Nano AIE probe as well as preparation method and application thereof

Abstract

The invention relates to a nano AIE probe as well as a preparation method and application thereof. The probe is synthesized by taking a compound A as a raw material and F-127 as a surfactant through a nano precipitation method; the structural general formula of the compound A is # imgabs0, wherein R is # imgabs1 # imgabs2 #, and the preparation method comprises the following steps: S1, preparing the compound A; and S2, preparing the nano AIE probe. The nano AIE probe has AIE characteristics, excellent phototoxicity, good cancer cell specificity, low cell dark toxicity and good biocompatibility, can well distinguish cancer cells from normal cells, can be applied to tumor treatment as a photosensitizer, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a nanometer AIE probe, a preparation method thereof and an application thereof. Background Art

[0002] Traditional organic luminescent materials emit bright fluorescence in dilute solutions. However, in concentrated solutions or solids, aggregation-induced quenching (ACQ) severely limits their practical application in complex aqueous environments or solid matrices. To address this problem of diminished fluorescence in traditional fluorescent materials at high concentrations or in the solid state, researchers have explored various approaches, such as chemical modification, the introduction of steric groups, or the design of specialized molecular structures to inhibit intermolecular π-π stacking, thereby reducing energy transfer and non-radiative transitions. However, these approaches often have limited effectiveness and fail to fundamentally address the problem. In 2001, while studying 1-methyl-1,2,3,4,5-pentaphenylsiloxane (HPS), a team led by Academician Tang Benzhong unexpectedly discovered that the molecule, while nearly luminescent in dilute solution, exhibits strong fluorescence in the aggregated or solid state. This phenomenon, in stark contrast to the ACQ effect of traditional fluorescent materials, overturned conventional understanding of fluorescent materials, quickly attracted widespread attention from the global scientific community, and thus pioneered the new research field of AIE.

[0003] Aggregation-induced emission (AIE) is a new type of fluorescent material developed in recent years. Its fluorescence emission is weak in the dispersed state, but it can show a significant fluorescence enhancement effect in the aggregated state. The emergence of AIE materials effectively solves the problem that traditional fluorescent molecules are difficult to apply in solid state or at high concentration due to the ACQ effect. With its unique photostability, high fluorescence quantum yield and excellent structural controllability, AIE materials provide new research ideas and technical solutions for fluorescence analysis and detection, bioimaging, optoelectronic devices and other fields, and promote the innovative development of fluorescent materials science. Based on its complex molecular structure, AIE photosensitizers can achieve efficient light-mediated generation of ROS. Although some AIE molecules exhibit poor solubility and cytotoxicity, biocompatibility and cell targeting can be improved by using appropriate nanomaterials.

[0004] Cancer cells differ significantly from normal cells in many ways, and these differences serve as crucial insights for the design of specific probes. Regarding cell surface markers, cancer cells often overexpress certain proteins or glycoproteins. For example, prostate-specific membrane antigen (PSMA) is expressed at much higher levels on prostate cancer cells than on normal prostate cells. This provides an ideal target for the design of specific probes targeting prostate cancer. Regarding their internal cellular environment, cancer cells exhibit unique metabolic characteristics. Compared to normal cells, cancer cells have an unusually robust glycolytic metabolism, preferring to obtain energy through glycolysis even in the presence of oxygen. This phenomenon is known as "aerobic glycolysis" or the "Warburg effect." This unique metabolic pathway alters the pH and redox state of the cancer cell's internal environment. For example, the microenvironment within cancer cells is typically more acidic than that of normal cells. This property can be exploited to design probes that specifically respond to acidic environments. When such a polymer enters the cancer cell, the acidic environment within the cancer cell causes structural changes in the polymer, generating a detectable signal and enabling specific identification of the cancer cell.

[0005] Based on this, a fluorescent probe with AIE properties, excellent phototoxicity and good cancer cell specificity can be constructed. Summary of the Invention

[0006] Based on this, it is necessary to provide a nano-AIE probe, its preparation method and application to address the defects and shortcomings of the existing technology.

