Perylene monoimidogen NIR-II fluorescent dye, nano-particles and preparation method and application of perylene monoimidogen NIR-II fluorescent dye
By developing perylene monoimidyl NIR-II fluorescent dye to prepare nanoparticles, the complex synthesis and poor therapeutic effects of existing photothermal therapy drugs have been solved, and efficient NIR-IIa fluorescence imaging and photothermal conversion have been achieved, which can accurately diagnose and treat lung cancer.
Patent Information
- Application Number
- CN202411715049.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-02-25
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The existing photothermal therapeutic drugs have problems such as complex synthesis, absorption wavelength is located in the NIR-I region, and imaging wavelength is only located in the NIR-IIa' region, which has poor treatment effect.
A perylene monoimidogenic NIR-II fluorescent dye was developed to prepare nanoparticles through specific synthetic methods, with a maximum absorption peak of 1053 nm and a maximum fluorescence emission peak of 1129 nm, which can extend to the NIR-IIa (1300-1400 nm) region.
It has achieved excellent NIR-IIa fluorescence imaging capability under 980 nm laser, can achieve high resolution imaging diagnosis, and has a photothermal conversion efficiency of 40.3% under 1064 nm laser irradiation, and can effectively kill cancer cells.
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Figure CN119504800A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of photothermal therapy agent preparation, and specifically relates to a perylene monoimide-based NIR-II fluorescent dye, nanoparticles, and a preparation method and application thereof. Background Art
[0002] Cancer is one of the major diseases that seriously endanger human health. Traditional cancer treatments, including radiotherapy, chemotherapy, and surgery, are often accompanied by extensive side effects. Therefore, research on non-invasive and non-invasive treatments has always been a hot topic in medical-related fields. Phototherapy has been widely used in cancer tumors in recent years due to its advantages such as high safety and high selectivity. During phototherapy, non-toxic phototherapeutic agents can be activated under light to selectively kill cancer cells without causing serious side effects. Through the careful design of phototherapeutic agents and good control of light exposure to lesions (such as tumor tissue), phototherapeutic agents can reduce the side effects of traditional chemotherapy and radiotherapy. Photothermal therapy (PTT), as a type of phototherapy, relies on nanomaterials with photothermal effect to convert light into heat to kill cancer cells, which can effectively avoid side effects on normal cells.
[0003] Accurate imaging diagnosis of cancer sites is of great significance for the diagnosis and treatment of cancer. Traditional near-infrared region I (NIR-I, 650-900 nm) fluorescence imaging technology has shortcomings such as low tissue penetration depth, low spatial resolution and low signal-to-noise ratio. In order to meet this challenge, in the past decade, people have developed a new imaging method that uses near-infrared region II fluorescence (NIR-II, 1000-1700 nm) imaging technology to achieve deeper tissue penetration, higher resolution and low background noise. Among them, the NIR-II region can be divided into: NIR-IIa' (1000-1300 nm), NIR-IIa (1300-1400 nm), NIR-IIb (1500-1700 nm), and the imaging resolution quality increases with increasing wavelength. The photothermal therapy drugs that have been reported so far still have problems such as complex synthesis, absorption wavelength in the NIR-I region, imaging wavelength only in the NIR-IIa' region, and unsatisfactory treatment effect. Summary of the invention
[0004] The purpose of the present invention is to provide a perylene monoimide-based NIR-II fluorescent dye, nanoparticles, and a preparation method and application thereof.
[0005] The perylene monoimide-based NIR-II fluorescent dye is composed of two parts: perylene monoimide and a polymethine skeleton, and its structural formula is as follows: .
