A carboxylesterase-activated mitochondrial-targeting fluorescent probe with photodynamic therapy properties, and preparation method and use thereof

Abstract

This invention discloses a carboxylesterase-activated mitochondrial-targeting fluorescent probe with photodynamic therapy properties, its preparation method, and its uses. The structure of the mitochondrial-targeting fluorescent probe is shown below. The mitochondrial-targeting fluorescent probe CML of this invention can specifically respond to different concentrations of carboxylesterases (CEs), with a detection limit as low as 0.30 U / L. Simultaneously, this probe can specifically target mitochondria, and through structural optimization (replacing the methyl group on the indole salt with a butyl group), its mitochondrial anchoring ability is significantly superior to that of methyl-substituted derivatives. Cytotoxicity experiments confirmed that the probe has good biocompatibility; confocal fluorescence imaging experiments showed that this probe can effectively monitor the fluorescence changes of endogenous and exogenous CEs in HepG2 cells. Most importantly, after being specifically activated by carboxylesterases, the probe can efficiently generate reactive oxygen species under light conditions, thereby exerting a photodynamic therapeutic effect on cancer cells.

Description

Technical Field

[0001] This invention relates to a carboxylesterase-activated mitochondrial-targeting fluorescent probe with photodynamic therapy properties, its preparation method, and its uses. Background Technology

[0002] Cancer is a major global disease that seriously threatens human health, characterized by high incidence and mortality rates. Current main clinical treatments include surgical resection, chemotherapy, and radiotherapy. While significant progress has been made, considerable limitations remain: surgery is highly dependent on the surgeon's experience, and chemotherapy and radiotherapy are prone to systemic toxicity, damage to normal tissues, and drug resistance. Therefore, developing novel cancer treatment strategies that are more targeted, have fewer side effects, and combine diagnostic and therapeutic functions is of significant clinical need and scientific value.

[0003] Photodynamic therapy (PDT), as a non-invasive, light-driven treatment, has shown promising application prospects in cancer treatment due to its advantages such as no significant cross-resistance and minimal side effects. PDT uses specific wavelengths of light to excite photosensitizers, generating free radicals such as superoxide anions and hydroxyl radicals via type I electron transfer, or generating singlet oxygen via type II energy transfer, thereby inducing tumor cell death. However, traditional photosensitizers generally suffer from poor targeting, insufficient tissue penetration depth, and low signal-to-noise ratio, making it difficult to achieve precise activation and efficient treatment.

[0004] Near-infrared (NII) fluorescent probes for small organic molecules have become important tools for bioimaging and disease detection due to their deep tissue penetration, high signal-to-noise ratio, and excellent sensitivity and selectivity. By responding to specific signals in the tumor microenvironment, such as abnormal viscosity, enzyme overexpression, and altered levels of active substances, probes can be selectively activated, simultaneously outputting fluorescent signals and initiating therapeutic functions, providing a feasible approach for building an integrated diagnostic and therapeutic system. Carboxylesterases (CEs), as important hydrolases and key enzymes in drug metabolism, are widely distributed in the liver and plasma, participating in the hydrolytic metabolism of esters, amides, and other compounds. Their abnormal expression is closely related to the occurrence and development of hyperlipidemia, fatty liver, and various tumors, making them highly valuable biomarkers for disease diagnosis. Therefore, constructing fluorescent probes that specifically respond to CEs is of great significance for the early detection and precise tracing of related diseases.

[0005] Currently, integrated probes combining mitochondrial targeting, carboxylesterase response, near-infrared fluorescence imaging, and photodynamic therapy are still relatively scarce. Conventional probes struggle to simultaneously achieve highly selective recognition of CEs, precise mitochondrial localization, real-time fluorescence imaging, and efficient photodynamic killing. Therefore, developing a multifunctional probe capable of targeting mitochondria, specifically responding to carboxylesterases, and simultaneously achieving fluorescence imaging and photodynamic therapy is of great significance for improving the accuracy of tumor diagnosis and the precision of treatment, promoting the development of integrated diagnostic and therapeutic technologies, and providing new ideas and technical support for the early diagnosis and efficient treatment of cancer. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a mitochondrial-targeting fluorescent probe activated by carboxylesterases and possessing photodynamic therapy properties, along with its preparation method and applications. Through molecular structure design, this invention constructs a multifunctional probe that exhibits a specific fluorescent on-off response to carboxylesterases (CEs) and can precisely target mitochondria, thus combining multiple functions such as mitochondrial targeting, carboxylesterase-specific recognition, real-time fluorescence imaging, and efficient photodynamic therapy.