[0007] A nano-AIE probe is synthesized by nanoprecipitation using compound A as a raw material and F-127 as a surfactant; the general structural formula of compound A is as follows:

[0008]

[0009] Where R is

[0010] like Figure 1 As shown, a method for preparing the nano-AIE probe as described above comprises the following steps:

[0011] S1. Dissolve the reactants and potassium tert-butoxide in ethanol, then add compound 1 to react to obtain compound A;

[0012] S2. Compound A and F-127 obtained in S1 are dissolved in dimethyl sulfoxide to obtain solution A. Ultrapure water is then added to solution A and ultrasonically mixed to obtain solution B. Solution B is added to a dialysis bag for dialyzation to obtain a nano-AIE probe.

[0013] As a preferred embodiment, the reactant in S1 is 2-(4-(pyridin-4-yl)phenyl)acetonitrile or 4-pyridineacetonitrile.

[0014] As a preferred embodiment, when the reactant is 2-(4-(pyridin-4-yl)phenyl)acetonitrile, the molar ratio of the reactant, the potassium tert-butoxide and the compound 1 is 103:98:34.

[0015] As a preferred embodiment, when the reactant is 4-pyridineacetonitrile, the molar ratio of the reactant, the potassium tert-butoxide and the compound 1 is 3:3:1.

[0016] As a preferred embodiment, the structural formula of the compound 1 is as follows:

[0017]

[0018] As a preferred embodiment, the mass ratio of the compound A to the F-127 in S2 is 1:10;

[0019] As a preferred embodiment, the volume ratio of the solution A to the ultrapure water is 1:10.

[0020] As a preferred embodiment, after the S1 reaction is completed, the product is purified by an eluent, wherein the eluent is prepared by mixing petroleum ether and ethyl acetate in a volume ratio of 10-40:1.

[0021] The nano-AIE probe as described above is used as a tumor-specific fluorescent tracer and photosensitizer in the field of tumors.

[0022] Beneficial effects of the present invention: The nano-AIE probe prepared by the preparation method of the present invention has AIE characteristics, excellent phototoxicity and good cancer cell specificity, has low cytotoxicity and good biocompatibility, and can well distinguish cancer cells from normal cells. It can be used as a tumor-specific fluorescent tracer and photosensitizer in the field of tumors and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the synthesis process of the AIE probe of the present invention;

[0024] Figure 2 This is a high-resolution mass spectrum of compound AIE-1 of the present invention;

[0025] Figure 3 This is a high-resolution mass spectrum of compound AIE-2 of the present invention;

[0026] Figure 4 is a hydrogen nuclear magnetic resonance spectrum of the compound AIE-1 of the present invention;

[0027] Figure 5 is the H NMR spectrum of compound AIE-2 of the present invention;

[0028] Figure 6 This is a graph showing the aggregation fluorescence enhancement effect of the compound AIE-1 of the present invention in DMSO solutions with different water contents;

[0029] Figure 7 This is a graph showing the aggregation fluorescence enhancement effect of the compound AIE-2 of the present invention in DMSO solutions with different water contents;

[0030] Figure 8 A comprehensive diagram of the quantitative experiment of the nanoprobe AIE NPs of the present invention (wherein, a is the standard curve of compound AIE-1, b is the UV absorption graph of nanoprobe AIE-1 NPs in DMSO solution, c is the standard curve of compound AIE-2, and d is the UV absorption graph of nanoprobe AIE-2 NPs in DMSO solution);

[0031] Figure 9 The ultraviolet absorption and fluorescence spectra of the nanoprobe AIE NPs of the present invention (wherein, a is the ultraviolet absorption spectrum of the nanoprobe AIE-1NPs, b is the fluorescence spectrum of the nanoprobe AIE-1NPs, c is the ultraviolet absorption spectrum of the nanoprobe AIE-2NPs, and d is the fluorescence spectrum of the nanoprobe AIE-2NPs);

[0032] Figure 10 The results of the MTT experiment of the nanoprobe AIE-1NPs of the present invention;

[0033] Figure 11 The results of the MTT experiment of the nanoprobe AIE-2NPs of the present invention;

