Laser-activated near-infrared two-region chemiluminescent molecule and preparation method thereof

Abstract

The invention relates to the technical field of chemiluminescent probes, and discloses a laser-activated near-infrared two-region chemiluminescent molecule and a preparation method thereof, and the molecular structural formula of the laser-activated near-infrared two-region chemiluminescent molecule is shown in the specification. The near-infrared two-region chemiluminescent molecule provided by the invention not only has a high signal-to-noise ratio (SBR) of near-infrared two-region chemiluminescence, but also responds to beta-galactase in a tumor microenvironment, subsequently realizes photoacoustic activated deep tissue imaging through laser irradiation, and selectively opens a signal in a specific tumor focus to achieve a treatment effect on tumors.

Description

Technical Field

[0001] This invention relates to the field of chemiluminescence probe technology, specifically to a laser-activated near-infrared II chemiluminescent molecule and its preparation method. Background Technology

[0002] Chemiluminescence is a phenomenon in which excited states are generated and photons are emitted through chemical reactions. Because it does not require external light source excitation and can effectively reduce autofluorescence and scattering interference, it has broad application prospects in the biomedical field. However, traditional chemiluminescent probes mainly emit light in the visible light region of 400 to 650 nanometers, which limits their resolution and penetration depth in biological organisms.

[0003] To overcome these limitations, chemiluminescent probes in the near-infrared (NIR) window, especially the second near-infrared window (NIR-II, 900 to 1700 nm), have become a research hotspot. Imaging in the NIR-II region offers advantages such as lower photon absorption and scattering, higher spatial resolution, and deeper tissue penetration, making it suitable for long-term in vivo imaging.

[0004] Activable optical probes (AOPs) can trigger signal changes in response to specific biomarkers (such as reactive oxygen species, nitrogen species, pH, and enzymes), typically exhibiting a higher signal-to-noise ratio (SBR) than normally-on probes. Current AOP design methods primarily rely on short-range energy or charge transfer mechanisms, with signal output dependent on fluorescence, inevitably subject to interference from tissue autofluorescence. In recent years, afterglow imaging technology has emerged as a promising imaging modality due to its ability to emit long-lived luminescent signals after photoexcitation ceases, minimizing tissue autofluorescence and increasing tissue penetration depth. A common afterglow imaging method involves generating singlet oxygen (a singlet oxygen donor) under laser irradiation using a photosensitizer. 1 O2), generated 1 O2 and afterglow substrate ( 1 The O2 receptor reacts to form a high-energy intermediate, which then slowly degrades and emits a residual signal.

[0005] Currently, several problems urgently need to be addressed in the field of medical research: First, imaging resolution and signal-to-noise ratio are limited by autofluorescence background; second, photon scattering still exists in deep tissues, causing imaging resolution to decrease significantly with increasing tissue depth; furthermore, third, insufficient probe brightness or limited injection dose may result in a low signal-to-noise ratio, affecting the clear identification of small lesions. Currently, chemiluminescent probes are mainly in the NIR-I region, with limited research in the NIR-II region, highlighting the urgent need to develop NIR-II CL probes with high biocompatibility and strong signal-to-noise ratio. Summary of the Invention

[0006] To address the aforementioned technical problems, the main objective of this invention is to provide a laser-activated near-infrared II chemiluminescent molecule and its preparation method.

[0007] To achieve the above objectives, on the one hand, the present invention provides a laser-activated near-infrared II chemiluminescent molecule, the molecular structure of which is as follows: .

[0008] According to a second aspect of the present invention, the present invention also provides a method for preparing laser-activated near-infrared II chemiluminescent molecules, comprising the following steps: S1. 2-Bromo-5-hydroxybenzaldehyde, trimethyl orthoformate and tetrabutylammonium bromide were dissolved in anhydrous methanol and reacted at room temperature. After the reaction was completed, the mixture was purified to obtain compound 1.

[0009] S2. Under an inert atmosphere, compound 1, imidazole and tert-butyldimethylchlorosilane were dissolved in anhydrous dichloromethane and reacted at room temperature. After the reaction was completed, compound 2 was obtained by purification.

[0010] S3. Compound 2 and trimethyl phosphite were dissolved in anhydrous dichloromethane and reacted under anhydrous and oxygen-free conditions at -10~0 °C. Then titanium tetrachloride was added dropwise to continue the reaction. After the reaction was completed, the reaction was quenched with saturated sodium bicarbonate solution and then purified to obtain compound 3.