[0007] This invention provides a fluorescent probe capable of specific quantitative fluorescence detection of carboxylesterases in solution systems and within the mitochondria of living cells. It features high selectivity, rapid and efficient detection, and excellent biocompatibility. Furthermore, upon response to carboxylesterases, the probe efficiently generates reactive oxygen species under illumination, thereby achieving photodynamic killing of tumor cells. This makes it a high-performance, therapeutically integrated fluorescent probe.

[0008] This invention uses anoxane derivatives that are easy to prepare, modify, and have low cytotoxicity as the fluorophore. A control fluorescent probe, CM, was designed and synthesized by introducing an indole salt cation unit and an acetyl group, a carboxylesterase-specific recognition site. Upon interaction with carboxylesterase, the acetyl group undergoes enzymatic hydrolysis and detachment, converting to CM-OH, resulting in a significant enhancement of the fluorescence signal. To further improve the probe's mitochondrial targeting and anchoring capabilities, based on the property that long-chain, lipid-soluble alkyl groups can enhance binding to the mitochondrial membrane lipid bilayer through hydrophobic interactions, this invention optimizes the structure of the control probe CM: replacing the methyl group on the indole salt with a butyl group and adjusting the alkyl chain length, ultimately obtaining the target probe CML. Experimental results show that CML can specifically react with carboxylesterase; after the acetyl group is hydrolyzed and CML-OH is generated, the fluorophore is released, exhibiting a significant fluorescence-on response. Simultaneously, experimental verification shows that both CML-OH and the control product CM-OH possess good reactive oxygen species (ROS) generation capabilities under light conditions, providing a solid foundation for their further application in photodynamic therapy.

[0009] The mitochondrial-targeting fluorescent probe of this invention, abbreviated as CML, has the following structure:

[0010] .

[0011] The method for preparing the mitochondrial-targeting fluorescent probe of the present invention includes the following steps:

[0012] Step 1: Under nitrogen protection, 20 mL of N,N-dimethylformamide (DMF) and 50 mL of dichloromethane (DCM) were mixed thoroughly and stirred in an ice bath. Then, 20 mL of phosphorus tribromide was slowly added dropwise, followed by the addition of 10 mL of cyclopentanone dropwise after 2 hours. After the addition was complete, the mixture was allowed to react at room temperature for approximately 6 hours. After the reaction was complete, the reaction solution was slowly quenched by adding ice water, and the pH of the system was adjusted to neutral with potassium carbonate powder. The organic phase was washed with water several times, dried, and insoluble solid impurities were filtered out. Finally, the filtrate was concentrated under reduced pressure to obtain a dark brown oily intermediate 1.

[0013] Step 2: 8.2 g of intermediate 1, 2-hydroxy-4-methoxybenzaldehyde, and potassium carbonate were added together to 37 mL of N,N-dimethylformamide (DMF), and the mixture was reacted for 12 h. After the reaction was completed, the reaction solution was extracted with dichloromethane (DCM); the organic phase was collected and dried, and the solvent was removed by rotary evaporation under reduced pressure to obtain the crude product. The crude product was separated by column chromatography (ethyl acetate: petroleum ether = 1:2, v / v) to obtain a yellow solid intermediate 2;

[0014] Step 3: Dissolve 3.20 g of intermediate 2 in 20 mL of dichloromethane (DCM). Under nitrogen protection, place the system in an ice bath, and then slowly add 17.5 mL of boron tribromide. After the addition is complete, stir for 1 h, then remove the ice bath and allow the reaction to continue at room temperature for 12 h. After the reaction is complete, cool the system in an ice bath, slowly add water to quench the reaction, and then add methanol to fully dissolve the system. Concentrate the mixture under reduced pressure to remove the solvent. After a large amount of solid precipitates, filter and dry to obtain yellow solid intermediate 3.