[0034] Figure 12 Figure 2 is a diagram of the cellular uptake of the nanoprobe AIE-1 NPs of the present invention (scale bar: 20 μm);

[0035] Figure 13 Figure 2 is a diagram of the cellular uptake of the nanoprobe AIE-2NPs of the present invention (scale bar: 20 μm);

[0036] Figure 14 Figure 2 is a fluorescence intensity diagram of the cell uptake of the nanoprobe AIE NPs of the present invention (where a is the nanoprobe AIE-1 NPs and b is the nanoprobe AIE-2 NPs);

[0037] Figure 15 CLSM images of the nanoprobe AIE-1 NPs of the present invention taken up by different cancer cells and normal cells (scale bar: 20 μm);

[0038] Figure 16 CLSM images of the nanoprobe AIE-2NPs of the present invention taken up by different cancer cells and normal cells (scale bar: 20 μm);

[0039] Figure 17 Figure 2 is a fluorescence intensity extraction diagram of the nanoprobe AIE NPs of the present invention taken up by different cancer cells and normal cells (where a is the nanoprobe AIE-1 NPs and b is the nanoprobe AIE-2 NPs);

[0040] Figure 18 CLSM images of the nanoprobe AIE NPs of the present invention being taken up in a mixture of L929 and HeLa cells (a is the nanoprobe AIE-1 NPs, b is the nanoprobe AIE-2 NPs, scale bar: 20 μm). DETAILED DESCRIPTION

[0041] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0042] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0043] Example 1

[0044] Preparation of compound 1

[0045] S11. Under nitrogen protection, 4-methoxyaniline (1.23 g, 10 mmol), 1-bromo-4-iodobenzene (7.075 g, 25 mmol), cuprous iodide (0.285 g, 1.5 mmol), potassium tert-butoxide (2.24 g, 20 mmol) and 1,10-phenanthroline (0.27 g, 1.5 mmol) were added to 85 mL of toluene and dissolved at room temperature for 24 h. The solvent was removed in vacuo, and dichloromethane and water were added for extraction. The organic phase was collected, dried, and filtered. After removing the solvent in vacuo, the resulting solid was mixed with petroleum ether and ethyl acetate in a ratio of 40:1 to 10:1, and separated by column chromatography to obtain 2.1 g of a white solid product (i.e., Compound I). The yield was 38.89%;

[0046] S12. Under nitrogen protection, compound I (0.866 g, 2 mmol), (5-formylthiophene-2-yl)boric acid (0.936 g, 6 mmol) and potassium carbonate (2.76 g, 20 mmol) prepared by S11 were dissolved in a mixture of 5 mL of toluene and 5 mL of methanol. After 2 min, PdCl2 (dppf) (0.042 g, 0.06 mmol) was added and refluxed at 75 ° C for 12 h. DCM was then added to start extraction, the organic phase was collected, and dried over anhydrous sodium sulfate. After removing the solvent in vacuo, the resulting solid was mixed with petroleum ether and ethyl acetate in a ratio of 40:1 to 10:1, and separated by column chromatography to obtain 0.630 g of a light yellow solid product (i.e., compound 1). The yield was 42.42%.

[0047] Example 2

[0048] Preparation of compound AIE-1

[0049] Under nitrogen, 2-(4-(pyridin-4-yl)phenyl)acetonitrile (0.2 g, 1.03 mmol) and potassium tert-butoxide (0.11 g, 0.98 mmol) were dissolved in 75 mL of ethanol and stirred for 10 min. Compound 1 (0.17 g, 0.34 mmol) prepared in Example 1 was then added, and the mixture was refluxed at 80°C for 7 h. DCM was added and extracted, and the organic phase was collected and dried over anhydrous sodium sulfate. After removing the solvent in vacuo, the resulting solid was mixed with petroleum ether and ethyl acetate in a ratio of 40:1 to 10:1 and separated by column chromatography to obtain 0.268 g of the product (i.e., compound AIE-1) as a pale yellow solid. The yield was 61.37%.

