Lysosome targeting probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a lysosome targeting probe and a preparation method and application thereof. The structural formula of the lysosome targeting probe is shown as a formula (I): # imgabs0 #, in the formula (I), A is selected from an oxygen atom and a sulfur atom, # imgabs1 # R1 is selected from a hydrogen atom or an aromatic ring group, R2 is selected from a cyano group or a group shown as a formula (II), R3 is selected from an aromatic ring group, and R4 is selected from a chain alkyl group with 1-20 hydrogen atoms or carbon atoms. The compound can specifically target lysosome in tumor cells, emit bright fluorescence, have a good fluorescence co-localization effect with the lysosome in the tumor cells and can be used for lysosome imaging, the fluorescence intensity of the compound is in positive correlation with the viscosity of the lysosome in living cells such as the tumor cells, and the fluorescence intensity of the compound is in positive correlation with the viscosity of the lysosome in the living cells such as the tumor cells. The viscosity change condition of lysosome in living cells can be monitored under various conditions; the compound can be used as a photosensitizer and a treatment sensitizer, specifically ablates tumors through photodynamic therapy (PDT), recovers the sensitivity of tumor cells to radiotherapy and chemotherapy, and enhances the treatment effect of radiotherapy and chemotherapy.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a lysosome targeting probe and a preparation method and application thereof. Background Art

[0002] Drug resistance is a major challenge in clinical tumor treatment and one of the main factors leading to the failure of cancer treatment. It may lead to rapid progression or recurrence of cancer or even death of patients. It is one of the most challenging problems faced by cancer patients and their families, medical staff and cancer researchers. Overcoming drug resistance, improving the clinical treatment effect of cancer, and ultimately improving patient survival are the goals of cancer treatment, but they are also extremely challenging. The development of tumor treatment resistance antagonists has great clinical application value and is expected to bring new hope to clinical tumor treatment. Using these preparations, the sensitivity of tumor cells to treatments such as radiotherapy and chemotherapy can be restored, thereby improving their efficacy. Taking advantage of the advantages of combined therapy is a potential effective strategy to break the treatment bottleneck. Therefore, the development of therapeutic sensitizers that can be integrated and adapted to conventional treatments (radiotherapy and chemotherapy) to overcome treatment resistance is expected to bring new breakthroughs to tumor treatment.

[0003] Lysosomes are important organelles that play key roles in secretion, degradation, and signal transduction, regulating cell proliferation, differentiation, metabolism, and apoptosis. In tumor cells, lysosomes undergo a variety of changes associated with malignant transformation, including changes in lysosomal number, morphology, polarity, and viscosity, as well as changes in lysosomal protein expression. Lysosomes play a vital role in tumor development and therapeutic resistance, and are also effective potential targets for the development of therapeutic sensitizers. The development of therapeutic sensitizers that can specifically target tumor cell lysosomes while minimizing side effects on normal tissues can provide a more effective strategy for overcoming drug resistance in tumor treatment. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the present invention provides a lysosome targeting probe and a preparation method and application thereof to solve the problem of drug resistance in tumor treatment.

[0005] To achieve the above objectives, the scheme of this application is as follows:

[0006] In a first aspect, the present invention provides a fluorescent compound (i.e., a lysosome targeting probe), the structural formula of the fluorescent compound is shown in formula (I):

[0007]

[0008]

[0009] In formula (I), A is selected from oxygen atom, sulfur atom, R1 is selected from a hydrogen atom or an aromatic ring group; R2 is selected from a cyano group or a group represented by formula (II); R3 is selected from an aromatic ring group; and R4 is selected from a hydrogen atom or a chain alkyl group having 1 to 20 carbon atoms.

[0010] Optionally, R1 is selected from any one of the groups represented by formulae (1) to (35), and R3 is selected from any one of the groups represented by formulae (31) to (35):

[0011]

[0012] Among them, R5 is selected from a chain alkyl or cycloalkyl group having 1 to 20 carbon atoms and 0 or some carbon atoms are substituted by oxygen atoms, alkenyl groups, alkynyl groups, aryl groups, carbonyl groups, hydroxyl groups, amino groups, carboxyl groups, cyano groups, nitro groups or ester groups; R6 is selected from a chain alkyl or cycloalkyl group having 1 to 20 carbon atoms and 0 or some carbon atoms are substituted by oxygen atoms, alkenyl groups, alkynyl groups, aryl groups, carbonyl groups, hydroxyl groups, amino groups, carboxyl groups, cyano groups, nitro groups or ester groups; R7 is selected from a chain alkyl or cycloalkyl group having 1 to 20 carbon atoms and 0 or some carbon atoms are substituted by oxygen atoms, alkenyl groups, alkynyl groups, aryl groups, carbonyl groups, hydroxyl groups, amino groups, carboxyl groups, cyano groups, nitro groups or ester groups.

[0013] Optionally, A is selected from R1 is selected from a hydrogen atom, R2 is selected from a cyano group, R3 is selected from a group represented by formula (35), and R4, R5 and R7 are all selected from a methyl group, that is, the structure of the compound represented by formula (I) is as shown in formula (III):

[0014]

[0015] In a second aspect, the present invention also provides a method for preparing the fluorescent compound as described above, comprising the following steps:

[0016] The compound with active α-hydrogen atom and the compound containing aldehyde group are dissolved in a solvent, and reflux reaction is carried out in a protective gas atmosphere under the action of a catalyst to prepare the fluorescent compound.

[0017] For example, when the structural formula of the fluorescent compound is as shown in formula (III), the compound having an active α-hydrogen atom may include the following compounds:

[0018]

[0019] The aldehyde group-containing compounds include the following compounds:

[0020]

[0021] Optionally, the solvent is selected from at least one of anhydrous ethanol, methanol, acetonitrile, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide and dimethyl sulfoxide.

[0022] Optionally, the catalyst is selected from at least one of amine, pyridine, piperidine and morpholine.

