Mitochondrial targeting anti-cancer drug based on cyanine and lonidamine as well as preparation method and application of mitochondrial targeting anti-cancer drug

Abstract

The invention discloses a mitochondrial targeted anti-cancer drug based on cyanine and lonidamine and a preparation method and application thereof, and belongs to the technical field of drugs for treating tumors. Cyanine molecules and 3-mercaptobenzoic acid are subjected to a nucleophilic substitution reaction to prepare a cyanine derivative; the preparation method comprises the following steps: carrying out a coupling reaction on a cyanine derivative and a selenocystamine compound to prepare a cyanine-connexon conjugate with an amino terminal; the cyanine-connexon conjugate and lonidamine are subjected to a coupling reaction, and the targeted anti-cancer drug is obtained. According to the mitochondrial targeting anti-cancer drug based on cyanine and lonidamine and the preparation method and application thereof, after the drug targets tumor cell mitochondria, cyanine molecules generate a photodynamic effect and a photothermal effect at the same time under the irradiation of near-infrared light, ROS and high temperature are generated in situ, the mitochondrial structure and function are directly damaged, and the drug can be used for preparing the mitochondrial targeting anti-cancer drug based on cyanine and lonidamine. The ROS also synchronously triggers diselenide bond breakage, lonidamine is accurately released, and mitochondrial dysfunction and metabolic crisis are further aggravated.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology for treating tumors, and in particular to a mitochondrial-targeted anticancer drug based on cyanine and chlordamine, its preparation method, and its application. Background Technology

[0002] Cancer stem cells are a subset of cells in tumor tissues with the potential for self-renewal, proliferation, and multi-lineage differentiation. Numerous studies have confirmed that cancer stem cells are key drivers mediating tumor development, progression, recurrence, metastasis, and treatment resistance. Therefore, developing therapeutic drugs that can specifically target and eliminate cancer stem cells is not only a current frontier focus in cancer research but also a promising breakthrough direction for overcoming the limitations of traditional therapies, preventing tumor recurrence, and ultimately achieving a radical cure for cancer, possessing significant clinical implications and research prospects.

[0003] Mitochondria are not only the energy metabolism center of cells, but also key signaling hubs regulating the origin, plasticity, and tumor recurrence and metastasis driven by tumor stem cells. Tumor stem cells highly rely on mitochondrial oxidative phosphorylation for energy and maintain their stemness and resistance to chemotherapy drugs through a sophisticated regulatory mechanism involving reactive oxygen species (ROS) and mitophagy. Developing novel mitochondrial-targeting agents by optimizing the structure and function of known drugs already on the market or in clinical trials has become one of the core strategies for precise intervention in tumor stem cells. This strategy utilizes the characteristic of positively charged compounds specifically enriching against their concentration gradient due to the high negative membrane potential of the mitochondrial inner membrane. By structurally modifying small molecule drugs to endow them with mitochondrial targeting capabilities, the designed drug molecules are efficiently enriched in the mitochondria of tumor stem cells. This achieves specific clearance of tumor cells and tumor stem cells by inducing mitochondrial membrane potential collapse, energy metabolism disorders, promoting excessive ROS production, or interfering with mitochondrial DNA replication and repair.

[0004] Lonidamine (LND), a classic energy metabolism regulator, interferes with the energy supply of tumor cells by inhibiting hexokinase II and disrupting mitochondrial function. However, its low bioavailability, poor tumor targeting, and limited mitochondrial accumulation efficiency, especially in tumor stem cells where it is difficult to achieve effective therapeutic concentrations, limit its clinical efficacy as a single agent. Therefore, how to specifically deliver higher concentrations of lonidamine to the mitochondria of tumor cells and tumor stem cells to enhance its anti-tumor efficacy has become a crucial scientific problem urgently needing to be solved in the field of tumor metabolic therapy.

[0005] Photodynamic therapy (PDT) and photothermal therapy (PTT) are effective therapeutic strategies that utilize the unique photophysical properties of photosensitizers to induce tumor cell death. PDT induces oxidative stress-mediated tumor cell apoptosis or necrosis by exciting photosensitizers to generate reactive oxygen species under specific wavelengths of light. Photothermal therapy utilizes photothermal agents to efficiently convert absorbed light energy into heat energy, inducing protein denaturation or thermal ablation of tumor cells through localized high temperatures. Cyanide dyes, in particular, not only possess excellent near-infrared fluorescence imaging capabilities but also demonstrate significant potential in enhancing the efficacy of PDT and PTT due to their remarkable tumor selectivity and mitochondrial enrichment capacity. They not only possess precise targeting capabilities for tumor cells but also exert a synergistic anti-tumor effect combining PDT and PTT under specific wavelengths of light excitation, making them a powerful and multifunctional therapeutic agent in the field of cancer treatment. Most importantly, cyanine dyes have mitochondrial targeting properties, and the mitochondria of tumor cells and tumor stem cells are highly sensitive to PDT, PTT and their synergistic combination therapy. They can further activate the body's anti-tumor immune response through the immunogenic cell death (ICD) effect, thereby effectively eradicating tumor cells and tumor stem cells.

