Organic afterglow nano material as well as preparation method and application thereof

Abstract

The invention discloses an organic afterglow nano material as well as a preparation method and application thereof, and belongs to the technical field of nano materials. The preparation method of the organic afterglow nano material provided by the invention comprises the following steps: preparing an afterglow substrate; the afterglow substrate and NIR-II fluorescent molecules 970 are dissolved in acetonitrile, and FDA is obtained; the FDA, 4-iodine methyl phenylboronic acid pinacol ester and K2CO3 are dissolved in DMF (Dimethyl Formamide), and FZA is obtained; fZA, zinc phthalocyanine and phospholipid-amino polyethylene glycol are dissolved in tetrahydrofuran, ultrasonic treatment is performed for co-encapsulation, separation and collection are performed, and the organic afterglow nano material is obtained. According to the preparation method provided by the invention, the organic afterglow nano material can be endowed with high specificity and controllable afterglow imaging of FZ970 only under the action of ONOO <->, and the FZ970 is basically non-toxic to cells, has a key effect in afterglow luminescence in a tumor microenvironment, and has a wide application prospect in biological imaging.

Description

Technical Field

[0001] This application belongs to the technical field of nanomaterials, and particularly relates to an organic afterglow nanomaterial, its preparation method and application. Background Art

[0002] Optical imaging technology has attracted extensive attention in detecting various physiological or pathological processes from the microscopic to the macroscopic level due to its characteristics such as real-time analysis, high sensitivity, high spatial resolution, and non-invasive visualization. Compared with the commonly used visible light and NIR-I (750 - 900 nm) fluorescence imaging technologies, NIR-II has a longer emission wavelength (1000 - 1700 nm), which can significantly reduce the influence of light scattering and autofluorescence effects when penetrating biological tissues, resulting in a deeper detection depth and higher spatial resolution. However, photoluminescence imaging suffers from severe light scattering, tissue absorption, and autofluorescence, leading to a high signal-to-noise ratio (SBR) and low sensitivity. Afterglow luminescence uses a probe as an "energy storage battery", and after the energy excitation stops, photons are slowly released from the afterglow probe defects that store energy. This imaging method avoids the interference of autofluorescence in the sample, greatly reduces the background signal, and significantly improves the imaging signal-to-noise ratio.

[0003] Current afterglow materials are divided into two types: inorganic afterglow materials and organic afterglow materials. Among them, most inorganic afterglow materials are composed of rare-earth metal-doped alkali metal aluminates, which have potential problems such as biological toxicity and biological metabolism. Organic afterglow materials are co-doped afterglow nanomaterials with semiconductor polymers and photosensitizers as initiators and afterglow luminescent substances as luminescent substrates. However, the existing organic afterglow imaging materials have the following problems: First, most of the materials are in the visible light region or NIR-I region, resulting in a lower penetration depth and resolution in biological organisms; second, the afterglow materials have structural inertia and insufficient response sites, which affects their wide application. Summary of the Invention

[0004] This application discloses an organic afterglow nanomaterial, its preparation method and application, aiming to solve the technical problems of the organic afterglow imaging material having a lower penetration depth and resolution in biological organisms and insufficient response sites.

[0005] To achieve the above object, the technical solution of this application is:

[0006] The first aspect of this application provides a preparation method of an organic afterglow nanomaterial, and the preparation method includes:

[0007] Prepare an afterglow substrate;

[0008] Dissolve the afterglow substrate and the NIR-II fluorescent molecule 970 in acetonitrile to obtain FDA;

[0009] Dissolve the said FDA, 4-iodomethylphenylboronic acid pinacol ester and K2CO3 in N,N-dimethylformamide to obtain FZA;

[0010] Dissolve the said FZA, zinc phthalocyanine and phospholipid-amino polyethylene glycol in tetrahydrofuran, perform co-encapsulation by ultrasonic treatment, separate and collect to obtain the organic afterglow nanomaterial.