[0015] Step 4: Take 1.60 mL of 2,3,3-trimethylindole and excess 1-iodobutane, and add them together to acetonitrile; reflux at 80 °C for 24 hours. After the reaction is completed, cool to room temperature, perform column chromatography, and dry the resulting mixture under vacuum to obtain solid intermediate 5.

[0016] Step 5: In a single-necked flask, add 5 mL of acetic anhydride, followed by intermediate 3 (214.22 mg, 1 mmol), intermediate 5 (259.41 mg, 1.2 mmol), and 250 mg of potassium carbonate. The reaction was carried out at 25 °C for 12 h, and the reaction was monitored by TLC. After the reaction was completed, the reaction solution was extracted with dichloromethane (DCM). The extraction operation was repeated 3 times. The organic phase was collected and dried, and separated by silica gel column chromatography (methanol:dichloromethane = 1:20, v / v) to obtain a black solid CML.

[0017] Step 6: Take 3.20 mL of 2,3,3-trimethylindole and excess iodomethane, and add them together to acetonitrile; place the system at 80°C and react overnight. After the reaction is complete, cool to room temperature first, then slowly add diethyl ether to the system and stir. Finally, dry the resulting mixture under vacuum to obtain purple intermediate 4.

[0018] Step 7: In a single-necked flask, 214.22 mg of intermediate 3, 209.29 mg of intermediate 4 and 250 mg of potassium carbonate were added sequentially to acetic anhydride. The reaction was carried out at 25 °C for 12 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was extracted with dichloromethane (DCM). The extraction operation was repeated 3 times. The organic phase was collected and dried. The solution was separated by silica gel column chromatography (methanol:dichloromethane = 1:20, v / v) to obtain CM black solid.

[0019] Step 8: Dissolve 454.24 mg CML in methanol under an ice-water bath at 0℃, add 0.2 mM potassium carbonate solution, stir at room temperature, detect by TLC, and record the experimental data. After the reaction is complete, remove the methanol solvent by vacuum distillation, extract the aqueous phase with dichloromethane, dry the combined organic extracts with anhydrous sodium sulfate, concentrate the product under reduced pressure, and purify by column chromatography (methanol:dichloromethane = 1:20, v / v) to obtain CML-OH black solid.

[0020] Step 9: Dissolve 412.19 mg of CM in methanol under an ice-water bath at 0℃, add 0.2 mM potassium carbonate solution, stir at room temperature, detect by TLC, and record the experimental data. After the reaction is complete, remove the methanol solvent by vacuum distillation, extract the aqueous phase with dichloromethane, dry the combined organic extracts with anhydrous sodium sulfate, concentrate the product under reduced pressure, and purify by column chromatography (methanol:dichloromethane = 1:20, v / v) to obtain CM-OH black solid.

[0021] The synthesis route is shown below:

[0022]

[0023] The present invention relates to the application of fluorescent probes in the preparation of CE detection reagents and / or photodynamic therapy drug formulations.

[0024] The detection reagent is in I 710nm The fluorescence intensity at the point is linearly related to the concentration of CEs.

[0025] The fluorescent probe CML of this invention uses an oxanthracene derivative that is easy to prepare, modify, and has low cytotoxicity as its fluorophore. By introducing an indole salt cation unit and an acetyl group, a carboxylesterase-specific recognition site, CML was designed and synthesized. CML can specifically react with carboxylesterase; after the acetyl group is hydrolyzed and removed, CML-OH is generated, releasing the fluorophore and exhibiting a significant fluorescence-on response. The mechanism of its response to CEs is illustrated below:

[0026]

[0027] The detection method is as follows:

[0028] The fluorescent probe CML of this invention was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe stock solution with a concentration of 2 mM. 15 μL of the above probe stock solution was transferred and added to 3 mL of phosphate-buffered saline (PBS) containing different concentrations of carboxylesterases (CEs) to prepare a CML test system with a final concentration of 10 μM. The UV-Vis absorption and fluorescence emission spectra of CML were then measured. Experimental results showed that as the concentration of CEs in the system increased, the characteristic absorption peak of the probe CML shifted significantly, while the fluorescence intensity gradually increased. After the probe CML reacted with CEs, the fluorescence signal was enhanced by more than 12 times compared to before the reaction, exhibiting typical on-response fluorescence behavior and possessing high-sensitivity fluorescence detection capability. Further linear fitting results showed that the fluorescence intensity of the probe CML and the concentration of CEs exhibited a good linear relationship within a certain range, with a correlation coefficient R0. 2 =0.99, indicating that it can achieve precise detection of carboxylesterases. Cell-level experiments confirmed that the probe CML can efficiently respond to endogenous carboxylesterases in HepG2 cells, enabling clear, high signal-to-noise ratio fluorescence imaging. Simultaneously, the probe CML exhibits excellent mitochondrial targeting ability and good mitochondrial co-localization imaging; compared with the control probe CM, CML has better anchoring stability within mitochondria and is less affected by changes in mitochondrial membrane potential, enabling long-term stable mitochondrial tracking.

