Near-infrared second-region small organic molecule fluorescent probe with photoacoustic effect as well as preparation method and application of near-infrared second-region small organic molecule fluorescent probe
By designing and synthesizing near-infrared II organic small molecule fluorescent probes with photoacoustic effects, the problems of low imaging sensitivity and insufficient resolution in existing technologies have been solved, realizing efficient photoacoustic and fluorescence imaging, especially for the early diagnosis of osteosarcoma.
Patent Information
- Application Number
- CN202511224980.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-29
- Publication Date
- 2025-12-12
AI Technical Summary
Existing clinical imaging techniques have low imaging sensitivity and potential hazards in the diagnosis of cancer, especially osteosarcoma. Near-infrared dual-zone fluorescence imaging has low spatial resolution in deep tissue imaging, and there is a lack of high-performance near-infrared II fluorophores suitable for photoacoustic imaging.
A class of near-infrared II organic small molecule fluorescent probes with photoacoustic effects were designed and synthesized. Trimeric structures were generated through Schiff base reaction to prepare near-infrared II organic small molecule fluorescent probe compounds, which were then encapsulated in nanoparticles for photoacoustic and fluorescence imaging.
It achieves high-resolution deep tissue imaging, has good biocompatibility and optical properties, and exhibits excellent photoacoustic and fluorescence imaging effects. It is suitable for the early diagnosis of cancers such as osteosarcoma. The synthesis route is simple and free of heavy metal catalysis.
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Figure CN121108157A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fluorescent dye preparation technology, specifically relating to a class of near-infrared II organic small molecule fluorescent probes with photoacoustic effects, their preparation methods, and their applications in biomedical photoacoustic / fluorescence imaging. Background Technology
[0002] Cancer (also known as malignant tumors) seriously threatens human health. Osteosarcoma is a malignant bone tumor that mostly occurs in children and adolescents aged 10-25. If early diagnosis is not timely, patients will face the risk of amputation or even death when the disease is in its middle or late stages or when the tumor has metastasized.
[0003] Existing clinical imaging technologies suffer from low imaging sensitivity and potential harm to the human body. Currently, near-infrared dual-zone fluorescence imaging has rapidly become a research hotspot in the diagnosis and treatment of diseases such as cancer due to its non-invasive, non-surgical, and highly sensitive advantages. However, due to the scattering of excitation and emission light, fluorescence imaging has low spatial resolution in deep tissue imaging. To overcome this limitation, a novel imaging technology—photoacoustic imaging—has emerged. Photoacoustic imaging combines the advantages of optical and acoustic imaging, offering better penetration than pure optical imaging and higher resolution than traditional ultrasound imaging. Its image resolution can reach the micrometer scale, enabling high-resolution deep tissue imaging. However, the application of near-infrared II fluorophores in fluorescence and photoacoustic imaging of tumors such as osteosarcoma remains limited. Therefore, the design and synthesis of high-performance NIR-II small molecule fluorophores is essential. Summary of the Invention
[0004] To address the shortcomings of existing technologies, the present invention aims to provide a novel near-infrared II organic small molecule fluorescent dye probe with photoacoustic imaging effects, high photostability, and good biocompatibility, and to use it as a photoacoustic and fluorescence imaging contrast agent for tumors to achieve early diagnosis of cancers such as osteosarcoma.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: Firstly, a class of near-infrared II region organic small molecule fluorescent probe compounds with photoacoustic effects is provided, with the general structural formula shown in formula (1) below: (1) in: Group R is selected from , , , , , One or more of the following; Group R1 is Or H, where n1 is an integer from 0 to 3, and the group R2 is selected from... , , , One of them; Groups R3 and R4 are Or H, and R3 and R4 are not both H, n2 is 0 or 1, n3 is an integer from 1 to 6, and X is element C or N; Group R5 is Or H, where n2 is 0 or 1, and n3 is an integer from 1 to 6.