[0006] The preparation method of the perylene monoimide-based NIR-II fluorescent dye is as follows: (1) dissolving a perylene monoimide compound and liquid bromine in a molar ratio of 1:1-2 in chloroform, and reacting them in a sealed container under ice bath conditions for 2-5 hours; extracting with an aqueous solution of NaOH, and drying the organic phase to obtain a bromoperylene monoimide Py-1; (2) Py-1, tert-butyl carbazate, sodium tert-butoxide, tri(dibenzylideneacetone)dipalladium and 2,2'-bis(diphenylphosphine)-1,1'-binaphthyl in a molar ratio of 1:1-1.5:4-4.5:0.1-0.2:0.2-0.4 are dissolved in anhydrous tetrahydrofuran, and reacted at 70-90°C for 10-20 hours under nitrogen protection; after the reaction is completed, the reaction is cooled to room temperature, and the organic solvent is removed by vacuum distillation. The crude product is separated and purified by a chromatographic column to obtain a Boc-protected perylene monoimide derivative Py-2; (3) dissolving Py-2 and trifluoroacetic acid in anhydrous dichloromethane at a molar ratio of 1:6-10, and reacting at room temperature for 1-3 hours; adding saturated sodium bicarbonate aqueous solution to the reaction solution for extraction, and separating the organic phase by column chromatography to obtain hydrazone-substituted perylene monoimide Py-3; (4) dissolving Py-3 and 3-methyl-2-butanone in a molar ratio of 1:2-3 in glacial acetic acid, and reacting under reflux at 100-140° C. for 10-15 hours; precipitating the reaction solution with saturated saline, filtering, washing the precipitate with deionized water, and separating and purifying by column chromatography to obtain perylene monoimide and indoline Py-4; (5) Py-4 and 1,3-propane sultone in a molar ratio of 1:4-6 are dissolved in 1,2-dichloroethane, and deoxygenated by freezing-evacuating-thawing with liquid nitrogen for three times, reacting at 160-200° C. for 20-30 hours under nitrogen protection, precipitating and washing with methyl tert-butyl ether, and separating and purifying by column chromatography to obtain a quaternary ammonium perylene monoimide indoline derivative Py-5; (6) 1-cyclohexene-1-carboxaldehyde and Py-5 in a molar ratio of 1:2-2.5 are dissolved in a mixed solvent of acetic acid and acetic anhydride, and then sodium acetate is added. The mixture is reacted at 100-140°C for 12-24 hours under nitrogen protection, precipitated and washed with methyl tert-butyl ether, and separated and purified by column chromatography to obtain a perylene monoimide NIR-II fluorescent dye.
[0007] The preparation method of the perylene monoimide NIR-II fluorescent dye nanoparticles is as follows: dissolving the perylene monoimide NIR-II fluorescent dye and an anticancer drug in DMF to obtain a mixed solution; dissolving an amphiphilic polymer in deionized water, adding the mixed solution under ultrasonic conditions, and continuing ultrasonication for 3-10 minutes; dialyzing to remove the organic solvent, and ultrafiltration and concentration to obtain nanoparticles.
[0008] The anticancer drug is osimertinib.
[0009] The amphiphilic polymer is phospholipid-polyethylene glycol-epidermal growth factor peptide.
[0010] The maximum absorption peak of the perylene monoimide-based NIR-II fluorescent dye nanoparticles prepared above in aqueous solution is at 1053 nm, the maximum fluorescence emission peak is at 1129 nm, and can extend to the NIR-IIa (1300-1400 nm) region.
[0011] The prepared perylene monoimide-based NIR-II fluorescent dye nanoparticles are used as near-infrared second-zone fluorescent imaging contrast agents with an excitation wavelength of 980 nm.
[0012] The application of the prepared perylene monoimide-based NIR-II fluorescent dye nanoparticles in the preparation of cancer photothermal therapeutic agents. The laser irradiation wavelength is 1064 nm.
