Liver / mitochondria dual-targeting carboxylesterase fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a liver / mitochondria dual-targeting carboxylesterase fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of medicines. The probe is a compound LDM-CA, has the liver and mitochondria dual-targeting characteristic, and shows high selectivity, sensitivity and strong binding affinity to CEs. In-vitro cell experiments show that the LDM-CA has excellent mitochondrial targeting ability in the HCC cells and specific fluorescence opening response to CEs in the cells, and can clearly distinguish the HCC cells from normal liver cells or non-liver cancer cells. In-vivo imaging of a mouse model shows that no matter in-tumor injection or intravenous injection, the LDM-CA can effectively visualize the HCC tumor and draw the edge of the tumor, has the prospect of serving as a powerful molecular tool for early HCC diagnosis and image-guided surgery, and also provides a valuable means for studying the action of mitochondrial CEs activity in HCC pathogenesis at the subcellular level.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, and particularly relates to a liver / microsome dual-targeted carboxylesterase fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is a primary liver cancer with high mortality and is one of the main causes of cancer-related deaths. At present, clinical practice relies on imaging techniques such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET) and ultrasound (US) to detect focal lesions in the liver. Although these methods play a key role in the diagnosis of liver cancer, they still have certain limitations. Specifically, they have low sensitivity to small lesions with a diameter of less than 1 centimeter, and often cannot reliably distinguish between benign regenerative nodules and malignant tumors. Therefore, some early HCC cases are still not detected or misdiagnosed. In addition, current imaging methods cannot provide real-time assessment of tumor biological activity, thus failing to meet the clinical needs for accurate diagnosis, dynamic monitoring of treatment response and guidance for surgical resection. Therefore, there is an urgent need to develop a new HCC diagnostic tool with high specificity, high sensitivity and real-time monitoring capability.

[0003] In the field of medical diagnosis, biomarkers provide a promising direction for accurate diagnosis of HCC. During the development of HCC, fluctuations in the expression levels of liver-specific biomarkers can directly reflect the malignant transformation of hepatocytes. Among them, carboxylesterases (CEs) are key hydrolytic enzymes that are mainly enriched in the liver, and their abundant presence in liver tissue makes them important biomarkers for assessing liver function and diagnosing liver-related diseases, including HCC. The expression level of CEs is closely related to the malignant degree of liver cancer, and its abnormal regulation may directly participate in the occurrence of liver cancer. There are significant limitations in the early detection methods of CEs. Techniques such as qRT-PCR and Western blot can quantify protein expression, but have limited sensitivity and selectivity, are easily affected by endogenous interferents in complex samples, and cannot reflect actual enzyme activity. In contrast, ultraviolet spectrophotometry, capillary electrophoresis and liquid chromatography can measure enzyme activity, but usually cannot perform real-time monitoring in vivo. Therefore, it is of great significance to develop a high-sensitivity and high-specificity in vivo CEs activity detection method, which provides a potential tool for early diagnosis of liver cancer, assessment of disease severity and guidance for surgical resection.

[0004] Fluorescent probe technology can visualize cellular and molecular events in real time with high sensitivity and specificity, showing great potential in overcoming the limitations of traditional diagnostic methods. In recent years, although many fluorescent probes for CEs detection have been reported, there are still some shortcomings in the existing probes. For example, the recognition groups of most CEs probes are mainly based on ester bonds, which can lead to cross-reactions with acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) substrates, thereby affecting the accuracy of CEs activity detection. In addition, a major challenge of current CEs detection methods is the lack of target specificity. The widespread presence of ester bonds in various tissues and organs can lead to off-target effects, reducing the accuracy of liver imaging. This lack of specificity undermines the accuracy of liver disease diagnosis, as signals can come from non-liver tissues, leading to false positives or misleading results. Therefore, there is an urgent need to develop enzyme-responsive fluorescent probes with liver-specific targeting to improve the accuracy and precision of liver disease diagnosis.