[0007] Furthermore, the fluorescence emission wavelength of the compound is 1000–1200 nm, and the wavelength corresponding to the maximum photoacoustic intensity is 600–900 nm.
[0008] Secondly, a method for preparing the aforementioned near-infrared II fluorescent organic small molecules with photoacoustic effects is provided, comprising the following steps: 1) The mixture of compound 8 and hexanone octahydrate cyclohexane was heated and stirred in an organic solvent until completely dissolved, cooled to room temperature, and the solvent was removed under reduced pressure; 2) The residue was washed with methanol, purified by column chromatography, and all green components were collected and dried under vacuum. 3) The resulting mixture was dissolved in THF (tetrahydrofuran), and excess chloroaniline was added to the solution. The mixture was stirred at room temperature for 30-90 minutes, filtered, and the filtrate was collected and dried under vacuum. 4) The crude product was purified by column chromatography to obtain an organic fluorescent small molecule compound (1); The structural formula of compound 8 is ; The reaction pathway described above is as follows: , in: Group R is selected from , , , , , One or more of the following; Group R1 is Or H, where n1 is an integer from 0 to 3, and the group R2 is selected from... , , , One of them; Groups R3 and R4 are Or H, and R3 and R4 are not both H, n2 is 0 or 1, n3 is an integer from 1 to 6, and X is element C or N; Group R5 is Or H, where n2 is 0 or 1, and n3 is an integer from 1 to 6.
[0009] Furthermore, the organic solvent is selected from any one or more mixed solvents selected from formic acid, acetic acid, propionic acid, ethyl acetate, acetone, methyl ethyl ketone, ethanol, isopropanol, and dichloromethane.
[0010] Furthermore, the solvent used for column chromatography in steps 2) and 4) is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 10:1.
[0011] Further, in step 1), the molar ratio of compound 8 to hexanone cyclohexane is 3-3.5:1, for example 3:1, 3.1:1, 3.2:1, 3.3:1, 3.4:1, 3.5:1.
[0012] Further, in step 1), the stirring temperature is 70~90℃, for example, 70℃, 73℃, 76℃, 80℃, 83℃, 87℃, 90℃; the stirring time is 4~8 h, for example, 4h, 5h, 6h, 7h, 8h.
[0013] Furthermore, in step 3), the mass of the added chloroaniline is not less than 0.5 times the mass of compound 8 in step 1), for example, 0.8 times, 0.9 times, 1 time, 1.1 times, or 1.2 times the mass of compound 8.
[0014] Thirdly, a fluorescent probe nanoparticle is provided, comprising the aforementioned near-infrared II region organic small molecule fluorescent probe compound, and an encapsulation matrix encapsulating the near-infrared II region organic small molecule fluorescent probe compound, wherein the encapsulation matrix is selected from any one of polyethylene glycol and its derivatives, polyether F127, polypeptides, nucleic acid aptamers, folic acid, proteins, and cell membranes.
[0015] Furthermore, the particle size range of the nanoparticles is 90~400nm.
[0016] Furthermore, the encapsulating matrix is selected from polyethylene glycol derivatives, such as DSPE-mPEG. 3.4k .
[0017] Fourthly, a method for preparing the aforementioned fluorescent probe nanoparticles is provided, comprising the following steps: 1) Mix and dissolve the above-mentioned near-infrared II organic small molecule fluorescent probe compound (Formula 1) with the encapsulation matrix and organic solvent, wherein the ratio of the near-infrared II organic small molecule fluorescent probe compound, the encapsulation matrix and the organic solvent is 1 mg: 1~10 mg: 1~10 mL; 2) Add the obtained mixed solution to deionized water, disperse it in time, and control the temperature of the deionized water at 0-5℃. After the mixed solution is completely turned into a blue transparent solution, remove the organic solvent in the solution under an inert gas flow environment. 3) The obtained solution is subjected to at least one ultrafiltration centrifugation operation to retain compounds with a molecular weight of 30 kDa or less, thereby obtaining the target nanoparticles.