[0011] S4. Compound 3 was dissolved in anhydrous THF, and diisopropylaminolithium was added dropwise under anhydrous and oxygen-free conditions at -70~80 °C to carry out the reaction. Then 2-adamantanone was added and reacted at room temperature. After the reaction was completed, the mixture was purified to obtain compound 4.

[0012] S5. Compound 4 was dissolved in anhydrous THF, and n-butyllithium was added dropwise under anhydrous and oxygen-free conditions at -70~80 ℃ to carry out the reaction. Then DMF was added to react at room temperature. After the reaction was completed, the reaction was quenched with saturated ammonium chloride solution, and then purified to obtain compound 5.

[0013] S6. Compound 5, imidazole and tert-butyldimethylchlorosilane were dissolved in anhydrous dichloromethane and reacted at room temperature. After the reaction was completed, the mixture was purified to obtain compound 6.

[0014] S7. Compound 5-1 was dissolved in anhydrous THF, and n-butyllithium was added dropwise under anhydrous and oxygen-free conditions at -70~80 °C to carry out the reaction. Then, compound 6 was added and reacted at room temperature. After the reaction was completed, the reaction was quenched with saturated ammonium chloride solution, and then purified to obtain compound 7.

[0015] S8. Compound 7 was dissolved in anhydrous dichloromethane. Dibutylaluminum hydroxide was added dropwise at -70~80 ℃ under anhydrous and oxygen-free conditions for 10~60 minutes. The reaction was then moved to room temperature and the reaction was continued. After the reaction was completed, the mixture was quenched with ice water and purified to obtain compound 8.

[0016] S9. Compound 7 and MnO2 were dispersed in ethyl acetate and reacted at 60-90 °C. After the reaction was completed, the mixture was filtered and evaporated under reduced pressure to obtain compound 9.

[0017] S10. Compound 9 was dissolved in dichloromethane, and tetrabutylammonium fluoride was added at room temperature to carry out the reaction. After the reaction was completed, the mixture was purified to obtain compound 10.

[0018] S11. Compound 10 was dissolved in anhydrous acetonitrile, and CsCO3 was added at -10~0 °C to react. Then, compound 11-1 and Na2SO4 were added and reacted for 1~5 minutes. The reaction was then moved to room temperature and the reaction continued. After the reaction was completed, compound 11 was obtained by purification.

[0019] S12. Under an inert atmosphere, compound 11 and rhodamine salt were dissolved in ethanol and refluxed at 60-90 °C. After the reaction was completed, compound 12 was obtained by purification.

[0020] S13. Compound 12 is dissolved in methanol, sodium methoxide is added and reacted at room temperature. After the reaction is completed, compound 13 is obtained by purification, which is the laser-activated near-infrared II chemiluminescent molecule. .

[0021] As a further preferred embodiment of the present invention, in step S1, the feed ratio of 2-bromo-5-hydroxybenzaldehyde, trimethyl orthoformate, and tetrabutylammonium bromide is 1 eq: 1.1-2 eq: 0.01 eq; in step S2, the feed ratio of compound 1, imidazole, and tert-butyldimethylchlorosilane is 1 eq: 3 eq: 2 eq; in step S3, the feed ratio of compound 2, trimethyl phosphite, and titanium tetrachloride is 1 eq: 1.5 eq: 1.5 eq; in step S4, the feed ratio of compound 3, 2-adamantanone, and lithium diisopropylaminodimethylamine is 1 eq: 1.5 eq: 1.5-2 eq; in step S5, the feed ratio of compound 4, DMF, and n-butyllithium is 1 eq: 2 eq: 3.5-4 eq. In step S6, the feed ratio of compound 5, imidazole, and tert-butyldimethylchlorosilane is 1 eq: 1.5-2 eq: 1.2-1.3 eq; In step S7, the feed ratio of compound 5-1, compound 6, and n-butyllithium is 1 eq: 0.5 eq: 1-1.5 eq; In step S8, the feed ratio of compound 7 and dibutylaluminum hydroxide is 1 eq: 3.2-4 eq; Raw materials: In step S9, the feed ratio of compound 8 and manganese dioxide (MnO2) is 1 eq: 5-6 eq; In step S10, the feed ratio of compound 9 and tetrabutylammonium fluoride is 1 eq: 1.5-2 eq; In step S11, the feed ratio of compound 10, cesium carbonate (CsCO3), compound 11-1, and anhydrous sodium sulfate (Na2SO4) is 1 eq: 2 eq: 2 eq: 2eq; In step S13, the feed ratio of compound 12 and rhodamine salt is 1 eq: 0.6-0.7 eq; In step S13, the feed ratio of compound 12 and sodium methoxide is 1 eq: 5 eq.