[0023] Optionally, the protective gas is selected from at least one of nitrogen, helium and argon.

[0024] Optionally, the temperature of the reflux reaction is 80-120°C.

[0025] Optionally, after the reflux reaction, the following step is further included: purification.

[0026] In the present application, the purification includes: filtration, washing, column chromatography separation and purification, and drying.

[0027] In a third aspect, the present invention also provides use of the fluorescent compound as described above or the fluorescent compound prepared according to the method as described above in lysosome targeted imaging.

[0028] The fluorescent compound of the present invention has excellent aggregation-induced emission (AIE) properties, that is, it emits weak light in solution, but the emission is significantly enhanced in the aggregated state.

[0029] The fluorescent compound of the present invention can specifically target lysosomes in tumor cells, emit bright fluorescence, and have a good fluorescence co-localization effect with lysosomes in tumor cells, can be used for lysosome imaging, and has the characteristics of high fluorescence intensity and high reactive oxygen species (ROS) generation rate.

[0030] The fluorescence intensity of the fluorescent compound of the present invention is positively correlated with the viscosity of lysosomes in living cells such as tumor cells. It can monitor the viscosity changes of lysosomes in living cells under various conditions (such as inflammation and autophagy) and can effectively distinguish tumor cells from normal cells.

[0031] In a fourth aspect, the present invention also provides use of the fluorescent compound as described above or the fluorescent compound prepared according to the method as described above in the preparation of a drug for assisting photodynamic therapy (PDT) for tumor treatment.

[0032] In the present application, tumors include but are not limited to the following types: lung cancer, liver cancer, breast cancer, colorectal cancer, pancreatic cancer, osteosarcoma, brain cancer, kidney cancer, thyroid cancer, lymphoma, bile duct cancer, uterine cancer, prostate cancer, and colon cancer.

[0033] Under the irradiation of light source, the fluorescent compound of the present invention can promote the generation of a large amount of ROS in tumor cells, trigger lysosomal membrane permeabilization (LMP) and lysosomal rupture, and show excellent PDT effect. In addition, the compound has good biocompatibility and small toxic and side effects.

[0034] In a fifth aspect, the present invention also provides use of the fluorescent compound as described above or the fluorescent compound prepared according to the method as described above as a radiation sensitizer.

[0035] Under the irradiation of light source, the fluorescent compound of the present invention can significantly enhance the radiotherapy effect by inducing ROS generation and accelerating cell apoptosis, showing the potential as a radiosensitizer.

[0036] In a sixth aspect, the present invention also provides use of the fluorescent compound as described above or the fluorescent compound prepared according to the method as described above as a chemosensitizer.

[0037] Under the irradiation of light source, the fluorescent compound of the present invention significantly enhances the chemotherapy effect by inducing ROS generation, triggering ROS-mediated LMP and accelerating the apoptosis of drug-resistant tumor cells, showing the potential as a chemotherapy sensitizer.

[0038] The fluorescent compound of the present invention can be used as a photosensitizer and a therapeutic sensitizer to specifically ablate tumors through PDT, restore the sensitivity of tumor cells to radiotherapy and chemotherapy, and enhance the therapeutic effect of radiotherapy and chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0040] Figure 1 is the NMR spectrum, Figure 1 A is the H NMR spectrum, Figure 1 B is the NMR carbon spectrum;

[0041] Figure 2 This is the result of the photophysical property test. Figure 2 A is the UV-visible absorption spectrum, Figure 2 B is the fluorescence emission spectrum, Wavelength represents wavelength, Molar absorptivity represents molar absorption coefficient, and PL intensity represents fluorescence intensity;

[0042] Figure 3 The results of the lysosome-specific cell imaging test and the lysosome viscosity-specific response test are shown in Figure 1. Figure 3 A is the result of lysosome-specific cell imaging test. Figure 3 B is the result of lysosomal viscosity-specific response test, scale bar: 20 μm;

[0043] Figure 4 This is a graph showing the test results for monitoring changes in lysosomal viscosity under pathological conditions;

[0044] Figure 5 This is a graph showing the results of a cell autophagy monitoring test;

[0045] Figure 6 This is a diagram of the tumor cell identification test results;

[0046] Figure 7 The results of Part 8.1 (A) and Part 8.2 (B, C, D) in the PDT photosensitizer (i.e., photodynamic therapy photosensitizer) test are shown in Figure 1. Scale bar: 20 μm.

[0047] Figure 8 This is a diagram showing the results of the study on the mechanism of selective targeted therapy of tumor cell lysosomes. Scale bar: 20 μm.

[0048] Fig. 9 The figure is the result of in vivo PDT efficacy test; Tumor volume means tumor volume, Time (day) means time (day), the same below;

[0049] Fig.10 This is the result of the radiotherapy sensitization test. Survival fraction indicates the cell survival fraction;

[0050] Fig.11 The results of Section 12.1 (A), Section 12.2 (B), and Section 12.3 (C) in the chemotherapy sensitization test are shown in Figure 12. Cellviability indicates cell viability, and the scale bar is 20 μm.

[0051] Fig.12 This is the result of part 12.4 in the chemotherapy sensitization test, scale bar: 20μm;

[0052] Fig.13 This is the result diagram of Section 12.5 in the chemotherapy sensitization test;

[0053] Fig.14 This is the result diagram of part 12.6 in the chemotherapy sensitization study. DETAILED DESCRIPTION

[0054] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0055] In the following description, numerous details are discussed to provide a more thorough explanation of embodiments of the present invention; however, it is apparent to one skilled in the art that embodiments of the present invention may be practiced without these specific details.