[0018] Furthermore, the organic solvent is selected from any one or more mixed solvents selected from formic acid, acetic acid, propionic acid, ethyl acetate, acetone, methyl ethyl ketone, ethanol, isopropanol, and dichloromethane.
[0019] Furthermore, the ratio of the near-infrared II region organic small molecule fluorescent probe compound, the encapsulating matrix, and the organic solvent is 1 mg: 5 mg: 1 mL.
[0020] Fifthly, the application of the aforementioned near-infrared II fluorescent imaging probe compounds and / or fluorescent probe nanoparticles is provided for the preparation of near-infrared II imaging contrast agents for tumors; especially photoacoustic imaging contrast agents for osteosarcoma.
[0021] Sixthly, the application of the aforementioned near-infrared II fluorescence imaging probe compounds and / or fluorescence probe nanoparticles is provided for the preparation of photoacoustic therapy drugs for osteosarcoma.
[0022] The technical advantages of this invention compared to existing technologies are as follows: 1. This invention uses the Schiff base formation reaction of amino and ketone to polymerize three traditional benzothiadiazole-based near-infrared II organic molecules to synthesize new near-infrared II organic molecules. The proposed trimer structure is a new design concept for NIR-II organic small molecule structures. In the future, this method can be used to design and synthesize more superior NIR-II small molecules. 2. The NIR-II fluorescent small molecule designed and synthesized in this invention as described in general formula (1) has good biocompatibility, optical properties, photoacoustic properties and photothermal properties, is non-toxic, has good biocompatibility, is easily absorbed and metabolized by organisms, and realizes dual-modal imaging of fluorescence and photoacoustic in osteosarcoma mice. 3. The synthesis route of this invention is simple, there are no hazardous chemical raw materials, and no heavy metal catalysis is used, so it has high industrial application prospects; 4. In biomedical imaging experiments, the probe was found to have excellent photoacoustic imaging and fluorescence effects, good specificity, and broad application prospects. Attached Figure Description
[0023] Appendix Figure 1 This is the synthetic route for the near-infrared II organic small molecule compound 1a with photoacoustic effects in Example 1.
[0024] Appendix Figure 2Nuclear magnetic resonance (NMR) of compound 1a obtained in Example 1 1 H spectrum.
[0025] Appendix Figure 3 The compound 1a obtained in Example 1 13 C-spectrum.
[0026] Appendix Figure 4 This is a schematic diagram of the preparation method of the fluorescent probe in Example 2.
[0027] Appendix Figure 5 The absorption spectrum, emission spectrum, and photoacoustic signal intensity spectrum of the fluorescent probe 1a dots obtained in Example 2 are shown, where a) is the absorption spectrum of the fluorescent probe 1a dots; b) is the emission spectrum of the fluorescent probe 1a dots; and c) is the photoacoustic signal intensity spectrum of the fluorescent probe 1a dots.
[0028] Appendix Figure 6 The diagram shows the test results of the photoacoustic signal penetration ability of the fluorescent probe 1a dots obtained in Example 2 to biological tissue, where a) is the result of photoacoustic intensity attenuation with penetration depth, and b) is the experimental setup diagram.
[0029] Appendix Figure 7 The image shows the DLS particle size distribution of the fluorescent probe 1a dots nanoparticles obtained in Example 2.
[0030] Appendix Figure 8 This is a comparison of the photostability of the fluorescent probe 1a dots and ICG in water as described in Example 3.
[0031] Appendix Figure 9 This is a comparison of the photoacoustic intensity of the fluorescent probe 1a dots and ICG in water as described in Example 3.
[0032] Appendix Figure 10 This is a near-infrared 2D fluorescence imaging image of the fluorescent probe 1a dots obtained in Example 2 on an orthotopic osteosarcoma mouse model.