[0029] More importantly, the probe CML, after activation by carboxylesterase, can efficiently generate reactive oxygen species under light conditions, thus possessing excellent photodynamic therapy (PDT) capabilities. Reactive oxygen species identification results indicate that CML, after activation by CEs, can simultaneously generate singlet oxygen (…) under light. 1 O2) and superoxide anion (O2· - This drug combines type I and type II photodynamic therapy, inducing mitochondrial damage, apoptosis, and necrosis in cancer cells through multiple pathways, significantly enhancing the photodynamic killing effect. Under dark conditions, CML produces almost no ROS, exhibits extremely low cytotoxicity, and demonstrates good biosafety; while under light, it can rapidly and efficiently exert its photodynamic killing effect, achieving integrated diagnosis and treatment.

[0030] In summary, this invention provides a mitochondrial-targeted, carboxylesterase-responsive multifunctional fluorescent probe, CML. This probe not only achieves highly sensitive and selective on-feed fluorescence detection and intracellular visualization of carboxylesterases, but also simultaneously generates singlet oxygen and superoxide anions upon enzyme activation, enabling precise and controllable photodynamic therapy. It holds significant application potential in the early diagnosis of carboxylesterase-related diseases, mitochondrial-targeted imaging, and synergistic photodynamic therapy. Attached Figure Description

[0031] Figure 1 (a) is the UV absorption spectrum of probes CML, CML+CEs and CML-OH; (b) is the fluorescence emission spectrum of probes CML and CML+CEs.

[0032] Figure 2 The UV fluorescence titration test was performed on the reaction process of CML with different concentrations of CEs at different reaction times. (a) Changes in UV absorption spectrum of CML (10 μM) and different concentrations of CEs; (b) Changes in fluorescence emission spectrum of CML (10 μM) and different concentrations of CEs.

[0033] Figure 3 (a) shows the changes in fluorescence emission spectra after the reaction of CML (10 μM) with different concentrations of CEs (0-20 U / L); (b) shows a good linear relationship between the reaction of CML (10 μM) with different concentrations of CEs (0-20 U / L) and the concentration of CEs (R0). 2 =0.996), and the limit of detection is 0.30 U / L.

[0034] Figure 4 The time response of the probe CML to CEs was investigated to explore the sensitivity of CML to CEs.

[0035] Figure 5 It is a selective test for CML. Figure 5 (a) shows the reaction of CML (10 μM) with Mg. 2+ Na + Fe 3+ Cu 2+ NH4 + SO4 2- S 2- ClO - I - HPO4 2- Changes in fluorescence intensity after the reaction of H2O2, Hcy, GSH, Cys, GGT, and CEs; Figure 5 (b) shows the fluorescence intensity changes of CML (10 μM) after reacting with NTR, AChE, TYR, β-gal, and CEs, respectively.

[0036] Figure 6 (a) is the fluorescence emission spectrum of DCFH (5 μM) under illumination; (b) is the fluorescence emission spectrum of probe CML-OH (10 μM) and DCFH (5 μM) under illumination; the ability of CML-OH to generate reactive oxygen species was investigated.

[0037] Figure 7 (a) is the fluorescence emission spectrum of SOSG (5 μM) under illumination; (b) is the fluorescence emission spectrum of probe CML-OH (10 μM) and SOSG (5 μM) under illumination; (c) is the fluorescence emission spectrum of DHR123 (5 μM) under illumination; (d) is the fluorescence emission spectrum of probe CML-OH (10 μM) and DHR123 (5 μM) under illumination. The fluorescence emission spectra of probe CML-OH (10 μM) and DHR123 (5 μM) under illumination were used to investigate the ability of CML-OH to generate singlet oxygen and superoxide anions.

