Cascaded activated fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescent probes, in particular to a cascade activated fluorescent probe as well as a preparation method and application thereof. The fluorescent probe has a structure as shown in a general formula (I): the diagnosis and treatment integrated fluorescent probe is obtained by combining an indoline group with an oxidative dehydrogenation reaction mechanism and an organic functional group with a distorted configuration, so that a near-infrared two-region fluorescent signal can be quickly activated under the action of ROS (reactive oxygen species), and a tumor focus is positioned; the system can be used as a light-activated guiding radar, can selectively illuminate a tumor area by using high space-time controllability of light, activates and amplifies the phototherapy activity according to needs, realizes effective killing of in-vivo and in-vitro tumor cells, and has important significance for precise tumor phototherapy under dual-mode imaging guidance.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and specifically to a cascade-activated fluorescent probe, a preparation method and application thereof, and more specifically to a fluorescent probe that responds to active oxygen and light cascade and has tumor localization-guided light-activated amplified phototherapy activity, and a preparation method and application thereof. Background Art

[0002] Photoactivated diagnostic and therapeutic technology, a significant breakthrough in precision medicine, leverages its advantages of non-invasiveness, precise spatiotemporal controllability, in situ activation, and low toxicity and side effects to activate fluorescent signals and phototherapeutic activity under illumination with specific wavelengths, providing a new strategy for tumor diagnosis and treatment. In recent years, a series of photoactivated fluorescent molecules based on different photochemical reaction mechanisms have been reported. However, these molecules generally rely on a single photoresponse mechanism, leaving the photoactivated molecules in a dark state at the tumor lesion site, making it impossible to pre-diagnose the tumor's location. Consequently, phototherapy requires the use of exogenous imaging techniques such as X-ray computed tomography, ultrasound, magnetic resonance imaging, photoacoustic imaging, single-photon emission computed tomography, and positron emission tomography. However, these techniques suffer from poor spatiotemporal resolution, low sensitivity, radiation hazards, and long signal collection times.

[0003] Fluorescence imaging has the advantages of high spatiotemporal resolution, in situ, real-time, and non-invasive, and is particularly suitable for biomedical research. However, the activation light source of most photoactivated molecules is ultraviolet light or blue light, and the emission wavelength is in the visible light region or the near-infrared region I (NIR-I, 700-900nm). There are defects such as shallow tissue penetration depth, strong biological tissue absorption and scattering, and fluorescence self-quenching in the aggregated state, which limits the application of photoactivated diagnostic molecules in in vivo tumor diagnosis and treatment. Aggregation-induced luminescence molecules can overcome the defects of fluorescence self-quenching and decreased phototherapeutic activity in the aggregated state. Therefore, the development of aggregation-induced near-infrared region II (NIR-II) diagnostic probes that integrate tumor localization and photoactivation is expected to break through the bottleneck that the photoactivated probes themselves cannot achieve tumor diagnosis and treatment.

[0004] The level of ROS in the tumor microenvironment is higher than that in normal tissue. ROS-activated NIR-II fluorescent probes can locate tumor lesions by fluorescent lighting, thereby achieving accurate diagnosis of tumors. However, ROS in tumors varies greatly from individual to individual and varies significantly in time and space, which can result in only partial activation of ROS-activated probes and poor phototherapy effects. Therefore, constructing ROS and light cascade-activated NIR-IIAIE probes, activating fluorescence signals through ROS in the tumor microenvironment, pre-locating the tumor site, and then selectively amplifying phototherapy activity at the tumor site through imaging-guided light activation is very important for accurate and effective tumor diagnosis and treatment. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies in the prior art, the present invention aims to provide a cascade-activated fluorescent probe and a preparation method and application thereof.