[0033] Appendix Figure 11 This is a live photoacoustic image of the fluorescent probe 1a dots in an orthotopic osteosarcoma mouse model as described in Example 2. Detailed Implementation
[0034] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention. The specific structures of compounds such as 8a involved in the embodiments are shown in the appendix. Figure 1 The reaction formula in the text.
[0035] Example 1: Preparation of compound 1a As attached Figure 1The reaction scheme shown involved heating compound 8a (0.33 g, 0.331 mmol) with hexane octahydrate (0.035 g, 0.111 mmol) in acetic acid (10 mL) to 80°C and stirring for 6 h. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was washed with methanol and further purified by column chromatography on silica gel using a solvent mixture (petroleum ether:ethyl acetate, v / v). All green fractions were collected and dried under vacuum. The mixture was dissolved in THF (15 mL), and excess chloroaniline (0.319 g, 1.297 mmol) was added to the solution. The mixture was stirred at room temperature for 1 h, and excess chloroaniline was removed by filtration. The filtrate was collected and dried under vacuum. Purification was then performed by column chromatography on silica gel using a solvent mixture (petroleum ether:ethyl acetate = 10:1 v / v). The product was a dark green solid (26.9 mg, 8% yield, two steps).
[0036] The structural results of compound 1a are as follows: 1 H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 8.3 Hz, 9H), 8.01 (d, J = 8.3 Hz,9H), 7.22 – 6.95 (m, 54H), 6.80 (d, J = 8.3 Hz, 12H), 4.22 – 4.11 (m, 12H), 2.93 – 2.76 (m, 12H), 2.62 – 2.47 (m, 12H), 1.03 – 0.93 (m, 14H), 0.03 (s, 54H). 13 C NMR (101 MHz, CDCl3) δ 173.1, 154.1, 147.7, 147.1, 147.1, 144.3,138.2, 136.2, 134.7, 130.9–130.1, 130.1–128.6, 127.6, 126.3, 125.9, 123.7,120.1, 62.7, 36.1, 29.7, 17.4, 1.1. MS (MALDI-TOF) m / z calcd for C 180 H 180 N 18 O 12 S3Si6, 3049.2, found, 3050.8. Example 2: Compound 1a was prepared as near-infrared II fluorescent probes 1a dots, and its photoacoustic properties were tested. In this embodiment, compound 1a was prepared into near-infrared II fluorescent probe nanoparticles (1a dots) with a concentration of 1 mg / mL using a nanoprecipitation method for near-infrared II fluorescence imaging studies of 143B tumors. The specific preparation steps are as follows: First, weigh 1 mg of compound 1a and 5 mg of encapsulation matrix DSPE-mPEG. 3.4k The two were mixed and added to 1 mL of tetrahydrofuran, and dissolved completely under sonication. Then, 10 mL of deionized water was placed in an ice-water bath (temperature controlled at 0-5℃). Under strong sonication, the tetrahydrofuran solution was slowly added dropwise to the deionized water using a cell disruptor. After the mixture completely turned into a blue transparent solution, the organic solvent was removed from the solution under an inert gas flow environment.
[0037] Next, using 50 mL ultrafiltration centrifuge tubes with a molecular weight cutoff of 30 kDa, ultrafiltration centrifugation was performed five times in a refrigerated centrifuge to remove excess DSPE-mPEG. 3.4k The target probe, 1a dots nanoparticles, was ultimately obtained. The specific synthesis scheme is attached. Figure 4 .
[0038] Dynamic light scattering (DLS) measurements showed that the average particle size of the probe 1a dots nanoparticles was approximately 120 nm (e.g., ...). Figure 7 As shown in the figure, its maximum absorption peak is located at 864 nm. When the excitation wavelength is 800 nm, its maximum emission peak is located at 1192 nm, as shown in the figure. Figure 5 As shown. Take a 0.5 mg / mL 1a dots aqueous solution, purchase fresh chicken breast, and cut the chicken breast into pieces with a cross-sectional area of 1 cm². 2 Multiple small pieces, each 1 mm thick, were stacked sequentially with varying numbers of small chicken pieces. Each time, the bottom layer of chicken pieces was injected with the same concentration and volume of 1a dots aqueous solution. The photoacoustic signal within the capillary tube was detected using a photoacoustic imager, and a graph showing the change in photoacoustic signal intensity as a function of imaging depth was plotted. The results are as follows: Figure 6 As shown.