[0050] Example 3

[0051] Preparation of compound AIE-2

[0052] Under nitrogen, 4-pyridineacetonitrile (0.036 g, 0.31 mmol) and potassium tert-butoxide (0.034 g, 0.30 mmol) were dissolved in 75 mL of ethanol and stirred for 10 min. Compound 1 (0.05 g, 0.10 mmol) prepared in Example 1 was then added and refluxed at 80°C for 12 h. DCM was added for extraction, and the organic phase was collected and dried over anhydrous sodium sulfate. The solvent was removed in vacuo, and the resulting solid was mixed with petroleum ether and ethyl acetate in a ratio of 40:1 to 10:1 and separated by column chromatography to obtain 0.062 g of the product (i.e., compound AIE-2) as a pale yellow solid. The yield was 57.47%.

[0053] Example 4

[0054] Preparation of AIE-1 NPs probes

[0055] 1 mg of compound AIE-1 prepared in Example 2 was dissolved in 1 mL of DMSO, followed by the addition of 10 mg of F-127 and sonication to obtain a transparent mixed solution. 1 mL of the transparent mixed solution and 10 mL of ultrapure water were added in a ratio of 1:10, and sonication was performed for 2 minutes (at a power ratio of 25%) to obtain a sonicated solution. The sonicated solution was then dialyzed into a dialysis bag (molecular weight 3500), with the ultrapure water changed three times every hour and three times every two hours. After 24 hours of dialysis, the AIE-1 NPs probe was obtained and stored in a 4°C refrigerator for later use.

[0056] Example 5

[0057] Preparation of AIE-2NPs probes

[0058] 1 mg of compound AIE-2 prepared in Example 3 was dissolved in 1 mL of DMSO, followed by the addition of 10 mg of F-127 and sonication to obtain a transparent mixed solution. 1 mL of the transparent mixed solution and 10 mL of ultrapure water were added in a ratio of 1:10, and sonication was performed for 2 minutes (at a power ratio of 25%) to obtain a sonicated solution. The sonicated solution was then dialyzed into a dialysis bag (molecular weight 3500), with the ultrapure water changed three times every hour and three times every two hours. After 24 hours of dialysis, the AIE-2 NPs probe was obtained and stored in a 4°C refrigerator for later use.

[0059] Test Example 1

[0060] Design, synthesis and characterization of nano-AIE probes

[0061] 1) Compound AIE-1 prepared in Example 2 and Compound AIE-2 prepared in Example 3 were detected using a mass spectrometer. The results were as follows: Figure 2-Figure 3 As shown;

[0062] 2) Compound AIE-1 prepared in Example 2 and Compound AIE-2 prepared in Example 3 were subjected to hydrogen spectrum detection using a magnetic nuclear resonance spectrometer. The results were as follows: Figure 4-Figure 5 shown.

[0063] Depend on Figure 2-Figure 5 It can be seen that with 4-methoxytriphenylamine skeleton as electron donor, two thiophene groups were introduced to extend conjugation and electron donor, 2-(4-(pyridin-4-yl)phenyl)acetonitrile and 4-pyridineacetonitrile were used as electron acceptors, respectively, and F-127 was used as surfactant. Based on the ICT mechanism, ADA-structured probe AIE-1NPs and probe AIE-2NPs were successfully constructed.

[0064] Test Example 2

[0065] AIE property analysis

[0066] A certain amount of compound AIE-1 prepared in Example 2 and compound AIE-2 prepared in Example 3 were respectively dissolved in a DMSO solution prepared by mixing water and DMSO in certain proportions, specifically 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99% (the percentage of water in the DMSO solution). Fluorescence emission curves were measured using the optimal wavelength for excitation. The emission curves of compound AIE-1 and compound AIE-2 at different proportions were measured. The results are shown in FIG. Figure 6-Figure 7 shown.

