A lysosome-targeting probe and a preparation method and application thereof

Abstract

The application belongs to the technical field of biotechnology, 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 in formula (I). In formula (I), A is selected from an oxygen atom and a sulfur atom, R1 is selected from a hydrogen atom and an aromatic ring group, R2 is selected from a cyano group and a group shown in formula (II), R3 is selected from an aromatic ring group, and R4 is selected from a hydrogen atom and an alkyl group with a carbon atom number of 1-20. The compound of the application can specifically target lysosomes in tumor cells, emit bright fluorescence, and has a good fluorescence co-localization effect with lysosomes in tumor cells. The compound can be used for lysosome imaging, and the fluorescence intensity thereof is positively correlated with the viscosity of lysosomes in living cells such as tumor cells. The compound can be used for monitoring the viscosity change of lysosomes in living cells under various conditions. The compound can be used as a photosensitizer and a treatment sensitizer, specifically ablates tumors through photodynamic therapy (PDT), restores the sensitivity of tumor cells to radiotherapy and chemotherapy, and enhances the treatment effect of radiotherapy and chemotherapy.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to a lysosomal targeting probe, its preparation method, and its application. Background Technology

[0002] Drug resistance is a major challenge in clinical oncology treatment and a leading factor in cancer treatment failure. It can cause rapid cancer progression or recurrence, and even patient death, making it one of the most challenging problems faced by cancer patients and their families, healthcare professionals, and cancer researchers. Overcoming drug resistance, improving clinical treatment outcomes, and ultimately improving patient survival rates are goals of cancer treatment, but also extremely challenging. Developing anti-drug antagonists for tumor therapy has significant clinical application value and holds promise for bringing new hope to clinical oncology treatment. These agents can restore the sensitivity of tumor cells to treatments such as radiotherapy and chemotherapy, thereby improving their efficacy. Utilizing the advantages of combination therapy is a potentially effective strategy for breaking through treatment bottlenecks. Therefore, developing therapeutic sensitizers that can be integrated and adapted to conventional treatment methods (radiotherapy and chemotherapy) to overcome treatment resistance holds promise for a new breakthrough in cancer treatment.

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

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

[0005] To achieve the above objectives, the solution proposed in this application is as follows:

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

[0007]

[0008]

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

[0010] Optionally, R1 is selected from any one of the groups shown in formulas (1) to (35), and R3 is selected from any one of the groups shown in formulas (31) to (35):

[0011]

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

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

[0014]

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

[0016] The fluorescent compound is prepared by dissolving a compound with an active α-hydrogen atom and a compound containing an aldehyde group in a solvent and then refluxing them under a protective gas atmosphere in the presence of a catalyst.

[0017] For example, when the fluorescent compound has the structural formula shown in formula (III), the following compounds can be listed as having an active α-hydrogen atom:

[0018]

[0019] The following compounds contain an aldehyde group:

[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 amines, pyridines, piperidines, and morpholines.

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

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

[0025] Optionally, after the reflux reaction, the process further includes the step of purification.

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

[0027] Thirdly, the present invention also provides the application of the fluorescent compound as described above or the fluorescent compound prepared according to the method described above in lysosomal targeted imaging.

[0028] The fluorescent compounds of the present invention have excellent aggregation-induced emission (AIE) properties, that is, they emit 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 has a good fluorescence co-localization effect with lysosomes in tumor cells. It 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 between tumor cells and normal cells.

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

[0032] In this 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 colorectal cancer.

[0033] Under light source irradiation, the fluorescent compound of this invention can induce the production of large amounts of ROS in tumor cells, triggering lysosomal membrane permeation (LMP) and lysosomal rupture, exhibiting excellent PDT effect. Furthermore, this compound has good biocompatibility and low toxicity.

[0034] Fifthly, the present invention also provides the use of the fluorescent compound as described above or the fluorescent compound prepared according to the method described above as a radiosensitizer.

[0035] Under light source irradiation, the fluorescent compounds of the present invention can significantly enhance the radiotherapy effect by inducing ROS generation and accelerating cell apoptosis, demonstrating their potential as radiosensitizers.