[0005] It was found through retrieval that the invention patent application with publication number CN119569711A discloses a fluorescent probe ZZD synthesized with an xanthene group as a fluorescent parent to specifically recognize carboxylic esterase, and its structure is as shown below:

[0006]

[0007] In the above-mentioned invention patent application, the quinoline salt part in the probe ZZD enables it to target mitochondria, and the ZD structure after response can be located in lipid droplets; moreover, the presence of AChE has no effect on the fluorescent probe, and the probe exhibits ultra-high selectivity and good response ability in living cells, as well as superior dual-organelle targeting ability. However, there is currently no related report on obtaining a fluorescent probe with liver and mitochondrial dual-targeting properties by introducing a cholic acid group on the rhodamine basic framework. SUMMARY

[0008] The technical problem to be solved by the present application is to provide a liver / microsome dual-targeting carboxylic esterase fluorescent probe obtained by introducing a cholic acid group on the rhodamine basic framework, as well as a preparation method and application thereof. The fluorescent probe CEs exhibits high selectivity, sensitivity and strong binding affinity, has excellent mitochondrial targeting ability in HCC cells and specific fluorescent opening response to intracellular CEs, and can clearly distinguish HCC cells from normal liver cells or non-liver cancer cells.

[0009] To solve the above technical problems, the following technical solutions are adopted in the present application:

[0010] A liver / microsome dual-targeting carboxylic esterase fluorescent probe is denoted as compound LDM-CA, and its structure is as shown in the following formula:

[0011]

[0012] The preparation method of the liver / microsome double-targeting carboxylesterase fluorescent probe comprises the following steps: reacting compound LDM and cholic acid (CA) in an organic solvent, removing the solvent from the reaction product to obtain the target crude product.

[0013]

[0014] In the method, the organic solvent can be one or a combination of two or more selected from N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc) and dimethyl sulfoxide (DMSO). Further preferably, it is N,N-dimethylformamide. The amount of the organic solvent is determined according to the need, and it is appropriate to be able to sufficiently dissolve the raw materials participating in the reaction. Specifically, based on 1 mmol of compound LDM, the total amount of organic solvent used for all raw materials is usually 2-6 mL.

[0015] In the method, the solvent in the reaction product is removed by using the existing conventional method. Specifically, water and dichloromethane can be added to the reaction product for extraction, and the organic phase is collected and dried to remove the solvent. Preferably, before the extraction operation, the reaction product is reduced in pressure and most of the solvent is removed by rotary evaporation.

[0016] In the method, the reaction can be carried out with or without heating, that is, the reaction temperature can be room temperature or between room temperature and the boiling point of the organic solvent. Further preferably, it is carried out at room temperature.

[0017] In order to reduce or avoid the oxidation of the phenolic group in compound LDM, the reaction is preferably carried out under an atmosphere protection, such as under the protection of inert atmosphere of nitrogen, argon or helium. In the preparation method, the compound LDM involved can be synthesized according to the existing literature (Dyes and Pigments 2021, 188, 109229; Journal of Materials Chemistry B 2017, 5, 3377).

[0018] The crude product of compound LDM-CA prepared by the above method also includes the step of purifying the prepared target crude product. Specifically, the existing conventional purification method can be used to purify it to improve the purity of compound LDM-CA, such as using silica gel column chromatography to purify the crude product. The eluent used in the chromatography can be a mixed solvent composed of dichloromethane and methanol in a volume ratio of 20:1-50:1.

[0019] The applicant found through experiments that the probe LDM-CA has high selectivity, sensitivity and strong binding affinity to CEs. LDM-CA has excellent mitochondrial targeting ability in HCC cells and specific fluorescence opening response to intracellular CEs, and can clearly distinguish HCC cells and normal liver cells. In addition, in vivo imaging in a mouse model shows that LDM-CA can effectively visualize HCC tumors and outline tumor margins, whether intratumoral injection or intravenous injection.

[0020] Based on the above findings, the present application includes the use of the above-mentioned liver / mitochondrial dual-targeting carboxyesterase fluorescent probe in the preparation of a carboxyesterase detection reagent; or in the preparation of a hepatocellular carcinoma diagnostic reagent; or in the imaging of carboxyesterase in cells or organisms.