[0067] Depend on Figure 6 As can be seen, compound AIE-1 exhibits weak luminescence in pure DMSO, with a peak at 648 nm. As the water content gradually increases, the fluorescence intensity of compound AIE-1 in DMSO solution decreases, then increases, and then decreases again. When the water content increases from 0% to 20%, the fluorescence intensity of compound AIE-1 gradually decreases and reaches a minimum. In a 20% water content H2O / DMSO mixture, compound AIE-1 maintains high dispersion and undergoes rotation, vibration, and stretching, dissipating excited-state energy to a certain extent through nonradiative processes. However, as the water content increases to 50%, the fluorescence emission of compound AIE-1 begins to significantly enhance due to increased molecular aggregation and reduced nonradiative dissipation. Notably, the emission wavelength of compound AIE-1 exhibits a slight redshift, but the fluorescence intensity significantly increases, reaching over 1.2 times the initial fluorescence intensity and reaching its maximum at this point. Generally, increasing molecular aggregation enhances π-π interactions, leading to a redshift in fluorescence. For compound AIE-1, water acts as a poor solvent, while DMSO acts as a good solvent. Both significantly affect its luminescence properties. As the amount of poor solvent increases, the aggregation of compound AIE-1 molecules increases, leading to an increase in fluorescence intensity. Subsequently, as the water content further increases, the fluorescence intensity slowly decreases.

[0068] Depend on Figure 7As can be seen, when dissolved in pure DMSO, compound AIE-2 molecules exhibit weak fluorescence emission. Subsequently, when water is introduced into the system and the water content is increased to 30%, the fluorescence intensity of compound AIE-2 molecules is observed to significantly weaken and reach a minimum. This is because the introduction of water molecules affects the interactions between compound AIE-2 molecules, changing the dissipation mechanism of compound AIE-2's excited state energy to a non-radiative mode, resulting in a decrease in fluorescence intensity. As the water content further increases, the fluorescence intensity of compound AIE-2 begins to increase. When the water content reaches 50%, the fluorescence intensity of compound AIE-2 reaches its maximum, even exceeding four times the fluorescence intensity measured in pure DMSO solution. This is because at a water content of 50%, compound AIE-2 molecules begin to form aggregates, resulting in a significant increase in fluorescence intensity, a typical AIE phenomenon. The fluorescence intensity slightly decreases in DMSO solutions with a water content of 50%-70%, while it slightly increases in DMSO solutions with a water content of 70%-80%. This is due to the change in the aggregate morphology or size of compound AIE-2 dye.

[0069] In summary, under a certain water content, both compound AIE-1 and compound AIE-2 can form aggregates that are conducive to fluorescence emission, exhibiting significant AIE effect.

[0070] Test Example 3

[0071] Quantitative experiments

[0072] An appropriate amount of the compound AIE-1 powder prepared in Example 2 was accurately weighed using an analytical balance and dissolved in a certain amount of DMSO solution to prepare a solution with a concentration of 1 mM. The solution was then diluted to 2, 4, 6, 8, and 10 μM compound AIE-1 standard solutions, respectively. The absorption spectrum of the compound AIE-1 standard solution was detected at 200 nm-800 nm, and a corresponding standard curve was plotted. Subsequently, the probe AIE-1 NPs was dispersed in the DMSO solution to prepare a 50 μg / mL probe AIE-1 NPs detection solution, and the absorption spectrum of the probe AIE-1 NPs detection solution was tested at 200 nm-800 nm.

[0073] An appropriate amount of the compound AIE-2 powder prepared in Example 3 was accurately weighed using an analytical balance and dissolved in a certain amount of DMSO solution to prepare a solution with a concentration of 1 mM. The solution was then diluted to 2, 4, 6, 8, and 10 μM compound AIE-2 standard solutions, and the absorption spectrum of the compound AIE-1 standard solution was detected at 200 nm-800 nm, and the corresponding standard curve was drawn. Subsequently, the probe AIE-2 NPs were dispersed in the DMSO solution to prepare a 50 μg / mL probe AIE-2 NPs detection solution. The absorption spectrum of the probe AIE-2 NPs detection solution was tested at 200 nm-800 nm. The results are as follows: Figure 8 shown.