[0006] The object of the present invention is achieved by the following technical solution: a cascade-activated fluorescent probe having a structure shown in general formula (I):

[0007]

[0008] Among them, R 1 is any one of hydrogen, halogen, alkyl, alkoxy, perfluoroalkyl, aryloxy, alkenyl, alkynyl, cycloalkyl, alkylthio, amide, amino, monoalkylamino, dialkylamino, diarylamino, triarylvinyl, carboxamide, hydroxyl, thiol, aryl or heteroaryl;

[0009] P is absent, furyl, thienyl, selenophenyl, epoxyfuryl, epoxythienyl, epoxyselenophenyl, isobenzofuranyl, benzo[C]thienyl, benzo[C]selenophenyl, aryl, or heteroaryl;

[0010] Q is absent, furyl, thienyl, selenophenyl, epoxyfuryl, epoxythienyl, epoxyselenophenyl, isobenzofuranyl, benzo[C]thienyl, benzo[C]selenophenyl, benzoxadiazolyl, benzothiadiazolyl, benzoselenadiazolyl, aryl, or heteroaryl;

[0011] A is any one of dihydropyridyl, dihydroquinolinyl, 2,3,3-trimethyl-3H-dihydroindolinyl, 1,1,2-trimethyl-1H-benzo[e]dihydroindolinyl, 2,3,3-trimethyl-3H-benzo[g]dihydroindolinyl, and benzo[c,d]dihydroindolinyl.

[0012] Preferably, A is any one of 2,3,3-trimethyl-3H-dihydroindole, 1,1,2-trimethyl-1H-benzo[e]dihydroindole, 2,3,3-trimethyl-3H-benzo[g]dihydroindole or benzo[c,d]dihydroindole. Their structural formulas are as follows:

[0013]

[0014] Among them, R 2 It is any one of a saturated or partially saturated alkyl group containing 1 to 16 carbon atoms, a sulfonate group connected to a saturated or partially saturated alkyl group containing 1 to 16 carbon atoms, a carboxylate group connected to a saturated or partially saturated alkyl group containing 1 to 16 carbon atoms, an aryl group or a heteroaryl group.

[0015] More preferably, the R 1is hydrogen; P is phenyl; Q is thienyl, and the fluorescent probe has a structure shown in general formula (II):

[0016]

[0017] wherein A is any one of 2,3,3-trimethyl-3H-indolinyl, 1,1,2-trimethyl-1H-benzo[e]indolinyl, 2,3,3-trimethyl-3H-benzo[g]indolinyl, and benzo[c,d]indolinyl.

[0018] More preferably, the A is 2,3,3-trimethyl-3H-dihydroindole, and the fluorescent probe has a structure represented by the general formula (III):

[0019]

[0020] Among them, R 2 It is any one of a saturated or partially saturated alkyl group containing 1 to 16 carbon atoms, a sulfonate group connected to a saturated or partially saturated alkyl group containing 1 to 16 carbon atoms, a carboxylate group connected to a saturated or partially saturated alkyl group containing 1 to 16 carbon atoms, an aryl group or a heteroaryl group.

[0021] Preferably, the R 2 is a methyl group, and the fluorescent probe has a structure shown in general formula (IV):

[0022]

[0023] Preferably, the fluorescent probe is a cascade-activated near-infrared region II AIE probe, which is also a molecular probe for tumor localization guidance light activation amplification of phototherapy activity; the cascade-activated near-infrared region II AIE probe refers to an AIE probe that activates near-infrared region II fluorescence signals and phototherapy activity by ROS and light cascade activation; the near-infrared region II AIE probe that is activated by ROS and light cascade activation refers to an infrared region II AIE probe that is cascade-activated by hydroxyl radicals and light of 660 nm wavelength.

[0024] Another object of the present invention is achieved by the following technical solution: a method for preparing a cascade-activated fluorescent probe, comprising reacting a compound represented by general formula (V) with sodium borohydride in an organic solvent to prepare the fluorescent probe;

[0025]

[0026] Wherein, M is any one of 2,3,3-trimethyl-3H-indolium salt, 1,1,2-trimethyl-1H-benzo[e]indolium salt, 2,3,3-trimethyl-3H-benzo[g]indolium salt or benzo[c,d]indolium salt. Their structural formulas are as follows:

[0027]

[0028] Among them, R 2 As defined above.

[0029] The structural formula of the sodium borohydride is

[0030] More preferably, the M is 2,3,3-trimethyl-3H-indolium salt.