[0011] Preferably in combination with the first aspect, the preparation of the afterglow substrate includes the following steps of formula I:

[0012]

[0013] 4-Methoxybenzenethiol reacts with ethyl acetoacetate to form compound a-1;

[0014] Compound a-1 reacts with BBr3 to form compound a-2;

[0015] Compound a-2 reacts with ethyl bromoacetate and K2CO3 to form compound a-3;

[0016] Compound a-3 reacts with malononitrile to form compound a-4;

[0017] Compound a-4 is hydrolyzed to form compound a-5;

[0018] Compound a-5 reacts with 3-hydroxy-2-(norbornene-5-yl)benzopyran-4-one to obtain the afterglow substrate.

[0019] Preferably in combination with the first aspect, dissolve the said NIR-II fluorescent molecule 970 and the afterglow substrate in acetonitrile, including the following steps of formula II:

[0020]

[0021] Preferably in combination with the first aspect, dissolve the said FDA, 4-iodomethylphenylboronic acid pinacol ester and K2CO3 in DMF, including the following steps of formula III:

[0022]

[0023] Preferably in combination with the first aspect, when dissolving the said FZA, zinc phthalocyanine and phospholipid-amino polyethylene glycol in tetrahydrofuran, the temperature is 0-5 °C and the time is 5-10 min.

[0024] Preferably in combination with the first aspect, the intensity of the ultrasonic treatment is 100-200 W / cm 2 , and the time is 1-5 min.

[0025] Preferably, in combination with the first aspect, after dissolving the FZA, zinc phthalocyanine, and phospholipid-amino polyethylene glycol in tetrahydrofuran, an ultrafiltration tube with a molecular weight cut-off of 30 kDa is used for ultracentrifugation to concentrate the nanoparticle solution, and ultracentrifugation is carried out at a speed of 4000 - 6000 rpm for 10 - 15 min.

[0026] The second aspect of the present application provides an organic afterglow nanomaterial prepared by the preparation method described in the first aspect.

[0027] The third aspect of the present application provides the application of the organic afterglow nanomaterial prepared by the preparation method described in the first aspect or the organic afterglow nanomaterial described in the second aspect in bioimaging.

[0028] Compared with the prior art, the advantages or beneficial effects of the embodiments of the present application at least include:

[0029] The preparation method provided by the present application encapsulates the NIR-II fluorescent molecule 970 and the afterglow substrate with 4-iodomethylphenylboronic acid pinacol ester, zinc phthalocyanine, and phospholipid-amino polyethylene glycol in sequence to prepare an organic afterglow nanomaterial. Under US irradiation, the generated 1 O2 oxidizes FDA, leading to the formation of high-energy dioxetane intermediates. The phenylborate groups present in the organic afterglow nanomaterial can protect the intermediates to maintain chemical inertness and prevent the activation of spontaneous afterglow. Only in the presence of ONOO - , the phenylborate groups are selectively cleaved to release phenolate anions, which helps chemical excitation and initiates an efficient chemiluminescence resonance energy transfer process, ultimately generating NIR-II afterglow emission. This "double-lock" controlled activation strategy utilizes the synergistic interaction of US and ONOO - to achieve highly specific and controllable afterglow imaging, and has broad application prospects in bioimaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] To more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0031] Figure 1 1H NMR spectrum of FZA provided by the embodiment of the present application;

[0032] Figure 2 13C NMR spectrum of FZA provided by the embodiment of the present application;

[0033] Figure 3 MALDI-TOF-MS spectrum of FDA provided by the embodiment of the present application;

[0034] Figure 4 TEM and particle size distribution diagrams of FD970 provided by the embodiments of the present application;

[0035] Figure 5 TEM and particle size distribution diagrams of FZ970 provided by the embodiments of the present application;

[0036] Figure 6 NIR-II spectral test diagram of FD970 provided by the embodiments of the present application;

[0037] Figure 7 Response experiment diagrams of FZ970 to different solutions provided by the embodiments of the present application;