[0006] However, there are currently no reports of using cyanin and chlordamine together to treat tumors. Summary of the Invention

[0007] The purpose of this invention is to provide a mitochondrial-targeted anticancer drug based on cyanine and chlordamine, its preparation method, and its application. The prepared drug can efficiently target the mitochondria of tumor cells. Under near-infrared light irradiation, cyanine molecules simultaneously generate photodynamic and photothermal effects, producing ROS and high temperature in situ. This not only directly damages the structure and function of mitochondria, but ROS also simultaneously triggers the breaking of diselenium bonds, precisely releasing chlordamine, further aggravating mitochondrial dysfunction and metabolic crisis. This achieves a synergistic effect of photodynamic therapy, photothermal therapy, and metabolic inhibition, and further activates the body's anti-tumor immune response through the ICD effect, achieving selective killing of tumor cells and tumor stem cells and targeted treatment of tumors.

[0008] To achieve the above objectives, the present invention provides a mitochondrial-targeted anticancer drug based on cyanin and chlordamine, wherein the targeted anticancer drug uses the compound represented by Formula I as its active ingredient. Formula I; Where R is one of methyl, sulfonic acid, and carboxyl groups, and n = 0-10.

[0009] Preferably, the targeted anticancer drug further includes pharmaceutically acceptable additives of Formula I, pharmaceutically acceptable carriers, and excipients.

[0010] Further, pharmaceutically acceptable excipients include microcrystalline cellulose, lactose, mannitol, starch, sucrose, glucose, dextran, sodium chloride, phosphate, citrate, croscarmellose sodium, hydroxypropyl methylcellulose, sodium carboxymethyl starch, magnesium stearate, silica, medium- and long-chain triglycerides, propylene glycol, polyethylene glycol, polylactic acid, polylactic acid-glycolic acid copolymer, polyvinylpyrrolidone, ethyl oleate, polysorbates, poloxamer, polyoxyethylene castor oil, cyclodextrin and its derivatives, polyvinyl alcohol, and chitosan; Pharmaceutically acceptable carriers include liposomes, polymer micelles, polymer nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers, polymer vesicles, exosomes, nanoemulsions, dendritic macromolecules, protein nanoparticles, nanocrystals, nanogels, metal-organic framework carriers, covalent organic framework carriers, upconversion nanoparticles, microspheres, microvesicles, microemulsions, hydrogels, and microneedles.

[0011] Furthermore, the dosage form of the targeted anticancer drug is one of the following: injection, powder for injection, oral preparation, spray, tablet, capsule, suppository, and patch.

[0012] This invention also provides a method for preparing a mitochondrial-targeted anticancer drug based on cyanin and chlordamine, the synthetic route of which is as follows: ; Where X is one of chlorine, bromine, and iodine; The preparation of targeted anticancer drugs specifically includes the following steps: S1. A nucleophilic substitution reaction was carried out between cyanine molecules and 3-mercaptobenzoic acid in DMF to obtain a reaction solution. The reaction solution was washed, centrifuged, separated and purified, and then vacuum dried to prepare cyanine derivatives. S2. The cyanine derivative obtained in S1 was dissolved in methanol solution, and a selenocysteine ​​compound was added to undergo a coupling reaction. After the reaction was completed, the mixture was separated and purified to prepare an amino-terminated cyanine-linker conjugate. S3. Clonidamine is dissolved in a methanol solution, and then the cyanine-linker conjugate obtained in S2 is added to react with clodinidamine to obtain a targeted anticancer drug.

[0013] Preferably, in S1, the molar ratio of cyanine molecules to 3-mercaptobenzoic acid is 1:(2-4), the nucleophilic substitution reaction temperature is 20-30℃, the reaction time is 40-50h, the reaction is carried out under light with stirring, the reaction solution is washed with ether and centrifuged 3-5 times, the precipitate is collected, and the precipitate is separated and purified by column chromatography. The chromatographic solvent for column chromatography is CH3OH / CH2Cl2, the volume ratio of CH3OH / CH2Cl2 is 1:3, and the solution is dried under vacuum at room temperature in the dark to obtain the cyanine derivative.