[0031] More preferably, the R 2 It is a methyl group.

[0032] Preferably, the molar ratio of the compound represented by general formula (V) to sodium borohydride is 1:1 to 1:20; the organic solvent is at least one of acetonitrile, N,N-dimethylformamide, tetrahydrofuran, methanol, ethanol or dimethyl sulfoxide; preferably methanol, ethanol; more preferably methanol; the reaction temperature is -80 to 65°C, preferably -80 to 25°C, more preferably 0°C; and the reaction time is 5 to 60 min.

[0033] Another object of the present invention is achieved through the following technical solution: the use of a cascade-activated fluorescent probe in the preparation of anti-tumor drugs and / or in vivo fluorescence imaging. The cascade-activated AIE probe of the present invention can activate a near-infrared second-region fluorescence signal in response to ROS in the tumor microenvironment, localizing the tumor. Acting as a light-activated radar, it guides light selectively to the tumor region, activating and amplifying phototherapeutic activity on demand, and effectively eliminating the tumor.

[0034] Preferably, the tumor comprises cervical cancer cells, breast cancer cells, ovarian cancer cells, lung cancer cells, or skin cancer cells; and the drug is a drug for photodynamic therapy or photothermal therapy. Photodynamic therapy and photothermal therapy utilize the high spatiotemporal controllability of near-infrared light to selectively irradiate superficial tumors or to irradiate deep tumor tissues via fiber optic delivery.

[0035] The beneficial effects of the present invention are: 1. The cascade-activated near-infrared second-region AIE probe of the present invention can achieve a specific response to hydroxyl radicals based on the chemical reaction mechanism of oxidative dehydrogenation of dihydroindole; at the same time, the activated molecules can efficiently generate hydroxyl radicals under near-infrared light irradiation, cyclically activate AIE molecules; and the photoactivation efficiency is positively correlated with the concentration of the activated molecules.

[0036] 2. The cascade-activated near-infrared zone II AIE probe of the present invention activates the near-infrared zone II fluorescence signal and phototherapy activity under the action of ROS in the tumor microenvironment, locates the tumor area, and serves as a guiding "radar" for light activation; utilizing the high spatiotemporal controllability of light, the tumor area is selectively illuminated, and the ROS-preactivated molecules can efficiently generate ROS, self-replenish to the tumor area, cyclically activate the molecular probes that have not yet been transformed, and amplify the phototherapy activity; ultimately achieving the effect of "localization first, then amplification of phototherapy", realizing the effective killing of tumor cells in vivo and in vitro, which is of great significance for precise tumor phototherapy under the guidance of dual-mode imaging.

[0037] 3. The cascade-activated near-infrared region II AIE probe of the present invention, under the cascade activation of hydroxyl free radicals and 660 nm laser, can achieve effective phototherapy of tumors under imaging guidance, while having good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 These are the hydrogen and carbon nuclear magnetic resonance spectra of compound III-1 in deuterated dimethyl sulfoxide.

[0039] Figure 2 This is the high-resolution mass spectrum of compound III-1.

[0040] Figure 3 These are the hydrogen and carbon nuclear magnetic resonance spectra of compound IV-1 in deuterated dimethyl sulfoxide.

[0041] Figure 4 This is the high-resolution mass spectrum of compound IV-1.

[0042] Figure 5 (A) Normalized UV-visible absorption spectra of TT-DHIn and TT-In; (B) Fluorescence spectrum of TT-In; (C) Fluorescence spectra and (D) relative fluorescence intensity of TT-In in different proportions of toluene.

[0043] Figure 6 (A) Reactive oxygen species generation of TT-In; (B) Superoxide anion generation; (C) Hydroxyl radical generation; (D) Thermal imaging at different powers; (E) Different concentrations of TT-In under 660nm laser (power 0.5W / cm 2 ) Temperature change curve under irradiation; (F) Photothermal cycle diagram.