[0038] Figure 8 Variation diagram of the afterglow intensity of FZ970 with the concentration of ONOO- solution provided by the embodiments of the present application;

[0039] Figure 9 Cytotoxicity test diagrams of FZ970 at different concentrations provided by the embodiments of the present application;

[0040] Figure 10 Cytotoxicity test diagrams of FZ970 under the action of US at different concentrations provided by the embodiments of the present application;

[0041] Figure 11 Afterglow intensity diagrams of 4T1 cells after co-incubation with different concentrations of FZ970 provided by the embodiments of the present application;

[0042] Figure 12 NIR-II afterglow imaging intensity diagrams at the tumor sites in tumors of FD970 and FZ970 in mice provided by the embodiments of the present application. Detailed implementation manners

[0043] Next, the technical solutions in the embodiments of the present application will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0044] In the following description of this embodiment, the term "and / or" is used to describe the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: the case of A alone, the case of B alone, and the case of both A and B existing simultaneously. Where A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after.

[0045] In the following description of this embodiment, the term "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or its similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0046] Those skilled in the art should understand that in the following description of the embodiments of this application, the sequence numbers do not imply the order of execution. Some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, without constituting any limitation to the implementation process of the embodiments of this application.

[0047] The terms used in the embodiments of this application are only for the purpose of describing specific embodiments, and are not intended to limit this application. The singular forms "a" and "the" used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0048] It should be noted that all raw materials and reagents in the embodiments of this application are purchased on the market or prepared by conventional methods well-known to those skilled in the art.

[0049] In a first aspect, the embodiments of this application provide a method for preparing an organic afterglow nanomaterial, and the preparation method includes:

[0050] Preparing an afterglow substrate;

[0051] Dissolving the afterglow substrate and the NIR-II fluorescent molecule 970 in acetonitrile to obtain FDA;

[0052] Dissolving the FDA, 4-iodomethylphenylboronic acid pinacol ester, and K2CO3 in N,N-dimethylformamide to obtain FZA;

[0053] Dissolving the FZA, zinc phthalocyanine, and phospholipid-amino polyethylene glycol in tetrahydrofuran, performing co-encapsulation by ultrasonic treatment, separating and collecting to obtain the organic afterglow nanomaterial.

[0054] Among them, under US radiation, the generated 1 O2 oxidizes FDA, resulting in the formation of high-energy dioxetane intermediates. The phenylborate groups present in the organic afterglow nanomaterial can protect the intermediates to maintain chemical inertness and prevent the activation of spontaneous afterglow. Only in ONOO -In the presence of [substance], the phenylborate group is selectively cleaved to release the phenolate anion, which helps to chemically activate and initiate an efficient chemiluminescence resonance energy transfer process, ultimately generating NIR-II afterglow emission. This "double-lock" controlled activation strategy utilizes the synergistic interaction of US and ONOO - to achieve highly specific and controllable afterglow imaging, showing broad application prospects in biological imaging.

[0055] It should be noted that the NIR-II fluorescent molecule 970 used as a raw material in this application can be prepared according to the preparation process disclosed in the article "Design and synthesis of a small molecular NIR-II chemiluminescence probe for in vivo-activated H2S imaging, PNAS 2023 Vol.120 No.8 e2205186120": Using 4-bromotriphenylamine and Cu(NO3)2·3H2O as raw materials, dissolving them in acetic anhydride, and stirring at room temperature for 10 h to obtain an orange solid compound. Subsequently, the new compound is stirred with bis(pinacolato)diboron, potassium acetate, and Pd(dppf)Cl2 at 85 °C for 36 h to obtain a yellow solid compound. Subsequently, Pd(PPh3)4 is added to the mixture of benzobisthiadiazole, potassium carbonate, toluene, and [substance], and the new compound obtained after stirring at 80 °C for 72 h is obtained. Zinc powder and NH4Cl are dispersed in a mixed solvent of 90% methanol and dichloromethane, stirred at room temperature for 6 h, and the solvent is removed under vacuum. Then, the obtained brown oil, N-sulfinylaniline, and trimethylchlorosilane are dissolved in anhydrous pyridine. The solution is stirred at 83 °C for 24 h, and then separated and purified to obtain the NIR-II fluorescent molecule 970. The preparation process is shown in Formula IV:

[0056]

[0057] In the examples of this application, the preparation of the afterglow substrate includes the following steps of Formula I:

[0058]

[0059] 4-Methoxythiophenol reacts with ethyl acetoacetate to form compound a-1; compound a-1 reacts with BBr3 to form compound a-2; compound a-2 reacts with ethyl bromoacetate and K2CO3 to form compound a-3; compound a-3 reacts with malononitrile to form compound a-4; compound a-4 is hydrolyzed to form compound a-5; compound a-5 reacts with 3-hydroxy-2-(norbornene-5-yl)benzopyran-4-one to obtain the afterglow substrate (DPBs).

[0060] It should be noted that 3-hydroxy-2-(norbornene-5-yl)benzopyran-4-one can be prepared according to the steps of Formula V disclosed in the article "Ultrasound-Activated NIR Chemiluminescence for Deep Tissue and Tumor Foci Imaging, Anal. Chem. 2023, 95, 11219-11226":

[0061]

[0062] In the embodiments of the present application, dissolving the NIR-II fluorescent molecule 970 and the afterglow substrate in acetonitrile includes the following steps of Formula II:

[0063]

[0064] In the embodiments of the present application, dissolving the FDA, 4-iodomethylphenylboronic acid pinacol ester, and K2CO3 in DMF includes the following steps of Formula III:

[0065]

[0066] In the embodiments of the present application, when dissolving the FZA, zinc phthalocyanine, and phospholipid-amino polyethylene glycol in tetrahydrofuran for reaction, the temperature is preferably 0-5°C and the time is preferably 5-10 min. The intensity of the ultrasonic treatment is preferably 100-200 W / cm 2 , and the time is preferably 1-5 min. A 30 kDa ultrafiltration tube can be used to separate and concentrate nanomaterials with a particle size of about 100 nm, removing small molecule impurities. Ultracentrifugation is carried out at 4000-6000 rpm for 10-15 min to collect the purified nanomaterials. The molar ratio of the FZA, ZnPc to DSPE-PEG2000 is preferably (5-40):3:(100-200), more preferably 10:3:100.

[0067] It should be noted that the afterglow nanoparticles (FZ970) are composed of an NIR-II fluorophore (970), FZA responsive to ONOO - , an afterglow initiator zinc phthalocyanine (ZnPc), and DSPE-PEG 2000 . For the first time, NIR-II afterglow-guided sonodynamic therapy responsive to ONOO - is achieved. In the nanoparticles (FD970) without a connected responsive group, the afterglow initiator zinc phthalocyanine (ZnPc) generates 1O2 is used to connect FDA with the NIR-II fluorophore (970) through an amide bond to achieve chemiluminescence resonance energy transfer (CRET), generating NIR-II afterglow luminescence at 950 nm, which is consistent with the wavelength of the NIR-II fluorescence signal generation.

[0068] The second aspect of this application provides the organic afterglow nanomaterial prepared by the preparation method described in the first aspect. Based on the above preparation process, the organic afterglow nanomaterial can be endowed with the property that only in the presence of ONOO - can the afterglow luminescence be specifically activated to generate NIR-II afterglow luminescence at 950 nm.

[0069] The third aspect of this application provides the application of the organic afterglow nanomaterial prepared by the preparation method described in the first aspect or the organic afterglow nanomaterial described in the second aspect in bioimaging. Among them, based on the above organic afterglow nanomaterial, only in the presence of ONOO - can the afterglow luminescence be specifically activated to generate NIR-II afterglow luminescence at 950 nm, which has broad application prospects in bioimaging.