[0056] (I) Preparation and structural characterization of the compound represented by formula (III)

[0057] The compound represented by formula (III) (hereinafter referred to as QM-DMAC) is prepared by the following specific steps:

[0058]

[0059] 1.1 mmol of compound 1 (the structure of compound 1 is shown above, commercially available) and 1.1 mmol of compound 2 (the structure of compound 2 is shown above, commercially available) are dissolved in 5 mL of anhydrous acetonitrile liquid, and several drops of piperidine (i.e., Piperidine) are added to the anhydrous acetonitrile liquid as a catalyst, and mixed to obtain a solution;

[0060] The above solution was reacted at 80° C. under nitrogen protection overnight. After the reaction was completed, a precipitate was precipitated and filtered. The filter cake was washed with acetonitrile, separated and purified by column chromatography, and dried in vacuo to obtain 52 mg of an orange powder product QM-DMAC (i.e., the compound represented by formula (III), the chemical reaction formula is shown above), with a yield of 10%.

[0061] The product was characterized by nuclear magnetic resonance spectrometer (Bruker Avance 600MHz). Figure 1 shown.

[0062] like Figure 1 As shown, the nuclear magnetic hydrogen spectrum characterization data of the product is: 1 H NMR (600MHz, CDCl3) δ9.17 (dd, J=8.5, 1.2Hz, 1H), 7.83 (d, J=8.3Hz, 2H), 7.81-7.78 (m, 1H), 7.63 (d, J=8.6Hz, 1H), 7.52-7.49 (m, 1H), 7.48 (dd, J=7.6, 1.5Hz, 2H), 7.44 -7.41 (m, 3H), 7.19 (s, 1H), 7.16 (d, J=15.8Hz, 1H), 7.00 (td, J=8.2, 7.7, 1.6Hz, 2H) , 6.95 (td, J=7.5, 1.3Hz, 2H), 6.30 (dd, J=8.1, 1.1Hz, 2H), 3.96 (s, 3H), 1.70 (s, 6H) ( Figure 1 A); the NMR carbon spectrum characterization data of the product is: 13C NMR (150MHz, CDCl3) δ153.7, 148.1, 143.0, 140.6, 139.4, 138.9, 134.6, 133.4, 132.1, 130.3, 130.1, 126. 9, 126.4, 125.4, 124.9, 121.4, 121.1, 120.9, 119.8, 118.8, 116.1, 114.0, 108.0, 52.8, 37.0, 36.03, 31.2( Figure 1 B). The results show that the compound represented by formula (III) was successfully prepared in the present application.

[0063] (II) Photophysical property test

[0064] The product obtained in step (I) was subjected to UV-visible absorption spectrum detection using a UV-visible spectrometer. The specific steps were as follows: using dimethyl sulfoxide (DMSO) as solvent, the product was prepared to a concentration of 1.0×10 -5 mol / L QM-DMAC solution was placed in a cuvette and the UV-visible absorption spectrum was detected using a UV-visible spectrometer. The results were as follows: Figure 2 As shown in A;

[0065] The fluorescence emission spectrum of the product obtained in (I) was detected by using a fluorescence spectrometer. The specific steps were as follows: dimethyl sulfoxide and water were mixed in different proportions to obtain liquids with water volume contents of 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% and 90%, respectively; the above liquids were used to prepare products with a concentration of 1.0×10 -5 mol / L QM-DMAC solution, and the fluorescence emission spectrum of each QM-DMAC solution was detected by fluorescence spectrometer. The results are as follows Figure 2 As shown in B.

[0066] Depend on Figure 2 It can be seen that the maximum absorption peak of QM-DMAC in dimethyl sulfoxide is located at a wavelength of 445nm ( Figure 2 A). The fluorescence signal of QM-DMAC in dimethyl sulfoxide is weak. As the water content in the system increases, the fluorescence intensity increases significantly ( Figure 2 B), the reason is that as the water content in the system increases, the solubility of QM-DMAC in the system gradually decreases, and then aggregates are formed, the intramolecular motion is restricted, and the aggregation-induced emission (AIE) effect is activated. This result shows that the compound of the present invention has excellent aggregation-induced emission (AIE) properties, that is, it emits weak fluorescence in solution, but the luminescence is significantly enhanced in the aggregated state.

[0067] (III) Lysosome-specific cell imaging assay

[0068] The product obtained in (a) was imaged using tumor cells for live cell imaging, and then analyzed using a laser confocal microscope. The specific steps are as follows:

[0069] A549 cells were mixed with the cell culture medium containing the product (QM-DMAC, 20 μM) prepared in step (i) (meaning that the final concentration of QM-DMAC in the system was 20 μM), and incubated for 60 min at 37°C, 21% O2 and 5% CO2 cell culture conditions, and then a commercially available lysosomal dye (Lysotracker Deep Red, LTDR 50 nM) was added for co-staining imaging and incubated for 20 min. The results are shown in FIG. Figure 3 As shown in A.

[0070] Depend on Figure 3 As shown in Figure A, good fluorescence colocalization can be observed between lysosomes and QM-DMAC, suggesting that QM-DMAC can be used to monitor lysosomes in tumor cells.

[0071] (IV) Lysosomal viscosity-specific response test

[0072] In order to evaluate the specific responsiveness of QM-DMAC to lysosomal viscosity, the antifungal drug Monensin and the clinical drug Dexamethasone, which can increase intracellular viscosity, were used to conduct a specific responsiveness test of lysosomal viscosity. The specific steps are as follows:

[0073] The antifungal drug monensin (20 μM) and the clinical drug dexamethasone (100 μM) were cultured with A549 cells at 37°C, 21% O2 and 5% CO2 for 60 minutes to increase the intracellular viscosity and induce cell dysfunction. After washing, QM-DMAC was added to the system to make the final concentration of QM-DMAC in the system 20 μM, and incubated at 37°C, 21% O2 and 5% CO2 for 60 minutes. The fluorescence intensity in the tumor cells was observed. The results are as follows: Figure 3 As shown in B;

[0074] Depend on Figure 3 B shows that as the lysosome viscosity increases, the fluorescence increases. This result shows that there is a positive correlation between the fluorescence intensity of QM-DMAC and the lysosome viscosity in tumor cells.