[0074] Depend on Figure 8 a and Figure 8 b shows that the maximum absorption of the standard solution of compound AIE-1 at 475 nm was 0.072, 0.15, 0.225, 0.305, and 0.362, respectively, at 2, 4, 6, 8, and 10 μM. The linear fitting yielded y = 0.0368x + 0.0023, R 2 =0.9971. Subsequently, the probe AIE-1 NPs solution detected a maximum absorption of 0.431 at a wavelength of 475 nm. Substituting y = 0.0368x + 0.0023 into the equation, we obtain x = 11.65 μmol / L. Since M(AIE-1) = 848.0550 g / molL, this translates to 9.88 μg / mL of AIE-1 molecules in a 50 μg / mL AIE-1 NPs solution.

[0075] Depend on Figure 8 c and Figure 8 d It can be seen that the standard working curve of the standard solution of compound AIE-2 obtained by linear fitting is y=0.0568x+0.0016, R 2 =0.9999. This indicates good linearity in the AIE-2 solution range of 2μmol / L-10μmol / L. Subsequently, the probe AIE-2 NPs detection solution detected a maximum absorption of 0.462 at a wavelength of 505nm. By substituting y = 0.0568x + 0.0016 into the equation, we obtain x = 8.10μmol / L. Since M(AIE-2) = 695.8590g / moL, after conversion, it is calculated that a 50μg / mL AIE-2 NPs solution system contains 5.64μg / mL AIE-2 molecules.

[0076] Test Example 4

[0077] UV-visible absorption and fluorescence spectra of probe AIE-1NPs and probe AIE-2NPs

[0078] The probe AIE-1NPs prepared in Example 4 and the probe AIE-2NPs prepared in Example 5 were dissolved in deionized water to prepare 10, 20, 30, 40, and 50 μg / mL test solutions, and deionized water was used as a blank control. The absorbance of the test solutions was measured at 200nm-800nm. At the same time, the fluorescence spectrum of 50μg / mL AIENPs was tested using a fluorescence spectrometer. The results are as follows: Figure 13 shown.

[0079] like Figure 9 As shown in a, the maximum absorption wavelength of AIE-1NPs in water is 470nm. As the concentration increases, the absorbance of AIE-1NPs in water increases. At the same time, the absorbance value of 50μg / mL AIE-1NPs was preliminarily determined as the dosing concentration for subsequent experiments. Figure 9 As shown in b, the maximum emission wavelength of the probe AIE-1NPs at 50 μg / mL is 650 nm, showing a large Stokes shift (180 nm for AIE-1NPs). Figure 9 As shown in c, the absorbance of probe AIE-2NPs in water increases with the increase of concentration; Figure 9 As shown in Figure d, when the probe AIE-2 NPs concentration is 50 μg / mL, the peak is distinct and broad, with a maximum absorption wavelength at 520 nm and a maximum emission wavelength at 630 nm, indicating a Stokes shift of 110 nm for AIE-2 NPs. Both AIE-1 NPs and AIE-2 NPs exhibit large Stokes shifts. Numerous studies have shown that fluorescent probes with large Stokes shifts can mitigate interference from native protein fluorescence, ensuring detection efficiency and sensitivity. Therefore, AIE-1 NPs and AIE-2 NPs have potential applications in fluorescent labeling, bioimaging, and other fields.

[0080] Test Example 5

[0081] Cytotoxicity assay

[0082] Preparation of AIE-1 NPs experimental solution: AIE-1 NPs prepared in Example 4 were dissolved in PBS solution, and AIE-1 NPs experimental solutions with concentrations of 25, 50, 100, 150, and 200 μg / mL were prepared using PBS solution;

[0083] Preparation of AIE-2NPs experimental solution: AIE-2NPs prepared in Example 5 were dissolved in PBS solution, and AIE-2NPs experimental solutions with concentrations of 25, 50, 100, 150, and 200 μg / mL were prepared using PBS solution;