[0021] Further, the liver / mitochondrial dual-targeting carboxyesterase fluorescent probe can be used in one or more of the following processes:

[0022] for observing and monitoring the dynamic changes and distribution of endogenous carboxyesterase or exogenous carboxyesterase in cells or organisms; or

[0023] for visualizing the dynamic changes and distribution of carboxyesterase in cells or organisms; or

[0024] for specific imaging of hepatocellular carcinoma in cells or organisms by intratumoral injection or intravenous injection.

[0025] Compared with the prior art, the present application provides a probe LDM-CA obtained by introducing a cholic acid group on a rhodamine basic framework, wherein the cholic acid group contained therein imparts liver targeting ability, the lipophilic cationic group is used to achieve precise mitochondrial localization, and the intramolecular ester bond serves as a CEs response site, so that the resulting probe has liver and mitochondrial dual-targeting properties; and the probe exhibits high selectivity, sensitivity and strong binding affinity to CEs. In vitro cell experiments show that LDM-CA has excellent mitochondrial targeting ability in HCC cells and specific fluorescence opening response to intracellular CEs, and can clearly distinguish HCC cells and normal liver cells or non-liver cancer cells. In addition, in vivo imaging in a mouse model shows that LDM-CA can effectively visualize HCC tumors and outline tumor margins, whether intratumoral injection or intravenous injection, highlighting its great potential in guiding precise surgical resection. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The LDM-CA prepared for Example 1 of the present application has the following 1 H NMR spectrum (600MHz, DMSO).

[0027] Figure 2High resolution mass spectrum of LDM-CA prepared for Example 1 of the present invention. 13 C NMR spectrum (150 MHz, DMSO).

[0028] Figure 3 High resolution mass spectrum of LDM-CA prepared for Example 1 of the present invention.

[0029] Figure 4 In vitro response and selectivity of LDM-CA to CEs. (a) Absorption spectra of LDM-CA with or without CEs. (b) Fluorescence spectra obtained by adding CEs (0-9 U) to LDM-CA. (c) Correlation of fluorescence intensity at 633 nm with CEs concentration. (d) Time-dependent fluorescence response of LDM-CA to CEs. (e) Selectivity of LDM-CA to CEs over various interfering species. Solvent: DMSO / PBS = 9 / 1 (V / V), pH = 7.4, incubation condition: 37 °C, λex / λem= 557 nm / 633 nm, slit: 5 / 5 nm, error bars represent ± SD (n = 3).

[0030] Figure 5 Response mechanism and spectral characteristics of LDM-CA to HCIO. (a) HRMS spectrum of the reaction product after incubation of LDM-CA with CEs. (b) HPLC analysis of LDM-CA, LDM, and the mixture obtained after incubation of LDM-CA with CEs. (c) Simulation of the interaction between LDM-CA and CEs. (d) DFT calculation of the corresponding energy levels of the frontier molecular orbitals of LDM-CA and LDM.

[0031] Figure 6 Viability, fluorescence images, and fluorescence intensity of LDM-CA in different cells. (a) Cell viability assay of LDM-CA in LO2 and HepG2 cells. (b) Fluorescence imaging of LO2 cells after treatment with LDM-CA (10 μΜ) under the regulation of exogenous CEs and CEs levels induced by inhibitors. (c) Mean fluorescence intensity of LO2 cells in (b). (d) Fluorescence imaging of different cell lines after incubation with LDM-CA (10 μΜ). (e) Mean fluorescence intensity of different cells in (d). Scale bar: 20 μm. Error bars represent mean ± SD (n = 3).

[0032] Figure 7 Co-localization of LDM-CA with mitochondria in LO2 cells. (a) Representative fluorescence images of LO2 cells co-stained with 10 μΜ LDM-CA (red channel) and 0.5 μΜ Mito-Tracker Green (green channel). (b) Fluorescence signal intensity distribution along the red line indicated in the merged image. Scale bar: 20 μm. Error bars represent mean ± SD (n = 3).

[0033] Figure 8 Fluorescent imaging of LDM-CA for the detection of CEs in zebrafish in vivo. (a) Representative confocal fluorescent images of zebrafish. (b) Quantitative analysis of the relative fluorescence intensity of the images in (a). Scale bar: 20 μm. Error bars represent mean ± SD (n = 3).