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

[0037] Under light source irradiation, the fluorescent compounds of the present invention significantly enhance the chemotherapy effect by inducing ROS generation, triggering ROS-mediated LMP, and accelerating apoptosis of drug-resistant tumor cells, demonstrating their potential as chemotherapy sensitizers.

[0038] The fluorescent compounds of the present invention can be used as photosensitizers and therapeutic sensitizers, and can enhance the therapeutic effects of radiotherapy and chemotherapy by specifically ablating tumors through PDT and restoring the sensitivity of tumor cells to radiotherapy and chemotherapy. Attached Figure Description

[0039] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0040] Figure 1 This is the NMR spectrum. Figure 1 A is the hydrogen NMR spectrum. Figure 1 B is the carbon NMR spectrum;

[0041] Figure 2 This is a graph showing the results of the photophysical property detection. Figure 2 A is the ultraviolet-visible absorption spectrum. Figure 2 B is the fluorescence emission spectrum, where Wavelength represents the wavelength, Molar absorptivity represents the molar absorptivity, and PL intensity represents the fluorescence intensity.

[0042] Figure 3 Figures showing the results of lysosome-specific cell imaging assay and lysosome viscosity-specific response assay. Figure 3 Figure A shows the results of a lysosome-specific cell imaging assay. Figure 3 B shows the results of the lysosome viscosity-specific response test. Scale bar: 20μm.

[0043] Figure 4 The image shows the detection results of lysosomal viscosity changes under pathological conditions.

[0044] Figure 5 The results of the autophagy assay were shown in the figure.

[0045] Figure 6 Image showing the results of the tumor cell identification test;

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

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

[0048] Figure 9 This is a graph showing the results of an in vivo PDT efficacy trial; Tumor volume represents tumor volume, and Time (day) represents time (number of days), the same applies below;

[0049] Figure 10 This is a graph showing the results of a radiotherapy sensitization test. The Survival fraction represents the cell survival fraction.

[0050] Figure 11 The results are shown in Parts 12.1 (A), 12.2 (B), and 12.3 (C) of the chemotherapy sensitization assay. Cellviability represents cell viability. Scale bar: 20 μm.

[0051] Figure 12 This is a partial result diagram of the chemotherapy sensitization test, section 12.4. Scale bar: 20μm.

[0052] Figure 13 This is a partial result diagram from the chemotherapy sensitization test (section 12.5).

[0053] Figure 14 This is a partial result diagram of the chemotherapy sensitization test, section 12.6. Detailed Implementation

[0054] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0055] Numerous details are explored in the following description to provide a more thorough explanation of embodiments of the invention; however, it will be apparent to those skilled in the art that embodiments of the invention may be practiced without these specific details.

[0056] (I) Preparation and structural characterization of the compound shown in formula (III)

[0057] The compound shown in formula (III) (hereinafter referred to as QM-DMAC) is prepared by the following 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) were dissolved in 5 mL of anhydrous acetonitrile liquid. A few drops of piperidine were added to the anhydrous acetonitrile liquid as a catalyst, and the mixture was stirred to obtain a solution.

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

[0061] The product was characterized using nuclear magnetic resonance spectroscopy (Bruker Avance 600MHz), and the results are as follows. Figure 1 As shown.

[0062] like Figure 1 As shown, the 1H NMR characterization data of the product are as follows: 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 carbon NMR characterization data of the product are as follows: 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). This result indicates that the compound shown in formula (III) was successfully prepared in this application.

[0063] (II) Photophysical property testing

[0064] The product obtained in step (I) was analyzed by UV-Vis absorption spectroscopy. The specific steps were as follows: using dimethyl sulfoxide (DMSO) as solvent, the product was prepared to a concentration of 1.0 × 10⁻⁶. -5 A mol / L QM-DMAC solution was prepared, and the solution was placed in a cuvette. The UV-Vis absorption spectrum was detected using a UV-Vis spectrometer. The results are as follows: Figure 2 As shown in A;

[0065] The fluorescence emission spectrum of the product obtained in step (I) was detected 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 product was then prepared using these liquids to achieve a concentration of 1.0 × 10⁻⁶. -5 The fluorescence emission spectra of each QM-DMAC solution (mol / L) were detected using a fluorescence spectrometer, and 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 445 nm. Figure 2 A). QM-DMAC exhibits weak fluorescence in dimethyl sulfoxide, but the fluorescence intensity significantly increases with increasing water content in the system. Figure 2 (B) The reason is that as the water content in the system increases, the solubility of QM-DMAC gradually decreases, leading to the formation of aggregates. Intramolecular movement is restricted, thereby activating the aggregation-induced emission (AIE) effect. This result indicates that the compounds of the present invention possess excellent aggregation-induced emission (AIE) properties, i.e., they emit weak fluorescence in solution, but the emission is significantly enhanced in the aggregated state.