[0075] (V) Monitoring lysosomal viscosity changes under pathological conditions

[0076] In order to evaluate the ability of QM-DMAC to monitor changes in lysosomal viscosity under pathological conditions, the present application uses inflammatory inducers to stimulate changes in cell state (inflammation can cause an increase in intracellular viscosity) to evaluate the ability of QM-DMAC to monitor changes in lysosomal viscosity under pathological conditions. The specific steps are as follows:

[0077] A549 cells were cultured with 20 μg / mL lipopolysaccharide (LPS), an inflammatory inducer, at 37°C, 21% O2, and 5% CO2 for 2 h, and then 20 μM QM-DMAC was added (meaning that the final concentration of QM-DMAC in the system was 20 μM, experimental group), and incubated at 37°C, 21% O2, and 5% CO2 for 1 h. A control group was set up, in which the same amount of PBS was used instead of LPS. The fluorescence signal was detected using the above method. The results are shown in FIG. Figure 4 shown.

[0078] Depend on Figure 4 It can be seen that the fluorescence intensity in the tumor cells of the experimental group was significantly enhanced compared with the control group. This result shows that QM-DMAC has excellent ability to monitor changes in lysosomal viscosity under pathological conditions.

[0079] (VI) QM-DMAC monitoring of cell autophagy

[0080] 20 μM QM-DMAC was added to A549 tumor cells, incubated at 37°C, 21% O2 and 5% CO2 for 1 h, and the fluorescence signal was detected by the above method; autophagy induction: 10 μg / mL autophagy inducer rapamycin (i.e., Rapamycin) was added in advance for 12 h to induce cell autophagy, and then 20 μM QM-DMAC was added, and incubated at 37°C, 21% O2 and 5% CO2 for 1 h, and the fluorescence signal was detected by the above method; autophagy inhibition: 100 μM autophagy inhibitor 3-methyladenine (3-MA, i.e., 3-Methyladenine) and 10 μg / mL autophagy inducer rapamycin (i.e., Rapamycin) were added in advance and incubated for 12 h, and then 20 μM QM-DMAC was added, and incubated at 37°C, 21% O2 and 5% CO2 for 1 h, and the fluorescence signal was detected by the above method. The results are as follows: Figure 5 As shown; Merge is the overlay of the fluorescence channel image and the bright field image.

[0081] Depend on Figure 5 It can be seen that treatment with autophagy inducers can induce autophagy in tumor cells, leading to increased lysosomal viscosity and a corresponding increase in the fluorescence of QM-DMAC. Adding autophagy inhibitors can inhibit the autophagy process, without further enhancement of fluorescence, and keeping the lysosomal viscosity stable.

[0082] (VII) Tumor cell identification

[0083] The lysosome viscosity of tumor cells is higher than that of normal cells, and can be used as a potential biomarker for detecting tumors. In this application, the lysosome viscosity response probe QM-DMAC is used to stain tumors and normal cells respectively. The specific steps are as follows:

[0084] 20 μM QM-DMAC was added to tumor cells (4T1 cells, A431 cells, Cal-27 cells and HeLa cells) and normal cells (NIH3T3 cells, 16HBE cells, HT22 cells and NCM460 cells) of different types and sources to stain the tumor cells and normal cells. The fluorescence signal was detected by the above method. The results are shown in FIG. Figure 6 shown.

[0085] Depend on Figure 6 It can be seen that after QM-DMAC staining, the fluorescence signal of tumor cells is significantly enhanced compared with that of normal cells. This result shows that QM-DMAC can effectively distinguish tumor cells from normal cells.

[0086] (VIII) PDT photosensitizer (i.e. photodynamic therapy photosensitizer)

[0087] 8.1 Reactive oxygen species (ROS) detection

[0088] The ROS indicator DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) was used to evaluate the ROS generation ability of QM-DMAC in tumor cells after white light irradiation. The specific steps are as follows:

[0089] A549 cells were divided into QM-DMAC+L+NAC group (NAC represents ROS scavenger N-acetylcysteine), QM-DMAC+L group, QM-DMAC group, illumination group (L), and control group (Control). In the QM-DMAC+L group, 20 μM QM-DMAC was added to the tumor cells and incubated for 60 min, cultured for 60 min at 37°C, 21% O2 and 5% CO2, and then irradiated with white light from LED lamp for 30 min; the QM-DMAC group was not irradiated with white light; the illumination group was only irradiated with white light; and the control group was not treated with any treatment. In addition, 10 μM NAC was added to the QM-DMAC+L+NAC group in advance and incubated at 37°C, 21% O2 and 5% CO2 for 1 hour, and then QM-DMAC was added for incubation; subsequently, 10 μM DCFH-DA (i.e., 2,7-dichlorofluorescein diacetate) was added to the tumor cells of the five groups and incubated for 30 minutes. The fluorescence signal was detected using the above method. The results are as follows Figure 7 As shown in A.

[0090] Depend on Figure 7A shows that the fluorescence signal of the QM-DMAC+L group was significantly enhanced, while the fluorescence signals of the control group and the QM-DMAC group alone were not significantly enhanced, indicating that QM-DMAC can produce a large amount of ROS in tumor cells after illumination. The fluorescence signal of the QM-DMAC+L+NAC group was also not significantly enhanced. This result shows that NAC can effectively inhibit the increase of ROS induced by QM-DMAC and light.

