A tumor cell mitochondrion targeting agent with fluorescent co-localization function and a preparation method thereof

Abstract

This specification provides a fluorescent co-localization mitochondrial targeting agent for tumor cells and its preparation method, belonging to the biomedical field. The method uses triphenylamine as a raw material, extends the π-conjugated backbone with a benzothiadiazole bridging unit, employs pyridinium salt as a strong electron acceptor, and connects different alkyl chains to synthesize a mitochondrial targeting agent with aggregation-induced emission properties. This fluorescent mitochondrial targeting agent, TBPMI, not only exhibits good biosafety but also excellent mitochondrial selectivity and targeting precision in tumor cells, with a co-localization coefficient as high as 0.96 with commercial mitochondrial probes. Simultaneously, the prepared fluorescent mitochondrial targeting agent TBPMI demonstrates superior aggregation-induced emission performance, significantly higher than that of the traditional commercial photosensitizer, Bengal rose red. It can be said that the newly prepared fluorescent mitochondrial targeting agent TBPMI provides a new material for precise localization at the organelle level under fluorescence imaging guidance.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a tumor cell mitochondrial targeting agent with fluorescence co-localization function and its preparation method. Background Technology

[0002] Malignant tumors are a major public health problem worldwide and one of the leading causes of death. Over the past 40 years, the cancer mortality rate has not decreased significantly, primarily due to a lack of accurate diagnosis and effective treatment methods. Current cancer treatments mainly include surgical resection and drug therapy. However, the drawbacks of chemotherapy, such as drug resistance and toxic side effects, the individualization of immunotherapy, and the lag in feedback on treatment efficacy, pose significant obstacles to improving cancer treatment outcomes. Clinically, there is an urgent need for a highly sensitive monitoring method to achieve better therapeutic effects and fewer side effects.

[0003] Fluorescence imaging (FLI) has been recognized as a non-invasive and effective method for cancer diagnosis due to its low cost, high sensitivity, outstanding temporal resolution and good consistency [2]. In diagnostic technology, near-infrared (NIR) fluorescence imaging is a non-invasive and essential diagnostic tool with significant advantages such as low cost, real-time imaging and high sensitivity [3]. However, traditional organic fluorescent probes, such as rhodamine, fluorescein, and coumarin, usually exhibit strong non-specific background signal interference under non-aqueous conditions. When a large number of traditional fluorophores with aromatic and planar chemical structures aggregate at the target site, their emission will decrease or even quench fluorescence, leading to false negative results. In 2001, Tang Benzhong et al. observed aggregation-induced emission (AIE) while studying the luminescence behavior of rotor-rich methylpentylsiloxane and proposed the concept of AIE. AIE is used to describe a class of molecules that show weak or no emission in a monodisperse state, but greatly enhance emission when aggregated or in a solid state. Compared to traditional fluorophores, AIE probes exhibit greater advantages in detection and imaging due to the intramolecular motion restriction mechanism of AIE, including limited intramolecular rotation and limited group vibration.

[0004] Organelles are structures distributed within the cytoplasm, possessing a specific morphology, and playing crucial roles in cellular physiological activities. Organelles include the cell membrane, endoplasmic reticulum, mitochondria, lysosomes, and Golgi apparatus, each with its specific function. Therefore, designing and synthesizing materials with sub-organelle targeting capabilities might enable precise targeted therapy of cells. The cell membrane, the interface separating different media and components within and outside the cell, is primarily composed of a phospholipid bilayer. It is the first step in cellular uptake and is essential for maintaining cell integrity. Mitochondria, located within the cell membrane, serve as the site of cellular energy metabolism and play an irreplaceable role in cellular survival. Summary of the Invention

[0005] The purpose of this invention is to provide a tumor cell mitochondrial targeting agent with fluorescence co-localization function and its preparation method.

[0006] This preparation method uses triphenylamine as a raw material, extends the π-conjugated backbone with benzothiadiazole bridging units, uses pyridinium salt as a strong electron acceptor, and connects different alkyl chains to synthesize a mitochondrial targeting agent with aggregation-induced emission properties. This fluorescent mitochondrial targeting agent TBPMI not only has good biosafety, but also has good mitochondrial selectivity and targeting precision in tumor cells, with a colocalization coefficient of up to 0.96 with commercial mitochondrial probes.