[0022] According to a third aspect of the present invention, the present invention also provides a near-infrared II chemiluminescent lipid platform, which employs the above-described laser-activated near-infrared II chemiluminescent molecules.

[0023] As a further preferred technical solution of the present invention, the near-infrared II chemiluminescent lipid platform is obtained by encapsulating the near-infrared II chemiluminescent molecules and the photosensitizer protoporphyrin PPIX in a set ratio through liposomes.

[0024] A near-infrared II chemiluminescent lipid platform, characterized in that it employs the laser-activated near-infrared II chemiluminescent molecule as described in claim 1.

[0025] As a further preferred embodiment of the present invention, the preparation method of the near-infrared II chemiluminescent lipid platform includes: Weigh out 1,2-bis-(9Z-octadecenoyl)- snGlyceryl-3-phosphate choline, cholesterol, and DSPE-PEG-2000 were dissolved in ethanol at a ratio of 6.8:3.8:0.7. Then, 0.1666 mg of compound 13 and 0.0096 mg of protoporphyrin PpIX were weighed and dissolved in ethanol. After mixing, the mixture was mixed with sodium citrate buffer solution by ethanol injection. After standing overnight at 0-4°C, the mixture was centrifuged at 2000-3000 rpm for 5-10 min using a 20-30 KD ultrafiltration tube. After removing the ethanol, the mixture was stored at 4°C protected from light.

[0026] Compared with the prior art, the present invention can achieve the following beneficial effects: The near-infrared II chemiluminescent molecule provided by this invention generates singlet oxygen under laser irradiation, oxidizing the double bond into an epoxy group, and then emits near-infrared II chemiluminescence through spontaneous decomposition. Compared with fluorescence, it is free from background signal interference and has ultra-high resolution and signal-to-noise ratio.

[0027] The laser-activated near-infrared II chemiluminescent lipid platform provided by this invention comprises protoporphyrin IX (PPIX) as the photosensitizer moiety, a β-galactosidase responsive group, and a phenoxy-dioxane precursor containing rhodamine dye as the photoresponsive moiety. Under laser irradiation, singlet oxygen (… 1 O2 acts as an "intermediary" for the enol ether bond in the photoresponsive part, and then emits near-infrared II light through spontaneous decomposition. Near-infrared II chemiluminescent molecules not only possess the high signal-to-noise ratio (SBR) of near-infrared II chemiluminescence, but also respond to β-galactosidase in the tumor microenvironment. Subsequent laser irradiation enables photoacoustic-activated deep tissue imaging, selectively activating signals in specific tumor lesions, and exhibiting a significant killing effect on ovarian cancer cells. Attached Figure Description

[0028] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0029] Figure 1 This is a circuit diagram of the laser-activated near-infrared II chemiluminescent molecule synthesis in Example 1.

[0030] Figure 2 The NMR spectrum of compound 5-1 is shown.

[0031] Figure 3 This is the NMR spectrum of compound 6.

[0032] Figure 4 This is the NMR spectrum of compound 7.

[0033] Figure 5 This is the NMR spectrum of compound 8.

[0034] Figure 6This is the NMR spectrum of compound 9.

[0035] Figure 7 This is the NMR spectrum of compound 10.

[0036] Figure 8 This is the NMR spectrum of compound 11.

[0037] Figure 9 This is the NMR spectrum of compound 12.

[0038] Figure 10 This is the NMR spectrum of compound 13.

[0039] Figure 11 (a) UV absorption spectrum and (b) TEM (transmission image) of the final product PLC-3-β-gal.

[0040] Figure 12 (a)TEM (close-up) and PLC-3 (enzyme-free response) of the final product PLC-3-β-gal with irradiation time of 660 nm laser (200 mW) 1 Diagram showing the generation of O2.

[0041] Figure 13 (a) cellular safety assessment of the final product PLC-3-β-gal and (b) assessment of the material's therapeutic efficacy under 660 nm laser irradiation.

[0042] Figure 14 Chemiluminescence imaging of the final product PLC-3-β-gal cells.

[0043] The objectives, features, and advantages of this invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0044] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0045] Unless otherwise defined, the technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which this invention pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; and the experimental methods described are conventional methods.