[0038] Figure 8 This is a cytotoxicity test for CML.

[0039] Figure 9 shows the results of the mitochondrial colocalization experiment of the target probe CML and the control probe CM. It also shows the colocalization comparison experiment results of cells after CCCP treatment; the influence of long chains on mitochondrial anchoring ability was investigated.

[0040] Figure 10 (a) is a test of CML for detecting endogenous and exogenous CEs. AEBSF can inhibit CE activity. CEs and AEBSF were added first, followed by CML (10 μM) and incubation for 30 min before confocal imaging. (b) is a bar graph of the corresponding fluorescence intensity, which explored the ability of CML to monitor intracellular CEs.

[0041] Figure 11The study investigated the ability of intracellular CML to generate ROS under light by detecting different ROS species in HepG2 cells after various treatments using different indicators.

[0042] Figure 12 The study investigated the photodynamic therapy effect of CML on HepG2 cells by analyzing calcein AM / PI staining under different treatment conditions. Detailed Implementation

[0043] The present invention will be further illustrated by the following examples.

[0044] Example 1: Synthesis of CML

[0045] In a single-necked flask, 5 mL of acetic anhydride was added, followed by compound 3 (214.22 mg, 1 mmol), compound 5 (259.41 mg, 1.2 mmol), and 250 mg of potassium carbonate. The reaction was carried out at 25 °C for 12 h, and the reaction was monitored by TLC. After the reaction was completed, the reaction solution was extracted with dichloromethane (DCM). The extraction was repeated 3 times. The organic phase was collected and dried, and separated by silica gel column chromatography (methanol:dichloromethane = 1:20, v / v) to obtain a black solid CML.

[0046] 1 H NMR (400 MHz, DMSO-d6) δ 8.19 (d, J = 15.0 Hz, 1H), 7.82 (d, J =7.2 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.56 (td, J = 8.7, 2.0 Hz, 2H), 7.50 –7.46 (m, 2H), 7.34 (s, 1H), 7.12 (dd, J = 8.4, 2.2 Hz, 1H), 6.49 (d, J = 14.9Hz, 1H), 4.45 (t, J = 7.2 Hz, 2H), 2.99 – 2.87 (m, 4H), 2.31 (s, 3H), 1.73 (s, 7H), 1.46 – 1.37 (m, 3H), 0.93 (d, J = 7.4 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 178.26, 169.48, 164.51, 152.59, 152.27, 142.98, 141.77, 140.36, 130.17,129.52, 128.82, 128.16, 126.55, 123.46, 120.30, 120.10, 114.29, 111.27,107.15, 51.30, 45.55, 30.21, 27.49, 21.43, 20.00, 14.25.

[0047] Example 2: Optical titration, linearity, and response time of CML in solvent in response to CEs

[0048] To investigate the specific response of the probe CML to carboxylesterases (CEs), we measured the UV absorption spectra of CML, the reaction system of CML and CEs, and the reaction product CML-OH, as shown in Figure 1(a). After the reaction of CML with CEs, the highest UV absorption peak of CML significantly red-shifted from 565 nm to 645 nm, and this position coincided with the characteristic absorption peak of CML-OH. Figure 1 As shown in (b), when the excitation wavelength is 630 nm, the fluorescence intensity of CML at 710 nm after CE response is significantly enhanced compared with the untreated group, indicating that the probe CML can produce an effective response to carboxylesterase.

[0049] We further investigated the UV and fluorescence spectra of the fluorescent probe CML before and after the response to CEs, as shown in Figure 2(a). When the CE concentration increased in a gradient of 40 U / L within the range of 0-160 U / L, the characteristic absorption peak of CML at around 565 nm gradually decreased and disappeared, while a new absorption peak appeared at around 645 nm. The overall absorption spectrum showed a significant red shift with increasing CE concentration. The corresponding fluorescence titration results are shown in Figure 2(b). With increasing CE concentration (0-160 U / L), the fluorescence signal of CML at 705 nm gradually increased. These results further confirm that the probe can respond efficiently to carboxylesterases.