[0091] 8.2 Evaluation of the therapeutic effect of QM-DMAC on tumor cells and normal cells

[0092] The present application further uses MTT to evaluate the therapeutic effect of QM-DMAC on tumor cells and normal cells, and the specific steps are as follows:

[0093] Tumor cells A549 or normal cells 16HBE were inoculated in two 96-well plates at a density of 6000 cells and cultured overnight; one group of 96-well plate cells was the control group without light treatment (Dark group); the other group was the white light treatment group (White Light group); the culture medium was replaced with fresh cell culture medium containing different QM-DMAC (0, 5, 10, 20, and 30 μM) and incubated for 1 hour; after incubation in the light group, the cells were exposed to LED white light for 30 minutes, and the Dark group plates were placed in the dark as a control; after the treatment, all 96-well plates were placed in a cell culture incubator at 37°C, 21% O2 and 5% CO2 for 24 hours, and then incubated with cell culture medium containing 10% MTT solution for 1 hour, replaced with DMSO solution, and analyzed with an enzyme marker after shaking; NAC was pre-added to the tumor cells of the group treated under white light irradiation, and the cell activity was detected again after the same treatment for 24 hours; the cell viability test results of QM-DMAC in normal cells 16HBE, tumor cells A549, and tumor cells A549 pretreated with NAC for 1 hour were as follows Figure 7 B, 7C and 7D.

[0094] Depend on Figure 7 B and 7C show that, under the condition of no light, after 24h of QM-DMAC staining, the viability of tumor cells and normal cells remained above 95% even when the QM-DMAC concentration was as high as 30 μM. This result shows that QM-DMAC has low dark toxicity.

[0095] Depend on Figure 7 B, 7C and 7D show that under white light irradiation, the vitality of normal cells remains at around 90% ( Figure 7 B); Under the same irradiation conditions, tumor cells showed dose-dependent cytotoxicity - cell viability gradually decreased with increasing concentration, among which, QM-DMAC concentration of 20μM caused significant cell death ( Figure 7C); After pretreatment with NAC, the phototoxicity of QM-DMAC to tumor cells was reduced ( Figure 7 D). These results indicate that QM-DMAC can be used for photodynamic therapy of tumor cells.

[0096] (IX) Mechanism of selective targeted therapy for tumor cell lysosomes

[0097] Based on the targeting of QM-DMAC to tumor cell lysosomes and its ability to induce ROS generation by white light, this application uses a lysosomal integrity index (acridine orange, AO) to evaluate the integrity of tumor cell lysosomal membranes to verify the targeted destruction ability of QM-DMAC on lysosomes. The specific steps are as follows:

[0098] A549 cells were divided into control group (Control), light alone group (L), QM-DMAC group, treatment group QM-DMAC+L, and QM-DMAC+L+NAC group. Lysosomal integrity was detected by AO staining. In the QM-DMAC+L+NAC group, 10 μM NAC, then pre-cultured at 37°C, 21% O2 and 5% CO2 for 1h, then added 20μM QM-DMAC, incubated at 37°C, 21% O2 and 5% CO2 for 1h, then irradiated with white light, the parameters were the same as above, then added AO (10μM) and incubated for 30min, and the fluorescence signal was detected by confocal microscopy; the difference between the control group (Control) and the QM-DMAC+L+NAC group was that the corresponding volume of PBS buffer was used to replace QM-DMAC and NAC, respectively, and no light treatment was performed; the difference between the single light group (L) and the QM-DMAC+L+NAC group was that the corresponding volume of PBS buffer was used to replace QM-DMAC and NAC, respectively; the difference between the treatment group QM-DMAC+L group and the QM-DMAC+L+NAC group was that the same volume of PBS buffer was used to replace NAC, and the results were as follows Figure 8 shown.

[0099] Depend on Figure 8 It can be seen that green fluorescence was observed in the cytoplasm / nucleus of the control group (Control), the light-only group (L), the QM-DMAC group, and the QM-DMAC+L+NAC group, and red fluorescence was observed in the lysosomes, indicating that the integrity of the lysosomes remained intact; on the contrary, tumor cells incubated with QM-DMAC and then irradiated with white light showed a significant decrease in the red fluorescence intensity of AO. This result shows that the lysosomal membrane of the QM-DMAC+L group in the treatment group was damaged and ruptured after light irradiation. For the QM-DMAC+L+NAC group, the red fluorescence in the lysosomes was still maintained after light irradiation, which shows that NAC can alleviate light-induced lysosomal rupture.

[0100] (X) In vivo PDT efficacy

[0101] The in vivo PDT efficacy of QM-DMAC was evaluated using a tumor-bearing mouse (BALB / c) model. The specific steps were as follows:

[0102] A549 cell-bearing mouse model: BALB / c nude mice (4 weeks old, 15-18 g) were purchased from Nanfang Hospital of Southern Medical University and cultured at a density of 2×10 6 A549 cells / 100 μL were subcutaneously injected into each nude mouse, and subcutaneous tumors were grown to about 150 mm. 3 , a tumor-bearing mouse (BALB / c) model was obtained;

[0103] Tumor-bearing BALB / c nude mice were randomly divided into a control group (Control), a light-only group (L), a QM-DMAC group, and a treatment group (QM-DMAC+L). In the QM-DMAC+L group, QM-DMAC (20 mM, 100 μL / 200 mm 3 Tumor), then irradiated with white light for 30 min every four days; the difference between the control group (Control) and the QM-DMAC+L group is that the corresponding volume of PBS buffer was used instead of QM-DMAC, and no light treatment was performed; the difference between the light-only group (L) and the QM-DMAC+L group is that the corresponding volume of PBS buffer was used instead of QM-DMAC; the difference between the QM-DMAC group and the QM-DMAC+L group is that no light treatment was performed, and the tumor volume of mice was monitored every 4 days, tumor volume = (tumor length) × (tumor width) 2 / 2, the result is Fig. 9 A and 9B.

[0104] Depend on Fig. 9 A and 9B show that the light alone group (L) and the QM-DMAC group had no significant inhibition on tumor growth, while the QM-DMAC+L group had a significant inhibitory effect on tumor growth. This result shows that QM-DMAC has a highly effective tumor PDT treatment effect.