[0046] Example 1 like Figure 1 The synthetic circuit shown in this embodiment provides a method for preparing laser-activated near-infrared II chemiluminescent molecules, as detailed below: S1, Synthesis steps of compound 1 1. Dissolve 2-bromo-5-hydroxybenzaldehyde (10 g, 49.7 mmol), trimethyl orthoformate (5.8 g, 54.7 mmol), and tetrabutylammonium bromide (TBAB, 239.6 mg, 0.497 mmol) in anhydrous methanol (100 mL).

[0047] 2. Stir the reaction at room temperature for 12-14 hours under nitrogen protection.

[0048] 3. After the reaction is complete, concentrate under reduced pressure, wash with water and extract with ethyl acetate.

[0049] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 1.

[0050] S2, Synthesis steps of compound 2 1. Compound 1 (5 g, 20.2 mmol), imidazole (4.1 g, 60.7 mmol) and tert-butyldimethylchlorosilane (6.1 g, 40.4 mmol) were dissolved in anhydrous dichloromethane (50 mL).

[0051] 2. Stir the reaction at room temperature under nitrogen protection for 1-2 hours, and monitor the reaction progress by TLC.

[0052] 3. After the reaction is complete, wash with water and extract with dichloromethane.

[0053] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 2.

[0054] S3, synthesis steps of compound 3 1. Compound 2 (3 g, 8.3 mmol) and trimethyl phosphite (1.5 g, 12.4 mmol) were dissolved in anhydrous dichloromethane (50 mL).

[0055] 2. Stir the reaction for 15 minutes under anhydrous and oxygen-free conditions at 0°C.

[0056] 3. Slowly add titanium tetrachloride (2.3 g, 12.4 mmol) dropwise, and continue stirring the reaction for 0.5-1 hour.

[0057] 4. After the reaction is complete, quench the reaction with saturated sodium bicarbonate solution and extract with dichloromethane.

[0058] 5. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 3.

[0059] S4, Synthesis steps of compound 4 1. Compound 3 (2 g, 4.6 mmol) was dissolved in anhydrous THF (15 mL), and diisopropylaminolithium (3.4 mL, 2 mol / mL) was added dropwise under anhydrous and oxygen-free conditions at -78°C.

[0060] 2. After stirring for 30 minutes, add dropwise 2-adamantanone (1.4 g, 9.2 mmol) (dissolved in anhydrous THF).

[0061] 3. Stir the reaction at room temperature for 2-3 hours and monitor the reaction progress by TLC.

[0062] 4. After the reaction is complete, wash with water and extract with ethyl acetate.

[0063] 5. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 4.

[0064] S5, Synthesis steps of compound 5 1. Compound 4 (1.5 g, 3.2 mmol) was dissolved in anhydrous THF (20 mL), and n-butyllithium (2.6 mL, 2.5 M in hexane) was added dropwise under anhydrous and oxygen-free conditions at -78°C.

[0065] 2. After stirring for 30 minutes, add DMF (1 mL) dropwise.

[0066] 3. Stir the reaction at room temperature for 2-3 hours and monitor the reaction progress by TLC.

[0067] 4. After the reaction is complete, quench the reaction with saturated ammonium chloride solution and extract with ethyl acetate.

[0068] 5. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 5.

[0069] S6, Synthesis steps of compound 6 1. Compound 5 (2 g, 6.7 mmol), imidazole (0.912 g, 13.4 mmol) and tert-butyldimethylchlorosilane (1.312 g, 8.71 mmol) were dissolved in anhydrous dichloromethane (20 mL).

[0070] 2. Stir the reaction at room temperature for 1-2 hours and monitor the reaction progress by TLC.

[0071] 3. After the reaction is complete, wash with water and extract with dichloromethane.

[0072] 4. Combine the organic layers, dry and concentrate them, and purify them by silica gel column chromatography to obtain compound 6.

[0073] S7, Synthesis steps of compound 7 1. Dissolve methyl 4-bromobut-2-enoate (3 g, 16.7 mmol) and trimethyl phosphite (2.70 g, 21.7 mmol) in anhydrous toluene (40 mL).

[0074] 2. The reaction was carried out under nitrogen protection at 120°C with reflux and stirring for 20 hours, and the reaction progress was monitored by TLC.

[0075] 3. After the reaction is complete, concentrate under reduced pressure, wash with saturated sodium chloride solution and extract with ethyl acetate.

[0076] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 5-1.

[0077]

[0078] 5. Compound 5-1 (1 g, 4.85 mmol) was dissolved in anhydrous THF (20 mL), and n-butyllithium (2.0 mL, 2.5 M in hexane) was added dropwise under anhydrous and oxygen-free conditions at -78°C.