[0050] Furthermore, the fluorescence response of CML to low concentrations of sulfatase (0-20 U / L) was investigated, and linear fitting was performed on the fluorescence intensity of CML at different CE concentrations. As shown in Figure 3(b), the fluorescence intensity of CML at 710 nm showed a good linear relationship with the CE concentration, with a correlation coefficient reaching R0. 2 =0.996. According to the detection limit formula... Fitting the data revealed that the detection limit of CML in response to carboxylesterase was 0.30 U / L, indicating that the probe CML is highly sensitive to carboxylesterase.

[0051] To further evaluate the response kinetics of probe CML to CEs, a time-dependent fluorescence detection experiment was conducted. As shown in Figure 4, in the reaction system of 10 μM CML and 160 U / L CEs, fluorescence signals were recorded every 1 min. The results showed that the fluorescence intensity increased rapidly in the first 15 min, and then gradually stabilized. Based on this, the optimal detection time for CML was determined to be 15 min, further demonstrating that probe CML has a rapid and sensitive response to carboxylesterases.

[0052] Example 3: Selectivity experiment of probe CML

[0053] The intracellular environment typically contains various metal ions, non-metal ions, and enzymes that may affect the probe. To eliminate interference from these substances and verify whether the fluorescent probe can accurately detect carboxylesterases, we performed selectivity experiments on these potentially influencing substances. Various analytes (such as...) were added to the fluorescent probe CML. Figure 5 (a): 1. Blank; 2. Mg 2+ 3. Na + ; 4. Fe 3+ 5. Cu 2+ 6. NH4 + 7. SO4 2- ; 8. S 2- 9. ClO - 10. I - 11. HPO4 2- ;12. H2O2; 13. Hcy; 14. GSH; 15. Cys; 16. GGT; 17. CEs; Figure 5 (b): 1. NTR; 2. AChE; 3. TYR; 4. β-gal; 5. CEs), and the fluorescence changes of the probes were tested. The test results showed that after the probe CML reacted with carboxylesterase, the fluorescence was significantly enhanced, while for many other substances, the fluorescence remained basically unchanged, indicating that these substances had little response to the probe. For some common biological enzymes, only a weak change in fluorescence signal occurred after the addition of probe CML, which was negligible compared to the fluorescence signal in response to carboxylesterase. Therefore, we can infer that the fluorescent probe CML exhibits high selectivity for carboxylesterase.

[0054] Example 4: The ability of CML-OH to generate reactive oxygen species (ROS) after exposure to different light durations.

[0055] To verify that the product CML-OH generated by the probe CML in response to carboxylesterase possesses the ability to generate reactive oxygen species (ROS) photoinducedly, this study evaluated its ROS generation ability using a pretreated fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA). First, to eliminate the interference of DCFH itself on the experimental results, a concentration of 20 mW / cm² was used. 2 The fluorescence spectrum changes of DCFH were recorded under different illumination times. As shown in Figure 6(a), there was almost no significant fluorescence enhancement at 525 nm within the 0-6 min illumination range, indicating that the pretreated DCFH was fluorescently stable under these conditions and did not interfere with ROS detection. Based on this, CML-OH was mixed with the pretreated DCFH, and the fluorescence spectrum changes were recorded at different times under the same illumination conditions. The results are shown in Figure 6(b). With prolonged illumination time, the fluorescence intensity at 525 nm increased significantly, indicating that CML-OH can effectively generate ROS under illumination and has the potential to be used as a photosensitizer for photodynamic therapy.

[0056] Example 5: The ability of CML-OH to generate singlet oxygen and superoxide anions after different light exposure times