[0070] Next, the technical solutions of this application will be further elaborated in combination with specific embodiments.

[0071] Example 1

[0072] This example provides a preparation method for the A1-organic afterglow nanomaterial (FZ970), which specifically includes:

[0073] S101: At 90 °C, 4-methoxythiophenol and ethyl acetoacetate are dissolved in preheated polyphosphoric acid and stirred for 30 min. After completion, the reaction mixture is cooled to room temperature and processed. The anhydrous dichloromethane solution of the obtained yellow solid compound is cooled to 0 °C, and BBr3 is added dropwise. The reaction mixture is stirred at room temperature for 12 h. After completion, ice water is added to precipitate a gray solid. After purification, a pale yellow solid compound is obtained. Subsequently, it is refluxed overnight with ethyl bromoacetate and K2CO3 in an acetonitrile solution. After the obtained product is heated and stirred with malononitrile in acetic anhydride at 140 °C for 5 h, the new product obtained by hydrolysis is covalently linked with 3-hydroxy-2-(norbornene-5-yl)benzopyran-4-one to obtain the afterglow substrate (DPBs);

[0074] S102: Dissolve DPBs, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and 1-hydroxy-7-azabenzotriazole (HOAT) in DCM and DMF. Then add N,N-diisopropylethylamine (DIEA). Under a nitrogen atmosphere, stir the mixture at 0 °C for 15 min. Subsequently, dropwise add a solution of the NIR-II fluorescent molecule 970 in DCM / DMF (1:1, v / v) to the mixture. Stir the reaction at room temperature for 24 h and purify to obtain a new organic compound (FDA);

[0075] S103: Dissolve FDA, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl iodide, and K2CO3 in DMF. Stir the reaction mixture at 0 °C for 12 h. After completion, extract the reaction mixture with dichloromethane, wash with a NaCl solution, and dry with anhydrous Na2SO4. Then purify the crude product by silica column chromatography using dichloromethane / methanol as the eluent to obtain a dark green solid organic compound (FZA);

[0076] S104: Co-encapsulate FZA, ZnPc with DSPE-PEG 2000 The molar ratio of FZA, ZnPc to DSPE-PEG 2000 is preferably 10:3:100 and dissolve in tetrahydrofuran (THF) to form a clear solution, and then rapidly inject it into deionized water under sonication at 100 - 200 W / cm 2 Ultrasonicate the mixture. Then, blow nitrogen into the solution at 55 - 65 °C to remove THF. Wash the resulting aqueous solution three times with deionized water by ultrafiltration centrifugation (Amicon centrifugal filter, 30 kDa cut-off, 4000 - 6000 rpm, 10 - 15 min). The final solution of FZ970 is obtained in PBS buffer (1×, pH 7.4, 1 mL).

[0077] Meanwhile, to verify the comprehensive performance of the organic afterglow nanomaterials prepared in the above examples, this application provides the following comparative examples for detailed elaboration.

[0078] Comparative Example 1

[0079] This comparative example provides a preparation method, component ratio, preparation operation, and process parameters of the B1-organic afterglow nanomaterial (FD970), which are basically the same as those in Example 1, except that:

[0080] In this comparative example, dissolve the prepared FDA, zinc phthalocyanine, and DSPE-PEG 2000 in tetrahydrofuran for reaction, dissolve in tetrahydrofuran (THF) to form a clear solution, and then rapidly inject it into deionized water under strong sonication. The mixture is sonicated at 100 - 200 W / cm2 Ultrasonic treatment. Then, nitrogen gas was blown into the solution at 55 - 65 °C to remove THF. The resulting aqueous solution was washed three times with deionized water by ultrafiltration centrifugation (Amicon centrifugal filter, 30 kDa cut-off, 4000 - 6000 rpm, 10 - 15 min), and the final solution of FD970 was obtained in PBS buffer (1×, pH 7.4, 1 - 2 mL) to obtain the B1-organophosphorescent nanomaterial (FD970).