[0084] The experiment was carried out in 96-well plates, with an initial seeding density of 6 × 103 HeLa cells / well. After 12 hours of incubation, the culture medium in each well was replaced with 100 μL of AIE-1NPs experimental solution and AIE-2NPs experimental solution, respectively. One group of 96-well plates was kept in the dark as a control group. After 3 hours of administration, the other group of plates was placed under white light (30 mW / cm 2 ) irradiation. Then, the cells were incubated for another 24 hours. Subsequently, MTT solution was added to the 96-well plate and reacted for 4 hours. After that, the culture was terminated and the culture medium in the well was carefully aspirated. 140 μL of dimethyl sulfoxide was added to each well, and the crystals were fully dissolved at low speed on an oscillating table for 20 minutes to dissolve the blue formazan crystals produced by cell metabolism. The 96-well plate was then placed in a microplate reader and the absorbance OD value was read at 490 nm. The experiment was repeated 3 times, and the corresponding cell survival rate was obtained by the following formula:

[0085] Cell viability (%) = OD experimental group / OD control group

[0086] Among them, the OD control group is the absorbance of the blank control group without adding probe AIE NPs; the specific results are as follows Figure 10 and Figure 11 shown.

[0087] Depend on Figure 10 It can be seen that in the absence of light, after AIE-1NPs were incubated with HeLa cells for 3 hours, no growth inhibition was observed. At the same time, with the increase of the concentration of AIE-1NPs, the cell survival rate remained at about 100%. However, when AIE-1NPs were incubated with HeLa cells for 3 hours and then subjected to 30mW / cm 2 Irradiation with cool white light produced varying degrees of growth inhibition on HeLa cells. The degree of growth inhibition increased with increasing illumination time. Notably, even a 25 μg / mL solution of AIE-1 NPs reduced cell survival to approximately 12% after 30 minutes of illumination. This demonstrates that AIE-1 NPs exhibit low dark toxicity and strong phototoxicity.

[0088] Depend on Figure 11 As can be seen, the non-illumination group showed no cell growth inhibition at any concentration, and the cell survival rate was around 100%. This indicates that AIE-2 NPs have very low dark toxicity. In the 10-min illumination group, cell survival rate decreased slightly, reaching 67% at 200 μg / mL. In the 15-min illumination group, cell survival rate showed a more significant downward trend, dropping to 45% at 100 μg / mL. In the 20-min illumination group, the downward trend was even more pronounced. Cell survival rate decreased with increasing illumination time. This indicates that AIE-2 NPs have strong phototoxicity, making them suitable for use as photosensitizers in the field of oncology.

[0089] Test Example 6

[0090] Cellular uptake experiments

[0091] HeLa cells were seeded in a confocal cell culture dish and incubated for 12 h. The old culture medium was replaced with fresh culture medium containing AIE-1 NPs prepared in Example 4 and AIE-2 NPs prepared in Example 5 (at a concentration of 50 μg / mL), and the cells were incubated for 0.5, 1, 2, 3, and 4 h, respectively. Subsequently, confocal fluorescence imaging of HeLa cells was performed using a confocal microscope. The results are shown in FIG. Figure 12 、 Figure 13 and Figure 14 shown.

[0092] Depend on Figure 12 and Figure 14 As shown in Figure a, the imaging results of the time-dependent fluorescence changes in HeLa cells after incubation with AIE-1 NPs. After 0.5 hours of incubation with HeLa cells, weak fluorescence appeared in the cells; after 1 hour, stronger fluorescence was already visible in the cells; after 2 hours, the fluorescence in the cells was very obvious, showing a punctate distribution, indicating that the probe AIE-1 NPs had entered the cells; after 3 hours, the intensity of the punctate fluorescence reached its maximum and approached saturation. After 4 hours, the fluorescence intensity was not much different from that at 3 hours. The experimental results show that the probe AIE-1 NPs can effectively enter HeLa cells and image cancer cells. At the same time, the cells incubated with AIE-1 NPs have a good morphology, demonstrating that the probe AIE-1 NPs have low cytotoxicity.