[0014] Preferably, in S2, the selenocystamine compound is one of selenocystamine and selenocystamine hydrochloride, the molar ratio of cyanine derivative to selenocystamine compound is 1:(2-4), the coupling reaction temperature is 20-30℃, the reaction time is 20-25h, the reaction is carried out under light with stirring, and the product obtained from the reaction is separated and purified by column chromatography. The chromatographic solvent for column chromatography is CH3OH / CH2Cl2, and the volume ratio of CH3OH to CH2Cl2 is 1:4.

[0015] Preferably, in S2, before adding the selenocysteine ​​compound, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinic acid are added first, and the molar ratio of cyanine derivative, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinic acid is 1:(4-6):1.

[0016] Preferably, in S3, the molar ratio of chlordamine to cyanine-linker coupling compound is (2-4):1, the coupling reaction temperature is 20-30℃, the reaction time is 20-25h, the reaction is carried out under light with stirring, and the product obtained from the reaction is separated and purified by column chromatography. The chromatographic solvent for column chromatography is CH3OH / CH2Cl2, and the volume ratio of CH3OH to CH2Cl2 is 1:4.

[0017] Preferably, in S3, before the cyanine-linker conjugate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinic acid are added first, and the molar ratio of chlordamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinic acid is (2-4):(2-4):1.

[0018] This invention also provides the application of a mitochondrial-targeted anticancer drug based on cyanin and chlordamine in the preparation of a tumor treatment drug.

[0019] The present invention also provides the application of a mitochondrial-targeted anticancer drug based on cyanin and chlordamine in the preparation of a drug that kills tumor stem cells.

[0020] Furthermore, tumors include one of the following: breast cancer, lung cancer, liver cancer, prostate cancer, head and neck tumors, brain tumors, colon cancer, ovarian cancer, melanoma, and pancreatic cancer.

[0021] Therefore, the present invention employs the above-mentioned mitochondrial-targeted anticancer drug based on cyanin and chlordamine, its preparation method, and its application, which has the following beneficial effects: (1) A novel chlordamine derivative was obtained by coupling cyanine molecules with chlordamine. Due to the significant tumor selectivity and mitochondrial enrichment ability of cyanine molecules, the chlordamine derivative can selectively target the mitochondria of tumor cells and tumor stem cells, thereby improving the tumor targeting and bioavailability of chlordamine.

[0022] (2) When cyanine molecules are excited by near-infrared light, they can generate a high efficiency photodynamic effect, generating a large amount of reactive oxygen species in situ at the tumor site, which precisely triggers the breaking of the diselenium bond in the prodrug, thereby achieving on-demand and controllable release of chlordamine. This design ensures that the drug delivery has high spatiotemporal specificity and target selectivity, and achieves high-precision selective killing of tumor cells and tumor stem cells through a dual targeting mechanism.

[0023] (3) Cyanide molecules can simultaneously generate efficient photodynamic and photothermal effects, generating a large amount of reactive oxygen species in situ at the tumor site and causing local high temperature, directly destroying the structure and function of mitochondria, and using the generated reactive oxygen species to trigger the precise breaking of diselenium bonds, releasing chlordamine in a controlled manner. This drug further aggravates the mitochondrial metabolic disorder of tumor cells, triggering an energy metabolism crisis, thereby forming a powerful synergistic anti-tumor effect among photodynamic therapy, photothermal therapy and metabolic intervention; in addition, this treatment process can also effectively induce immunogenic cell death, thereby activating the body's own anti-tumor immune response and enhancing the systemic clearance ability of tumors; based on the above multi-mechanism synergistic effect, this invention can not only selectively kill ordinary tumor cells, but also has a significant inhibitory effect on tumor stem cells with strong drug resistance and tumorigenicity, realizing precise targeted therapy for tumors and providing a new technology path with translational potential for clinical tumor treatment.

[0024] (4) Cyanide dyes have near-infrared fluorescence imaging capabilities, which can monitor and provide feedback on the distribution of drugs in the body in real time and non-invasively. Under the precise guidance of diagnostic information, the timing and plan of treatment can be dynamically optimized, ensuring the accuracy and timeliness of treatment. This provides an innovative solution for achieving precise and efficient individualized treatment and has good application prospects.