[0081] To verify the chemical structure of the organophosphorescent nanoparticles prepared in the examples, NMR characterization was performed on them and the intermediate products, and the test results are as Figures 1-3 shown. Among them, Figure 1 is the 1 1H NMR spectrum of FZA; Figure 2 is the 13 13C NMR spectrum of FZA; Figure 3 is the MALDI-TOF-MS spectrum of FDA.

[0082] According to Figure 1 it can be seen that for the 1H NMR spectrum of FZA, 1 1H NMR (400 MHz, DMSO-d6) δ 10.28 (s, 1H), 8.36 (s, 1H), 8.15 (d, J = 8.4 Hz, 4H), 8.16 - 8.04 (m, 6H), 8.02 - 7.96 (m, 6H), 7.96 (s, 2H) 7.68 - 7.46 (m, 8H), 7.15 - 7.09 (m, 10H), 5.25 (s, 4H), 4.88 (s, 4H), 3.22 (s, 2H), 3.20 (s, 6H), 2.09 - 1.68 (m, 24H), 1.24 (s, 24H), which can be well corresponded to each chemical group.

[0083] According to Figure 2 it can be seen that for the 13C NMR spectrum of FZA, 13 13C NMR (101 MHz, CDCl3) δ 176.2, 162.6, 153.6, 152.1, 142.7, 140.3, 136.0, 132.8, 130.4, 130.3, 129.9, 129.4, 128.0, 126.3, 125.3, 124.9, 121.2, 85.1, 54.4, 50.8, 46.5, 46.1, 40.8, 37.1, 30.1, 24.9, 22.2, which can be well corresponded to each chemical group.

[0084] According to Figure 3 it can be seen that for the MALDI-TOF-MS spectrum of FDA, the molecular formula of FDA is C 136 H120 B2N 12 O 12 S4, [M+H] + The molecular weight is 2263.8204, which is basically consistent with the actually calculated 2263.8218.

[0085] To verify the appearance morphology of the prepared organic afterglow nanoparticles in the examples, the prepared organic afterglow nanoparticles were characterized by transmission electron microscopy (TEM) and particle size. The particle size of the nanoparticles was analyzed by dynamic light scattering (DLS) experiments, and the test results are as Figure 4 and Figure 5 shown. Among them, Figure 4 is the TEM and particle size distribution diagram of FD970; Figure 5 is the TEM and particle size distribution diagram of FZ970.

[0086] According to Figure 4 it can be seen that FD970 is in the form of nanoparticles, the particle size of the nanoparticles is relatively uniform, the particle size distribution is a normal distribution, and the particle size is about 100 nm.

[0087] According to Figure 5 it can be seen that FZ970 is in the form of nanoparticles, the particle size of the nanoparticles is relatively uniform, the particle size distribution is a normal distribution, and the particle size is about 100 nm.

[0088] To verify the in vitro afterglow imaging of FD970 prepared in the examples, under the action of US (1 - 2 W cm -2 , 50% duty cycle, 1 - 2 min), the stock solution of FD970 was taken for afterglow luminescence spectrum test. Under an 808 nm laser (800 - 1200 ms, 500 - 1500 mA), the stock solution of FD970 was taken for NIR-II fluorescence spectrum test and compared with the afterglow luminescence spectrum. The results are Figure 6 shown that according to the spectral comparison, the peaks of both afterglow luminescence (AGL) and fluorescence luminescence (FL) are near 970 nm, and the wavelengths match, indicating that FD970 can produce NIR-II afterglow luminescence.

[0089] To verify the afterglow imaging effect and sonodynamic therapy effect of the prepared organic afterglow nanoparticles (FZ970) in the examples, the following tests were carried out on the prepared organic afterglow nanomaterial (FZ970) in the examples.