[0034] Figure 9 In vivo imaging of LDM-CA after intratumoral injection in HCC-bearing mice. (a) Time-dependent in vivo fluorescent imaging of LDM-CA after intratumoral injection in healthy mice or HCC-bearing mice. (b) Clear fluorescent boundary of LDM-CA at the tumor margin provides clear guidance for surgical resection after local dissection to expose the tumor site.

[0035] Figure 10 Biodistribution and tumor-targeting ability of LDM-CA. (a) Non-invasive in vivo fluorescent imaging of LDM-CA (100 μΜ, 100 μί) at the indicated time points after intravenous injection in tumor-bearing mice. (b) Quantitative ex vivo fluorescent imaging of the excised tumor and major organs at the end of the study. (c) Distribution of LDM-CA was verified by confocal microscopy imaging of tumor and organ sections. DETAILED DESCRIPTION

[0036] In order to better explain the technical solutions of the present application, the present application will be further described in detail below in conjunction with the embodiments, but the embodiments of the present application are not limited thereto.

[0037] The compound LDM involved in the following examples was prepared according to the following synthetic route:

[0038]

[0039] Compound 1, i.e. 2-(4-diethylamino-2-hydroxybenzoyl)benzoic acid (2.5 g, 8 mmol) and compound 2, i.e. 1,6-dihydroxynaphthalene (1.3 g, 8 mmol) were dissolved in trifluoroacetic acid (CF3COOH, 21 mL, 42 mmol) and the resulting mixture was refluxed for 24 hours; after completion of the reaction, the solvent was evaporated and the resulting red solid was purified by silica gel column chromatography using DCM / MeOH (40 / 1, v / v) as eluent to obtain compound LDM as a red solid (3.23 g, yield: 85%). 1H NMR (600 MHz, CDC13) δ 8.22 (s, 1H), 8.13 (d, J = 7.3 Hz, 1H), 7.59 (t, J = 6.9 Hz, 2H), 7.10 (dd, J = 15.1, 8.0 Hz, 2H), 7.00 (d, J = 8.9 Hz, 1H), 6.91 (s, 1H), 6.85 (d, J = 9.0 Hz, 1H), 6.70 (d, J = 8.7 Hz, 1H), 6.56 (s, 1H), 6.42 (d, J = 9.3 Hz, 1H), 3.31 (d, J = 7.7 Hz, 4H), 1.14 (t, J = 7.3 Hz, 6H). 13 C NMR (150 MHz, CDC13) δ 170.86, 158.56, 154.42, 151.55, 149.76, 137.32, 133.35, 129.99, 129.75, 126.68, 125.68, 124.40, 123.91, 123.23, 119.17, 117.30, 111.94, 110.94, 110.31, 97.13, 45.00, 12.66. HRMS (ESI + )m / z, calcd for C 28 H 24 NO4 + [M] + : 438.1700, found: 438.1690.

[0040] Example 1: Preparation of compound LDM-CA

[0041]