[0079] 6. After stirring for 30 minutes, add compound 6 (1 g, 2.43 mmol) dropwise.

[0080] 7. Stir the reaction at room temperature for 2 hours and monitor the reaction progress by TLC.

[0081] 8. After the reaction is complete, quench the reaction with saturated ammonium chloride solution and extract with ethyl acetate.

[0082] 9. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 7.

[0083] S8, Synthesis steps of compound 8 1. Compound 7 (1 g, 4.85 mmol) was dissolved in anhydrous dichloromethane (20 mL) and dibutylaluminum hydroxide (DIBAL-H, 15.6 mL, 15.6 mmol) was added dropwise under anhydrous and oxygen-free conditions at -78°C.

[0084] 2. Stir for 30 minutes, then move to room temperature and stir for another 1-2 hours.

[0085] 3. After the reaction is complete, quench with ice water and wash with 0.5 M HCl and saturated sodium chloride solution.

[0086] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 8.

[0087] S9, Synthesis steps of compound 9 1. Compound 7 (1 g, 2.14 mmol) and MnO2 (0.931 g, 10.7 mmol) were dispersed in ethyl acetate (20 mL).

[0088] 2. Stir at 80℃ for 3-4 hours.

[0089] 3. The mixture was filtered and evaporated under reduced pressure to obtain a liquid, which finally yielded compound 9.

[0090] S10, Synthesis steps of compound 10 1. Compound 9 (1 g, 2.15 mmol) was dissolved in dichloromethane (20 mL), and tetrabutylammonium fluoride (16.1 mL, 0.2 M in THF) was added dropwise at room temperature.

[0091] 2. Stir continuously while dripping, and monitor the reaction progress using TLC.

[0092] 3. After the reaction is complete, wash with saturated ammonium chloride solution and extract with dichloromethane.

[0093] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 10.

[0094] S11, Synthesis steps of compound 11 1. Dissolve compound 10 (0.1 g, 0.2 mmol) in anhydrous acetonitrile (5 mL) and incubate at 0 °C for 5 minutes.

[0095] 2. Add CsCO3 (0.13 g, 0.4 mmol), react for 10 minutes, then add the following compound 11-1 (0.225 g, 0.4 mmol) and Na2SO4 (0.056 g, 0.4 mmol), react for 2 minutes, then move to room temperature and stir for 12 hours.

[0096]

[0097] 3. The reaction progress was monitored by TLC, the mixture was washed with saturated ammonium chloride solution, and extracted with ethyl acetate.

[0098] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 11.

[0099] S12, Synthesis steps of compound 12 1. Compound 11 (0.1 g, 0.13 mmol) and rhodamine salt (0.040 g, 0.085 mmol) were dissolved in ethanol (10 mL).

[0100] 2. Under nitrogen protection, the reaction was refluxed at 80°C for 12 hours, and the reaction progress was monitored by TLC.

[0101] 3. After the reaction is complete, wash with saturated sodium chloride solution and extract with ethyl acetate.

[0102] 4. Combine the organic layers, dry and concentrate, and purify by silica gel column chromatography to obtain compound 12.

[0103] S13, Synthesis steps of compound 13 1. Compound 12 (0.1 g, 0.087 mmol) was dissolved in methanol (5 mL), sodium methoxide (0.0235 g, 0.43 mmol) was added, and the mixture was stirred at room temperature for 30 minutes.

[0104] 2. Monitor the reaction progress using TLC.

[0105] 3. After neutralizing the reaction mixture with 2N hydrochloric acid, the mixture was purified by HPLC to obtain compound 13.

[0106] The above-mentioned compounds 5-1 and compounds 6 to 13 1 The characterization results of H NMR are as follows Figures 2 to 10 This demonstrates the successful synthesis of the near-infrared II chemiluminescent molecule of Formula 13.

[0107] Example 2 This embodiment provides a method for preparing a near-infrared II chemiluminescent lipid platform, as detailed below: Compound 13 and the photosensitizer protoporphyrin PpIX were encapsulated in liposomes at a specific ratio to assemble the near-infrared II chemiluminescent lipid platform PLC-3-β-gal, as follows: Weigh out 1,2-bis-(9Z-octadecenoyl)- sn 3-glycerol-3-phosphocholine (DOPC, 0.5354 mg), cholesterol (0.1469 mg), and DSPE-PEG-2000 (0.196 mg) were dissolved in 20 μL of ethanol at a ratio of 6.8:3.8:0.7. Then, 0.1666 mg of the above compound 13 and 0.0096 mg of protoporphyrin PpIX (ratio 10:1) were weighed and dissolved in 20 μL of ethanol. After mixing, the mixture was mixed with 300 μL of sodium citrate buffer solution by ethanol injection. After standing overnight at 4°C, the mixture was centrifuged at 3000 rpm for 10 min using a 15 mL 30 KD ultrafiltration tube. After removing the ethanol, the mixture was stored at 4°C in the dark to obtain the assembly PLC-3-β-gal.