[0044] Figure 7 (A) Schematic diagram of ROS and light cascade activation of TT-DHIn; laser light at 660 nm (0.2 W / cm 2) irradiation of TT-DHIn and TT-DHIn+MB (B) relative absorption at 554 nm and (C) relative PL intensity at 1000 nm; (D) at 660 nm laser (0.2 W / cm 2 ) irradiated by 660 nm laser (0.2 W / cm 2 ) containing different ratios of TT-DHIn (E) relative absorption at 554 nm and (F) relative PL intensity at 1000 nm; (G) at 660 nm laser (0.2 W / cm 2 ) Near-infrared zone II imaging of MDA-MB-231 cells containing different ratios of TT-DHIn under irradiation; (H) CLSM images of MDA-MB-231 cells co-treated with TT-DHIn and MB under 638 nm laser irradiation (40% power); (I) CLSM images of MDA-MB-231 multicellular tumor spheroids treated with TT-DHIn before and after 638 nm laser irradiation (40% power).

[0045] Figure 8 (A) Relative absorption intensity of TT-DHIn+NaN3 and TT-DHIn+Trolox containing 20% ​​TT-In at 554 nm as a function of illumination time under 660 nm laser irradiation; (B) UV-visible absorption spectra, (C) fluorescence spectra, (D) relative PL intensity at 1000 nm, and (E) NIR-II fluorescence imaging of TT-DHIn after treatment with different ROS or RNS; (F) UV-visible absorption spectra, (G) relative absorption at 554 nm, (H) fluorescence spectra, and (I) relative fluorescence intensity at 1000 nm of TT-DHIn treated with different concentrations of ·OH.

[0046] Figure 9 (A) Intracellular reactive oxygen species generation in different treatment groups; (B) Live-dead staining of cells in different treatment groups; (C) Cell viability of breast cancer cells (MDA-MB-231) and (D) normal breast cells (MCF-10A) after treatment with different concentrations of probes.

[0047] Figure 10 (A) In vivo NIR-II imaging of tumor-bearing mice, (B) Changes in tumor temperature over time in tumor-bearing mice under light irradiation, (C) Changes in tumor volume over time in nude mice, (D) Photos of tumors in each group of mice after 21 days of treatment, (E) Hematoxylin-eosin staining and TUNEL images of tumor sections.

[0048] Figure 11 This is the hematoxylin-eosin staining image of the fluorescent probe TT-DHIn.

[0049] Figure 12 Diagram of the mechanism of the fluorescent probe TT-DHIn in tumor and imaging applications. DETAILED DESCRIPTION

[0050] In order to facilitate the understanding of those skilled in the art, the following embodiments and accompanying drawings are provided. Figures 1 to 12 The present invention is further described, and the contents mentioned in the embodiment are not intended to limit the present invention.

[0051] Example 1 Preparation of cascade-activated NIR-IIAIE probe

[0052] Compound I-1 (213 mg, 0.6 mmol) and compound II-1 (158 mg, 0.5 mmol) were added to 10 mL of anhydrous ethanol, refluxed under nitrogen protection, and stirred for 10 hours. Cooled to room temperature, the solvent was dried and dissolved in acetone, 3 mL of saturated KPF6 solution was added, and the reaction was stirred for 6 hours. Subsequently, the acetone was dried, the precipitate was filtered, washed with water, and dried in a vacuum drying oven. Finally, compound III-1 (TT-In) was separated by silica gel column (DCM: MeOH = 20: 1) with a yield of 88%. The nuclear magnetic resonance hydrogen spectrum and carbon spectrum of the compound III-1 in deuterated dimethyl sulfoxide are shown as follows Figure 1 shown. 1 H NMR(500MHz, DMSO-d6)δ8.68(d,J=15.7Hz,1H),8.23(d,J=4.1Hz,1H),7.89–7.83(m,2H),7.76–7.70(m,3H),7.63–7.55(m, 2H),7.38(t,J=7.9Hz,4H),7.24–7.11(m,7H),7.00–6.96(m,2H),4.62(q,J=7.2Hz,2H),1.78(s,6H),1.43(t,J=7.2Hz,3H). 13 C NMR (126 MHz, DMSO-d6) δ 179.81, 154.54, 148.97, 146.22, 146.09, 143.52, 140.53, 140.35, 138.24, 129.86, 129.06, 128.74, 127.47, 125.41, 125.27, 124.43, 123.03, 121.30, 114.49, 108.80, 51.72, 41.51, 25.77, 13.53. The high-resolution mass spectrum of compound III-1 is shown in FIG. Figure 2 HRMS (ESI): m / z [M] + calcd for C 36 H 33N2S:525.2359,found:525.2355.