[0042] To compound LDM (1.3 g, 3 mmol) and cholic acid (1.3 g, 3.2 mmol) were dissolved in DMF (15 mL), the resulting mixture was stirred at room temperature under nitrogen protection for 6 hours. After the reaction was completed, most of the solvent was removed by rotary evaporation, and the mixture was extracted with water and dichloromethane to remove the remaining solvent, the organic phase was collected, dried, and the resulting red solid was purified by silica gel column chromatography (using DCM / MeOH (40:1, v / v) as eluent) to obtain LDM-CA as a red solid (1.5 g, yield 60%). Melting point 155-170 °C. 1H NMR (600MHz, DMSO-d6) δ8.64(d,J=9.1Hz,1H),8.05(d,J=7.6Hz,1H),7.80–7.70(m,3H),7.57(d,J=8.9Hz,1H),7.51(d,J=9.1Hz,1H),7.28(d,J= 7.6Hz,1H),6.77(d,J=7.2Hz,2H),6.56(t,J=10.1Hz,2H),4.32(d,J=4.3 Hz,1H),4.18–4.15(m,1H),4.02(s,1H),3.82(s,1H),3.62(s,1H),3.42– 3.38(m,5H),3.21–3.15(m,1H),3.07(d,J=7.4Hz,3H),2.68(d,J=10.2H z,1H),2.57(d,J=7.7Hz,1H),2.29–2.12(m,2H),1.85(d,J=9.6Hz,2H),1 .82–1.77(m,3H),1.65(d,J=14.1Hz,2H),1.45–1.42(m,4H),1.36(s,2H) ,1.23(d,J=8.5Hz,4H),1.02(d,J=5.4Hz,3H),0.81(s,3H),0.62(s,3H)( Figure 1 ). 13 C NMR(150MHz,DMSO-d6)δ172.28,152.84,151.71,149.94,149.29,146.55,135.62,134.53,130.12,128 .59,126.07,124.70(d,J=16.4Hz),123.69,122.80,122.32,121.03,118.82,112.54,109.19,104.27, 97.22,83.40,70.98,70.41,66.22,46.00,45.79,45.67,43.76,41.50,41.38,35.29,35.02,34.86,34 .36,30.63(d,J=17.0Hz),30.39,28.52,27.29,26.20,22.78,22.59,16.98,12.34(d,J=5.0Hz),8.67(( Figure 2 HRMS (ESI+) analysis of LDM-CA showed the molecular ion [M]. + At m / z 828.4468 (calculated value C) 52 H 62 NO8 + The value of 828.4470 is in good agreement with the theoretical value.Figure 3 ).

[0043] Example 2: Preparation of compound LDM-CA

[0044] Example 1 was repeated, except that the reaction was not protected by nitrogen.

[0045] Finally, a red solid was obtained with a yield of 34%. It was characterized by nuclear magnetic hydrogen spectrum, carbon spectrum and high resolution mass spectrum to be compound LDM-CA.

[0046] Example 3: Preparation of compound LDM-CA

[0047] Example 1 was repeated, except that DMSO was used instead of DMF, and the reaction was carried out at 60°C until the reaction was complete.

[0048] Finally, a red solid was obtained with a yield of 22%. It was characterized by nuclear magnetic hydrogen spectrum, carbon spectrum and high resolution mass spectrum to be compound LDM-CA.

[0049] Experimental Example 1: In vitro response and detection of CEs by the probe LDM-CA according to the present application

[0050] Response performance. The response performance of the probe LDM-CA to CEs was evaluated in an in vitro system. As shown in Fig. Figure 4 a, after the addition of CEs, LDM-CA showed a clear ultraviolet absorption peak at 557 nm. Fluorescence measurement further showed that the LDM-CA solution showed almost no fluorescence emission in the absence of CEs, while after incubation with CEs, a strong fluorescence signal gradually appeared at 633 nm Figure 4 b). The fluorescence intensity steadily increased with the increase of the concentration of CEs, showing a good linear relationship in the range of 0-9 U / mL (R 2 = 0.9927) Figure 4 c). The regression equation was fitted as y = 47.275 + 83.665 x [CEs], the standard deviation σ = 0.6203, and the detection limit was determined to be 2.22 x 10-2 U / mL. These results showed that LDM-CA had high sensitivity to CEs and had the potential to linearly detect CEs in vitro.

[0051] Response time. In the case where the concentration of CEs was fixed at 9 U / mL, the effect of different substrate (LDM-CA) concentrations on the catalytic hydrolysis rate was studied. As shown in Fig. Figure 4 d, during the reaction period of 60 minutes, the fluorescence intensity showed an approximately linear increase, which preliminarily reflected the kinetic behavior of the enzymatic reaction.