[0093] Depend on Figure 13 and Figure 14 b shows the imaging results of the changes in intracellular fluorescence over time after HeLa cells were incubated with AIE-2NPs. Compared with the HeLa cell group incubated without AIE-2NPs, the fluorescence of the experimental group incubated for 0.5h was weak; after incubation for 1h, a brighter red fluorescence could be clearly observed; after 2h, the fluorescence in the cells further enhanced; after 3h, the fluorescence in the cells reached its peak; after 4h, the fluorescence in the cells basically no longer enhanced. Since the fluorescence intensity basically reached the maximum after 3h, 3h was selected as the optimal incubation time for AIE-2NPs. In general, under physiological conditions, both probes AIE-1NPs and probes AIE-2NPs can successfully penetrate the cell membrane and enter the cell. At the same time, the optimal incubation time for AIE-1NPs and AIE-2NPs was determined to be 3h.

[0094] Test Example 7

[0095] Cellular uptake experiments in complex environments

[0096] The AIE-1NPs prepared in Example 4 and the AIE-2NPs prepared in Example 5 were subjected to cell uptake experiments with human lung cancer (A549) cells, colon adenocarcinoma (Caco-2) cells, human breast cancer (MDA-MB-231) cells, HeLa cells, mouse mononuclear macrophage leukemia (Raw 264.7) cells, and mouse connective tissue fibroblast (L929) cells. The experimental results are shown in Figure 2. Figure 15 、 Figure 16 、 Figure 17 and Figure 18 shown.

[0097] like Figure 15 and Figure 16 As shown, all cancer cell lines were successfully stained. In sharp contrast, the fluorescence signal obtained from normal cell lines was very weak. The fluorescence intensity extracted from cancer cells was about 1 times higher than that of normal cells ( Figure 17 In addition, if L929 cells and HeLa cells were subcultured and treated with AIE-1NPs or AIE-2NPs (50μg / mL), HeLa cells could still be stained by AIE nanoparticles, producing strong red fluorescence, while L929 cells only showed negligible fluorescence ( Figure 18 The above results indicate that both AIE-1NPs and AIE-2NPs can effectively distinguish normal cells from cancer cells, showing their potential as tumor-specific fluorescent tracers, which is beneficial to the production of intracellular reactive oxygen species and PDT effect in vitro.

[0098] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0099] The above-described embodiments merely represent several implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A nano-AIE probe, characterized in that: The product was synthesized by nanoprecipitation method using compound A as raw material and F-127 as surfactant; the general structural formula of compound A is as follows: Where R is 2. A method for preparing a nano-AIE probe according to claim 1, characterized in that: The following steps are involved: S1. Dissolve the reactants and potassium tert-butoxide in ethanol, then add compound 1 to react to obtain compound A; S2. Compound A and F-127 obtained in S1 are dissolved in dimethyl sulfoxide to obtain solution A. Ultrapure water is then added to solution A and ultrasonically mixed to obtain solution B. Solution B is added to a dialysis bag for dialyzation to obtain a nano-AIE probe.

3. The method for preparing the nano-AIE probe according to claim 2, wherein The reactant in S1 is 2-(4-(pyridin-4-yl)phenyl)acetonitrile or 4-pyridineacetonitrile.

4. The method for preparing the nano-AIE probe according to claim 3, wherein When the reactant is 2-(4-(pyridin-4-yl)phenyl)acetonitrile, the molar ratio of the reactant, the potassium tert-butoxide, and the compound 1 is 103:98:

34.

5. The method for preparing the nano-AIE probe according to claim 3, wherein When the reactant is 4-pyridineacetonitrile, the molar ratio of the reactant, the potassium tert-butoxide and the compound 1 is 3:3:

1.

6. The method for preparing the nano-AIE probe according to claim 2, wherein: The structural formula of the compound 1 is as follows:

7. The method for preparing the nano-AIE probe according to claim 2, wherein: The mass ratio of the compound A in S2 to the F-127 is 1:10; the volume ratio of the solution A to the ultrapure water is 1:

10.

8. The method for preparing the nano-AIE probe according to claim 2, wherein: After the S1 reaction is completed, the product is purified by an eluent, wherein the eluent is prepared by mixing petroleum ether and ethyl acetate in a volume ratio of 10-40:

1.

9. The nano-AIE probe according to claim 1 is used as a tumor-specific fluorescent tracer and photosensitizer in the field of tumors.

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