[0025] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0026] Figure 1 This is a synthetic route diagram of IR-dSe-LND in Example 1 of the present invention; Figure 2This is the 1H NMR spectrum of the IR-dSe-LND of Example 1 of the present invention; Figure 3 This is the absorption and fluorescence spectrum of the IR-dSe-LND of Example 1 of the present invention; Figure 4 This is a graph showing the change in singlet oxygen produced by the IR-dSe-LND aqueous solution under 808nm laser irradiation in Example 1 of the present invention over time. Figure 5 This is a graph showing the temperature change of the IR-dSe-LND aqueous solution in Example 1 of the present invention under 808nm laser irradiation. Figure 6 These are the phototoxicity / dark toxicity test results of IR-dSe-LND on HepG2 cells under darkness and 808nm laser irradiation, respectively, according to Example 1 of this invention. Figure 7 This is a laser confocal microscope and colocalization analysis diagram of the IR-dSe-LND targeting effect on mitochondria of tumor cells in Example 1 of the present invention, where the scale bar is 20 μm; Figure 8 This is a graph showing the changes in intracellular reactive oxygen species levels in tumor cells caused by IR-dSe-LND in Example 1 of the present invention, where the scale bar is 20 μm; Figure 9 This is a graph showing the effect of IR-dSe-LND on mitochondrial membrane potential of tumor cells in Example 1 of the present invention, where the scale bar is 20 μm; Figure 10 This is an evaluation diagram of the ability of IR-dSe-LND to kill H22 liver cancer stem cells in Example 1 of the present invention, where the scale bar is 50 μm. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0028] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0029] Example 1 When n=4, this invention provides a mitochondrial-targeted anticancer drug based on cyanin and chlordamine, wherein the targeted anticancer drug uses the compound shown in Formula II as the active ingredient. Formula II; The synthetic route of Formula II is as follows Figure 1 As shown, the specific steps include: S1. IR783 (0.50 g, 0.67 mmol) and 3-mercaptobenzoic acid (0.308 g, 2.0 mmol) were dissolved in 10.0 mL of DMF and stirred at room temperature in the dark for 48 h. Excess diethyl ether was added to the reaction solution for washing and centrifugation three times. The final precipitate was collected and purified by column chromatography (CH3OH / CH2Cl2 = 1 / 3, v / v). The precipitate was then dried under vacuum at room temperature in the dark to obtain the solid powder product IR783-Ph-COOH.

[0030] S2. Dissolve IR783-Ph-COOH (260.8 mg, 0.3 mmol) in 10.0 mL of methanol solution, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (287.6 mg, 1.5 mmol) and N-hydroxysuccinimide (34.6 mg, 0.3 mmol). 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride first reacts with the carboxyl group of IR783-Ph-COOH to generate an intermediate. The intermediate immediately reacts with N-hydroxysuccinimide to form a stable and highly reactive NHS ester. After stirring at room temperature for 30 min to fully activate the carboxyl group of IR783-Ph-COOH, a methanol solution (5.0 mL) of selenocysteine ​​hydrochloride (287.1 mg, 0.9 mmol) is slowly added dropwise to the mixed solution. The reaction is carried out at room temperature in the dark with stirring for 24 h. Selenocysteine ​​hydrochloride contains two primary amines. The synthesized NHS ester reacts with one of the primary amines of selenocysteine ​​hydrochloride to form a stable amide bond. After separation and purification by column chromatography (CH3OH / CH2Cl2=1 / 4, v / v), the product is dried under vacuum at room temperature in the dark to obtain a solid powder product IR-dSe. The terminus of the generated IR-dSe is a primary amine.

[0031] S3. Weigh out 289.1 mg (0.9 mmol) of chlordamine and dissolve it in 5.0 mL of methanol solution. Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (172.5 mg, 0.9 mmol) and N-hydroxysuccinimide (34.5 mg, 0.3 mmol). 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride first reacts with the carboxyl group of chlordamine to form an intermediate. This intermediate immediately reacts with N-hydroxysuccinimide to form a stable and highly reactive NHS ester. Stir at room temperature for 30 min to fully activate the carboxyl group of chlordamine. Then, slowly add 5.0 mL of methanol solution of IR-dSe (351.0 mg, 0.3 mmol) to the mixed solution. Stir the reaction at room temperature in the dark for 24 h. The generated NHS ester reacts with the primary amine of IR-dSe to form a stable amide bond. The product was purified by column chromatography (CH3OH / CH2Cl2=1 / 4, v / v), and dried under vacuum at room temperature in the dark to obtain a solid powder product IR-dSe-LND.

[0032] Figure 2 The image shows the 1H NMR spectrum of the IR-dSe-LND prepared in Example 1. The 1H NMR data indicates that the IR-dSe-LND was successfully prepared.

[0033] I. Optical performance testing of the IR-dSe-LND prepared in Example 1: The prepared IR-dSe-LND was dissolved in DMSO, and DMSO was used as a reference to test its absorption and fluorescence spectra. The absorption spectrum was measured using a UV-Vis spectrophotometer. The results are as follows: Figure 3 As shown, by Figure 3 It can be seen that the aqueous solution of IR-dSe-LND exhibits strong near-infrared light absorption in the wavelength range of 600-900 nm, with a maximum absorption peak at 811 nm, and shows the characteristic absorption peak of LND at 299 nm. The characteristic fluorescence emission peak of the aqueous solution of IR-dSe-LND is located at 825 nm, which is in the near-infrared region I, making it suitable for fluorescence imaging in living organisms.

