Composition capable of selectively anchoring endoplasmic reticulum of PLD1 high-expression tumor cell and application of composition

Abstract

The invention provides a composition capable of selectively anchoring a PLD1 high-expression tumor cell endoplasmic reticulum and application of the composition, and belongs to the technical field of biological medicines. The composition capable of selectively anchoring the endoplasmic reticulum of the PLD1 high-expression tumor cell is composed of oxoTCO capable of being catalyzed and integrated to phospholipid by phospholipase D1 and an aggregation-induced emission molecule Tz-TBR quenched by tetrazine, in the tumor cell with high-expression phospholipase D1, oxoTCO is firstly integrated to cell membrane phospholipid, then the Tz-TBR and the oxoTCO on the phospholipid are subjected to a biological orthogonal reaction, and the endoplasmic reticulum of the tumor cell with high-expression phospholipase D1 is obtained. The fluorescent light is transferred to the endoplasmic reticulum through phospholipid while the fluorescent light is turned on, and the endoplasmic reticulum is induced to generate visual stress under illumination. The method has the beneficial effects that 1, the preparation is simple; 2, the tumor cell selectivity is high; and 3, the endoplasmic reticulum targeting injury effect is obvious.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a composition that can selectively anchor the endoplasmic reticulum of PLD1-overexpressing tumor cells and its application. Background Technology

[0002] Bioorthogonal techniques are powerful tools in chemical biology and disease diagnosis and treatment research. Due to their high kinetics, selectivity, and stability under complex biological conditions, bioorthogonal techniques have been widely used to elucidate the functions of intrinsic biomolecules in biological processes, thereby improving the specificity of reagents in clinical diagnosis and enhancing the efficiency of drugs in disease treatment. Recently, this technology has been introduced into the field of precision medicine, showing great potential in subcellular targeted therapy, which can improve treatment outcomes and minimize side effects.

[0003] As crucial components for maintaining cellular function, organelles have been identified as key targets for disease treatment. In recent years, various organelle-targeted therapies have emerged for cancer treatment, including those targeting cell membrane integrity disruption, mitochondrial metabolic interference, endoplasmic reticulum (ER) oxidative stress induction, lysosomal membrane permeability, and nuclear DNA damage. Some studies have combined bioorthogonal techniques with these organelle-targeted therapies to improve drug selectivity and reduce side effects. However, research on using bioorthogonal techniques for organelle-targeted therapies remains limited and primarily focuses on the cell membrane, mitochondria, and lysosomes. The ER is vital for protein synthesis, lipid metabolism, and signal transduction. Inducing ER stress to lead to cell death is an effective method for inducing immunogenic cell death (ICD), which can significantly enhance tumor immunogenicity, thus making it suitable for treating "cold" tumors. However, currently reported organelle-targeting bioorthogonal techniques mainly rely on small molecules or peptides lacking tumor selectivity, thus limiting off-target effects. Therefore, developing novel bioorthogonal therapeutic systems that combine organelle specificity and tumor selectivity is crucial for advancing precision cancer treatment. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a composition that can selectively anchor the endoplasmic reticulum of PLD1-overexpressing tumor cells and its application. In the composition that can selectively anchor the endoplasmic reticulum of PLD1-overexpressing tumor cells prepared using the fluorescent substances of the present invention, under the catalysis of PLD1, oxoTCO first integrates onto the cell membrane phospholipids. Subsequently, Tz-TBR undergoes a bioorthogonal reaction with oxoTCO on the phospholipids, achieving fluorescence activation while simultaneously reaching the endoplasmic reticulum via phospholipid transport, and selectively inducing a visible stress response in the endoplasmic reticulum of tumor cells under light irradiation.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a fluorescent substance having the structure shown in Formula I:

[0007]

[0008] This invention also provides a method for preparing the fluorescent substances described in the above technical solution, comprising the following steps:

[0009] 4-bromo-N,N-bis(4-methoxyphenyl)aniline, bis(pinacol)diboron, Pd(dppf)Cl2, AcOK and an organic solvent were mixed and subjected to a first substitution reaction to obtain the first substituted product;

[0010] The first substitution product, 7-bromo-2,3-dihydrothiopheno[3,4-b][1,4]dioxane-5-carboxaldehyde, Pd(PPh3)4, an inorganic alkaline solution, and an organic solvent were mixed to carry out a second substitution reaction to obtain the second substitution product;

[0011] The second substituted product, rhodanine-N-acetic acid, AcONH4 and an organic solvent were mixed and subjected to a third substituted reaction to obtain the third substituted product.