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

[0068] The product obtained in step (I) was subjected to live-cell imaging using tumor cells, followed by analysis using laser confocal microscopy. The specific steps are as follows:

[0069] A549 cells were mixed with cell culture medium containing the product obtained in step (I) (QM-DMAC, 20 μM) (meaning the final concentration of QM-DMAC in the system was 20 μM), and incubated at 37°C, 21% O2, and 5% CO2 for 60 min. Then, commercially available lysosomal dye (Lysotracker Deep Red, LTDR 50 nM) was added for co-staining and imaging, and incubated for 20 min. The results are as follows: Figure 3 As shown in Figure 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) Lysosome viscosity-specific response test

[0072] To evaluate the specific responsiveness of QM-DMAC to lysosomal viscosity, the antifungal drug monensin, which can increase intracellular viscosity, and the clinical drug dexamethasone were used. This application conducted a specific responsiveness test on lysosomal viscosity, the specific steps of which are as follows:

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

[0074] Depend on Figure 3 As shown in Figure B, fluorescence intensity increases with increasing lysosomal viscosity. This result indicates a positive correlation between the fluorescence intensity of QM-DMAC and the lysosomal viscosity in tumor cells.

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

[0076] To evaluate the ability of QM-DMAC to monitor lysosomal viscosity changes under pathological conditions, this application uses an inflammation inducer to stimulate changes in cell state (inflammation causes an increase in intracellular viscosity) to evaluate the ability of QM-DMAC to monitor lysosomal viscosity changes under pathological conditions. The specific steps are as follows:

[0077] A549 cells were cultured with 20 μg / mL of the inflammation inducer lipopolysaccharide (LPS) at 37°C, 21% O2, and 5% CO2 for 2 h. Then, 20 μM QM-DMAC (meaning the final concentration of QM-DMAC in the system was 20 μM, experimental group) was added, and the cells were incubated at 37°C, 21% O2, and 5% CO2 for 1 h. A control group was also included, using the same amount of PBS instead of LPS. Fluorescence signals were detected using the above method, and the results are as follows: Figure 4 As shown.

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

[0079] (vi) QM-DMAC monitoring of autophagy

[0080] A549 tumor cells were incubated with 20 μM QM-DMAC at 37°C, 21% O2, and 5% CO2 for 1 h, and fluorescence signals were detected using the above method. For autophagy induction, cells were incubated with 10 μg / mL rapamycin for 12 h to induce autophagy, followed by incubation with 20 μM QM-DMAC at 37°C, 21% O2, and 5% CO2 for 1 h, and fluorescence signals were detected using the above method. For autophagy inhibition, cells were incubated with 100 μM 3-methyladenine (3-MA) ​​and 10 μg / mL rapamycin for 12 h, followed by incubation with 20 μM QM-DMAC at 37°C, 21% O2, and 5% CO2 for 1 h, and fluorescence signals were detected using the above method. The results are as follows: Figure 5 As shown; Merge is an overlay of the fluorescence channel image and the bright field image.

[0081] Depend on Figure 5 It is known that treatment with autophagy inducers can induce autophagy in tumor cells, leading to an increase in lysosomal viscosity and a corresponding increase in the fluorescence of QM-DMAC. Adding autophagy inhibitors can inhibit the autophagy process, and the fluorescence does not increase further, while the lysosomal viscosity remains stable.

[0082] (vii) Tumor cell identification

[0083] The lysosomal viscosity of tumor cells is higher than that of normal cells, which can serve as a potential biomarker for tumor detection. This application uses the lysosomal viscosity-responsive probe QM-DMAC to stain tumor 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 origins to stain tumor cells and normal cells, respectively. Fluorescence signals were detected using the method described above. The results are as follows: Figure 6 As shown.