[0090] I. ONOO - Responsiveness experiment

[0091] Prepare different RONS (ONOO - , Fe 2+ , Ca 2+ , ·OH, PBS, NO2 - , ClO- A solution of [substance] and H2O2 was diluted to the same concentration and then added to FZ970. Under the action of US, the NIR-II afterglow luminescence signal was detected. The above steps were repeated three times, and then the NIR-II afterglow intensity was analyzed. The test results Figure 7 are shown as follows.

[0092] According to Figure 7 the display, only under the action of ONOO - can the afterglow luminescence of FZ970 be specifically activated. The selective afterglow luminescence of FZ970 when exposed to various reactive oxygen and nitrogen species (20 μg mL -1 ) in PBS solution at pH 7.4.

[0093] Solutions of different concentrations of ONOO - were added to FZ970, and then afterglow imaging was performed. The test results Figure 8 are shown as follows. Figure 8 The results showed that the afterglow intensity increased with the increase in the concentration of the ONOO - solution. This once again demonstrated the effect of ONOO - on FZ970.

[0094] II. Cell experiments

[0095] Cytotoxicity and killing experiments: The cytotoxicity test was carried out by the standard cell counting kit-8 (CCK-8) test. 4T1 cells were seeded into 96-well plates (8×10 3 -1×10 4 cells per well), and then incubated at 37 °C for 24 h to allow the cells to attach. After that, different concentrations of FZ970 (0, 5, 10, 20, and 40, 80 μg / mL) were incubated with the cells at 37 °C for 24 h. The culture medium was removed, and the cells were washed twice with PBS buffer (10 mM, pH 7.4). 100 μL of the culture medium containing 10% CCK-8 was added to the wells and co-incubated for 1 h. The absorbance (OD) value at 450 nm was detected using a BioTek Synergy H1. The test results are as Figure 9 shown as follows.

[0096] According to Figure 9 it can be seen that FZ970 is basically non-toxic to cells.

[0097] 4T1 cells were seeded into 96-well plates (8×10 3 -1×10 4 cells per well), and then incubated at 37 °C for 24 hours to allow the cells to attach. The cell killing ability was tested under the action of US. The test results are as Figure 10 shown as follows.

[0098] According toFigure 10 It can be seen that with the increase in concentration, the killing ability of FZ970 against cells is enhanced.

[0099] FZ970 at different concentrations was co-incubated with 4T1 cells, and then afterglow imaging was performed. The test results are as Figure 11 shown.

[0100] According to Figure 11 it can be seen that the afterglow intensity increases with the increase in the concentration of ONOO - , indicating that ONOO - inside the cells can activate the afterglow signal of FZ970. It has better tissue penetration and is easy to achieve imaging of deep tissues.

[0101] III. In vivo imaging experiment

[0102] 1×10 6 -1×10 7 100 μL of 4T1 cells were subcutaneously injected into the legs of 6 - 8-week-old nude mice to establish a tumor model. After 14 days of feeding, obvious tumors appeared in the mice subcutaneously injected with 4T1 cells, which could be used for subsequent imaging experiments. The in-tumor afterglow imaging was performed on the tumor-bearing mice after intratumoral injection of FZ970(, and images were collected using a NIR-II small animal in vivo imaging system. After irradiating the tumor site with US (1.6 W cm -2 ) for 1 min, the afterglow luminescence intensity was recorded for 60 s through the NIR-II small animal in vivo imaging system under a 950 nm filter. After injecting FD970 ([FDA] = 100 μg mL -1 ) and FZ970 ([FZA] = 100 μg mL -1 ) into the tumor respectively, the afterglow signal in the tumor was continuously monitored by afterglow imaging, and the in-tumor afterglow luminescence intensities were compared and analyzed. The results are as Figure 12 shown.

[0103] According to Figure 12 it can be seen that, among them, a) the afterglow imaging diagrams of intratumoral injection of FD970 or FZ970, b) the afterglow intensity analysis diagrams; the NIR-II afterglow signal of FD970 remained basically unchanged within 30 min, while FZ970 showed a rapid increase in signal, reaching a peak at 6 min, and then the afterglow luminescence intensity maintained a stable state. These findings confirmed the key role of ONOO - in selectively activating the afterglow luminescence of FZ970 in the tumor microenvironment.