[0012] The third substituted product, methyltetraazineamine, EDCI, 1-hydroxybenzotriazole, diisopropylethylamine and organic solvent were mixed and subjected to a condensation acylation reaction to obtain the fluorescent substance.

[0013] Preferably, the temperature of the first substitution reaction is 100–120°C and the time is 6–10 h; the temperature of the second substitution reaction is 70–80°C and the time is 6–10 h; and the temperature of the third substitution reaction is 100–120°C and the time is 6–10 h.

[0014] This invention also provides the application of the fluorescent substances described in the above technical solution in the preparation of drugs for anchoring PLD1-highly expressed tumor cells in the endoplasmic reticulum.

[0015] This invention also provides a composition (also a biological orthogonal therapy system) that can selectively anchor the endoplasmic reticulum of PLD1-overexpressing tumor cells, comprising oxoTCO and the fluorescent substance described in the above-mentioned technical solution, wherein the oxoTCO has the structure shown in Formula II:

[0016]

[0017] Preferably, the molar ratio of oxoTCO to the fluorescent substance is 1000-5000:10-20.

[0018] The present invention also provides an aqueous composition comprising the composition described above and water.

[0019] Preferably, the concentration of oxoTCO in the aqueous solution of the composition is 1-5 mM, and the concentration of the fluorescent substance is 10-20 μM.

[0020] The present invention also provides the use of the composition described in the above-described technical solutions or the aqueous solution of the composition described in the above-described technical solutions in the preparation of antitumor drugs.

[0021] Preferably, the antitumor drug includes an anticervical cancer drug.

[0022] This invention provides a fluorescent substance having the structure shown in Formula I. Compared with the prior art, the fluorescent substance of this invention is a novel fluorescent substance that can be combined with oxoTCO to obtain a biological orthogonal therapy system that can selectively anchor the endoplasmic reticulum of PLD1-overexpressing tumor cells.

[0023] The present invention also provides a method for preparing the fluorescent substance, which is simple and easy to implement for industrial application.

[0024] This invention also provides a composition that can selectively anchor the endoplasmic reticulum (ER) of PLD1-overexpressing tumor cells. A bioorthogonal therapeutic system was designed and developed that can specifically anchor the ER of tumor cells. Under PLD1 catalysis, oxoTCO first integrates into cell membrane phospholipids. Subsequently, Tz-TBR undergoes a bioorthogonal reaction with the oxoTCO on the phospholipids, achieving fluorescence activation while simultaneously reaching the ER via phospholipid transport. Under illumination, it selectively induces a visible stress response in the ER of tumor cells. The composition of this invention exhibits high tumor cell selectivity and significant ER-targeted damage effect. Attached Figure Description

[0025] Figure 1 For Tz-TBR in d6-DMSO 1 H NMR spectrum;

[0026] Figure 2 For Tz-TBR in d6-DMSO 13 C NMR spectrum;

[0027] Figure 3 Fluorescence and reactive oxygen species generation performance of Tz-TBR with aqueous oxoTCO as the inoculum;

[0028] Figure 4 The results of PLD1 expression level detection in human tumor cells (human cervical cancer cells HeLa and human prostate cancer cells PC3) and human normal cells (human liver stellate cells LX-2 and human cardiomyocytes AC16) were compared.

[0029] Figure 5 To determine the subcellular organelle localization of the activated Tz-TBR within the cell;

[0030] Figure 6 To detect the activation effect of Tz-TBR in human tumor cells and normal cells;

[0031] Figure 7 In HeLa cells, activated Tz-TBR induces endoplasmic reticulum damage under light activation;

[0032] Figure 8 In HeLa cells, the activated Tz-TBR can effectively visualize the morphology of the endoplasmic reticulum before and after damage.