[0057] To systematically investigate the formation of CML-OH, a product of the probe CML after responding to carboxylesterase, under light conditions... 1 O2 and O2· - To assess their capabilities, we used singlet oxygen green fluorescent probe (SOSG) and dihydrorhodamine 123 (DHR123) as... 1 O2 and O2· - Specific fluorescent indication. SOSG itself is non-fluorescent, and... 1 The O2-specific reaction produces green fluorescence, with a maximum excitation wavelength of approximately 488 nm and a maximum emission wavelength of approximately 525 nm. For example... Figure 7 As shown in (a), no significant fluorescence enhancement was observed at 525 nm within the 0-6 min illumination range, indicating that SOSG itself has good stability and can be used as a [missing information - likely a reference material]. 1 An effective O2-capturing probe. Under the same test conditions, SOSG was mixed with CML-OH and subjected to light treatment, and the results were as follows. Figure 7 As shown in (b), the fluorescence intensity at 525 nm increases significantly with prolonged illumination, proving that CML-OH can effectively generate singlet oxygen under illumination. Similarly, DHR123 itself has no fluorescence, but reacts with O2· −The reaction produces green fluorescence. As shown in Figure 7(c), no significant enhancement of the fluorescence signal was observed at 525 nm within the 0-6 min illumination range, indicating that DHR123 is suitable for O2· - The fluorescence signal at 525 nm was significantly enhanced after DHR123 was mixed with CML-OH and treated with light, as the illumination time increased (Figure 7(d)), indicating that CML-OH can effectively generate superoxide anions under light.

[0058] Example 6: Cytotoxicity test of CML

[0059] Before using the probe CML in cell imaging studies, its dark toxicity and phototoxicity were systematically evaluated using the MTT assay. Using HepG2 cells as a model, CML concentrations of 0, 10, 20, and 30 μM were selected, and after incubation under dark conditions, different durations of light treatment were applied. Figure 8 As shown, under no light exposure, the cell viability of all concentration groups remained above 80%, with almost no significant cytotoxicity. Under the same light intensity, after 20 min of light exposure, the cell viability showed a significant decreasing trend with increasing probe concentration; when the light exposure time was further extended to 40 min, the cell-killing effect was more significant, and the viability further decreased. These results indicate that the probe CML exhibits extremely low cytotoxicity and excellent biocompatibility under no-light conditions, making it safe for use in biological applications such as cell imaging; while under light exposure, it can efficiently induce cell death, demonstrating good photodynamic therapy effects and possessing potential for PDT anti-tumor research.

[0060] Example 7: Mitochondrial Colocalization Experiment

[0061] To evaluate the mitochondrial targeting ability of the probe CML in HepG2 cells, the results are shown in Figure 9. By co-incubating the probe with Mito-Tracker, CML exhibited excellent mitochondrial targeting properties, with a co-localization coefficient of 0.95. In contrast, the probe CM also showed some mitochondrial targeting ability, with a co-localization coefficient of 0.94. To further investigate the long-term anchoring ability of the probe in mitochondria, 3-chlorophenylhydrazone (CCCP) was added to disrupt the mitochondrial membrane potential (MMP) after co-incubating HepG2 cells with the probe and Mito-Tracker. The results showed that after CCCP treatment, the co-localization coefficient of CM significantly decreased from 0.94 to 0.86; while CML maintained a high co-localization coefficient of 0.93, demonstrating stable anchoring within mitochondria unaffected by changes in mitochondrial membrane potential. This phenomenon is mainly attributed to the stronger hydrophobicity of CML.

[0062] Example 8: Response of probe CML to endogenous and exogenous CEs in cells

[0063] To investigate the fluorescence imaging performance of CML on endogenous and exogenous carboxylesterases at the live-cell level, this study designed three cell experiments: a control group (treated with only 10 μM CML probe), an exogenous experimental group (treated with exogenous carboxylesterase and CML probe), and an endogenous experimental group (pretreated with AEBSF for 1 h, then treated with 10 μM CML probe). After co-incubation for 30 min, unbound probes and impurities were removed by washing with PBS buffer, followed by fluorescence imaging detection. Figure 10 As shown in (a) and (b), compared with the control group, the red fluorescence signal in the exogenous experimental group was significantly enhanced; while the red fluorescence signal in the endogenous experimental group treated with the carboxylesterase inhibitor AEBSF was significantly weakened. The results further confirm that the probe CML can achieve precise fluorescence imaging of changes in endogenous carboxylesterase content at the live cell level.