[0104] Therefore, the method for preparing the organic afterglow nanomaterial provided by this application includes: preparing an afterglow substrate; dissolving the afterglow substrate and the NIR-II fluorescent molecule 970 in acetonitrile to obtain FDA; dissolving the FDA, 4-iodomethylphenylboronic acid pinacol ester, and K2CO3 in DMF to obtain FZA; dissolving the FZA, zinc phthalocyanine, and phospholipid-amino polyethylene glycol in tetrahydrofuran, and performing co-encapsulation by ultrasonic treatment, followed by separation and collection to obtain the organic afterglow nanomaterial. Only under the action of ONOO - can the afterglow luminescence of FZ970 be specifically activated, and it is basically non-toxic to cells and plays a key role in the afterglow luminescence in the tumor microenvironment.

[0105] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referred to each other, and each embodiment focuses on the differences from other embodiments.

[0106] The above embodiments are only used to illustrate the technical solutions of this application, rather than limiting this application; although this application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. A preparation method of an organic afterglow nanomaterial, characterized in that, The preparation method includes: Preparing the afterglow substrate; Dissolving the afterglow substrate and the NIR-II fluorescent molecule 970 in acetonitrile to obtain FDA; Dissolving the FDA, 4-iodomethylphenylboronic acid pinacol ester and K2CO3 in N,N-dimethylformamide to obtain FZA; Dissolving the FZA, zinc phthalocyanine and phospholipid-amino polyethylene glycol in tetrahydrofuran, performing co-encapsulation by ultrasonic treatment, separating and collecting to obtain the organic afterglow nanomaterial.

2. The preparation method of the organic afterglow nanomaterial according to claim 1, characterized in that, The preparation of the afterglow substrate includes the following steps of formula I: 4-Methoxythiophenol reacts with ethyl acetoacetate to generate compound a-1; Compound a-1 reacts with BBr3 to generate compound a-2; Compound a-2 reacts with ethyl bromoacetate and K2CO3 to generate compound a-3; Compound a-3 reacts with malononitrile to generate compound a-4; Compound a-4 is hydrolyzed to generate compound a-5; Compound a-5 reacts with 3-hydroxy-2-(norbornene-5-yl)benzopyran-4-one to obtain the afterglow substrate.

3. The preparation method of the organic afterglow nanomaterial according to claim 1, characterized in that, Dissolving the NIR-II fluorescent molecule 970 and the afterglow substrate in acetonitrile includes the following steps of formula II:

4. The preparation method of the organic afterglow nanomaterial according to claim 1, wherein Dissolving the FDA, 4-iodomethylphenylboronic acid pinacol ester and K2CO3 in DMF includes the following steps of formula III:

5. The preparation method of the organic afterglow nanomaterial according to claim 1, wherein When dissolving the FZA, zinc phthalocyanine and phospholipid-amino polyethylene glycol in tetrahydrofuran, the temperature is 0-5 °C and the time is 5-10 min.

6. The preparation method of the organic afterglow nanomaterial according to claim 1, wherein The intensity of the ultrasonic treatment is 100 - 200 W / cm 2 , and the time is 1 - 5 min.

7. The preparation method of the organic afterglow nanomaterial according to claim 1, characterized in that, After dissolving the FZA, zinc phthalocyanine and phospholipid-amino polyethylene glycol in tetrahydrofuran, use a 30 kDa ultrafiltration tube for ultracentrifugation to concentrate the nanoparticle solution, and perform ultracentrifugation at a speed of 4000-6000 rpm for 10-15 min.

8. An organic afterglow nanomaterial prepared by the preparation method according to any one of claims 1-7.

9. Use of an organic afterglow nanomaterial prepared by the preparation method according to any one of claims 1-7 or the organic afterglow nanomaterial according to claim 8 in biological imaging.

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