[0033] Figure 9 The activated Tz-TBR effectively inhibited HeLa cell activity. Detailed Implementation

[0034] This invention provides a fluorescent substance having the structure shown in Formula I:

[0035]

[0036] This invention also provides a method for preparing the fluorescent substances described in the above technical solution, comprising the following steps:

[0037] 4-bromo-N,N-bis(4-methoxyphenyl)aniline, bis(pinacol)diboron, Pd(dppf)Cl2, AcOK and an organic solvent were mixed and subjected to a first substitution reaction to obtain the first substituted product;

[0038] The first substitution product, 7-bromo-2,3-dihydrothiopheno[3,4-b][1,4]dioxane-5-carboxaldehyde, Pd(PPh3)4, an inorganic alkaline solution, and an organic solvent were mixed to carry out a second substitution reaction to obtain the second substitution product;

[0039] The second substituted product, rhodanine-N-acetic acid, AcONH4 and an organic solvent were mixed and subjected to a third substituted reaction to obtain the third substituted product.

[0040] The third substituted product, methyltetraazineamine, EDCI, 1-hydroxybenzotriazole, diisopropylethylamine and organic solvent were mixed and subjected to a condensation acylation reaction to obtain the fluorescent substance.

[0041] In this invention, the preparation principle of the fluorescent substance is shown in the following formula:

[0042]

[0043] Unless otherwise specified, all raw materials used in this invention are commercially available products in the field.

[0044] In this invention, 4-bromo-N,N-bis(4-methoxyphenyl)aniline, bis(pinacol)diboron, Pd(dppf)Cl2, AcOK and an organic solvent are mixed to carry out a first substitution reaction to obtain the first substituted product.

[0045] In this invention, the temperature of the first substitution reaction is preferably 100-120°C, specifically 100, 110 or 120°C, and the time is preferably 6-10 hours, specifically 6, 8 or 10 hours.

[0046] In this invention, the first substitution reaction is preferably carried out in nitrogen.

[0047] In this invention, the organic solvent is preferably toluene.

[0048] In this invention, the molar ratio of 4-bromo-N,N-bis(4-methoxyphenyl)aniline, bis(pinacol)diboron, Pd(dppf)Cl2 and AcOK is preferably 2:2.4:0.06:6.

[0049] In this invention, TLC is preferably used to monitor the progress of the first substitution reaction.

[0050] After the first substitution reaction is completed, the present invention preferably obtains the crude product by rotary evaporation, and then performs column chromatography purification to obtain the first substitution product.

[0051] After obtaining the first substituted product, the present invention mixes the first substituted product, 7-bromo-2,3-dihydrothiopheno[3,4-b][1,4]dioxane-5-carboxaldehyde, Pd(PPh3)4, an inorganic alkaline solution and an organic solvent to carry out a second substitution reaction to obtain the second substituted product.

[0052] In this invention, the temperature of the second substitution reaction is preferably 70-80°C, specifically 70, 75 or 80°C, and the time is preferably 6-10 hours, specifically 6, 8 or 10 hours.

[0053] In this invention, the second substitution reaction is preferably carried out in nitrogen.

[0054] In this invention, the organic solvent is preferably tetrahydrofuran (THF).

[0055] In this invention, the molar ratio of the first substituted product, 7-bromo-2,3-dihydrothiopheno[3,4-b][1,4]dioxane-5-carboxaldehyde, and Pd(PPh3)4 is preferably 1.78:2.14:0.018.

[0056] In this invention, the inorganic alkaline solution is preferably an aqueous solution of K2CO3, and the concentration of the aqueous solution of K2CO3 is preferably 2M.

[0057] In this invention, the preferred ratio of the first substituted product to the K2CO3 aqueous solution is 1.78 mmol: 4 mL.

[0058] In this invention, TLC is preferably used to monitor the progress of the second substitution reaction.

[0059] After the second substitution reaction is completed, the reaction temperature is preferably lowered to room temperature. Then, ethyl acetate (EA) is added to the reaction solution to extract the aqueous phase and collect the organic phase. The organic phase is concentrated by rotary evaporation to obtain the crude product, which is then purified by silica gel chromatography to obtain the second substitution product.

[0060] After obtaining the second substituted product, the present invention mixes the second substituted product, rhodanine-N-acetic acid, AcONH4 and an organic solvent to carry out a third substituted reaction to obtain the third substituted product.