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

[0086] (viii) PDT photosensitizers (i.e., photodynamic therapy photosensitizers)

[0087] 8.1 Detection of Reactive Oxygen Species (ROS)

[0088] The ability of tumor cells to generate ROS after white light irradiation was assessed using the ROS indicator DCFH-DA (2,7-dichlorodihydrofluorescein diacetate). The specific steps were as follows:

[0089] A549 cells were divided into four groups: QM-DMAC+L+NAC (NAC represents the ROS scavenger N-acetylcysteine), QM-DMAC+L, QM-DMAC, light-only group (L), and control group. In the QM-DMAC+L group, tumor cells were incubated with 20 μM QM-DMAC for 60 min, cultured at 37°C, 21% O2, and 5% CO2 for 60 min, and then irradiated with white light from an LED lamp for 30 min. The QM-DMAC group did not receive white light irradiation; the light-only group received only white light irradiation; and the control group received no treatment. In addition, the QM-DMAC+L+NAC group was pre-treated with 10 μM NAC and incubated at 37℃, 21% O2, and 5% CO2 for 1 h before being incubated with QM-DMAC. Subsequently, tumor cells in all five groups were incubated with 10 μM DCFH-DA (i.e., 2,7-dichlorofluorescein diacetate) for 30 min, and fluorescence signals were detected using the above method. The results are as follows. Figure 7 As shown in Figure A.

[0090] Depend on Figure 7As shown in Figure A, the fluorescence signal of the QM-DMAC+L group was significantly enhanced, while the fluorescence signals of the control group and the QM-DMAC-only group were not significantly enhanced, indicating that QM-DMAC can generate a large amount of ROS in tumor cells after light irradiation. The fluorescence signal of the QM-DMAC+L+NAC group was also not significantly enhanced, suggesting that NAC can effectively inhibit QM-DMAC and light-induced ROS increase.

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

[0092] This application further uses thiazolyl blue (MTT) to evaluate the therapeutic effect of QM-DMAC on tumor cells and normal cells. The specific steps are as follows:

[0093] Tumor cells (A549) or normal cells (16HBE) were seeded at a density of 6000 cells each in two 96-well plates and cultured overnight. One group of 96-well plates served as a control group (Dark group) without light treatment; the other group served as a white light treatment group (White group). The light group was treated with fresh cell culture medium containing different amounts of QM-DMAC (0, 5, 10, 20, and 30 μM) for 1 h. After incubation, the light-treated cells were exposed to LED white light for 30 min, while the dark-treated plates were placed in the dark as a control. After treatment, all 96-well plates were incubated at 37°C, 21% O2, and 5% CO2 for 24 h, then incubated with cell culture medium containing 10% MTT solution for 1 h, followed by replacement with DMSO solution. After shaking, the cells were analyzed using a microplate reader. NAC was pre-added to tumor cells in the white light-treated group at the same dosage, and cell viability was tested again after 24 h of the same treatment. The cell viability test results for QM-DMAC in normal 16HBE cells, tumor cells A549, and tumor cells A549 pretreated with NAC for 1 h are shown below. Figure 7 As shown in B, 7C and 7D.

[0094] Depend on Figure 7 As shown in B and 7C, under dark conditions, even at a QM-DMAC concentration as high as 30 μM, the viability of tumor cells and normal cells remained above 95% after 24 hours of QM-DMAC staining. This result indicates that QM-DMAC has low dark toxicity.

[0095] Depend on Figure 7 Based on B, 7C, and 7D, it can be seen that under white light irradiation, the viability of normal cells remains at around 90%. Figure 7 B); Under the same irradiation conditions, tumor cells exhibited dose-dependent cytotoxicity—cell viability gradually decreased with increasing concentration, and a QM-DMAC concentration of 20 μM led to significant cell death. Figure 7C); After NAC pretreatment, the photoinducible toxicity of QM-DMAC to tumor cells was reduced. Figure 7 (D). This result indicates that QM-DMAC can be used for photodynamic therapy of tumor cells.