[0052] Selectivity of LDM-CA towards CEs. To verify the specific response capability of LDM-CA towards CEs, we systematically evaluated the fluorescence behavior of LDM-CA in the presence of various potential interferents. Fifty-one substances that could potentially trigger fluorescence were tested under the same testing conditions, including various ions, small molecules, amino acids, and enzymes. Substance numbers 1 to 51 are as follows: Control, Na + , K + , Mg 2+ , Fe 3+ , Fe 2+ , Hg + , Zn 2+ , Ca 2+ , Cu 2+ , Al 3+ , Pb 2+ , Ag + , Cs 2+ , HCO3 - , Cl - , SO4 2- , COOH - , SCN - , OH - , NO2 - , CO3 2- , I - , NO3 - , HSO4 - , HS - , H - , H2O2, HCIO, homocysteine (Hcy), glutamine (Gln), glutathione (GSH), L-phenylalanine (L-Phe), histidine (His), threonine (Thr), serine (Ser), cysteine (Cys), valine (Val), isoleucine (Iso), proline (Pro), aspartic acid (ASP), lysine (Lys), glycine (Gly), L-tyrosine (L-Tyr), β-galactosidase (β-gal), lysozyme (Lyso), glucose oxidase (GOD), trypsin (Tryp), β-glucosidase (β-Glu), AChE, CEs. As shown in FIG. 1e, only CEs activated LDM-CA and induced a significant fluorescence signal, while none of the other substances caused a detectable response. It is worth noting that even AChE, which is homologous to CEs, failed to activate LDM-CA. These results indicate that LDM-CA exhibits high specificity and excellent selectivity in detecting CEs. Figure 4

[0053] Experimental Example 2: Recognition and response mechanism of the probe LDM-CA according to the present application

[0054] ​To elucidate the mechanism of CEs activating LDM-CA, we first analyzed the reaction product using HRMS. The molecular weight of LDM-CA was determined to be 828.4470 Figure 5 ). After incubation of LDM-CA with CEs, a peak at m / z 438.1709 Figure 5 a) appeared, which was consistent with the molecular weight of compound LDM. High-performance liquid chromatography (HPLC) analysis of the co-incubation mixture indicated that the main product was the fluorescent core structure LDM Figure 5 b). This result further confirmed the activation process of the probe by CEs, indicating that CEs selectively hydrolyzed the ester bond connecting the cholic acid moiety in LDM-CA. The cleavage of the cholic acid unit finally activated LDM-CA, converting it into the highly fluorescent compound LDM, which was consistent with our initial hypothesis about the mechanism of CEs-mediated activation of LDM-CA.

[0055] Subsequently, molecular docking simulation was performed to study the molecular recognition between LDM-CA and CEs (PDB ID: 1MX5). As shown in Figure 5 c, the surface model showed that the ligand was located in a cavity on the surface of the protein, and the cavity structure was complementary to the ligand, thus promoting binding. The three-dimensional model showed that LDM-CA formed five hydrogen bonds with four amino acid residues of CEs: LEU-1264, ASP-1260, TYR-1386, and LYS-1258. Notably, the distance between the carbonyl oxygen and TYR-1386 was only This was very conducive to nucleophilic attack. The binding energy of this docking calculation was -8.2 kcal / mol, indicating that there was strong binding affinity between LDM-CA and CEs.

[0056] To further study the mechanism of fluorescence change after LDM-CA was hydrolyzed by CEs, density functional theory (DFT) calculations were performed using Gaussian 16 and B3LYP / 6-31G(d,p) basis sets. As shown in Figure 5 d, the π-electron cloud of the lowest unoccupied molecular orbital (LUMO) of LDM-CA was mainly located in the cholic acid moiety, while the π-electron cloud of the highest occupied molecular orbital (HOMO) was concentrated on the fluorophore core (LDM). This distribution indicated the presence of a photo-induced electron transfer (PET) process from the electron-rich LDM core to the electron-deficient cholic acid moiety, resulting in efficient fluorescence quenching of LDM-CA. In the presence of CEs, CEs hydrolyzed the ester bond to release the fluorophore LDM. Therefore, the π-electron clouds of both LUMO and HOMO were concentrated on the LDM fluorophore, thus suppressing the PET process and restoring the fluorescence signal. These calculation results strongly supported the hydrolysis mechanism proposed above.

[0057] Experimental Example 3: Cell-level fluorescence imaging of the probe LDM-CA described in the present application

[0058] Cytotoxicity of LDM-CA. The cytotoxicity of LDM-CA was evaluated in LO2 (human normal hepatocytes) and HepG2 (human hepatocarcinoma cells) cell lines using CCK-8 method. As shown in Figure 6 a, LDM-CA exhibited good biocompatibility and low cytotoxicity in both cell types even at a concentration of 100 mM. Notably, it showed lower toxicity in normal hepatocytes (LO2 cells). These results indicated that LDM-CA has good biocompatibility, supporting its potential for subsequent bioimaging applications.