[0061] In this invention, the temperature of the third substitution reaction is preferably 100-120°C, specifically 100, 110 or 120°C, and the time is preferably 6-10 hours, specifically 6, 8 or 10 hours.

[0062] In this invention, the third substitution reaction is preferably carried out in nitrogen.

[0063] In this invention, the organic solvent is preferably glacial acetic acid.

[0064] In this invention, the molar ratio of the second substituted product, rhodanine-N-acetic acid, and AcONH4 is preferably 1.5:7.5:1.5.

[0065] In this invention, the preferred ratio of the second substituted product to glacial acetic acid is 1.5 mmol: 5 mL.

[0066] After the third substitution reaction is completed, it is preferable to restore the reaction temperature to room temperature, then add ice water to the reaction solution for filtration, leaving the filtrate to obtain the third substitution product.

[0067] After obtaining the third substituted product, the present invention mixes the third substituted product, methyltetraazineamine, EDCI, 1-hydroxybenzotriazole (HOBt), diisopropylethylamine (iPr2NEt) and an organic solvent to carry out a condensation acylation reaction to obtain the fluorescent substance.

[0068] In this invention, the molar ratio of the third substituted product, methyltetraazine, EDCI and 1-hydroxybenzotriazole (HOBt) is preferably 1.16:1.16:2.23:2.23.

[0069] In this invention, the organic solvent is preferably dichloromethane (DCM).

[0070] In this invention, the preferred ratio of the third substituted product, dichloromethane, and iPr2NEt is 1.16 mmol: 9 mL: 0.105 mL.

[0071] In this invention, the condensation acylation reaction is preferably carried out at room temperature with stirring and left to stand overnight.

[0072] In this invention, the third substituted product, methyltetraazine, EDCI, and 1-hydroxybenzotriazole are preferably introduced into a reaction flask. After purging the reaction flask with nitrogen three times, dichloromethane and iPr2NEt are added sequentially. The mixture is then stirred at room temperature and left overnight for condensation acylation. ddH2O is then added to the reaction mixture, and the organic phase is extracted three times. The combined organic extracts are dried under reduced pressure by rotary evaporation. Finally, the fluorescent substance is purified by silica gel column chromatography.

[0073] This invention also provides the application of the fluorescent substances described in the above technical solution in the preparation of drugs for anchoring PLD1-highly expressed tumor cells in the endoplasmic reticulum.

[0074] This invention also provides a composition (also a biological orthogonal therapy system) that can selectively anchor the endoplasmic reticulum of PLD1-overexpressing tumor cells, comprising oxoTCO and the fluorescent substance described in the above-mentioned technical solution, wherein the oxoTCO has the structure shown in Formula II:

[0075]

[0076] The present invention does not impose any particular limitation on the preparation method of the oxoTCO, and any preparation method well known to those skilled in the art can be used. The specific preparation principle is shown in the following formula:

[0077]

[0078] In this invention, the molar ratio of oxoTCO to fluorescent substances is 1000-5000:10-20.

[0079] The composition of the present invention is simple to prepare, has high selectivity for tumor cells, and exhibits significant endoplasmic reticulum-targeted damage.

[0080] The present invention also provides a method of applying the composition, preferably comprising the following steps: treating PLD1-overexpressing tumor cells with the oxoTCO; washing with PBS to remove unbound oxoTCO, then adding Tz-TBR to treat the cells; and irradiating the cells with a light source for 3 min.

[0081] The present invention also provides an aqueous composition comprising the composition described above and water.

[0082] In this invention, the concentration of oxoTCO in the aqueous solution of the composition is 1-5 mM, specifically 1, 2, 3, 4 or 5 mM, and the concentration of the fluorescent substance is 10-20 μM, specifically 10, 15 or 20 μM.

[0083] In this invention, the aqueous solution of the composition preferably also includes phospholipase D1 (PLD1), and the amount of PLD1 added to the aqueous solution of the composition is preferably 10 U / mL.

[0084] In this invention, the preparation method of the composition and the aqueous solution of the composition is preferably a conventional method for preparing the composition and the aqueous solution of the composition in the art.