[0105] The organs of mice in each group were further stained with hematoxylin-eosin (H&E). The specific steps were as follows: the main organs of mice were removed, embedded in paraffin, sliced, stained with hematoxylin-eosin after slicing, and imaged under an optical microscope (20×). The results are shown in Fig. 9 C. There was no obvious tissue damage or inflammation in the major organs of each group.

[0106] Depend on Fig. 9 It can be seen that QM-DMAC has significant therapeutic effect on PDT of tumors, good biocompatibility and small toxic side effects.

[0107] (XI) Radiotherapy sensitization

[0108] 11.1 The DCFH-DA assay was used to evaluate the efficiency of ROS generation in the “combination therapy” method (R+QM-DMAC+L) of PDT combined with low-dose radiotherapy (R). The specific steps were as follows:

[0109] A549 cells were divided into combined treatment group (R+QM-DMAC+L), ROS scavenger group (R+QM-DMAC+L+NAC), QM-DMAC+L group, R+QM-DMAC group, radiation alone group (R), and control group (Control).

[0110] ROS scavenger group (R+QM-DMAC+L+NAC) added 10 μM NAC to A549 cells and cultured them at 37°C, 21% O2 and 5% CO2 for 1 h, then added 20 μM QM-DMAC, then cultured at 37°C, 21% O2 and 5% CO2 for 1 h, then irradiated with white light from an LED white light lamp for 30 min to generate a large amount of ROS, and then treated with X-rays (R=4 Gy, dose rate: 1 Gy / min); the difference between the "combined treatment" group (R+QM-DMAC+L) and the ROS scavenger group was that the same volume of PBS buffer was used to replace NAC; the difference between the QM-DMAC+L group and the ROS scavenger group was that the corresponding volume of PBS buffer was used to replace NAC and no X-rays were performed ( R = 4Gy, dose rate: 1Gy / min) treatment; the difference between the radiation group (R) and the ROS scavenger group is that no white light irradiation was performed, and the corresponding volume of PBS buffer was used to replace QM-DMAC and NAC respectively; the difference between the control group (Control) and the ROS scavenger group is that the corresponding volume of PBS buffer was used to replace QM-DMAC and NAC respectively, and no X-ray treatment and white light irradiation were performed; after each group was treated, 10μM DCFH-DA was added and incubated for 30min, and the fluorescence signal was detected by flow cytometry. The results are shown in Fig.10 As shown in A.

[0111] Depend on Fig.10 A shows that compared with other treatment groups, the ROS level of tumor cells in the "combination therapy" group increased significantly; the ROS level of cells in the "combination therapy" group increased only slightly when pretreated with NAC. This result shows that under light conditions, QM-DMAC can promote cellular ROS generation and enhance the ROS generation effect of radiotherapy, while ROS scavengers can reverse the efficacy of "combination therapy".

[0112] 11.2 The potential of QM-DMAC to improve radiosensitivity was evaluated using a clone formation assay. The specific steps were as follows:

[0113] A549 cells were divided into a combined treatment group (R+QM-DMAC+L), a ROS scavenger group (R+QM-DMAC+L+NAC), a QM-DMAC+L group, a R+QM-DMAC group, a radiation group (R), and a control group (Control). The treatment of each group was the same as in 11.1, except that the ionizing radiation irradiation (dose rate: 1 Gy / min) was adjusted to 0, 2, 4, 6, 8, and 10 Gy doses. After irradiation, the cells were detached by trypsin treatment and seeded into cell culture dishes (about 200 to 6000 cells) and cultured for 12 to 15 days to allow colony formation. The colony-forming ability of tumor cells was evaluated by survival curve, and the results are shown in Figure 11. Fig.10 As shown in B;

[0114] Depend on Fig.10 B shows that the survival fraction was determined and calculated using the survival curve, and the radiosensitization effect of QM-DMAC was calculated by measuring the radiosensitization ratio (SER). The radiosensitization ratio (SER) of the "combined treatment" group (R+QM-DMAC+L) was 1.75, which was much higher than the SER value (1.45) of other treatment groups and the commonly used photosensitizer Ce6 (R+Ce6+L). This result shows that the radiosensitization effect of QM-DMAC is better than that of Ce6, because QM-DMAC can not only produce a large amount of ROS, but also trigger LMP. After adding the ROS scavenger NAC, the SER value of the R+QM-DMAC+L+NAC group was only 0.95. This result shows that increasing the level of cellular ROS is crucial to enhancing the radiosensitization effect of QM-DMAC. Compared with the radiation group (R) or the QM-DMAC+L group alone, the combined treatment of the R+QM-DMAC+L group showed a significant inhibitory effect on colony formation.

[0115] 11.3 The in vivo radiosensitization effect of QM-DMAC was evaluated using a tumor-bearing mouse model. The specific steps are as follows:

[0116] A549 tumor-bearing BALB / c nude mice were randomly divided into five groups, namely, the combined treatment group (R+QM-DMAC+L), the QM-DMAC+L group, the R+QM-DMAC group, the radiation group (R), and the control group (Control). PBS or QM-DMAC (20 mM, 100 μL / 200 mm 3 The light-treated group was exposed to light for 30 min every four days, and the combined treatment group was treated with X-rays (R = 4 Gy) after R+QM-DMAC+L light exposure, and the tumor volume of the mice was monitored. Tumor volume = (tumor length) × (tumor width) 2 / 2), the result is as follows Fig.10 C and 10D.

[0117] Depend on Fig.10 C and 10D show that compared with the control group (Control), the tumor volume of the QM-DMAC+L group and the "combination treatment" group (R+QM-DMAC+L) was significantly reduced, and the "combination treatment" group showed significantly enhanced anti-tumor activity. This result shows that under white light irradiation, QM-DMAC significantly enhances the anti-tumor effect of radiotherapy, further proving the potential of QM-DMAC as a radiosensitizer.

[0118] (XII) Chemosensitization

[0119] Cisplatin is a platinum-based chemotherapy drug widely used in clinical practice to treat various malignant tumors, but the existence of drug resistance poses a great challenge to the treatment effect, and lysosomes are believed to play an important role in the development of drug resistance.