[0034] II. Photodynamic performance testing of the IR-dSe-LND prepared in Example 1: An IR-dSe-LND aqueous solution (50 μg / mL, 200 μL) was added to a 96-well plate. The 96-well plate was placed in a dry incubator and incubated at a constant temperature of 25 °C. Near-infrared lasers with a wavelength of 808 nm were used under different laser power conditions (0, 0.3, 0.7, 1.0 W / cm²). 2 Irradiate continuously for 5 minutes, then use a singlet oxygen green fluorescent probe (SOSG). The generation of singlet oxygen was monitored, and the fluorescence signal was detected every 30 seconds. The kinetic curve of singlet reactive oxygen generation in IR-dSe-LND aqueous solution was plotted based on this. Ultrapure water was used as a negative control.

[0035] Photodynamic performance test results are as follows Figure 4 As shown, IR-dSe-LND aqueous solution can be significantly induced to produce a large amount of singlet oxygen under near-infrared laser irradiation, and the production of singlet oxygen shows dependence on laser power.

[0036] III. Photothermal performance testing of the IR-dSe-LND prepared in Example 1: IR-dSe-LND aqueous solutions of various concentrations (10, 20, 50, and 100 μg / mL) were added to 96-well plates. The 96-well plates were placed in a dry incubator and incubated at a constant temperature of 25°C using a near-infrared laser with a wavelength of 808 nm (0.5 W / cm²). 2The sample was continuously irradiated with IR-dSe-LND aqueous solution for 5 min, and the temperature was measured by a near-infrared thermal imager. The temperature value was recorded every 30 s, and the temperature change curve of IR-dSe-LND aqueous solution with different concentrations was plotted as a time. Ultrapure water was used as a negative control.

[0037] An IR-dSe-LND aqueous solution (50 μg / mL, 200 μL) was added to a 96-well plate. The 96-well plate was placed in a dry incubator and incubated at a constant temperature of 25 °C. An 808 nm near-infrared laser was used at different laser powers (0, 0.3, 0.5, 0.7, and 1.0 W / cm²). 2 The sample was continuously irradiated for 5.0 min under the specified conditions. The temperature of the sample was measured by a near-infrared thermal imager and the temperature value was recorded every 30 s. The temperature change curve of the IR-dSe-LND aqueous solution under irradiation with different powers of 808 nm laser was plotted. Ultrapure water was used as a negative control.

[0038] Photothermal performance test results are as follows Figure 5 As shown, where Figure 5 In the figure, A represents the temperature change curves of IR-dSe-LND aqueous solutions of different concentrations under 808 nm laser irradiation. Figure 5 Figure B shows the temperature change curves of IR-dSe-LND aqueous solutions under 808nm laser irradiation at different laser powers. The IR-dSe-LND aqueous solution exhibits a significant photothermal heating effect under near-infrared 808nm laser irradiation, and the temperature increase is significantly concentration- and power-dependent. Specifically, when the concentration of the IR-dSe-LND aqueous solution is 100μg / mL and irradiated with a laser (808nm) for 5 min, the solution temperature can increase by 33℃. Furthermore, for a 50μg / mL IR-dSe-LND aqueous solution, when the laser power increases from 0.3W / cm², the temperature rises significantly. 2 Increased to 1.5 W / cm 2 At that time, its temperature rise also increased significantly from 19.1℃ to 43.1℃.

[0039] IV. Evaluation of the tumor selectivity and killing effect of the IR-dSe-LND prepared in Example 1: HepG2 cells were planted at a density of 8.0 × 10⁶ cells per well. 3 Individual samples were seeded at a density of 100 μL of culture medium per well in 96-well plates. After incubation at 37°C and 5% CO2 for 12 h, the original culture medium was removed, and 200 μL of culture medium containing different concentrations of IR-dSe-LND and LND compounds were added to each well, followed by incubation for another 12 h. Except for the control group, the other experimental groups were treated with an 808 nm laser (power 1.0 W / cm²). 2Irradiate for 5 minutes. After laser irradiation, all cells are cultured for another 12 hours, and finally, cytotoxicity is assessed using the MTT assay.