[0085] The present invention also provides the use of the composition described in the above-described technical solutions or the aqueous solution of the composition described in the above-described technical solutions in the preparation of antitumor drugs.

[0086] In this invention, the antitumor drug includes an anticervical cancer drug.

[0087] The present invention does not impose any special limitation on the specific method of application, and any method known to those skilled in the art can be used.

[0088] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0089] Example 1

[0090] Tz-TBR synthesis:

[0091] (1) 4-Bromo-N,N-bis(4-methoxyphenyl)aniline (2 mmol), bis(pinacol)diboron (2.4 mmol), Pd(dppf)Cl2 (0.06 mmol), and AcOK (6 mmol) were dissolved in toluene (8 mL). The reaction mixture was then heated to 110 °C and maintained under nitrogen atmosphere for 8 h. The reaction progress was monitored by TLC, and the crude product was obtained by rotary evaporation after the reaction was complete. Finally, compound 7 (the first substituted product) was purified by column chromatography.

[0092] (2) Compound 7 (1.78 mmol), 7-bromo-2,3-dihydrothiopheno[3,4-b][1,4]dioxane-5-carboxaldehyde (2.14 mmol), and Pd(PPh3)4 (0.018 mmol) were dissolved in THF (6 mL), and then an aqueous solution of K2CO3 (2 M, 4 mL) was added. The reaction temperature was then raised to 75 °C and maintained under nitrogen atmosphere for 8 h. The reaction was monitored by TLC. After the reaction was complete, the reaction temperature was lowered to room temperature. Ethyl acetate was then added to the reaction solution to extract the aqueous phase and collect the organic phase. The organic phase was concentrated by rotary evaporation to obtain the crude product. Final purification was performed using silica gel chromatography to obtain compound 8 (i.e., the second substituted product).

[0093] (3) Compound 8 (1.5 mmol), rhodanine-N-acetic acid (7.5 mmol), and AcONH4 (4.5 mmol) were dissolved in glacial acetic acid (5 mL). The reaction temperature was then raised to 110 °C and carried out under nitrogen atmosphere for 8 h. After the reaction was completed, the reaction temperature was returned to room temperature. Finally, ice water was added to the reaction solution for filtration, and the filtrate was used to obtain compound 9 (i.e., the third-substituted product).

[0094] (4) Weigh compound 9 (1.16 mmol), methyltetraazine (1.16 mmol), EDCI (2.23 mmol), and HOBt (2.23 mmol) and introduce them into a reaction flask. After purging the flask three times with nitrogen, add DCM (9 mL) and iPr2NEt (105 μL) sequentially. The mixture is then stirred at room temperature and allowed to stand overnight. ddH2O is then added to the reaction mixture, and the organic phase is extracted three times. The combined organic extracts are dried under reduced pressure by rotary evaporation. Finally, Tz-TBR is purified by silica gel column chromatography.

[0095] Figure 1 For Tz-TBR in d6-DMSO 1 H NMR spectrum; Figure 2 For Tz-TBR in d6-DMSO 13 C NMR spectrum.

[0096] oxoTCO was prepared using conventional methods.

[0097] Figure 3To investigate the fluorescence and ROS-activating effect of oxoTCO on Tz-TBR using fluorescence spectroscopy and UV-Vis spectrophotometry, aqueous solutions of Tz-TBR (20 μM) with or without oxoTCO (400 μM) were prepared. The fluorescence spectra of both solutions were detected using fluorescence spectroscopy. 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a ROS indicator. First, DCFH-DA was activated with 0.01 M sodium hydroxide at room temperature for 30 min to generate 2′,7′-dichlorodihydrofluorescein (DCFH). Subsequently, DCFH (working concentration: 20 μM) was added to the Tz-TBR (20 μM) aqueous solutions with or without oxoTCO (400 μM). Then, the fluorescence spectra were measured using white light (100 mW / cm²). 2 The solution was irradiated for different durations to activate Tz-TBR. Finally, the total ROS level in the solution was assessed by detecting changes in the DCF (excitation light: 488 nm) fluorescence spectrum. The results confirmed that oxoTCO can effectively activate the fluorescence and ROS generation capabilities of Tz-TBR.