[0013] The perylene monoimide-based NIR-II fluorescent dye synthesized by the present invention has simple synthesis, clear structure, good water solubility, biocompatibility and stability, and its absorption and fluorescence emission spectra are both located in the near-infrared region II, and its fluorescence emission spectrum can extend to the NIR-IIa (1300-1400nm) region. The fluorescence quantum yield is 1.7% under 980 nm laser, and it has excellent NIR-IIa fluorescence imaging capability, which can achieve high-resolution imaging diagnosis of in situ lung cancer. The photothermal conversion efficiency under 1064 nm laser irradiation is 40.3%, which has excellent photothermal conversion performance, can efficiently kill cancer cells, and can perform accurate NIR-II light diagnosis and treatment of in situ lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] Figure 1 This is a synthetic flow chart of the Py1053 molecule in Example 1 of the present invention.
[0015] Figure 2 This is the hydrogen 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 the absorption and emission diagram of the Py1053 molecule in Application Example 1 of the present invention in dichloromethane.
[0018] Figure 5 Py1053 / Osi@DSPE-PEG in Application Example 2 of the present invention 2000 -Hydrated particle size and transmission electron microscopy image of GE11 nanoparticles.
[0019] Figure 6Py1053 / Osi@DSPE-PEG in Application Example 2 of the present invention 2000 -Absorption and emission spectra of GE11 nanoparticles in water.
[0020] Figure 7 Py1053 / Osi@DSPE-PEG in Application Example 2 of the present invention 2000 -Photothermal heating effect diagram of GE11 at different concentrations.
[0021] Figure 8 Py1053 / Osi@DSPE-PEG in Application Example 2 of the present invention 2000 -Photothermal stability effect diagram of GE11.
[0022] Fig. 9 Py1053 / Osi@DSPE-PEG in Application Example 3 of the present invention 2000 -GE11's (A) dark toxicity and (B) cell-killing effect under light conditions.
[0023] Fig.10 PCy / Osi@DSPE-PEG in Application Example 4 of the present invention 2000 -GE11 targeting effect on cell EGFR.
[0024] Fig.11 PCy / Osi@DSPE-PEG in Application Example 5 of the present invention 2000 -In vivo NIR-II fluorescence imaging of GE11.
[0025] Fig.12 PCy / Osi@DSPE-PEG in Application Example 6 of the present invention 2000 -In vivo photoacoustic imaging of GE11. DETAILED DESCRIPTION
[0026] Example 1 (1) Dissolve perylene monoimide (1.0 g, 2.08 mmol) in 200 mL of chloroform, then add 1.5 mL of liquid bromine, seal the reaction system to prevent leakage of liquid bromine, and place the reaction bottle in an ice-water bath for 3 hours. Add NaOH aqueous solution for extraction, separate the organic phase and the aqueous phase, and spin dry the organic phase to obtain an orange-red solid bromoperylene monoimide compound Py-1. The reaction yield is 99%.
[0027] (2) Py-1 (0.56 g, 1 mmol), tert-butyl carbazate (0.158 g, 1.2 mmol), sodium tert-butoxide (0.81 g, 4.1 mmol), tris(dibenzylideneacetone)dipalladium (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 completed, the mixture was cooled to room temperature and the organic solvent was removed by vacuum distillation to obtain a crude product as a purple solid. The crude product was separated and purified by chromatography to obtain a 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 mixture was reacted at room temperature for 2 hours. Saturated sodium bicarbonate aqueous solution was added to the reaction solution for extraction. After the organic phase was dried, 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, and then 4.4 mmol of 3-methyl-2-butanone was added, and then the temperature was slowly raised to 120°C for 12 hours. After the reaction was completed, the reaction solution was precipitated with saturated brine, filtered, and the precipitate was washed with deionized water, filtered and vacuum dried to obtain a red solid. The crude product was separated and purified by chromatography to obtain perylene monoimide and 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-propane sultone (0.913 g, 7.5 mmol) were added. The mixture was deoxygenated by freezing-evacuating-thawing with liquid nitrogen three times, and the mixture was reacted at 180 °C for 24 hours under nitrogen protection. The reaction solution was precipitated with methyl tert-butyl ether and the precipitate was washed. The crude product was separated and purified by chromatography to obtain the quaternary ammonium perylene monoimide indoline derivative Py-5 (0.47 g, 56%).