[0108] The chemiluminescent lipid platform was characterized and its performance was investigated, as detailed below.

[0109] (1) Speciation of compound PLC-3-gal: To measure the morphology, PLC-3-gal (2 µg / mL, 13 µL) was dropped onto a copper wire, negatively stained with 1% phosphotungstic acid, and dried overnight. Transmission electron microscopy (TEM) was used to scan the copper wire, obtaining TEM images. The images showed that the nanoparticles had a diameter between 140 and 160 nm.

[0110] (2) Determination of the photophysical properties of compound 13 / PLC-3-gal: Compound 13 in a 5% dimethyl sulfoxide (DMSO) / H₂O solution (10 µg / mL, 200 µL) and PLC-3-gal in an aqueous solution (10 µg / mL, 200 µL) were added to cuvettes, and their UV / Vis absorption was measured. The cuvettes were placed on a UV-Vis spectrometer, and the absorption spectra were measured. Figure 11 As shown in the ultraviolet absorption spectrum, the highest absorption peak of compound 13 is 672 nm. The presence of the characteristic peak of compound 13 in the ultraviolet absorption spectrum of PLC-3-gal nanoparticles indicates the successful preparation of PLC-3-gal.

[0111] Compound 13 in 5% dimethyl sulfoxide (DMSO) / H₂O solution (10 µg / mL, 200 µL) and PLC-3-gal in aqueous solution (10 µg / mL, 200 µL) were added to cuvettes respectively, and fluorescence absorption was measured. The cuvettes were placed in a steady-state transient spectrometer (FLS980), and fluorescence spectra were measured. The fluorescence emission spectra show that the highest emission peak of compound 13 under 808 nm excitation is at 971 nm.

[0112] (3) In vitro singlet oxygen of PLC-3-gal ( 1 Measurement of O2 production: Add 400 µL of 100 µM / mL and 10 µL of SOSG aqueous solution to a 1.5 mL centrifuge tube. Irradiate the tube with a 660 nm laser for the following durations (distance 5 cm): 0, 0.5, 1, 2, 5, 10, 15, 20 min, 0.2 W / cm². 2 Add the solution to a cuvette. Place the cuvette on a UV / Vis spectrometer and measure the absorption spectrum of the solution. Determine the formation of ROS by observing changes in its characteristic absorption peaks. The results are as follows: Figure 12 As shown.

[0113] (4) Cell culture: In terms of cell culture, SK-OV-3 cells (SK-OV-3 cell line purchased from National Identification Cell Culture Center (Shanghai, China)) were cultured in McCoy's 5A medium (containing 1% penicillin-streptomycin (PS) and 10% fetal bovine serum (FBS)) at 37°C and 5% CO2.

[0114] 1) Evaluation of PLC-3-β-gal cytotoxicity using the CCK-8 assay: SK-OV-3 cells were cultured in McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO2. Cells were seeded into 96-well plates at a density of 5 × 10³ cells / well and allowed to adhere for 24 hours. When the cells reached approximately 70% confluence, the medium was replaced with fresh serum-free McCoy's 5A medium containing different concentrations of PLC-3-β-gal nanoparticles (0, 10, 25, 50, or 75 μM). Incubation was continued for 24 hours to assess dose-dependent cytotoxicity. After treatment, the medium containing nanoparticles was aspirated, and the cells were gently washed three times with phosphate-buffered saline (PBS) to remove residual nanoparticles. Subsequently, 100 μL of fresh McCoy's 5A medium containing 10% (v / v) CCK-8 reagent was added to each well. Cells were incubated at 37°C for 1 hour to induce formaldehyde formation. The absorbance of each well was measured at 450 nm using a BioTek Synergy H1 microplate reader to assess the cytotoxicity of the material. After incubation of SK-OV-3 cells with different concentrations of PLC-3-β-gal for 24 h, even at concentrations as high as 50 μM / mL, over 80% of the SK-OV-3 cells remained viable. This indicates that PLC-3-β-gal exhibits low cytotoxicity and good biocompatibility. Figure 13 As shown.