[0096] (ix) Mechanism of selective targeted therapy to tumor cell lysosomes

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

[0098] A549 cells were divided into a control group (Control), a light-only group (L), a QM-DMAC group, a treatment group (QM-DMAC+L), and a QM-DMAC+L+NAC group. Lysosomal integrity was assessed using AO staining. The QM-DMAC+L+NAC group received 10 μM of [a specific treatment] on A549 tumor cells. NAC was pre-cultured at 37℃, 21% O2, and 5% CO2 for 1 h, followed by the addition of 20 μM QM-DMAC and incubation at 37℃, 21% O2, and 5% CO2 for 1 h. Then, it was irradiated with white light using the same parameters, followed by incubation with 10 μM AO for 30 min. Fluorescence signals were detected using confocal microscopy. The difference between the control group (Control) and the QM-DMAC+L+NAC group was that QM-DMAC and NAC were replaced with corresponding volumes of PBS buffer, and no light treatment was performed. The difference between the light-only group (L) and the QM-DMAC+L+NAC group was that QM-DMAC and NAC were replaced with corresponding volumes of PBS buffer, respectively. The difference between the treatment group (QM-DMAC+L) and the QM-DMAC+L+NAC group was that NAC was replaced with the same volume of PBS buffer. The results are as follows: Figure 8 As shown.

[0099] Depend on Figure 8 Green fluorescence was observed in the cytoplasm / nucleus of the control group, the light-only group (L), the QM-DMAC group, and the QM-DMAC+L+NAC group, while red fluorescence was observed in the lysosomes, indicating that the lysosomes remained intact. Conversely, tumor cells incubated with QM-DMAC and subsequently irradiated with white light showed a significant decrease in the intensity of the red fluorescence of AO. This result indicates that the lysosomal membrane in the QM-DMAC+L group was damaged and ruptured after light irradiation. However, in the QM-DMAC+L+NAC group, the red fluorescence in the lysosomes remained intact after light irradiation, suggesting 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 model (BALB / c). The specific steps were as follows:

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

[0103] Tumor-bearing BALB / c nude mice were randomly divided into a control group, a light-only group (L), a QM-DMAC group, and a treatment group (QM-DMAC+L). The QM-DMAC+L group received an intratumoral injection of QM-DMAC (20 mM, 100 μL / 200 mm). 3 The tumors were then irradiated with white light for 30 minutes every four days. The control group (Control) and the QM-DMAC+L group differed in that the QM-DMAC group used the corresponding volume of PBS buffer instead of PBS buffer and did not receive light treatment. The light-only group (L) and the QM-DMAC+L group differed in that the QM-DMAC group used the corresponding volume of PBS buffer instead of PBS buffer. The QM-DMAC group and the QM-DMAC+L group differed in that the QM-DMAC group did not receive light treatment. Tumor volume was monitored every four days, calculated as (tumor length) × (tumor width). 2 / 2, the result is as follows Figure 9 As shown in A and 9B.

[0104] Depend on Figure 9 As shown in A and 9B, the light-only group (L) and the QM-DMAC group had no significant inhibitory effect on tumor growth, while the QM-DMAC+L group had a significant inhibitory effect on tumor growth. These results indicate that QM-DMAC has a highly effective tumor phototherapy (PDT) effect.

[0105] Further hematoxylin-eosin (H&E) staining was performed on the organs of each group of mice. The specific steps were as follows: the main organs of the mice were removed, embedded in paraffin, sectioned, stained with hematoxylin-eosin, and imaged under an optical microscope (20×). The results are as follows. Figure 9 As shown in C. No obvious tissue damage or inflammation was observed in the major organs of each group.

[0106] Depend on Figure 9 It is known that QM-DMAC has significant therapeutic effect on tumor PDT, and has good biocompatibility and few toxic side effects.

[0107] (xi) Radiosensitization

[0108] 11.1 The efficiency of ROS generation in the "combination therapy" approach (R+QM-DMAC+L) of PDT combined with low-dose radiotherapy (R) was evaluated using the DCFH-DA assay. The specific steps were as follows:

[0109] A549 cells were divided into a combination therapy 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-only group (R), and a control group (Control).