[0053] Compound III-1 (196 mg, 0.3 mmol) was added to 20 mL of methanol and stirred rapidly at 0 ° C. Then, sodium borohydride (13 mg, 0.36 mmol) dissolved in ethanol was slowly added dropwise. After reacting for 10 minutes, the solvent was dried by spin drying, and 10 mL of ice water was slowly added to quench the sodium borohydride. Subsequently, it was extracted with DCM, the solvent was dried by spin drying, and the compound IV-1 (TT-DHIn) was separated by silica gel column (petroleum ether: DCM = 3: 1) with a yield of 68%. The H NMR spectrum and C NMR spectrum of the compound IV-1 in deuterated dimethyl sulfoxide are shown as follows. Figure 3 shown. 1 H NMR(500MHz,DMSO-d6)δ7.56(dd,J=8.8,2.3Hz,2H),7.35–7.30(m,5H),7.12–7.0 0(m,9H),6.98–6.94(m,2H),6.90(dd,J=15.6,1.7Hz,1H),6.62(t,J=7.3Hz,1H), 6.54–6.49(m,1H),5.99(dd,J=15.7,9.1Hz,1H),3.70(d,J=9.0Hz,1H),3.31–3.2 7(m,1H),3.03(dq,J=14.1,7.0Hz,1H),1.27(d,J=1.2Hz,3H),1.02–0.97(m,6H). 13 CNMR (126 MHz, DMSO-d6) δ 149.60, 147.34, 147.27, 142.45, 140.34, 138.89, 130.11, 128.42, 127.90, 127.82, 127.58, 126.99, 126.86, 124.80, 123.93, 123.64, 123.38, 122.15, 117.91, 107.74, 75.92, 44.43, 26.25, 24.58, 10.33. The high resolution mass spectrum of compound III-1 is shown in FIG. Figure 4 HRMS(ESI):m / z[M]+calcd for C 36 H 34 N2S:526.2443; found:526.2418.

[0054] The reaction formula is as follows:

[0055]

[0056] Example 2: Characterization of the photophysical properties of fluorescent probes TT-DHIn and TT-In

[0057] like Figure 5 As shown, the TT-DHIn and TT-In synthesized in Example 1 were subjected to UV-visible absorption spectroscopy and fluorescence spectroscopy detection, and the fluorescence intensity change of TT-In in different proportions of poor solvents was observed. Figure 5 A shows the UV-visible absorption spectra of the molecular probes TT-DHIn and TT-In. The maximum absorption peaks of TT-DHIn and TT-In are located at 383 nm and 554 nm, respectively. Compared with TT-DHIn, the maximum absorption wavelength of TT-In is red-shifted by 171 nm, which is very suitable for monitoring the conversion of TT-DHIn to TT-In via oxidative dehydrogenation. The emission tail of TT-In, which is well soluble in DMSO, is located in the NIR-II window ( Figure 5 B), which may be attributed to the strong donor-acceptor effect. As the amount of poor solvent (toluene) added increases from 0 to 90%, the fluorescence intensity increases by 3.9 times, showing a typical AIE characteristic ( Figure 5 C-5D).

[0058] Example 3: Evaluation of the Phototherapeutic Activity of the Fluorescent Probe TT-In

[0059] DCFH was used as an indicator to characterize the generation of reactive oxygen species (ROS) in PBS solution of TT-In synthesized in Example 1. Figure 6 As shown in A, after irradiation with 660nm laser for 50s, the fluorescence intensity of TT-In+DCFH group increased by 143 times, indicating that TT-In has excellent photodynamic activity. Figure 6 B and Figure 6 C respectively confirmed that TT-In can effectively generate type I ROS (O2 ·- and ·OH), indicating that TT-In has excellent type I photodynamic activity.