[0120] 12.1 In order to evaluate the targeting ability of QM-DMAC on drug-resistant tumor cells, this application uses QM-DMAC to perform live cell imaging on multi-drug-resistant tumor cells. The specific steps are as follows:

[0121] A549 / DDP cells were mixed with cell culture medium containing 20 μM QM-DMAC and incubated for 60 min at 37°C, 21% O2 and 5% CO2. A commercially available lysosomal dye (Lysotracker Deep Red, LTDR 50 nM) was then added for co-staining imaging and incubated for 20 min. Confocal microscopy was used for imaging observation. The results are shown in Figure 2. Fig.11 As shown in A.

[0122] Depend on Fig.11 A shows that QM-DMAC has good cell permeability and high-contrast lysosome-targeted imaging capabilities.

[0123] 12.2 Evaluate the enhanced chemotherapy effect of QM-DMAC under white light irradiation. The specific steps are as follows:

[0124] MTT detection of cell activity: Tumor cells A549 / DDP were inoculated at a density of 6000 cells in two 96-well plates for overnight culture. One group of 96-well plate cells was a non-light-treated control group (Dark group), and the other group was a white light-treated group (White Light group). The culture medium was replaced with fresh cell culture medium containing different QM-DMAC (0, 5, 10, 20, and 30 μM) and incubated for 1 hour. After incubation in the light group, the cells were exposed to LED white light for 30 minutes. The Dark group plates were placed in the dark as a control. After the treatment, all 96-well plates were placed in a cell culture incubator at 37°C, 21% O2 and 5% CO2 for 24 hours. Then, after incubation with a cell culture medium containing 10% MTT solution for 1 hour, it was replaced with a DMSO solution, shaken and analyzed with an enzyme reader. The results are as follows: Fig.11 As shown in B.

[0125] Depend on Fig.11 As shown in B, the drug-resistant tumor cells (A549 / DDP) exhibited dose-dependent cytotoxicity.

[0126] 12.3DCFH-DA method was used to detect the ROS generation capacity after illumination. The specific steps are as follows:

[0127] A549 / DDP tumor cells were divided into a combined treatment group (CDDP+QM-DMAC+L, CDDP represents cisplatin), a ROS scavenger group (CDDP+QM-DMAC+L+NAC), a CDDP+QM-DMAC group, a QM-DMAC+L group, a cisplatin group alone (CDDP), and a control group (Control). 10 μM ROS scavenger group (CDDP+QM-DMAC+L+NAC) was added to A549 / DDP tumor cells. NAC, and cultured at 37°C, 21% O2 and 5% CO2 for 1 h, then added 20 μM QM-DMAC, and then cultured at 37°C, 21% O2 and 5% CO2 for 1 h, then irradiated with white light from LED white light for 30 min to generate a large amount of ROS, and then treated with cisplatin CDDP (final concentration of 200 μM); the difference between the "combination treatment" group (CDDP+QM-DMAC+L) and the ROS scavenger group is that the same volume of PBS buffer was used instead of NAC; CDDP+QM-DMAC group The difference between the cisplatin group (CDDP) and the ROS scavenger group is that the corresponding volume of PBS buffer was used to replace NAC and no white light irradiation was performed; the difference between the cisplatin group (CDDP) and the ROS scavenger group is that no white light irradiation was performed, and the corresponding volume of PBS buffer was used to replace QM-DMAC and NAC respectively; the difference between the control group (Control) and the ROS scavenger group is that the corresponding volume of PBS buffer was used to replace QM-DMAC and NAC respectively, and no cisplatin CDDP treatment and white light irradiation were performed; after each group was treated, 10 μM DCFH-DA was added, and the cells were incubated at 37°C, 21% O2 and 5% CO2 for 30 minutes, and the fluorescence signal was detected by flow cytometry. The results are shown in FIG. Fig.11 As shown in C.

[0128] Depend on Fig.11 C shows that after receiving "combination therapy" (PDT combined with cisplatin treatment, CDDP+QM-DMAC+L), the level of ROS generation in drug-resistant tumor cells is higher. This result shows that under light conditions, QM-DMAC can promote cellular ROS generation and enhance the anti-tumor efficacy of CDDP treatment on drug-resistant tumor cells. ROS scavengers can reverse the effect of combined therapy-induced ROS generation. It also shows that cellular ROS levels are crucial for enhancing the effect of chemotherapy. ROS induces LMP, destroys the integrity of the lysosomal membrane, and causes the leakage of lysosomal contents. This process allows the anti-tumor drugs isolated in the lysosome to be released, thereby restoring their anti-tumor effects.

[0129] 12.4 To evaluate the ability of QM-DMAC to induce LMP in drug-resistant tumor cells, this application uses the lysosomal integrity index (AO) to observe the integrity of the lysosomal membrane of tumor cells. The specific steps are as follows:

[0130] A549 / DDP cells were divided into a combined treatment group (CDDP+QM-DMAC+L), a ROS scavenger group (CDDP+QM-DMAC+L+NAC), a QM-DMAC+CDDP group, a QM-DMAC+L group, a cisplatin group (CDDP), and a control group (Control). The treatment parameters of each group were the same as those in 12.3. After each group was treated, 10 μM AO was added and incubated at 37°C, 21% O2 and 5% CO2 for 30 min. The fluorescence signal was detected using a fluorescence confocal microscope. The results are shown in Fig.12 shown.

[0131] Depend on Fig.12 It can be seen that green fluorescence was observed in the cytoplasm / nucleus of the control group (Control), cisplatin alone group (CDDP) and CDDP+QM-DMAC group, and red fluorescence was observed in the lysosome, indicating that the integrity of the lysosome was good. In contrast, the QM-DMAC+L group and CDDP+QM-DMAC+L group showed a significant decrease in the red fluorescence intensity of AO. This result indicates that the treatment caused LMP and lysosomal membrane rupture, and NAC can alleviate light-induced LMP.