[0040] Cytotoxicity test results as follows Figure 6 As shown, IR-dSe-LND effectively kills HepG2 cells in a dose-dependent manner, with an IC50 value of [missing information]. 50 The value is 2.2 times lower than that of LND. Under near-infrared laser irradiation (1.0 W / cm²), 2 At 5.0 min, the cell survival rate of the IR-dSe-LND+NIR treatment group (near-infrared laser irradiation group) was significantly reduced (P<0.01), which confirms that the photodynamic / photothermal effect triggered by near-infrared laser combined with the chemotherapy effect of LND can effectively kill tumor cells.

[0041] V. Evaluation of the mitochondrial targeting effect of IR-dSe-LND prepared in Example 1 on tumor cells: HepG2 cells were fed at a concentration of 1.0 × 10⁻⁶. 5 Cells were seeded at a density of 1 / 24 wells in 24-well plates containing round coverslips and cultured for 12 hours to allow for full adhesion. The original culture medium was then removed and replaced with medium containing either LND or IR-dSe-LND solution (LND concentration 40 μg / mL), and incubated for 4 hours. Cells were then incubated using an 808 nm laser (power 1.0 W / cm²). 2 Cells were continuously irradiated for 5.0 min and incubated in an incubator for another 30 min. The culture medium was discarded, and the cells were washed three times with PBS buffer. Mito-Tracker fluorescent probe working solution diluted 1:2000 with serum-free DMEM medium was added, and the cells were incubated in a cell culture incubator in the dark for 30 min. After incubation, the supernatant was discarded, and the cells were washed with PBS buffer, fixed with 4% paraformaldehyde solution for 30 min, and finally stained with DAPI for 20 min. All samples were observed and images were acquired under a confocal fluorescence microscope.

[0042] Confocal fluorescence microscopy results are as follows Figure 7 As shown, compared with staining of mitochondrial probes, the IR-dSe-LND treatment group exhibited significant fluorescence enrichment in the mitochondria of tumor cells, and the fluorescence signals of the two groups highly overlapped, confirming the excellent mitochondrial targeting of IR-dSe-LND. After irradiation with 808nm near-infrared light, the fluorescence intensity in the mitochondria was further enhanced, suggesting that near-infrared light can promote the selective accumulation of IR-dSe-LND in mitochondria.

[0043] VI. Testing the changes in intracellular reactive oxygen species levels in tumor cells caused by IR-dSe-LND prepared in Example 1: HepG2 cells were planted at a density of 1.0 × 10⁶ cells per well.5 Cells were seeded at a density of [number] cells per well on round coverslips placed in 24-well plates. After 12 hours of incubation, the original culture medium was discarded and replaced with medium containing either LND or IR-dSe-LND solution (LND concentration 40 μg / mL), and incubated for another 6 hours. An 808 nm laser (power 1.0 W / cm²) was then used. 2 Cells were continuously irradiated for 5.0 min and then incubated in an incubator for 30 min. The culture medium was discarded, and the cells were washed three times with PBS buffer. DCFH-DA working solution diluted 1:1000 with serum-free DMEM was added, and the cells were incubated in a cell culture incubator in the dark for 30 min. After incubation, the supernatant was discarded, and the cells were washed with PBS buffer, fixed with 4% paraformaldehyde for 30 min, and finally stained with DAPI for 20 min. All samples were observed and images were acquired under a confocal fluorescence microscope.

[0044] The fluorescence confocal imaging results of intracellular reactive oxygen species (ROS) levels in tumor cells induced by IR-dSe-LND in Example 1 and the changes in intracellular ROS levels in tumor cells are shown in the figure below. Figure 8 As shown, where, Figure 8 In the figure, A represents the fluorescence confocal imaging result of the intracellular reactive oxygen species level in tumor cells caused by IR-dSe-LND in Example 1. Figure 8 Figure B shows the changes in intracellular reactive oxygen species (ROS) levels in tumor cells induced by IR-dSe-LND in Example 1. Compared with the PBS group, the relative fluorescence intensity of DCFH in HepG2 cells treated with LND showed no significant change, while obvious green fluorescence was observed in cells treated with IR-dSe-LND. Furthermore, the ROS fluorescence intensity in the IR-dSe-LND+NIR treatment group was significantly stronger than that in the IR-dSe-LND treatment group. These results indicate that IR-dSe-LND can generate a large amount of ROS in HepG2 cells under 808 nm near-infrared laser irradiation, demonstrating good photodynamic properties.