[0059] Detection of CEs at cellular level by LDM-CA. As shown in Figure 6 b and 6c, LDM-CA exhibited negligible fluorescence in LO2 cells, while strong fluorescence was observed in cells pretreated with CEs. This indicates that the probe can effectively cross the cell membrane and rapidly respond to trace intracellular CEs. When the reagent BNPP, which reduces the activity of intracellular CEs, was applied to esterase-pretreated LO2 cells, only weak fluorescence was detected. This result further confirmed that the fluorescence activation of LDM-CA is specifically triggered by intracellular CEs.

[0060] Distinguishing HCC cells. To evaluate the ability of LDM-CA to distinguish HCC cells from normal cells, a fluorescence-based assay was performed. After incubation with LDM-CA alone, normal cell lines (LO2 and BEAS-2B (human bronchial epithelial cells)) showed negligible fluorescence, indicating that LDM-CA remained in a non-fluorescent state in non-malignant cell environments. In contrast, the HCC cell line HepG2 exhibited a significant increase in fluorescence intensity. Notably, this signal was also stronger than that detected in A549 (human non-small cell lung cancer cells) Figure 6 d and 6e). In summary, these results indicate that LDM-CA can effectively distinguish HepG2 cells from normal cells (LO2, BEAS-2B) and non-HCC cancer cells (A549), highlighting its potential application in HCC diagnosis.

[0061] Mitochondrial targeting ability. To evaluate whether LDM-CA specifically localizes to mitochondria, LO2 cells were co-incubated with a commercially available mitochondrial tracer (Mito-Tracker Green) and LDM-CA. As shown in Figure 7 , the red channel shows the fluorescence signal from LDM-CA, while the green channel shows the corresponding distribution of Mito-Tracker Green. The Pearson correlation coefficient between the two channels was calculated to be 0.87, indicating that LDM-CA strongly co-localizes with mitochondria in LO2 cells, confirming its excellent mitochondrial targeting ability.

[0062] Experimental Example 4: Detection of CEs in vivo by the probe LDM-CA in zebrafish

[0063] Based on the aforementioned good experimental results, the ability of LDM-CA to detect CEs and its distribution in vivo in zebrafish were further tested to evaluate its potential for effective in vivo CEs imaging and possible application in HCC detection.

[0064] In the experimental group pretreated with exogenous CEs, a clear fluorescence signal was observed in the liver tissue of zebrafish, and the fluorescence was specifically concentrated in the liver area Figure 8 ). In contrast, almost no fluorescence signal was detected in zebrafish without CEs pretreatment. This finding indicates that LDM-CA remains in a stable "fluorescence-off state in vivo under physiological conditions without CEs activation, confirming its low background interference in living organisms. In summary, these results show that LDM-CA exhibits high sensitivity to CEs in vivo and can efficiently and specifically monitor CEs activity in living zebrafish. At the same time, the localization of fluorescence only in the liver further confirms that LDM-CA has excellent liver targeting properties, which lays a solid foundation for its subsequent application in HCC-related in vivo imaging studies.

[0065] Experimental Example 5: Fluorescence imaging of LDM-CA in mice in vivo

[0066] To systematically evaluate the performance of LDM-CA in detecting HCC in vivo and its potential to guide surgical resection, we conducted a series of experiments. As shown in Fig. Figure 9 a, after intratumoral injection of LDM-CA in HCC-bearing mice, the fluorescence signal in the tumor area was dynamically monitored for 30 minutes. The results showed that the fluorescence intensity gradually increased over time, and the entire HCC tumor could be effectively visualized within 5 minutes. Specifically, the tumor area exhibited obvious fluorescence, while the background signal in the mouse was very low, effectively reducing the interference that could affect the imaging quality. In contrast, after subcutaneous injection of LDM-CA in the corresponding anatomical site of the control mice, no significant fluorescence was observed. Moreover, by exposing the subcutaneous tumor through local dissection, it was found that the fluorescence induced by LDM-CA was uniformly distributed within the tumor, with a clear boundary from the surrounding normal tissue. No fluorescence was detected in the adjacent normal tissue Figure 9 b). These findings demonstrate that intratumoral injection of LDM-CA can achieve specific in vivo imaging of HCC and accurately distinguish the tumor margin from normal tissue, thereby providing key technical support for precise tumor delineation during surgical resection.