[0098] Figure 4 To detect the expression level of PLD1 in tumor cells and normal cells using Western blotting, HeLa cells, PC3 cells, AC16 cells, and LX-2 cells were cultured to the logarithmic growth phase and plated into 6-well plates. After reaching 80% confluence, cells were collected, and cellular proteins were extracted using radioimmunoprecipitation (RIPA) lysis buffer. Western blotting was then performed to detect the expression level of PLD1 in different cell lines. The experiment was performed in triplicate, and quantitative analysis was performed using ImageJ Fiji. Histograms were then used for statistical analysis. The results confirmed that the PLD1 expression level in tumor cells (HeLa and PC3) was significantly higher than that in normal cells (LX-2 and AC16).

[0099] Figure 5 To observe the subcellular organelle localization of the activated Tz-TBR using laser scanning confocal microscopy, HeLa cells were plotted at 6 × 10⁻⁶ cells / year. 4Cells were seeded at a density of cells / well in confocal culture dishes. After cell adhesion, cells were treated with drugs according to experimental groups. First, cells were cultured in blank medium containing 100 nM MPMA for 20 min to enhance PLD1 activity, while ER-tracker Green (1 μM), Mito-tracker Green (200 nM), or Lyso-tracker Green (100 nM) were added for organelle labeling. Next, cells were cultured again in blank medium containing 100 nM MPMA and 3 mM oxoTCO for 5 min. Then, cells were washed with PBS to remove unbound oxoTCO. Next, cells were cultured in blank medium containing Tz-TBR (10 μM) for 20 min. Finally, cells were observed and photographed using CLSM. The results confirmed that Tz-TBR was mainly localized in the endoplasmic reticulum.

[0100] Figure 6 To observe the activation effect of Tz-TBR in different cell types using laser scanning confocal microscopy, HeLa, PC3, LX-2, or AC16 cells were used at a concentration of 6 × 10⁻⁶. 4 Cells were seeded at a density of cells / well in confocal culture dishes. After cell adhesion, cells were treated with drugs according to experimental groups. First, cells were cultured in blank medium containing 100 nM PMA for 20 min to enhance PLD1 activity, while ER-tracker Green (1 μM) was added for ER labeling. Next, cells were cultured again in blank medium containing 100 nM PMA and 3 mMoxoTCO for 5 min. Then, cells were washed with PBS to remove unbound oxoTCO. Next, cells were cultured in blank medium containing Tz-TBR (10 μM) for 20 min. Finally, cells were observed and photographed using a CLSM. The results confirmed that Tz-TBR was activated only in tumor cells with high PLD1 expression.

[0101] Figure 7 To observe endoplasmic reticulum damage induced by the bioorthogonal therapy system using biotransmission electron microscopy (Bio-TEM), HeLa cells were seeded in 10 cm petri dishes. When the cell density reached 80%, PMA, oxoTCO, and Tz-TBR were added sequentially as described above. After 20 min, the cells were washed three times with PBS and then placed under light (100 mW / cm²). 2(3 min). After 4 h, the culture medium was discarded, cells were washed with PBS, and collected into 1.5 mL EP tubes using a cell scraper. The tubes were centrifuged at 2000 rpm for 10 min to enrich the cells at the bottom. The supernatant was discarded, and pre-chilled fixative (2.5 wt% glutaraldehyde) at 4 °C was slowly added along the tube wall. The tubes were then stored overnight at 4 °C. Subsequently, the samples were dehydrated, embedded, sectioned, and stained sequentially for ER morphological observation under TEM. The results confirmed that the bioorthogonal therapy system can effectively induce endoplasmic reticulum damage under photo-activated conditions.

[0102] Figure 8 To observe the co-localization of Tz-TBR with ER before and after ER injury using laser scanning confocal microscopy, HeLa cells were cultured at 6 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of cells / well in confocal culture dishes. After cell adhesion, cells were first cultured for 20 min in blank medium containing 100 nM PMA to enhance PLD1 activity, while ER-tracker Green (1 μM) was added for ER labeling. Next, cells were cultured again for 5 min in blank medium containing 100 nM PMA and 3 mM oxoTCO. Then, cells were washed with PBS to remove unbound oxoTCO. Subsequently, cells were cultured for 20 min in blank medium containing Tz-TBR (10 μM). Finally, observation and imaging were performed using laser scanning confocal microscopy before and after light activation, and co-localization analysis of Tz-TBR fluorescence and ER-tracker fluorescence before and after ER damage was performed using ImageJ Fiji software. The results confirmed that activated Tz-TBR can effectively label ER before and after ER damage.