[0115] 2) CCK-8 assay for the efficacy of PLC-3-β-gal photodynamic therapy (PDT): As described above, SK-OV-3 cells were seeded into 96-well plates. At 80% density, cells were incubated with 25 μM PLC-3-β-gal nanoparticles in serum-free McCoy's 5A medium for 6 hours to allow for nanoparticle internalization. After incubation, the medium was replaced with fresh PBS. Cells were irradiated with a 660 nm laser at an intensity of 0.2 W / cm² for 0, 3, 6, 9, 12, and 15 minutes, then returned to McCoy's 5A medium and incubated for another 12 hours to assess the therapeutic effect. After a 12-hour recovery period, the medium was aspirated, and cells were washed three times with PBS to remove debris. CCK-8 reagent (10% in McCoy's 5A medium) (100 μL / well) was added to each well, and the cells were incubated at 37°C for 1 hour. As described above, absorbance was measured at 450 nm. After 6 hours of incubation with PLC-3-β-gal, the cells were laser-excited (0.2 W / cm²). 2 After 10 minutes of illumination, the relative viability of standard CCK-8 cells was measured using an ELISA reader. Cells treated with PLC-3-β-gal and laser showed decreased viability with increasing illumination time. When the illumination time was 15 minutes, cell survival dropped to 20%. These results indicate that PLC-3-β-gal has good photodynamic therapy efficacy at the cellular level.

[0116] 3) PLC-3-β-gal cytoluminescence detection: As described above, SK-OV-3 cells were seeded into 96-well plates. At 80% density, cells were incubated with PLC-3-β-gal nanoparticles (concentrations: 0, 6.25, 12.5, 25, 50 μM) in serum-free McCoy's 5A medium for 6 hours to allow for nanoparticle internalization. After incubation, the medium was replaced with fresh PBS. Cells were irradiated with a 660 nm laser at an intensity of 0.2 W / cm² for 2 minutes, and then detected using a near-infrared II fluorescence imager. The results are as follows: Figure 14 As shown.

[0117] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. The scope of protection of the present invention is defined only by the appended claims.