[0007] To achieve the above objectives, the technical solution adopted by this invention is: a method for preparing a tumor cell mitochondrial targeting agent with fluorescence co-localization function, comprising the following steps:

[0008] (1) Synthesis of Intermediate 1: 4,7-Dibromo-2,1,3-benzothiadiazole, potassium carbonate, and tetra(triphenylphosphine)palladium were sequentially dissolved in tetrahydrofuran aqueous solution and stirred at room temperature for 1–5 h to obtain a reaction solution. After the reaction was completed, 4-boronic acid triphenylamine powder was slowly added to the reaction solution, and the mixture was heated at 50–100 °C for 10–15 h. After the reaction was completed, the mixture was precipitated with ethyl acetate and washed with distilled water. Finally, the precipitate was further purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1–15:1, v / v) to obtain Intermediate 1.

[0009] ;

[0010] (2) Synthesis of TBP: Under nitrogen protection, intermediates 1,4-borate pyridine, potassium carbonate, and tetrakis(triphenylphosphine)palladium were added sequentially to a mixture of toluene and methanol, and the temperature was slowly raised to 80–150 °C. The mixture was stirred for 10–15 h. After the reaction was completed by TLC monitoring, the mixture was extracted with ethyl acetate, and the organic layer was collected. The organic layer was then dried with anhydrous sodium sulfate and evaporated under reduced pressure to obtain a precipitate. Finally, the precipitate was further purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1–15:1, v / v) to obtain TBP.

[0011] ;

[0012] (3) Synthesis of TBP-1: TBP was dissolved in acetonitrile solution at 55–100 °C. Then, 1-bromo-2-(2-methoxyethoxy)ethane was added to the solution, and stirring was continued for 6–18 hours. The mixture was then concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol = 15:1–25:1, v / v) to obtain TBP-1.

[0013] ;

[0014] (4) Synthesis of the targeting agent TBPMI: Compound TBP-1 was dissolved in a mixture of acetone and methanol. Then, potassium hexafluorophosphate solution was slowly added dropwise to the reaction solution, and the mixture was stirred at 80–130 °C for 10–15 h. After the reaction was completed, the mixture was precipitated with ethyl acetate, and the precipitate was further purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1–5:1, v / v) to obtain the fluorescent targeting agent TBPMI.

[0015] .

[0016] Furthermore, in step (1), the volume ratio of tetrahydrofuran to water is 4:1, and the concentration of 4,7-dibromo-2,1,3-benzothiadiazole is 0.02-0.5 g / mL.

[0017] Furthermore, in step (1), the mass ratio of 4,7-dibromo-2,1,3-benzothiadiazole to potassium carbonate is 1:0.002 to 1:0.05, and the mass ratio of 4,7-dibromo-2,1,3-benzothiadiazole to tetra(triphenylphosphine)palladium is 1:0.006 to 1:0.09.

[0018] Furthermore, in step (2), the volume ratio of toluene to methanol is 1:1, and the concentration of intermediate 1 is 0.0125 to 0.167 g / mL.

[0019] Furthermore, in step (2), the mass ratio of intermediate 1 to pyridine 4-borate is 1:0.16 to 1:0.8, the mass ratio of intermediate 1 to potassium carbonate is 1:0.2 to 1:3, and the mass ratio of intermediate 1 to tetrakis(triphenylphosphine)palladium is 1:0.01 to 1:0.2.

[0020] Furthermore, the concentration of TBP in step (3) is 0.02 to 0.14 g / mL.

[0021] Furthermore, in step (3), the mass ratio of TBP to 1-bromo-2-(2-methoxyethoxy)ethane is 1:1.4 to 1:13.3.

[0022] Furthermore, in step (4), the volume ratio of acetone to methanol is 3:1, and the mass ratio of compound TBP-1 to potassium hexafluorophosphate is 1:1.1 to 1:4.2.

[0023] This invention also proposes a tumor cell mitochondrial targeting agent with fluorescence co-localization function prepared by the above preparation method.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] This preparation method uses triphenylamine as a raw material, extends the π-conjugated backbone with benzothiadiazole bridging units, uses pyridinium salt as a strong electron acceptor, and connects different alkyl chains to synthesize a mitochondrial targeting agent with aggregation-induced emission properties.

[0026] The fluorescent mitochondrial targeting agent TBPMI not only has good biosafety, but also good mitochondrial selectivity and targeting precision in tumor cells, with a colocalization coefficient of up to 0.96 with commercial mitochondrial probes.

[0027] Meanwhile, the prepared fluorescent mitochondrial targeting agent TBPMI has excellent aggregation-induced emission performance, which is significantly higher than that of the traditional commercial photosensitizer, Rosin Bengal, effectively solving the problem of fluorescence quenching of traditional targeting agents in the aggregation state.