[0110] The ROS scavenger group (R+QM-DMAC+L+NAC) involved adding 10 μM NAC to A549 cells and culturing them at 37°C, 21% O2, and 5% CO2 for 1 h. Then, 20 μM QM-DMAC was added, followed by culturing at 37°C, 21% O2, and 5% CO2 for another 1 h. Cells were then irradiated with white LED light for 30 min to generate a large amount of ROS, and subsequently treated with X-rays (R = 4 Gy, dose rate: 1 Gy / min). The difference between the "combination therapy" group (R+QM-DMAC+L) and the ROS scavenger group was that the same volume of PBS buffer was used instead of NAC. The difference between the QM-DMAC+L group and the ROS scavenger group was that the corresponding volume of PBS buffer was used instead of NAC, and X-ray treatment was not performed. Treatment was administered at a dose rate of 1 Gy / min (R = 4 Gy). The difference between the radiation-only group (R) and the ROS scavenger group was that R did not receive white light irradiation and used corresponding volumes of PBS buffer instead of QM-DMAC and NAC, respectively. The difference between the control group and the ROS scavenger group was that R also used corresponding volumes of PBS buffer instead of QM-DMAC and NAC, and did not receive X-ray treatment or white light irradiation. After treatment, each group was incubated with 10 μM CFH-DA for 30 min, and fluorescence signals were detected by flow cytometry. Results are as follows: Figure 10 As shown in Figure A.

[0111] Depend on Figure 10 As shown in Figure A, compared with other treatment groups, the ROS level of tumor cells in the "combination therapy" group was significantly increased; the ROS level of cells in the "combination therapy" group only increased slightly when pretreated with NAC. These results indicate that under light irradiation, 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 enhance radiosensitivity was evaluated using a clonogenic assay. The specific steps were as follows:

[0113] A549 cells were divided into a combination therapy 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-only group (R), and a control group (Control). Except for ionizing radiation irradiation (dose rate: 1 Gy / min) adjusted to 0, 2, 4, 6, 8, and 10 Gy doses (dose rate: 1 Gy / min), the treatment methods for each group were the same as in section 11.1. After irradiation, cells were isolated using trypsin and seeded into cell culture dishes (approximately 200–6000 cells per dish) and cultured for 12–15 days to allow colony formation. Survival curves were used to assess the colony-forming ability of tumor cells. The results are as follows: Figure 10 As shown in B;

[0114] Depend on Figure 10 As shown in Figure B, the survival fraction was determined and calculated using survival curves, and the radiosensitization effect of QM-DMAC was calculated by measuring the radiosensitization ratio (SER). The radiosensitization ratio (SER) of the "combination therapy" group (R+QM-DMAC+L) was 1.75, which was significantly higher than the SER values ​​of other treatment groups and the commonly used photosensitizer Ce6 (R+Ce6+L) (1.45). This result indicates that the radiosensitization effect of QM-DMAC is superior to that of Ce6 because QM-DMAC not only generates a large amount of ROS but also triggers LMP. After adding the ROS scavenger NAC, the SER value of the R+QM-DMAC+L+NAC group was only 0.95. This result indicates that increasing cellular ROS levels is crucial for enhancing the radiosensitization effect of QM-DMAC. Compared with the single radiation group (R) or the QM-DMAC+L group, the combination therapy of the R+QM-DMAC+L group showed a significant inhibitory effect on colony formation.

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

[0116] A549 tumor-bearing BALB / c nude mice were randomly divided into five groups: combined treatment group (R+QM-DMAC+L), QM-DMAC+L group, R+QM-DMAC group, radiation alone group (R), and control group (Control). The mice were injected intratumorally with either PBS or QM-DMAC (20mM, 100μL / 200mm). 3 (Tumor); The light irradiation group received 30 minutes of light irradiation every four days, while the combined treatment group received R+QM-DMAC+L light irradiation followed by X-ray treatment (R=4Gy), and the tumor volume of mice was monitored. Tumor volume = (tumor length) × (tumor width) 2 / 2), the result is as follows Figure 10 As shown in C and 10D.

[0117] Depend on Figure 10 As shown in C and 10D, compared with the control group, the tumor volume in both the QM-DMAC+L group and the "combination therapy" group (R+QM-DMAC+L) was significantly reduced, and the "combination therapy" group exhibited significantly enhanced antitumor activity. These results indicate that under white light irradiation, QM-DMAC significantly enhances the antitumor effect of radiotherapy, further demonstrating 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 considered to play an important role in the development of drug resistance.

