KR102350574B1 - Fluorescent probe compound having Hepatocellular carcinoma specificity and a Hepatocellular carcinoma detection fluorescence sensor comprising the same - Google Patents

Abstract

.The present invention relates to a hepatocellular carcinoma-specific fluorescent probe compound represented by the following [Formula 1] and a fluorescent sensor for detecting hepatocellular carcinoma comprising the same:

.

Description

Hepatocellular carcinoma specific fluorescence probe compound and fluorescence sensor for detecting hepatocellular carcinoma comprising the same

The present invention relates to a hepatocellular carcinoma-specific fluorescent probe compound and a fluorescent sensor for detecting hepatocellular carcinoma comprising the same.

Hepatocellular carcinoma (HCC) is one of the most common cancers with high mortality worldwide (Non-Patent Document 1). The 5-year survival rate of HCC patients is reported to be less than 15%, and, with the exception of liver transplantation, surgical resection is considered the preferred treatment option, but often incomplete surgical resection and consequent tumor recurrence due to limited visibility of the embedded tumor. Problematic (Non-Patent Document 2)

Since the incidence and related mortality of HCC are increasing worldwide, the development of a diagnostic method for effective detection of HCC is required. Conventionally, techniques such as positron emission computed tomography (PET/CT), magnetic resonance imaging (MRI), ultrasound and radiography have been used to detect cancer. However, they have the disadvantage of relying on a contrast agent that continuously emits a signal for imaging regardless of interaction with the target.

On the other hand, a fluorescence imaging probe has emerged as an alternative and powerful tool in the field of in vivo imaging, particularly in the measurement of intracellular pH changes, due to its excellent detection sensitivity and high spatial resolution (Non-Patent Document 3). However, the imaging quality in this technique is highly dependent on the photophysical parameters of the fluorescent dyes used. Recently, fluorescent dyes emitting in the near-infrared (NIR) region (700-900 nm) have gained great interest in the field of bioimaging due to two main advantages over dyes emitting in the visible region (Non-patent literature). 4). First, the biological sample has reduced absorption of NIR, which minimizes light damage to the biological sample and deepens tissue penetration by radiation. Second, NIR imaging minimizes the interference of autofluorescence, enabling a high signal-to-noise ratio. In this method, an active NIR dye (quenched state) is conjugated to a tumor-specific target ligand to help transport the dye to the intended site of action, and then reacts to the cancer microenvironment to activate the dye (Non-Patent Document 5) . This strategy can greatly improve the sensitivity and specificity in tumor detection and development of therapeutic agents (Non-Patent Document 6).

In the case of HCC, the galactose unit has been extensively studied as an active hepatocyte-targeting ligand because it can be readily recognized by the asialoglycoprotein receptor (ASGP-R) overexpressed in hepatocytes. However, under certain pathological conditions, such as cirrhosis, the overall binding ability of ASGP-R is remarkably inhibited by the inhibitor in serum, so that its efficacy may be reduced (Non-Patent Document 7). In addition, according to Kim et al. (Non-Patent Document 8), ASGP-Rs were found to be overexpressed in other types of cancer, such as colon cancer. Therefore, the development of a more efficient targeting ligand for HCC targeting is required.

18β-glycyrrhetinic acid (GA) has been widely used in medicine for the treatment of various liver-related diseases (Non-Patent Document 9). Interestingly, Negishi et al. demonstrated the existence of abundant receptors for GA on the hepatocyte membrane (Non-Patent Document 10), and these receptors are overexpressed in liver cancer cells than in normal cells (Non-Patent Document 11). In the past few years, many studies have reported that the carrier conjugated with GA exhibits greater accumulation in hepatocytes than the unconjugated (Non-Patent Document 12). However, most of these studies using GA as an active targeting ligand have focused only on drug delivery with a carrier accompanied by a namomaterial, and there is no report on a GA-modified NIR imaging probe based on a small molecule organic compound.

A. P. Venook, C. Papandreou, J. Furuse, L. L. de Guevara, Oncologist 2010, 15, 5-13. J. D. Yang, L. R. Roberts, Nat. Rev. Gastroenterol. Hepatol. 2010, 7, 448-458. G. Chen, Q. Fu, F. Yu, R. Ren, Y. Liu, Z. Cao, G. Li, X. Zhao, L. Chen, H. Wang, J. You, Anal. Chem. 2017, 89, 8509-8516. S. Acilefu, Angew. Chem. Int. Ed. 2010, 49, 9816-9818; Angew. Chem. 2010, 122, 10010-10012. M. H. Lee, J. H. Han, P. S. Kwon, S. Bhuniya, J. Y. Kim, J. Am. Chem. Soc. 2012, 134, 1316-1322. M. H. Lee, A. Sharma, M. J. Chang, J. Lee, S. Son, J. L. Sessler, C. Kang, J. S. Kim, Chem. Soc. Rev. 2018, 47, 28-52. R. J. Stockert, A. G. Morell, Hepatology 1983, 3, 750-757. A. Sharma, E. J. Kim, H. Shi, J. Y. Lee, B. G. Chung, J. S. Kim, Biomaterials 2018, 155, 145-151. M. P. Manns, H. Wedemeyer, A. Singer, N. Khomutjanskaja, H. P. Dienes, T. Roskams, R. Goldin, U. Hehnke, H. Inoue, J. Viral Hepat. 2012, 19, 537-546. M. Negishi, A. Irie, N. Nagata, A. Ichikawa, Biochim. Biophys. Acta 1991, 1066, 77-82. Z. Y. He, X. Zheng, X. H. Wu, X. R. Song, G. He, W. F. Wu, S. Yu, S. J. Mao, Y. Q. Wei, Int. J. Pharm. 2010, 397, 147-154. C. Zhang, W. Wang, T. Liu, Y. Wu, H. Guo, P. Wang, Q. Tian, Y. Wang, Z. Yuan, Biomaterials 2012, 33, 2187-2196.