[0060] The photothermal activity of TT-In synthesized in Example 1 under 660nm laser irradiation. Figure 6 C-6D, the temperature change of TT-In is correlated with the laser power and the concentration of TT-In. TT-In (200μM) is 2 Under 660nm laser irradiation, the temperature rises to above 95℃, indicating that TT-In has excellent photothermal activity. At the same time, TT-In shows good photostability ( Figure 6 E).

[0061] Example 4: Evaluation of ROS / light cascade activation performance of fluorescent probe TT-DHIn

[0062] Figure 7 A is a schematic diagram of the ROS and light cascade activation of TT-DHIn. Methylene blue (MB) was used as a photosensitizer to verify the responsiveness of the fluorescent probe TT-DHIn synthesized in Example 1 to ROS. 2 ) irradiated for 10 min, the absorption at 554 nm and the emission intensity at 1000 nm of the TT-DHIn+MB group increased by 9.4 and 14.5 times, respectively. This was mainly due to the oxidative dehydrogenation reaction of TT-DHIn under the action of ROS to convert it into TT-In( Figure 7 B-7C). At the same time Figure 7 NIR-II fluorescence imaging of D further confirmed the responsiveness of TT-DHIn to ROS.

[0063] By adding different proportions of TT-In to the TT-DHIn synthesized in Example 1, the response performance of TT-DHIn to light at different activation levels was simulated. Figure 7 As shown in E-7F, as the TT-In ratio increased from 0 to 40%, the absorption at 554 nm and the emission intensity at 1000 nm of TT-DHIn increased by 157 and 87.5 times, respectively; at the same time, NIR-II fluorescence imaging also showed similar results ( Figure 7 G), which is mainly due to the ROS generated during the photoactivation of TT-In oxidizing TT-DHIn to TT-In, confirming that the photoactivation performance of TT-DHIn is positively correlated with the concentration of TT-In.

[0064] The performance of the TT-DHIn synthesized in Example 1 in terms of ROS and light cascade activation at the cellular level was investigated. Figure 7 As shown in H, after the breast cancer cells MDA-MB-231 were co-treated with TT-DHIn and MB, the fluorescence intensity gradually increased with the extension of the illumination time. At the same time, the confocal images of the in vitro three-dimensional tumor spheres co-incubated with TT-DHIn further confirmed the cascade activation characteristics of TT-DHIn on ROS and light ( Figure 7 I), is particularly suitable for applications in tumor localization and light-activated anti-tumor therapy.

[0065] Example 5: Study on the ROS / light cascade activation mechanism of the fluorescent probe TT-DHIn

[0066] The activation mechanism of the ROS- and light-induced cascade-activated fluorescent probe TT-DHIn synthesized in Example 1 was studied. By adding different types of ROS quenchers to a TT-DHIn solution containing 20% ​​TT-In, it was verified whether TT-In was activated by type I or type II ROS. Figure 8As shown in Figure A, when the free radical quencher Trolox is added to the mixed system of TT-DHIn and TT-In, the absorption intensity of TT-DHIn at 554 nm remains unchanged; however, regardless of whether the singlet oxygen quencher NaN3 is added or not, the absorption intensity of TT-DHIn at 554 nm shows a similar enhancement, indicating that TT-DHIn is a free radical-induced oxidative dehydrogenation reaction.

[0067] The response mechanism of the ROS- and light-activated fluorescent probe TT-DHIn synthesized in Example 1 to free radicals was further studied. Figure 8 B-8E, ROS and light cascade activated fluorescent probe TT-DHIn showed a specific response to ·OH. At the same time, the absorption intensity of TT-DHIn at 554nm and the fluorescence intensity at 1000nm showed excellent ·OH concentration dependence ( Figure 8 F-8I).