[0120] 12.1 To evaluate the targeting ability of QM-DMAC against drug-resistant tumor cells, this application used QM-DMAC to perform live-cell imaging of multidrug-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 co-incubated at 37℃, 21% O2, and 5% CO2 for 60 min. Then, commercially available lysosomal dye (Lysotracker Deep Red, LTDR 50 nM) was added for co-staining and imaging incubation for 20 min. Imaging was then performed using a confocal microscope. The results are as follows: Figure 11 As shown in Figure A.

[0122] Depend on Figure 11 As can be seen from A, QM-DMAC has good cell permeability and high-contrast lysosomal targeting imaging capabilities.

[0123] 12.2 Under white light irradiation, the enhancement effect of QM-DMAC chemotherapy was evaluated. The specific steps were as follows:

[0124] MTT assay for cell viability: A549 / DDP tumor cells were seeded at a density of 6000 cells per well in two 96-well plates and cultured overnight. One group of 96-well plates served as a control group (Dark group) without light treatment, while the other group served as a white light treatment group (White Light group). The culture medium was replaced with fresh cell culture medium containing different amounts of QM-DMAC (0, 5, 10, 20, and 30 μM) and incubated for 1 h. After incubation, the cells in the light group were exposed to LED white light for 30 min, while the Dark group plates were placed in the dark as a control. After treatment, all 96-well plates were incubated in a cell culture incubator at 37°C, 21% O2, and 5% CO2 for 24 h. Then, the cells were incubated with cell culture medium containing 10% MTT solution for 1 h, replaced with DMSO solution, shaken, and analyzed using a microplate reader. The results are shown below. Figure 11 As shown in B.

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

[0126] 12.3 The DCFH-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 combination therapy group (CDDP+QM-DMAC+L, CDDP representing cisplatin), a ROS scavenger group (CDDP+QM-DMAC+L+NAC), a CDDP+QM-DMAC group, a QM-DMAC+L group, a cisplatin-only group (CDDP), and a control group. The ROS scavenger group (CDDP+QM-DMAC+L+NAC) was treated with 10 μM cisplatin added to A549 / DDP tumor cells. NAC was cultured at 37°C, 21% O2, and 5% CO2 for 1 hour, then 20 μM QM-DMAC was added, followed by 1 hour of culture at 37°C, 21% O2, and 5% CO2. The cells were then irradiated with LED white light for 30 minutes to generate significant amounts of ROS, and subsequently treated with cisplatin-CDDP (final concentration 200 μM). The difference between the "combination therapy" group (CDDP+QM-DMAC+L) and the ROS scavenger group was that the same volume of PBS buffer was used instead of NAC. The CDDP+QM-DMAC group... The difference between the ROS scavenger group and the control group was that the corresponding volume of PBS buffer was used instead of NAC and no white light irradiation was performed. The difference between the cisplatin-only (CDDP) group and the ROS scavenger group was that no white light irradiation was performed, and the corresponding volume of PBS buffer was used instead of QM-DMAC and NAC, respectively. The difference between the control group and the ROS scavenger group was that the corresponding volume of PBS buffer was used instead of QM-DMAC and NAC, respectively, and no cisplatin CDDP treatment or white light irradiation was performed. After treatment, 10 μM DCFH-DA was added to each group, and the mixture was incubated at 37°C, 21% O2, and 5% CO2 for 30 min. Fluorescence signals were detected by flow cytometry. The results are as follows: Figure 11 As shown in C.

[0128] Depend on Figure 11 C indicates that after receiving "combination therapy" (PDT combined with cisplatin, CDDP + QM-DMAC + L), the level of ROS generation in drug-resistant tumor cells was higher. This result suggests that under light exposure, QM-DMAC can promote cellular ROS generation and enhance the antitumor efficacy of CDDP treatment against drug-resistant tumor cells. ROS scavengers can reverse the ROS generation induced by combination therapy. This also demonstrates that cellular ROS levels are crucial for enhancing the efficacy of chemotherapy. ROS induces LMP, disrupting the integrity of the lysosomal membrane and leading to leakage of lysosomal contents. This process allows the release of antitumor drugs isolated within the lysosome, thereby restoring their antitumor 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 several groups: a combination therapy 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-only group (CDDP), and a control group. The treatment parameters for each group were the same as in section 12.3. After treatment, each group was incubated with 10 μM AO at 37°C, 21% O2, and 5% CO2 for 30 min. Fluorescence signals were detected using a fluorescence confocal microscope. The results are as follows: Figure 12 As shown.