Claims

1. A laser-activated near-infrared II chemiluminescent molecule, characterized in that, Its molecular structure is as follows: 。 2. A method for preparing laser-activated near-infrared II chemiluminescent molecules as described in claim 1, characterized in that, Includes the following steps: S1. 2-Bromo-5-hydroxybenzaldehyde, trimethyl orthoformate and tetrabutylammonium bromide were dissolved in anhydrous methanol and reacted at room temperature. After the reaction was completed, the mixture was purified to obtain compound 1. ； S2. Under an inert atmosphere, compound 1, imidazole and tert-butyldimethylchlorosilane were dissolved in anhydrous dichloromethane and reacted at room temperature. After the reaction was completed, compound 2 was obtained by purification. ； S3. Compound 2 and trimethyl phosphite were dissolved in anhydrous dichloromethane and reacted under anhydrous and oxygen-free conditions at -10~0 °C. Then titanium tetrachloride was added dropwise to continue the reaction. After the reaction was completed, the reaction was quenched with saturated sodium bicarbonate solution and then purified to obtain compound 3. ； S4. Compound 3 was dissolved in anhydrous THF, and diisopropylaminolithium was added dropwise under anhydrous and oxygen-free conditions at -70~80 °C to carry out the reaction. Then 2-adamantanone was added and reacted at room temperature. After the reaction was completed, the mixture was purified to obtain compound 4. ； S5. Compound 4 was dissolved in anhydrous THF, and n-butyllithium was added dropwise under anhydrous and oxygen-free conditions at -70~80 ℃ to carry out the reaction. Then DMF was added to react at room temperature. After the reaction was completed, the reaction was quenched with saturated ammonium chloride solution, and then purified to obtain compound 5. ； S6. Compound 5, imidazole and tert-butyldimethylchlorosilane were dissolved in anhydrous dichloromethane and reacted at room temperature. After the reaction was completed, the mixture was purified to obtain compound 6. ； S7. Compound 5-1 was dissolved in anhydrous THF, and n-butyllithium was added dropwise under anhydrous and oxygen-free conditions at -70~80 °C to carry out the reaction. Then, compound 6 was added and reacted at room temperature. After the reaction was completed, the reaction was quenched with saturated ammonium chloride solution, and then purified to obtain compound 7. ； S8. Compound 7 was dissolved in anhydrous dichloromethane. Dibutylaluminum hydroxide was added dropwise at -70~80 ℃ under anhydrous and oxygen-free conditions for 10~60 minutes. The reaction was then moved to room temperature and the reaction was continued. After the reaction was completed, the mixture was quenched with ice water and purified to obtain compound 8. ； S9. Compound 8 and MnO2 were dispersed in ethyl acetate and reacted at 60-90 °C. After the reaction was completed, the mixture was filtered and evaporated under reduced pressure to obtain compound 9. ； S10. Compound 9 was dissolved in dichloromethane, and tetrabutylammonium fluoride was added at room temperature to carry out the reaction. After the reaction was completed, the mixture was purified to obtain compound 10. ； S11. Compound 10 was dissolved in anhydrous acetonitrile, and CsCO3 was added at -10~0 °C to react. Then, compound 11-1 and Na2SO4 were added and reacted for 1~5 minutes. The reaction was then moved to room temperature and the reaction continued. After the reaction was completed, compound 11 was obtained by purification. ； S12. Under an inert atmosphere, compound 11 and rhodamine salt were dissolved in ethanol and refluxed at 60-90 °C. After the reaction was completed, compound 12 was obtained by purification. ； S13. Compound 12 is dissolved in methanol, sodium methoxide is added and reacted at room temperature. After the reaction is completed, compound 13 is obtained by purification, which is the laser-activated near-infrared II chemiluminescent molecule. 。 3. The method for preparing laser-activated near-infrared II chemiluminescent molecules according to claim 2, characterized in that, In step S1, the feed ratio of 2-bromo-5-hydroxybenzaldehyde, trimethyl orthoformate, and tetrabutylammonium bromide is 1 eq: 1.1-2 eq: 0.01 eq; In step S2, the feed ratio of compound 1, imidazole, and tert-butyldimethylchlorosilane is 1 eq: 3 eq: 2 eq; In step S3, the feed ratio of compound 2, trimethyl phosphite, and titanium tetrachloride is 1 eq: 1.5 eq: 1.5 eq; In step S4, the feed ratio of compound 3, 2-adamantanone, and lithium diisopropylaminodimethylamine is 1 eq: 1.5 eq: 1.5-2 eq; In step S5, the feed ratio of compound 4, DMF, and n-butyllithium is 1 eq: 2 eq: 3.5-4 In step S6, the feed ratio of compound 5, imidazole, and tert-butyldimethylchlorosilane is 1 eq: 1.5-2 eq: 1.2-1.3 eq; In step S7, the feed ratio of compound 5-1, compound 6, and n-butyllithium is 1 eq: 0.5 eq: 1-1.5 eq; In step S8, the feed ratio of compound 7 and dibutylaluminum hydroxide is 1 eq: 3.2-4 eq; Raw materials: In step S9, the feed ratio of compound 8 and manganese dioxide (MnO2) is 1 eq: 5-6 eq; in step S10, the feed ratio of compound 9 and tetrabutylammonium fluoride is 1 eq: 1.5-2 eq; in step S11, the feed ratio of compound 10, cesium carbonate (CsCO3), compound 11-1, and anhydrous sodium sulfate (Na2SO4) is 1 eq: 2 eq: 2 eq: 2 eq; in step S12, the feed ratio of compound 11 and rhodamine salt is 1 eq: 0.6-0.7 eq; in step S13, the feed ratio of compound 12 and sodium methoxide is 1 eq: 5 eq.

4. A near-infrared II chemiluminescent lipid platform, characterized in that, The laser-activated near-infrared II chemiluminescent molecule as described in claim 1 was used.

5. The near-infrared II chemiluminescent lipid platform according to claim 4, characterized in that, The near-infrared II chemiluminescent lipid platform is obtained by encapsulating the near-infrared II chemiluminescent molecules and the photosensitizer protoporphyrin PpIX in a set ratio through liposomes.

6. The near-infrared II chemiluminescent lipid platform according to claim 5, characterized in that, The preparation method of the near-infrared II chemiluminescent lipid platform includes: Weigh out 1,2-bis-(9Z-octadecenoyl)- sn Glyceryl-3-phosphate choline, cholesterol, and DSPE-PEG-2000 were dissolved in ethanol at a ratio of 6.8:3.8:0.

7. Then, 0.1666 mg of compound 13 and 0.0096 mg of protoporphyrin PpIX were weighed and dissolved in ethanol. After mixing, the mixture was mixed with sodium citrate buffer solution by ethanol injection. After standing overnight at 0-4°C, the mixture was centrifuged at 2000-3000 rpm for 5-10 min using a 20-30 KD ultrafiltration tube. After removing the ethanol, the mixture was stored at 4°C protected from light.

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