[0028] It can be said that the newly prepared fluorescent mitochondrial targeting agent TBPMI provides a new material for precise localization at the organelle level under fluorescence imaging guidance, and also provides a new idea for the development of targeted diagnostic platforms for breast cancer. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the synthesis of the fluorescent mitochondrial targeting agent TBPMI of the present invention;

[0030] Figure 2 The fluorescence emission spectra of the fluorescent mitochondrial targeting agent TBPMI of the present invention in DMSO / toluene mixed solutions with different toluene volume fractions are shown.

[0031] Figure 3 This is a molecular orbital energy level diagram of the fluorescent mitochondrial targeting agent TBPMI of the present invention;

[0032] Figure 4 The toxicity test of different concentrations of the fluorescent mitochondrial targeting agent TBPMI of the present invention on NIH-3T3 cells under dark (A) and white light irradiation (B) conditions;

[0033] Figure 5 H&E stained sections of major organ (heart, liver, spleen, lung, and kidney) tissues from mice after injection of the fluorescent mitochondrial targeting agent TBPMI of this invention;

[0034] Figure 6 This is a fluorescence confocal imaging image of NIH-3T3 and 4T1 cells after incubation of the fluorescent mitochondrial targeting agent TBPMI of the present invention with NIH-3T3 and 4T1 cells for different times;

[0035] Figure 7This is a laser confocal image of 4T1 cells and Mito Tracker Green (200 nM) after co-staining the fluorescent mitochondrial targeting agent TBPMI (5 μmol / L) of this invention with Mito Tracker Green (200 nM) at 37°C for 15 min.

[0036] Figure 8 This is a flowchart illustrating the preparation steps of the fluorescent mitochondrial targeting agent TBPMI of the present invention. Detailed Implementation

[0037] To enable those skilled in the art to better understand the technical solutions in this specification, the technical solutions in the embodiments of this specification will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this specification, and not all embodiments. Based on the embodiments in this specification, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this specification.

[0038] The invention will now be further described with reference to the accompanying drawings.

[0039] Example 1:

[0040] (1) Synthesis of Intermediate 1: 1.76 g of 4,7-dibromo-2,1,3-benzothiadiazole, 28 mg of potassium carbonate, and 60 mg of tetra(triphenylphosphine)palladium were dissolved sequentially in 25 mL of tetrahydrofuran aqueous solution and stirred at room temperature for 2 h. Then, 1.45 g of 4-boronic acid triphenylamine powder was slowly added to the reaction solution, and the mixture was heated at 80 °C for 12 h. After the reaction was complete, the mixture was precipitated with ethyl acetate and washed with distilled water. Finally, the precipitate was further purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1, v / v) to obtain 1 g of Intermediate 1.

[0041] (2) Synthesis of TBP: Under nitrogen protection, 1.52 g of intermediate 1,627 mg of pyridine 4-borate, 1.38 g of potassium carbonate, and 100 mg of tetra(triphenylphosphine)palladium were added sequentially to a mixture of 50 ml toluene and methanol, and the mixture was slowly heated to 110 °C and stirred for 12 h. After the reaction was completed as monitored by TLC, the mixture was extracted with ethyl acetate, and the organic layer was collected. The organic layer was then dried over anhydrous sodium sulfate and evaporated under reduced pressure to obtain the precipitate. Finally, the precipitate was further purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1, v / v) to obtain 0.5 g of TBP.

[0042] (3) Synthesis of TBP-1: 456 mg of compound TBP was dissolved in 10 mL of acetonitrile solution at 85 °C. Then, 1.35 mL of 1-bromo-2-(2-methoxyethoxy)ethane was added to the solution, and stirring was continued for 12 hours. The mixture was then concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol = 20:1, v / v) to obtain TBP-1.

[0043] (4) Synthesis of the target agent TBPMI: First, 127.6 mg of compound TBP-1 was dissolved in a mixture of 4 mL acetone and methanol. Then, 4 mL of potassium hexafluorophosphate solution (50 mg / mL) was slowly added dropwise to the reaction mixture, and the mixture was stirred at 100 °C for 12 hours. After the reaction was completed, the mixture was precipitated with ethyl acetate, and the precipitate was further purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1, v / v) to obtain 100 mg of the fluorescent target agent TBPMI.