[0132] 12.5 This application further uses Alexa 647Anti-gamma H2A.X antibody was used to immunostain drug-resistant tumor cells and flow cytometry analysis was performed. The specific steps are as follows:

[0133] A549 / DDP cells were divided into a combined treatment group (CDDP+QM-DMAC+L), a ROS scavenger group (CDDP+QM-DMAC+L+NAC), a CDDP+QM-DMAC group, a QM-DMAC+L group, a cisplatin group (CDDP), and a control group (Control). The treatment parameters of each group were the same as those in 12.3. After 16 h of treatment, the cells were fixed and permeabilized (4% paraformaldehyde), and then treated with 1 / 500 diluted Alexa Fluor Cells were labeled with 647Anti-gamma H2A.X antibody and analyzed by flow cytometry. Fig.13 shown.

[0134] Depend on Fig.13 It can be seen that the histone γ-H2AX fluorescence expression level in the combined treatment (CDDP+QM-DMAC+L) group was at the highest level. The reason is that after the integrity of the lysosomal membrane was destroyed, the CDDP stored in the lysosome was released into the cytoplasm, causing severe DNA damage and restoring the therapeutic function of CDDP.

[0135] 12.6 The present application further evaluates the in vivo chemosensitization efficacy of QM-DMAC in drug-resistant tumors, and the specific steps are as follows:

[0136] A549 / DDP tumor-bearing BALB / c nude mice were randomly divided into a combined treatment group (CDDP+QM-DMAC+L), a CDDP+QM-DMAC group, a QM-DMAC+L group, a cisplatin group (CDDP), and a control group (Control); the illumination group was illuminated for 30 min every four days, and the combined treatment group CDDP+QM-DMAC+L was illuminated and then received cisplatin treatment; the corresponding drugs were injected into the tumor (QM-DMAC: 20 mM, 100 μL / 200 mm 3 Tumor); The tumor volume of mice was monitored every four days, tumor volume = (tumor length) × (tumor width) 2 / 2. The result is Fig.14 shown.

[0137] Depend on Fig.14 It can be seen that the tumor volume of the QM-DMAC+L group and the combined treatment (CDDP+QM-DMAC+L) group was significantly reduced, and the anticancer activity of the CDDP+QM-DMAC+L group was particularly significant. This result shows that under white light irradiation, QM-DMAC can significantly enhance the anti-tumor effect of chemotherapy drugs, especially in drug-resistant tumors.

[0138] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A fluorescent compound, characterized in that: The structural formula of the fluorescent compound is shown in formula (I): In formula (I), A is selected from oxygen atom, sulfur atom, R1 is selected from a hydrogen atom or an aromatic ring group; R2 is selected from a cyano group or a group represented by formula (II); R3 is selected from an aromatic ring group; and R4 is selected from a hydrogen atom or a chain alkyl group having 1 to 20 carbon atoms.

2. The fluorescent compound according to claim 1, characterized in that R1 is selected from any one of the groups represented by formulae (1) to (35); R3 is selected from any one of the groups represented by formulae (31) to (35): In the formula, R5 is selected from a chain alkyl or cycloalkyl group having 1 to 20 carbon atoms and 0 or some of the carbon atoms are substituted by an oxygen atom, an alkenyl group, an alkynyl group, an aryl group, a carbonyl group, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group or an ester group; R6 is selected from a chain alkyl or cycloalkyl group having 1 to 20 carbon atoms and 0 or some of the carbon atoms are substituted by an oxygen atom, an alkenyl group, an alkynyl group, an aryl group, a carbonyl group, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group or an ester group; R7 is selected from a chain alkyl or cycloalkyl group having 1 to 20 carbon atoms and 0 or some of the carbon atoms are substituted by an oxygen atom, an alkenyl group, an alkynyl group, an aryl group, a carbonyl group, a hydroxyl group, an amino group, a carboxyl group, a cyano group, a nitro group or an ester group.

3. The fluorescent compound according to claim 2, characterized in that A is selected from R1 is selected from a hydrogen atom, R2 is selected from a cyano group, R3 is selected from a group represented by formula (35), and R4, R5 and R7 are all selected from a methyl group.

4. The method for preparing a fluorescent compound according to any one of claims 1 to 3, characterized in that: The following steps are involved: The compound with active α-hydrogen atom and the compound containing aldehyde group are dissolved in a solvent, and reflux reaction is carried out in a protective gas atmosphere under the action of a catalyst to prepare the fluorescent compound.

5. The method for preparing a fluorescent compound according to claim 4, characterized in that: The solvent is selected from at least one of anhydrous ethanol, methanol, acetonitrile, tetrahydrofuran, ethyl acetate, N,N-dimethylformamide and dimethyl sulfoxide; and / or, the catalyst is selected from at least one of amine, pyridine, piperidine and morpholine; And / or, the protective gas is selected from at least one of nitrogen, helium and argon; And / or, the temperature of the reflux reaction is 80-120°C.

6. The method for preparing a fluorescent compound according to claim 4, characterized in that: After the reflux reaction, the method further comprises the following steps: purification.

7. Use of the fluorescent compound according to any one of claims 1 to 3 or the fluorescent compound prepared according to the method according to any one of claims 4 to 6 in lysosomal targeted imaging.

8. Use of the fluorescent compound according to any one of claims 1 to 3 or the fluorescent compound prepared according to the method according to any one of claims 4 to 6 in the preparation of a drug for assisting photodynamic therapy of tumors.

9. Use of the fluorescent compound according to any one of claims 1 to 3 or the fluorescent compound prepared by the method according to any one of claims 4 to 6 as a radiosensitizer.

10. Use of the fluorescent compound according to any one of claims 1 to 3 or the fluorescent compound prepared according to the method according to any one of claims 4 to 6 as a chemosensitizer.

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