[0039] Example 3: Comparison of photostability between near-infrared II probe 1a dots and ICG Equal volumes of the probe 1a dots prepared in Example 2 and an aqueous solution of ICG were taken, and their initial fluorescence intensities were adjusted to be consistent. The samples were continuously irradiated with an 808 nm laser for 1 hour using a near-infrared II imager. Fluorescence images were captured and saved at 5, 10, 20, 30, 40, and 60 minutes after irradiation. Fluorescence intensity change curves were plotted based on the experimental data (see Appendix). Figure 8 The results showed that after one hour of continuous irradiation, the fluorescence signal intensity of probe 1a dots decreased by less than 10%, while the fluorescence signal of ICG decreased significantly, with a decrease of more than 65% after one hour. This indicates that the photostability of probe 1a dots is significantly better than that of ICG.
[0040] Example 4: Comparison of near-infrared II probe 1a dots and ICG photoacoustic intensity Probe 1adots and ICG aqueous solutions at concentrations of 20 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 500 μg / mL were prepared, and the photoacoustic intensity of each concentration sample at 850 nm was measured. Based on the experimental results, photoacoustic intensity curves of 1adots and ICG aqueous solutions at different concentrations were plotted (see Appendix). Figure 9 The results showed that the photoacoustic intensity of 1a dots was significantly higher than that of ICG at all tested concentrations. This indicates that the 1a dots probe has a significant advantage in enhancing photoacoustic performance.
[0041] Example 5: Near-infrared II in vivo imaging of mice with orthotopic osteosarcoma using fluorescent probe 1a dots Mice with the established orthotopic osteosarcoma model were injected via tail vein with a 1 mg / mL solution of probe 1a dots, and in vivo imaging data were collected at different time points to obtain fluorescence signal maps (see appendix). Figure 10 Experimental results showed that the fluorescence signal at the tumor site in mice gradually increased, reaching a maximum peak approximately 60 hours after injection, and then gradually decreased due to in vivo metabolism. This experiment demonstrates that the 1a dots probe can target tumor sites using the EPR effect, achieving efficient and dynamic near-infrared II fluorescence imaging.
[0042] Example 6: Photoacoustic in vivo imaging of mice with orthotopic osteosarcoma using fluorescent probe 1a dots Mice with established model were injected intravenously with 1 mg / mL of 1a dots via the tail vein. Photoacoustic signal maps were obtained by collecting imaging data at different time points (see attached image). Figure 11The study found that the photoacoustic signal intensity gradually increased from 0 to 36 hours, reaching its highest value at 36 hours. Subsequently, the signal intensity began to decrease due to metabolism in the body, indicating that 1a dots successfully achieved photoacoustic in vivo imaging of mice with osteosarcoma in situ.
[0043] The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.
Claims
1. A class of near-infrared II region organic small molecule fluorescent probe compounds with photoacoustic effects, characterized in that, The general structural formula is shown in equation (1) below: (1) in: Group R is selected from , , , , , One or more of the following; Group R1 is Or H, where n1 is an integer from 0 to 3, and the group R2 is selected from... , , , One of them; Groups R3 and R4 are Or H, and R3 and R4 are not both H, n2 is 0 or 1, n3 is an integer from 1 to 6, and X is element C or N; Group R5 is Or H, where n2 is 0 or 1, and n3 is an integer from 1 to 6.
2. The organic small molecule fluorescent probe compound according to claim 1, characterized in that, The compound has a fluorescence emission wavelength of 1000–1200 nm and a maximum photoacoustic intensity corresponding to a wavelength of 600–900 nm.