[0103] Figure 9 To evaluate the inhibitory effect of the bioorthogonal therapy system on HeLa cell activity using a cytotoxicity assay (CCK-8), HeLa cells were incubated at 3 × 10⁻⁶ cells / cells. 3 Cells were seeded at a density of cells / well in 96-well plates, and the drug was added after cell adhesion. In the dark toxicity assay, gradient concentrations of Tz-TBR were added to the cells, and the results were analyzed using the CCK-8 assay after 24 hours. In the phototoxicity assay, the "Tz-TBR+L" group cells were first stimulated with PMA (100 nM) for 25 min, then treated with gradient concentrations of Tz-TBR, and irradiated with light (100 mW / cm²) for 20 min. 2Cells in the "oxoTCO+Tz-TBR+L" group were treated with PMA (100 nM) for 20 min before being treated with gradient concentrations of Tz-TBR, followed by treatment with PMA (100 nM) combined with oxoTCO (3 mM) for 5 min. Phototoxicity was assessed using CCK-8 assays 24 h after cell exposure to light. The working solution for CCK-8 assays was prepared by diluting CCK-8 stock solution with serum-free culture medium at a volume ratio of 1:9. The culture medium in the 96-well plates was discarded, and 100 μL of working solution was added to each well. Cells were then incubated at 37°C for 1 h. Assays were performed using a microplate reader at a wavelength of 450 nm. The results confirmed that oxoTCO pretreatment effectively activated the tumor-suppressive capacity of Tz-TBR under photoactivation.

[0104] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fluorescent substance having the structure shown in Formula I:

2. The method for preparing the fluorescent substance according to claim 1, characterized in that, Includes the following steps: 4-bromo-N,N-bis(4-methoxyphenyl)aniline, bis(pinacol)diboron, Pd(dppf)Cl2, AcOK and an organic solvent were mixed and subjected to a first substitution reaction to obtain the first substituted product; The first substitution product, 7-bromo-2,3-dihydrothiopheno[3,4-b][1,4]dioxane-5-carboxaldehyde, Pd(PPh3)4, an inorganic alkaline solution, and an organic solvent were mixed to carry out a second substitution reaction to obtain the second substitution product; The second substituted product, rhodanine-N-acetic acid, AcONH4 and an organic solvent were mixed and subjected to a third substituted reaction to obtain the third substituted product. The third substituted product, methyltetraazineamine, EDCI, 1-hydroxybenzotriazole, diisopropylethylamine and organic solvent were mixed and subjected to a condensation acylation reaction to obtain the fluorescent substance.

3. The preparation method according to claim 2, characterized in that, The temperature of the first substitution reaction is 100–120°C and the time is 6–10 h; the temperature of the second substitution reaction is 70–80°C and the time is 6–10 h; the temperature of the third substitution reaction is 100–120°C and the time is 6–10 h.

4. The use of the fluorescent substance according to claim 1 in the preparation of a drug for anchoring PLD1-overexpressing tumor cells in the endoplasmic reticulum.

5. A composition for selectively anchoring the endoplasmic reticulum of PLD1-overexpressing tumor cells, characterized in that, Including oxoTCO and the fluorescent material of claim 1, wherein the oxoTCO has the structure shown in Formula II:

6. The composition according to claim 5, characterized in that, The molar ratio of oxoTCO to fluorescent substances is 1000–5000:10–20.

7. An aqueous solution of a composition, characterized in that, Includes the composition of claim 5 or 6 and water.

8. The aqueous solution of the composition according to claim 7, characterized in that, The concentration of oxoTCO in the aqueous solution of the composition is 1–5 mM, and the concentration of the fluorescent substance is 10–20 μM.

9. The use of the composition of claim 5 or 6 or the aqueous solution of the composition of claim 7 or 8 in the preparation of an antitumor drug.

10. The application according to claim 9, characterized in that, The anti-tumor drugs include anti-cervical cancer drugs.

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