[0045] VII. Investigating the effect of IR-dSe-LND prepared in Example 1 on the mitochondrial membrane potential of tumor cells: HepG2 cells were fed at a concentration of 1.0 × 10⁻⁶. 5 Cells were seeded at a density of 1 / 24 wells in 24-well plates with round coverslips and cultured for 12 hours to allow for full adhesion. The original culture medium was then removed and replaced with medium containing either LND or IR-dSe-LND aqueous solution (LND concentration 40 μg / mL). After further incubation for 6 hours, cells were incubated using an 808 nm laser (power 1.0 W / cm²). 2Cells were continuously irradiated for 5.0 min and incubated in an incubator for another 30 min. The culture medium was discarded, and the cells were washed three times with PBS buffer. Mito-Tracker Deep Red fluorescent probe working solution diluted 1:2000 with serum-free DMEM medium was added, and the cells were incubated in a cell culture in the dark for 30 min. After incubation, the supernatant was aspirated, and the cells were washed with PBS buffer, fixed with 4% paraformaldehyde solution for 30 min, and finally stained with DAPI for 20 min. All samples were observed and images were acquired under a confocal fluorescence microscope.

[0046] Since the accumulation of Mito-Tracker DeepRed within mitochondria depends on the mitochondrial membrane potential, it is used as an indicator probe of mitochondrial membrane potential. The confocal results of the effect of IR-dSe-LND on the mitochondrial membrane potential of tumor cells are shown below. Figure 9 As shown, where Figure 9 In the image, A represents a fluorescence imaging plot showing the effect of IR-dSe-LND on the mitochondrial membrane potential of tumor cells. Figure 9 Figure B shows the fluorescence emission spectrum of the effect of IR-dSe-LND on the mitochondrial membrane potential of tumor cells. Compared with the PBS, LND, and IR-dSe-LND treatment groups, the red fluorescence intensity of the mitochondrial probe in HepG2 cells treated with IR-dSe-LND+NIR was significantly reduced, indicating a significant decrease in mitochondrial membrane potential. This result demonstrates that IR-dSe-LND can effectively induce mitochondrial membrane potential collapse after 808 nm near-infrared laser irradiation, leading to severe mitochondrial dysfunction.

[0047] VIII. Evaluation of the killing effect of IR-dSe-LND prepared in Example 1 on tumor stem cells: H22 tumor stem cells were cultured in 96-well plates using a 3D soft fibrin glue-based system to form tumor spheroids. First, the H22 cell density was diluted to 1.0 × 10⁻⁶ cells using RPMI-1640 medium. 4Cells / mL, while fibrinogen was diluted to 2.0 mg / mL with T7 buffer (150 mM NaCl, 50 mM Tris, pH 7.4). An equal volume of the cell suspension and fibrinogen solution were mixed and gently pipetted to mix. Then, 1.0 μL of thrombin (0.1 U / μL) was added to each well of a pre-chilled 96-well plate, followed by 50 μL of the cell-fibrinogen mixture, and thoroughly mixed. The plate was incubated at 37°C for 20 min to form a colloid, after which 150 μL of RPMI-1640 complete medium was added to each well. The 96-well plate was placed in a cell culture incubator for continuous culture. When the tumor spheroids reached approximately 100 µm in diameter, the original medium was discarded and replaced with medium containing either LND or IR-dSe-LND solution (LND concentration 40 μg / mL). After culturing for another 12 h, the plate was subjected to an 808 nm laser (power 1.0 W / cm²). 2 The tumor spheres were irradiated for another 5.0 min, followed by incubation for 12 h. The tumor stem cell spheres were counted and photographed using an optical microscope, and their size was statistically analyzed.

[0048] Evaluation of the killing effect of IR-dSe-LND on tumor stem cells, as follows: Figure 10 As shown, where Figure 10 In the figure, A represents the killing effect of different treatment conditions on tumor stem cells. Figure 10 In this context, B represents the colony size under different treatment conditions. Figure 10 In the figure, C represents the colony count under different treatment conditions. As shown in the figure, after LND treatment, neither the volume nor the number of colonies of tumor stem cell spheres decreased significantly, indicating that LND had no significant inhibitory effect on their proliferation and activity. However, after IR-dSe-LND treatment, the number of tumor stem cell colonies decreased significantly, and the volume of stem cell spheres also decreased significantly, indicating that IR-dSe-LND can inhibit the growth of tumor stem cells to a certain extent. In the IR-dSe-LND+NIR group under 808nm laser irradiation, the number and volume of H22 tumor stem cell spheres decreased significantly further, indicating that IR-dSe-LND+NIR can effectively kill three-dimensional tumor stem cell spheres and significantly inhibit their proliferation ability.