[0068] Example 6: Evaluation of the Phototherapeutic Activity of the Fluorescent Probe TT-DHIn

[0069] The ROS generation performance of the ROS and light cascade activated fluorescent probe TT-DHIn synthesized in Example 1 was evaluated using DCFH-DA as an indicator. After co-staining with TT-DHIn and DCFH-DA, the ROS generation performance of the fluorescent probe TT-DHIn in cells was evaluated at 660 nm (0.5 W / cm 2 ) After 10 minutes of laser irradiation, a strong green fluorescence signal appeared in MDA-MB-231 cells, indicating a high ROS generation capacity ( Figure 9 A). Subsequently, MDA-MB-321 cells treated with TT-DHIn were irradiated with 660 nm laser for 10 min and then stained with PI (dead cell indicator) ( Figure 9 B); while green fluorescence (live cell indicator) was observed in the entire area in the control group, laser group and TT-DHIn group. The results showed that TT-DHIn can effectively kill tumor cells in vitro under 660nm laser irradiation. In addition, we also evaluated the phototherapy effect of TT-DHI under 660nm laser irradiation. Figure 9 As shown in Figure C, after 10 minutes of 660nm laser irradiation, the cell viability of MDA-MB-231 cells treated with TT-DHIn (10μM) decreased to 26%, while in the dark, MDA-MB-231 cells still maintained a high cell viability. Interestingly, even at a high concentration of 100μM, TT-DHIn still showed a high cell viability in normal breast cells MCF-10A, which proves that TT-DHIn has good biosafety ( Figure 9 D).

[0070] Example 7: In vivo imaging and anti-tumor activity evaluation of the fluorescent probe TT-DHIn

[0071] In order to verify the ROS and light cascade activation properties of the cascade-activated fluorescent probe TT-DHIn synthesized in Example 1 at the in vivo animal level, we established an MDA-MB-231 tumor nude mouse model. Figure 10 As shown in A, after injection of TT-DHIn, a bright NIR-II fluorescence signal was observed at the tumor site with a signal-to-noise ratio of up to 11.5. This is because under the activation of ROS in the tumor microenvironment, TT-DHIn is rapidly converted into TT-In through oxidative dehydrogenation. Therefore, the NIR-II fluorescence signal at the tumor site can be used as a "guiding radar" for light activation. By utilizing the high spatiotemporal controllability of light, the tumor is selectively irradiated with a 660nm laser for 5 minutes. Compared with before light activation, the NIR-II fluorescence intensity at the tumor site is significantly enhanced. This is because under 660nm laser irradiation (0.5W / cm 2 ), the ROS generated by TT-In in the tumor self-replenishes the ROS in the tumor area. In addition, the inactivated TT-DHIn in the tumor site can also be recycled and converted into TT-In, enhancing its phototherapy activity. It is worth noting that under 660nm laser irradiation for 10 minutes, the tumor temperature of mice treated with TT-DHIn reached 53.1℃ ( Figure 10 B), indicating excellent photothermal activity. In contrast, the control group showed small body temperature fluctuations. Subsequently, we systematically studied the in vivo anti-tumor effect of TT-DHIn. Figure 10 As shown in Figure C, compared with the other groups, the MDA-MB-231 nude mouse tumors treated with TT-DHIn and TT-In were effectively eliminated under 660nm laser irradiation. At the same time, tumor tissues of different treatment groups were dissected after 21 days of treatment. The photos of the ex vivo tumor tissues, hematoxylin-eosin staining and TUNEL staining further verified the significant phototherapy effect of the TT-DHIn + light irradiation group and the TT-In + light irradiation group on tumors ( Figure 10 D-10E), which was due to the efficient activation of phototherapeutic activity of TT-DHIn under ROS and 660 nm laser irradiation.

[0072] Example 8: Biosafety of the fluorescent probe TT-DHIn

[0073] Biosafety verification of the ROS and light cascade activated fluorescent probe TT-DHIn synthesized in Example 1. Figure 11 As shown in the hematoxylin-eosin staining of the fluorescent probe TT-DHIn, there is no obvious inflammation or damage, confirming that the ROS- and light-activated fluorescent probe TT-DHIn has good biosafety.

[0074] The above embodiments are preferred implementation schemes of the present invention. In addition, the present invention can also be implemented in other ways. Any obvious replacement without departing from the concept of the present invention is within the scope of protection of the present invention.