[0044] To verify the aggregation-induced emission properties of the fluorescent mitochondrial targeting agent TBPMI, we used fluorescence spectroscopy to measure its fluorescence intensity in DMSO / toluene mixed solutions with different volume ratios (toluene volume fraction 0%-99%). The results are as follows: Figure 2 As shown. When the volume fraction of toluene is 0%, the fluorescent mitochondrial targeting agent TBPMI shows almost no fluorescence. With the increase of the volume fraction of toluene, the fluorescence intensity of the fluorescent mitochondrial targeting agent TBPMI gradually increases. When the toluene content is 99%, the fluorescence emission intensity of TBPMI increases by 45 times, indicating that the fluorescent mitochondrial targeting agent TBPMI has excellent aggregation-induced emission performance. To better understand the photophysical properties of the fluorescent mitochondrial targeting agent TBPMI, we performed density function theory (DFT) calculations on it at the TD-PBE0 / TZVP level. Under the action of the polarizable continuum model (PCM), the HOMO and LUMO orbitals of TBPMI are concentrated on the TBP structure, and the additional alkyl chain does not significantly affect the HOMO-LUMO band gap ( Figure 3 The calculated maximum emission wavelength of TBPMI in DMSO is 624.69 nm, which further supports our initial design concept that TBPMI has good aggregation-induced emission physical properties in the dispersed state.

[0045] To investigate the biocompatibility of the fluorescent mitochondrial targeting agent TBPMI, we first used a CCK-8 assay kit to detect the absorbance of NIH-3T3 cells incubated with different concentrations (0 μmol / L, 0.1 μmol / L, 0.2 μmol / L, 0.5 μmol / L, 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, and 10 μmol / L) of TBPMI solution under dark and light conditions. The results are shown in Figure 4. Under dark conditions, the survival rate of NIH-3T3 fibroblasts co-incubated with a high concentration of TBPMI (10 μmol / L) remained around 80%. However, under light conditions (100 mW / cm²), the survival rate was significantly higher. 2 Under these conditions, the survival rate of NIH-3T3 fibroblasts co-incubated with high concentration TBPMI (10 μmol / L) remained greater than 60%, indicating that the prepared fluorescent mitochondrial targeting agent TBPMI did not exhibit significant cytotoxicity. Simultaneously, after tail vein injection of the fluorescent mitochondrial targeting agent TBPMI, sections of vital mouse organs (heart, liver, spleen, lung, and kidney) were prepared and stained with H&E. Figure 5 As can be seen, the H&E staining sections of the major organs (heart, liver, spleen, lung, and kidney) of the mice in the TBPMI treatment group showed no obvious pathological changes, indicating that TBPMI has no obvious systemic toxicity at therapeutic doses and has good biosafety.

[0046] Given that cancer cells have a higher negative membrane potential than normal cells, the positively charged pyridine salt in the synthesized fluorescent mitochondrial targeting agent TBPMI will enable them to target tumor cells. We co-incubated 4T1 tumor cells or normal cells (NIH-3T3) with the fluorescent mitochondrial targeting agent TBPMI for 5 seconds to 30 minutes, and observed the cell uptake using confocal microscopy. Figure 6 As shown, 4T1 cells exhibit stronger red fluorescence than NIH-3T3 cells, indicating that 4T1 tumor cells take up more TBPMI than NIH-3T3 cells, thus demonstrating that TBPMI has selective uptake capabilities on tumor cells.

[0047] To clarify the subcellular localization of TBPMI in 4T1 cells, laser confocal fluorescence microscopy was used to test the co-localization of TBPMI after cell fixation, and the results are shown in Figure 7. In the experiment, 4T1 cells were first incubated with 5 μmol / L TBPMI for 30 min, and then co-stained with 200 nM mitochondrial-specific probe Mito-Tracker Green. After fixation with 4% paraformaldehyde and DAPI nuclear staining, imaging showed that the red fluorescence of TBPMI (collection band 600-650 nm) and the green fluorescence of Mito-Tracker Green (collection band 500-550 nm) highly overlapped, with a Pearson correlation coefficient of 0.96, confirming that TBPMI can still stably localize in mitochondria after cell fixation, further verifying its excellent mitochondrial targeting specificity.

[0048] The above are merely preferred embodiments of the present invention and do not constitute any limitation on the present invention. Any equivalent substitutions or modifications made by those skilled in the art to the technical solutions and content disclosed in the present invention without departing from the scope of the present invention shall be deemed to have remained within the protection scope of the present invention.

