solution was precipitated with saturated saline, filtered, the precipitate was washed with deionized water, and purified by column chromatography to obtain perylene monoimide and indoline Py-4. (5) Py-4 and 1,3-propanesulfonyl lactone in a molar ratio of 1:4-6 were dissolved in 1,2-dichloroethane. The oxygen was removed by freezing-vacuuming-thawing three times with liquid nitrogen. The reaction was carried out at 160-200°C for 20-30 hours under nitrogen protection. The product was precipitated with methyl tert-butyl ether and washed. The product was purified by column chromatography to obtain quaternary ammonium perylene monoimide and indoline derivative Py-5. (6) Dissolve 1-cyclohexene-1-carboxaldehyde and Py-5 in a mixed solvent of acetic acid and acetic anhydride at a molar ratio of 1:2-2.5, then add sodium acetate, and react at 100-140°C for 12-24 hours under nitrogen protection. Precipitate with methyl tert-butyl ether and wash, then separate and purify by column chromatography to obtain perylene monoimide NIR-II fluorescent dye.

3. A method for preparing nanoparticles of a perylene monoimide-based NIR-II fluorescent dye, characterized in that, The specific operation of the preparation method is as follows: dissolve perylene imide-based NIR-II fluorescent dye and anticancer drugs in DMF to obtain a mixed solution; dissolve the amphiphilic polymer in deionized water, add the mixed solution under ultrasonic conditions, and continue ultrasonication for 3-10 minutes; remove the organic solvent by dialysis, and concentrate by ultrafiltration to obtain nanoparticles.

4. The preparation method according to claim 3, characterized in that, The anticancer drug in question is osimertinib.

5. The preparation method according to claim 3, characterized in that, The amphiphilic polymer is a phospholipid-polyethylene glycol-epidermal growth factor peptide.

6. The application of perylene monoimide-based NIR-II fluorescent dye nanoparticles prepared by the method according to any one of claims 3-5 as a near-infrared II fluorescence imaging contrast agent.

7. The application of perylene monoimide-based NIR-II fluorescent dye nanoparticles prepared by the method according to any one of claims 3-5 in the preparation of cancer photothermal therapy agents.

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