[0064] Example 9: Detection of reactive oxygen species (ROS) in CML in HepG2 cells

[0065] To investigate the photoinduced reactive oxygen species (ROS) generation capacity and ROS types in HepG2 cells after CML activation by endogenous carboxylesterase, this study used multiple ROS-specific fluorescent probes for detection: DCFH DA was used as the total ROS probe, and SOSG was used to detect singlet oxygen (SOSG). 1 O2), DHE is used to detect superoxide anion (O2· - ).like Figure 11 As shown, under dark conditions, cells in each group exhibited only negligible background fluorescence; however, after white light irradiation, the intracellular fluorescence intensity of CML activated by carboxylesterase was significantly enhanced. Furthermore, the CML combined with light irradiation group showed distinct characteristic green and red fluorescence after SOSG and DHE staining, respectively, confirming that CML can effectively produce fluorescence after carboxylesterase response. 1 O2 and O2· - The above results confirm that CML exhibits excellent photoinduced reactive oxygen species (ROS) generation capacity after activation by carboxylesterase, and can simultaneously generate both type I and type II photodynamic-related ROS, demonstrating great application potential in precision-targeted photodynamic therapy (PDT).

[0066] Example 10: Photodynamic Therapy Effect of CML in HepG2 Cells

[0067] To further verify the photocytotoxicity of CML activated by endogenous carboxylesterase, calcein and propidium iodide (PI) were used to determine cell viability under different treatment conditions. Live cells showed green fluorescence, while dead cells showed red fluorescence. After 60 minutes of white light irradiation (20 mW / cm²), the cells were... 2 After treatment with CML, HepG2 cells showed significant red fluorescence and minimal green fluorescence, while other groups still showed strong green fluorescence. Figure 12 This indicates that CML has significant PDT capabilities.

Claims

1. A mitochondrial-targeting fluorescent probe, abbreviated as CML, characterized in that... Its structure is as follows: 。 2. The method for preparing the fluorescent probe according to claim 1, characterized in that... Includes the following steps: Step 1: Under nitrogen protection, N,N-dimethylformamide and dichloromethane were mixed evenly and stirred in an ice bath. Phosphorus tribromide was then slowly added dropwise. After 2 hours, cyclopentanone was added dropwise. After the addition was complete, the mixture was moved to room temperature for reaction. After the reaction was completed, the reaction solution was slowly quenched by adding ice water. The pH of the system was adjusted to neutral with potassium carbonate powder. The organic phase was washed with water several times and dried. Insoluble solid impurities were filtered out. Finally, the filtrate was concentrated under reduced pressure to obtain a dark brown oily intermediate 1. Step 2: Intermediate 1, 2-hydroxy-4-methoxybenzaldehyde and potassium carbonate were added together to N,N-dimethylformamide and mixed for reaction; after the reaction was completed, the reaction solution was extracted with dichloromethane; the organic phase was collected and dried, and the solvent was removed by rotary evaporation under reduced pressure to obtain crude product, which was separated by column chromatography to obtain yellow solid intermediate 2; Step 3: Dissolve intermediate 2 in dichloromethane. Place the system in an ice bath under nitrogen protection, then slowly add boron tribromide. After the addition is complete, stir for 1 h, then remove the ice bath and move to room temperature to continue the reaction. After the reaction is complete, cool the system in an ice bath, slowly add water to quench the reaction, and then add methanol to fully dissolve the system. Concentrate the mixture under reduced pressure to remove the solvent. After a large amount of solid precipitates, filter and dry to obtain yellow solid intermediate 3. Step 4: Add 2,3,3-trimethylindole and 1-iodobutane together to acetonitrile, heat to reflux and react for 24 hours. After the reaction is completed, cool to room temperature, separate by column chromatography and vacuum dry to obtain solid intermediate 5. Step 5: Add acetic anhydride to a single-necked flask, followed by intermediate 3, intermediate 5 and potassium carbonate in sequence. React at 25 °C. After the reaction is complete, extract the reaction solution with dichloromethane, collect the organic phase and dry it. Separate it by silica gel column chromatography to obtain black solid CML. The synthesis route is shown below:

3. The use of the mitochondrial-targeting fluorescent probe of claim 1 in the preparation of CE detection reagents and / or photodynamic therapy drug formulations.

4. The application according to claim 3, characterized in that: The detection reagent is in I 710nm The fluorescence intensity at the point is linearly related to the concentration of CEs.

5. The application according to claim 3, characterized in that: The detection reagent targets mitochondria.

6. The application according to claim 3, characterized in that: The detection reagent, upon response to carboxylesterase, can simultaneously generate singlet oxygen and superoxide anions under light conditions, thus exhibiting both type I and type II photodynamic therapy effects.

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