3. A method for preparing a near-infrared II region organic small molecule fluorescent probe compound with photoacoustic effects as described in any one of claims 1 to 2, characterized in that, Includes the following steps: 1) The structural formula is The mixture of the compound and hexanone cyclohexane was heated in an organic solvent to 70-90°C and stirred continuously for 4-8 h, then cooled to room temperature and the solvent was removed under reduced pressure. 2) The residue was washed with methanol, purified by column chromatography, and all green components were collected and dried under vacuum. 3) The resulting mixture was dissolved in THF, and excess chloroaniline was added to the solution. The mixture was stirred at room temperature for 30-90 minutes, filtered, and the filtrate was collected and dried under vacuum. 4) The crude product was purified by column chromatography to obtain the organic small molecule fluorescent probe compound; in: Group R is selected from , , , , , One or more of the following; Group R1 is Or H, where n1 is an integer from 0 to 3, and the group R2 is selected from... , , , One of them; Groups R3 and R4 are Or H, and R3 and R4 are not both H, n2 is 0 or 1, n3 is an integer from 1 to 6, and X is element C or N; Group R5 is Or H, where n2 is 0 or 1, and n3 is an integer from 1 to 6.
4. The preparation method according to claim 3, characterized in that, The organic solvent is selected from any one or a mixture of formic acid, acetic acid, propionic acid, ethyl acetate, acetone, methyl ethyl ketone, ethanol, isopropanol, and dichloromethane.
5. The preparation method according to claim 3, characterized in that, The solvent used for column chromatography in steps 2) and 4) is a mixture of petroleum ether and ethyl acetate in a volume ratio of 10:
1.
6. A fluorescent probe nanoparticle, characterized in that, The invention includes the near-infrared II organic small molecule fluorescent probe compound with photoacoustic effect as described in any one of claims 1 to 2, or the near-infrared II organic small molecule fluorescent probe compound with photoacoustic effect obtained by the preparation method described in any one of claims 3 to 5, and an encapsulation matrix for encapsulating the near-infrared II organic small molecule fluorescent probe compound with photoacoustic effect, wherein the encapsulation matrix is selected from polyethylene glycol and its derivatives, polyether F127, polypeptides, nucleic acid aptamers, folic acid, proteins, and cell membranes.
7. The fluorescent probe nanoparticles according to claim 6, characterized in that, The particle size range of the nanoparticles is 90~400nm.
8. A method for preparing fluorescent probe nanoparticles according to any one of claims 6-7, characterized in that, Includes the following steps: 1) The near-infrared II organic small molecule fluorescent probe compound with photoacoustic effect as described in any one of claims 1 to 2, or the near-infrared II organic small molecule fluorescent probe compound with photoacoustic effect obtained by the preparation method described in any one of claims 3 to 5, is mixed and dissolved with an encapsulation matrix and an organic solvent, wherein the ratio of the near-infrared II organic small molecule fluorescent probe compound with photoacoustic effect, the encapsulation matrix, and the organic solvent is 1 mg: 1 to 10 mg: 1 to 10 mL; 2) Add the obtained mixed solution to deionized water, disperse it, and control the temperature of the deionized water at 0-5℃. After the mixed solution is completely turned into a blue transparent solution, remove the organic solvent in the solution under an inert gas flow environment. 3) The obtained solution is subjected to at least one ultrafiltration centrifugation operation to retain compounds with a molecular weight of 30 kDa or less, thereby obtaining the target nanoparticles.
9. The application of a near-infrared II fluorescence imaging probe compound according to any one of claims 1-2 or a fluorescent probe nanoparticle according to any one of claims 6-7, characterized in that, Used to prepare contrast agents for near-infrared II imaging of tumors.
10. The application of a near-infrared II fluorescence imaging probe compound according to any one of claims 1-2 or a fluorescent probe nanoparticle according to any one of claims 6-7, characterized in that, Used to prepare photoacoustic therapy drugs for osteosarcoma.