[0031] (6) Py-5 (0.133 g, 2 mmol) and 1-cyclohexene-1-formaldehyde (0.172 g, 1 mmol) 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), followed by the addition of sodium acetate (0.082 g, 1 mmol). 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
[0032] Py1053 (1 mg) and osimertinib (0.5 mg) were dissolved in DMF to prepare a mixed solution. 2000 -GE11 (5 mg) was dissolved in deionized water to prepare DSPE-PEG 2000 -GE11 aqueous solution; then, while ultrasonicating the DSPE-PEG2000-GE11 aqueous solution, the DMF solutions of Py1053 and osimertinib were quickly injected, and the mixed solution was ultrasonicated for 5 minutes, the DMF organic solvent was removed by dialysis, and the Py1053 / Osi@DSPE-PEG2000-GE11 nanoparticle aqueous solution was obtained by ultrafiltration and concentration.
[0033] Application Example 1 The dichloroethane solution of Py1053 was prepared, and the NIR-II dye IR26 (quantum efficiency in dichloroethane is 0.05%) was used as the standard. Using IR26 as the standard, the Py1053 / Osi@DSPE-PEG was calculated by the formula 2000 The fluorescence quantum yield of -GE11 is 1.7%.
[0034] Application Example 2 Preparation of different concentrations of Py1053 / Osi@DSPE-PEG 2000 -GE11 aqueous solution (1 mL) was illuminated using a 1064 nm laser (1.0 W / cm 2 ) for irradiation, and the temperature at different time points was recorded by an infrared camera. The photothermal calculation formula was used to calculate the temperature of Py1053 / Osi@DSPE-PEG 2000 -GE11 has a photothermal conversion efficiency of 40.3%.
[0035] Application Example 3 Py1053 / Osi@DSPE-PEG 2000-GE11 medium was co-cultured with NCl-H1975 cells for 24 hours. The medium was replaced with medium containing 10% CCK-8 and incubated for another 0.5 to 2 hours. Subsequently, the relative cell activity was measured using a microplate reader to test the Py1053 / Osi@DSPE-PEG 2000 Furthermore, the medium containing Pcy / Osi@GE11 was co-cultured with cells in a 96-well plate for 12 h, followed by irradiation with a 1064 nm laser (1.0 W / cm 2 ) irradiated for 10 minutes. The same CCK-8 assay was used to detect cell activity and test Py1053 / Osi@DSPE-PEG 2000 -GE11 cell killing effect. At the same time, calcein-propidium iodide cell death and viability staining was used to further verify Py1053 / Osi@DSPE-PEG 2000 -Cell-killing effect of GE11.
[0036] Application Example 4 NCl-H1975 cells were seeded into glass-bottom culture dishes and Py1053 / Osi@DSPE-PEG 2000 -GE11 nanoparticles were co-incubated for 12 hours. After incubation, the cells were fixed with 4% paraformaldehyde and blocked with 3% bovine serum albumin. The cells were then incubated with anti-EGFR monoclonal antibodies and imaged under a fluorescence microscope.
[0037] Application Example 5 Balb / c mice with orthotopic lung cancer were selected and injected with Py1053 / Osi@DSPE-PEG through the tail vein of the mice. 2000 -GE11 nanoparticles, with a dose of 100 μL / mouse (100 μM), were used to observe the brightness of the mouse lung tumor at different time points using a near-infrared zone II in vivo imager.
[0038] Application Example 6 Balb / c mice bearing NCl-H1975 cells were selected and injected with Py1053 / Osi@DSPE-PEG 2000 -GE11 nanoparticles, with a dose of 100 μL / mouse (100 μM), were used to observe the brightness of the mouse tumor location at different time points using an in vivo photoacoustic imager.