Claims

1. A cascade-activated fluorescent probe, characterized in that: The fluorescent probe has a structure shown in general formula (I): Among them, R 1 is any one of hydrogen, halogen, alkyl, alkoxy, perfluoroalkyl, aryloxy, alkenyl, alkynyl, cycloalkyl, alkylthio, amide, amino, monoalkylamino, dialkylamino, diarylamino, triarylvinyl, carboxamide, hydroxyl, thiol, aryl or heteroaryl; P is absent, furyl, thienyl, selenophenyl, epoxyfuryl, epoxythienyl, epoxyselenophenyl, isobenzofuranyl, benzo[C]thienyl, benzo[C]selenophenyl, aryl, or heteroaryl; Q is absent, furyl, thienyl, selenophenyl, epoxyfuryl, epoxythienyl, epoxyselenophenyl, isobenzofuranyl, benzo[C]thienyl, benzo[C]selenophenyl, benzoxadiazolyl, benzothiadiazolyl, benzoselenadiazolyl, aryl, or heteroaryl; A is any one of dihydropyridyl, dihydroquinolinyl, 2,3,3-trimethyl-3H-dihydroindolinyl, 1,1,2-trimethyl-1H-benzo[e]dihydroindolinyl, 2,3,3-trimethyl-3H-benzo[g]dihydroindolinyl, and benzo[c,d]dihydroindolinyl.

2. A cascade-activated fluorescent probe according to claim 1, characterized in that: The R 1 is hydrogen; P is phenyl; Q is thienyl, and the fluorescent probe has a structure shown in general formula (II): wherein A is any one of 2,3,3-trimethyl-3H-indolinyl, 1,1,2-trimethyl-1H-benzo[e]indolinyl, 2,3,3-trimethyl-3H-benzo[g]indolinyl, and benzo[c,d]indolinyl.

3. A cascade-activated fluorescent probe according to claim 2, characterized in that: The A is 2,3,3-trimethyl-3H-dihydroindole, and the fluorescent probe has a structure shown in the general formula (III): Among them, R 2 It is any one of a saturated or partially saturated alkyl group containing 1 to 16 carbon atoms, a sulfonate group connected to a saturated or partially saturated alkyl group containing 1 to 16 carbon atoms, a carboxylate group connected to a saturated or partially saturated alkyl group containing 1 to 16 carbon atoms, an aryl group or a heteroaryl group.

4. The cascade-activated fluorescent probe according to claim 3, characterized in that: The R 2 is a methyl group, and the fluorescent probe has a structure shown in general formula (IV):

5. The cascade-activated fluorescent probe according to claim 1, characterized in that: The fluorescent probe is a cascade-activated near-infrared second-region AIE probe, and is also a molecular probe that guides light activation and amplifies phototherapy activity for tumor localization.

6. The method for preparing a cascade-activated fluorescent probe according to claim 2, wherein: reacting the compound represented by general formula (V) with sodium borohydride in an organic solvent to prepare the fluorescent probe; Here, M is any one of 2,3,3-trimethyl-3H-indolium salt, 1,1,2-trimethyl-1H-benzo[e]indolium salt, 2,3,3-trimethyl-3H-benzo[g]indolium salt, or benzo[c,d]indolium salt.

7. The method for preparing a cascade-activated fluorescent probe according to claim 6, wherein: The M is 2,3,3-trimethyl-3H-indolium salt.

8. The method for preparing a cascade-activated fluorescent probe according to claim 6, wherein: The molar ratio of the compound represented by general formula (V) to sodium borohydride is 1:1 to 1:20; the organic solvent is at least one of acetonitrile, N,N-dimethylformamide, tetrahydrofuran, methanol, ethanol or dimethyl sulfoxide; the reaction temperature is -80 to 65°C, and the reaction time is 5 to 60 minutes.

9. Use of a cascade-activated fluorescent probe according to any one of claims 1 to 5 in the preparation of anti-tumor drugs and / or in vivo fluorescence imaging.

10. The use according to claim 9, characterized in that: The tumor includes a tumor formed by cervical cancer cells, breast cancer cells, ovarian cancer cells, lung cancer cells or skin cancer cells; and the drug is a drug for photodynamic therapy and photothermal therapy.

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