[0131] Depend on Figure 12 Green fluorescence was observed in the cytoplasm / nucleus of the control group, the cisplatin-only group (CDDP), and the CDDP+QM-DMAC group, and red fluorescence was observed in the lysosomes, indicating good lysosomal integrity. Conversely, the QM-DMAC+L group and the CDDP+QM-DMAC+L group showed a significant decrease in the red fluorescence intensity of AO. These results indicate that treatment led to LMP and lysosomal membrane rupture, and that NAC could alleviate light-induced LMP.

[0132] 12.5 This application further adopts Alexa Immunostaining and flow cytometry analysis of drug-resistant tumor cells with 647Anti-gamma H2A.X antibody were performed. The specific steps were as follows:

[0133] A549 / DDP cells were randomly assigned to four groups: a combination therapy 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-only group (CDDP), and a control group. Treatment parameters for each group were the same as in section 12.3. After 16 hours of treatment, cells were fixed and permeabilized (4% paraformaldehyde), then treated with Alexa at a 1 / 500 dilution. Cells were labeled with 647Anti-gamma H2A.X antibody and analyzed by flow cytometry. The results are as follows: Figure 13 As shown.

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

[0135] 12.6 This application further evaluated the in vivo chemosensitizing efficacy of QM-DMAC in drug-resistant tumors, specifically through the following steps:

[0136] A549 / DDP tumor-bearing BALB / c nude mice were randomly divided into a combination therapy group (CDDP+QM-DMAC+L), a CDDP+QM-DMAC group, a QM-DMAC+L group, a cisplatin-only group (CDDP), and a control group. The light-exposed groups received 30 minutes of light exposure every four days, while the CDDP+QM-DMAC+L combination therapy group received cisplatin treatment after light exposure. The corresponding drugs were administered via intratumoral injection (QM-DMAC: 20mM, 100μL / 200mm). 3 (Tumor); Tumor volume in mice was monitored every four days. Tumor volume = (tumor length) × (tumor width) 2 / 2. The result is as follows: Figure 14 As shown.

[0137] Depend on Figure 14 The results showed that tumor volume was significantly reduced in both the QM-DMAC+L group and the combination therapy (CDDP+QM-DMAC+L) group, with the CDDP+QM-DMAC+L group exhibiting particularly significant anti-cancer activity. These results indicate that under white light irradiation, QM-DMAC can significantly enhance the anti-tumor effect of chemotherapeutic 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 invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should 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 below: 。 2. The method for preparing the fluorescent compound according to claim 1, characterized in that, Includes the following steps: Compound 1 and Compound 2 were dissolved in a solvent and subjected to a reflux reaction under a protective gas atmosphere in the presence of a catalyst to obtain the fluorescent compound. The structural formulas of compounds 1 and 2 are shown below: ； 。 3. The method for preparing the fluorescent compound according to claim 2, 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 amines, pyridines, piperidines, and morpholines; 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.

4. The method for preparing the fluorescent compound according to claim 2, characterized in that, The reflux reaction is followed by the following step: purification.

5. The use of the fluorescent compound as described in claim 1 or the fluorescent compound prepared according to any one of claims 2 to 4 in lysosomal targeted imaging for non-disease diagnostic purposes.

6. The use of the fluorescent compound of claim 1 or the fluorescent compound prepared according to any one of claims 2 to 4 in the preparation of a medicament for adjuvant photodynamic therapy of tumors, wherein the tumor is selected from lung cancer.

7. The use of the fluorescent compound of claim 1 or the fluorescent compound prepared according to any one of claims 2 to 4 in the preparation of radiosensitizers.

8. The use of the fluorescent compound of claim 1 or the fluorescent compound prepared according to any one of claims 2 to 4 in the preparation of chemotherapeutic sensitizers.

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