Claims

1. A method for preparing a tumor cell mitochondrial targeting agent with fluorescence co-localization function, characterized in that, Includes the following steps: (1) Synthesis of Intermediate 1: 4,7-Dibromo-2,1,3-benzothiadiazole, potassium carbonate, and tetra(triphenylphosphine)palladium were sequentially dissolved in tetrahydrofuran aqueous solution and stirred at room temperature for 1–5 h to obtain a reaction solution. After the reaction was completed, 4-boronic acid triphenylamine powder was slowly added to the reaction solution, and the mixture was heated at 50–100 °C for 10–15 h. After the reaction was completed, the mixture was precipitated with ethyl acetate and washed with distilled water. Finally, the precipitate was further purified by silica gel column chromatography to obtain Intermediate 1. ； (2) Synthesis of TBP: Under nitrogen protection, intermediates 1, pyridine 4-borate, potassium carbonate, and tetrakis(triphenylphosphine)palladium were sequentially added to a mixture of toluene and methanol, and the temperature was slowly raised to 80–150 °C. The mixture was stirred for 10–15 h. After the reaction was completed by TLC monitoring, the mixture was extracted with ethyl acetate, and the organic layer was collected. The organic layer was then dried with anhydrous sodium sulfate and evaporated under reduced pressure to obtain a precipitate. Finally, the precipitate was further purified by silica gel column chromatography to obtain TBP. ； Synthesis of TBP-1: TBP was dissolved in acetonitrile solution at 55–100 °C. Then, 1-bromo-2-(2-methoxyethoxy)ethane was added to the solution, and stirring was continued for 6–18 hours. The mixture was then concentrated under reduced pressure and purified by silica gel column chromatography to obtain TBP-1. ； Synthesis of the targeting agent TBPMI: Compound TBP-1 was dissolved in a mixture of acetone and methanol. Then, potassium hexafluorophosphate solution was slowly added dropwise to the reaction mixture, and the mixture was stirred at 80–130 °C for 10–15 h. After the reaction was complete, the mixture was precipitated with ethyl acetate, and the precipitate was further purified by silica gel column chromatography to obtain the fluorescent targeting agent TBPMI. The colocalization coefficient of the targeting agent with a commercial mitochondrial probe was 0.

96. 。 2. The method for preparing a tumor cell mitochondrial targeting agent with fluorescence co-localization function as described in claim 1, characterized in that, In step (1), the volume ratio of tetrahydrofuran to water is 4:1, and the concentration of 4,7-dibromo-2,1,3-benzothiadiazole is 0.02-0.5 g / mL.

3. The method for preparing a tumor cell mitochondrial targeting agent with fluorescence co-localization function as described in claim 1, characterized in that, In step (1), the mass ratio of 4,7-dibromo-2,1,3-benzothiadiazole to potassium carbonate is 1:0.002 to 1:0.05, and the mass ratio of 4,7-dibromo-2,1,3-benzothiadiazole to tetra(triphenylphosphine)palladium is 1:0.006 to 1:0.

09.

4. The method for preparing a tumor cell mitochondrial targeting agent with fluorescence co-localization function as described in claim 1, characterized in that, In step (2), the volume ratio of toluene to methanol is 1:1, and the concentration of intermediate 1 is 0.0125 to 0.167 g / mL.

5. The method for preparing a tumor cell mitochondrial targeting agent with fluorescence co-localization function as described in claim 1, characterized in that, In step (2), the mass ratio of intermediate 1 to pyridine 4-borate is 1:0.16 to 1:0.8, the mass ratio of intermediate 1 to potassium carbonate is 1:0.2 to 1:3, and the mass ratio of intermediate 1 to tetrakis(triphenylphosphine)palladium is 1:0.01 to 1:0.

2.

6. The method for preparing a tumor cell mitochondrial targeting agent with fluorescence co-localization function as described in claim 1, characterized in that, The concentration of TBP in step (3) is 0.02 to 0.14 g / mL.

7. The method for preparing a tumor cell mitochondrial targeting agent with fluorescence co-localization function as described in claim 1, characterized in that, In step (3), the mass ratio of TBP to 1-bromo-2-(2-methoxyethoxy)ethane is 1:1.4 to 1:13.

3.

8. The method for preparing a tumor cell mitochondrial targeting agent with fluorescence co-localization function as described in claim 1, characterized in that, In step (4), the volume ratio of acetone to methanol is 3:1, and the mass ratio of compound TBP-1 to potassium hexafluorophosphate is 1:1.1 to 1:4.

2.

9. A tumor cell mitochondrial targeting agent with fluorescence co-localization function prepared by the preparation method according to any one of claims 1-8.

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