[0067] To further evaluate the ability of LDM-CA to reach the tumor site through blood circulation and achieve targeted imaging after intravenous administration, we conducted a tail vein injection experiment. As shown in Fig.Figure 10 a) As shown in Figure 2, in vivo imaging showed that LDM-CA accumulated specifically in the tumor area with strong fluorescence signals at 2 h post-injection. Subsequent dissection of mouse organs further confirmed that fluorescence was only concentrated in tumor tissue, and no signal was detected in major organs such as the heart, non-tumor liver, spleen, lung, or kidney Figure 10 b) Histological examination of tissue sections confirmed these observations Figure 10 c) In summary, these results indicate that LDM-CA can be accurately delivered to HCC tissue through the bloodstream after intravenous injection and activated fluorescence there, thus achieving non-invasive targeted imaging of HCC in vivo. This property indicates the potential of LDM-CA for precise HCC detection, preoperative localization, and dynamic monitoring.

[0068] In summary, we designed and synthesized a CEs-activated probe LDM-CA with dual targeting ability for liver and mitochondria for precise detection, dynamic monitoring, and surgical guidance of HCC. LDM-CA was rationally designed: cholic acid as a liver targeting moiety, a cationic group for mitochondrial localization, and an intramolecular ester bond as a specific response site. The probe showed high specificity and sensitivity for CEs, and molecular docking simulation further confirmed its strong binding affinity to the enzyme. Cell experiments demonstrated its excellent mitochondrial targeting and fluorescence-on ability in the presence of CEs in cells, thus producing a specific response in HCC cells. Zebrafish experiments showed that it had effective liver targeting in vivo, supporting its potential in HCC detection. Importantly, LDM-CA could achieve specific in vivo imaging of HCC by intratumoral or intravenous injection, accurately distinguishing tumor margins from normal tissue, thus providing key technical support for precise tumor resection. This study not only provides a powerful molecular tool for early HCC diagnosis and surgical guidance, but also provides valuable means for exploring the role of mitochondrial CEs activity in the development of HCC at the subcellular level.

Claims

1. A liver / mitochondrial dual-targeting carboxylesterase fluorescent probe, denoted as compound LDM-CA, has the following structure:

2. The method for preparing the liver / mitochondrial dual-targeting carboxylesterase fluorescent probe according to claim 1, characterized in that, Compound LDM and cholic acid were reacted in an organic solvent. The solvent was removed from the reaction mixture to obtain the crude product of the target compound.

3. The preparation method according to claim 2, characterized in that, The organic solvent is selected from one or a combination of two or more of N,N-dimethylformamide, N,N-dimethylacetamide and dimethyl sulfoxide.

4. The preparation method according to claim 2, characterized in that, The reaction temperature is room temperature, or between room temperature and the boiling point of the organic solvent.

5. The preparation method according to any one of claims 2 to 4, characterized in that, The reaction was carried out under a protected atmosphere.

6. The preparation method according to any one of claims 2 to 4, characterized in that, It also includes a step of purifying the crude target product obtained.

7. The use of the liver / mitochondrial dual-targeting carboxylesterase fluorescent probe according to claim 1 in the preparation of carboxylesterase detection reagents.

8. The use of the liver / mitochondrial dual-targeting carboxylesterase fluorescent probe according to claim 1 in the preparation of diagnostic reagents for hepatocellular carcinoma.

9. The application of the liver / mitochondrial dual-targeting carboxylesterase fluorescent probe of claim 1 in intracellular or in vivo carboxylesterase imaging.

10. The application according to claim 9, characterized in that, The liver / mitochondrial dual-targeting carboxylesterase fluorescent probe described herein can be used in one or more of the following processes: Used for observing and monitoring the dynamic changes and distribution of endogenous or exogenous carboxylesterases within cells or organisms; or Used for visualizing the dynamic changes and distribution of carboxylesterases within cells or organisms; or Specific imaging of hepatocellular carcinoma via intratumoral or intravenous injection.

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