[0049] Therefore, this invention employs the aforementioned mitochondrial-targeted anticancer drug based on cyanine and chlordamine, its preparation method, and its application. The prepared drug can efficiently target tumor cell mitochondria. Under near-infrared light irradiation, cyanine molecules simultaneously generate photodynamic and photothermal effects, producing ROS and high temperatures in situ. This not only directly damages mitochondrial structure and function, but ROS also simultaneously triggers the breaking of diselenium bonds, precisely releasing chlordamine, further exacerbating mitochondrial dysfunction and metabolic crisis. This achieves synergistic effects of photodynamic therapy, photothermal therapy, and metabolic inhibition, and further activates the body's anti-tumor immune response through the ICD effect, achieving selective killing of tumor cells and tumor stem cells and targeted tumor therapy.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A chlorin and lonidamide based mitochondrially targeted anticancer drug, characterized in that: Targeted anticancer drugs use compounds represented by Formula I as their active ingredients. Formula I; Where R is one of methyl, sulfonic acid, and carboxyl groups, and n = 0-10.

2. The anticancer drug based on chlorin and lonidamide for mitochondria targeting according to claim 1, characterized in that: Targeted anticancer drugs also include pharmaceutically acceptable additives of Formula I, pharmaceutically acceptable carriers, and excipients.

3. A method for preparing a pyropheophorbide and lonidamide-based mitochondrial-targeted anticancer drug according to any one of claims 1-2, characterized by: The synthetic route for targeted anticancer drugs is as follows: ； Where X is one of chlorine, bromine, and iodine; The preparation of targeted anticancer drugs specifically includes the following steps: S1. A nucleophilic substitution reaction was carried out between cyanine molecules and 3-mercaptobenzoic acid in DMF to obtain a reaction solution. The reaction solution was washed, centrifuged, separated and purified, and then vacuum dried to prepare cyanine derivatives. S2. The cyanine derivative obtained in S1 was dissolved in methanol solution, and a selenocysteine ​​compound was added to undergo a coupling reaction. After the reaction was completed, the mixture was separated and purified to prepare an amino-terminated cyanine-linker conjugate. S3. Clonidamine is dissolved in a methanol solution, and then the cyanine-linker conjugate obtained in S2 is added to react with clodinidamine to obtain a targeted anticancer drug.

4. The method for preparing a mitochondrial-targeted anticancer drug based on cyanin and chlordamine according to claim 3, characterized in that: In S1, the molar ratio of cyanine molecules to 3-mercaptobenzoic acid is 1:(2-4). The nucleophilic substitution reaction is carried out at a temperature of 20-30℃ for 40-50 h. The reaction is carried out under light and stirred. The reaction solution is washed with ether and centrifuged 3-5 times. The precipitate is collected and purified by column chromatography. The chromatographic solvent for column chromatography is CH3OH / CH2Cl2 with a volume ratio of 1:

3. The solution is dried under vacuum at room temperature in the dark to obtain the cyanine derivative.

5. The method for preparing a mitochondrial-targeted anticancer drug based on cyanin and chlordamine according to claim 3, characterized in that: In S2, the selenocystamine compound is one of selenocystamine and selenocystamine hydrochloride. The molar ratio of cyanine derivative to selenocystamine compound is 1:(2-4). The coupling reaction temperature is 20-30℃, the reaction time is 20-25h, and the reaction is carried out under light with stirring. The product obtained from the reaction is separated and purified by column chromatography. The chromatographic solution for column chromatography is CH3OH / CH2Cl2, and the volume ratio of CH3OH to CH2Cl2 is 1:

4.

6. The method for preparing a mitochondrial-targeted anticancer drug based on cyanin and chlordamine according to claim 3, characterized in that: In S2, before adding selenocysteine ​​compounds, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinic acid are added first. The molar ratio of cyanine derivative, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinic acid is 1:(4-6):

1.

7. The method for preparing a mitochondrial-targeted anticancer drug based on cyanine and chlordamine according to claim 3, characterized in that: In S3, the molar ratio of chlordamine to cyanine-linker coupling compound is (2-4):

1. The coupling reaction temperature is 20-30℃, the reaction time is 20-25h, and the reaction is carried out under light with stirring. The product obtained from the reaction is separated and purified by column chromatography. The chromatographic solution for column chromatography is CH3OH / CH2Cl2, and the volume ratio of CH3OH to CH2Cl2 is 1:

4.

8. The method for preparing a mitochondrial-targeted anticancer drug based on cyanin and chlordamine according to claim 3, characterized in that: In S3, before the cyanine-linker conjugate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinic acid are added first. The molar ratio of chlordamine, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinic acid is (2-4):(2-4):

1.

9. The use of a mitochondrial-targeted anticancer drug based on cyanin and chlordamine as described in any one of claims 1-2 in the preparation of a tumor treatment drug.

10. The use of a mitochondrial-targeted anticancer drug based on cyanin and chlordamine as described in any one of claims 1-2 in the preparation of a drug for killing tumor stem cells.

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