Claims
1. A perylene monoimide-based NIR-II fluorescent dye, characterized in that: The perylene monoimide-based NIR-II fluorescent dye consists of two parts: perylene monoimide and a polymethine skeleton, and its structural formula is as follows: 。 2. A method for preparing a perylene monoimide-based NIR-II fluorescent dye, characterized in that: The specific steps of the preparation method are: (1) dissolving a perylene monoimide compound and liquid bromine in a molar ratio of 1:1-2 in chloroform, and reacting them in a sealed container under ice bath conditions for 2-5 hours; extracting with an aqueous solution of NaOH, and drying the organic phase to obtain a bromoperylene monoimide Py-1; (2) Py-1, tert-butyl carbazate, sodium tert-butoxide, tri(dibenzylideneacetone)dipalladium and 2,2'-bis(diphenylphosphine)-1,1'-binaphthyl in a molar ratio of 1:1-1.5:4-4.5:0.1-0.2:0.2-0.4 are dissolved in anhydrous tetrahydrofuran, and reacted at 70-90°C for 10-20 hours under nitrogen protection; after the reaction is completed, the reaction is cooled to room temperature, and the organic solvent is removed by vacuum distillation. The crude product is separated and purified by a chromatographic column to obtain a Boc-protected perylene monoimide derivative Py-2; (3) dissolving Py-2 and trifluoroacetic acid in anhydrous dichloromethane at a molar ratio of 1:6-10, and reacting at room temperature for 1-3 hours; adding saturated sodium bicarbonate aqueous solution to the reaction solution for extraction, and separating the organic phase by column chromatography to obtain hydrazone-substituted perylene monoimide Py-3; (4) dissolving Py-3 and 3-methyl-2-butanone in a molar ratio of 1:2-3 in glacial acetic acid, and reacting under reflux at 100-140° C. for 10-15 hours; precipitating the reaction solution with saturated saline, filtering, washing the precipitate with deionized water, and separating and purifying by column chromatography to obtain perylene monoimide and indoline Py-4; (5) Py-4 and 1,3-propane sultone in a molar ratio of 1:4-6 are dissolved in 1,2-dichloroethane, and deoxygenated by freezing-evacuating-thawing with liquid nitrogen for three times, reacting at 160-200° C. for 20-30 hours under nitrogen protection, precipitating and washing with methyl tert-butyl ether, and separating and purifying by column chromatography to obtain a quaternary ammonium perylene monoimide indoline derivative Py-5; (6) 1-cyclohexene-1-carboxaldehyde and Py-5 in a molar ratio of 1:2-2.5 are dissolved in a mixed solvent of acetic acid and acetic anhydride, and then sodium acetate is added. The mixture is reacted at 100-140°C for 12-24 hours under nitrogen protection, precipitated and washed with methyl tert-butyl ether, and separated and purified by column chromatography to obtain a perylene monoimide NIR-II fluorescent dye.
3. A method for preparing nanoparticles of perylene monoimide-based NIR-II fluorescent dye, characterized in that: The specific operation of the preparation method is: dissolving the perylene monoimide NIR-II fluorescent dye and the anticancer drug in DMF to obtain a mixed solution; dissolving the amphiphilic polymer in deionized water, adding the mixed solution under ultrasonic conditions, and continuing ultrasonication for 3-10 minutes; dialysis to remove the organic solvent, and ultrafiltration concentration to obtain nanoparticles.
4. The preparation method according to claim 3, characterized in that: The anticancer drug is osimertinib.
5. The preparation method according to claim 3, characterized in that: The amphiphilic polymer is phospholipid-polyethylene glycol-epidermal growth factor peptide.
6. Use of the nanoparticles of perylene monoimide-based NIR-II fluorescent dye prepared by the method according to any one of claims 3 to 5 as near-infrared II region fluorescent imaging contrast agents.
7. Use of nanoparticles of perylene monoimide-based NIR-II fluorescent dye prepared by the method according to any one of claims 3 to 5 in the preparation of cancer photothermal therapeutic agents.