An object of the present invention is to provide a hepatocellular carcinoma-specific fluorescent probe compound having high selectivity for hepatocellular carcinoma and a fluorescent sensor for detecting hepatocellular carcinoma comprising the same.

The present invention in order to solve the above problems,

It provides a hepatocellular carcinoma-specific fluorescent probe compound represented by the following [Formula 1]:

.

.

According to the present invention, the fluorescent probe compound may be characterized in that it is captured into the hepatocellular carcinoma through the GA receptors-mediated endocytic pathway.

According to the present invention, the fluorescent probe compound may be characterized in that it is activated at an acidic pH condition of 2 to 6.

According to the present invention, the fluorescent probe compound may be characterized in that it emits fluorescence in the near-infrared region through spirolactam ring opening ^{(H +} -triggered spirolactam ring opening) in the lysosome among the cell organelles of the hepatocellular carcinoma.

According to the present invention, the hepatocellular carcinoma may be characterized in that it is HepG2 or Huh7 cells.

The present invention also provides a fluorescent sensor for detecting hepatocellular carcinoma comprising the fluorescent probe compound.

The fluorescent probe compound according to the present invention has excellent selectivity for hepatocellular carcinoma, and enables excellent detection sensitivity and high spatial resolution imaging without damaging or impacting cells, and thus can be usefully used in the field of liver cancer diagnosis. have.

1 is a UV/Vis (FIG. 1 (A)) of NIR-GA (compound represented by Formula 1) (5 μM) at various pH values in 30% EtOH in PBS (0.1 M, pH 7.4, 37 °C). , shows the fluorescence spectrum (FIG. 1(B)). The excitation and emission wavelengths were 709 nm and 733 nm, respectively. Each reading was recorded after incubation at 37 °C for 15 min.
Figure 2A) shows the fluorescence “off/on” cycle between pH 7.5 and 5.0 showing the reversibility of NIR-GA. B) shows the time-dependent fluorescence spectra of NIR-GA at pH 7.5 and 5.0. C) is a bar diagram showing the selectivity of NIR-GA for acids (eg, 1=pH 3.0, 2=Zn ^{2 +} , 3=Cu ^{2 +} , 4=Co ^{2 +} , 5=Ca ^{2 +} , 6= Fe ^{2 +} , 7=Mg ^{2 +} , 8=Ag ^{2 +} , 9=Ba ²⁺ , 10=Cd ^{2 +} , 11=Pb ^{2 +} , 12=Ni ^{2 +} , 13=Hg ^{2 +} , 14=Mn ^{2 +} , 15=Na ⁺ ). D) shows the fluorescence intensity according to irradiation time (1.5 h), showing the light stability of NIR-GA. The excitation and emission wavelengths were 709 nm and 733 nm, respectively.
Fig. 3 shows fluorescence microscopy images of normal cells (fibroblasts) and hepatocellular carcinoma cells (HepG2 and Huh7) incubated with various concentrations of NIR-GA as a result of intracellular uptake of NIR-GA. The excitation and emission wavelengths were 709 nm and 733 nm, respectively. Data are expressed as mean±SD (n=3).
4 shows the effect of free GA on the cell uptake behavior of NIR-GA. A) Fluorescence microscopy images of Huh7 cells pretreated with various concentrations of free GA [(1) to (4): 0, 5, 10 and 25 μM] before treatment with 20 μM NIR-GA. B) shows the corresponding mean fluorescence intensity of NIR-GA measured in Huh7 cells. The excitation wavelength was 639 nm and the emission was collected in the 700-1000 nm range. Images were acquired using Image J software. Data are expressed as mean±SD (n=3).
5 shows the results of MTT assay for cytotoxicity evaluation of NIR-GA against fibroblasts, Huh7 and HepG2 cell lines. Data are expressed as mean±SD (n=3).
6 shows confocal fluorescence microscopy images of Huh7 cells treated with NIR-GA, Mito-, ER-, and Lyso-Tracker green. Cells were co-labeled with 20 μM NIR-GA and 5 μM of each organelle marker. The plot in the upper right shows the intensity profile of the region of interest (indicated by the white bar) in the merged image. Images were acquired at 60 magnification with a confocal fluorescence microscope. Data are expressed as mean±SD (n=3).
7 shows absorbance plots of 5 μM NIR-GA for various pH values. Spectra were recorded in 30% EtOH in PBS (pH 7.4, 37 °C).
8 shows a plot of fluorescence intensity of 5 μM NIR-GA as a function of pH value. Spectra were recorded in 30% EtOH in PBS, pH 7.4, 37° C. Excitation and emission wavelengths were 709 nm and 733 nm, respectively.
9 is a bar graph showing the selective response of NIR-GA to acidic pH in the presence of various analytes: (1) pH = 3.0, (2) L-ascorbic acid, (3) L-Glutamic acid, ( 4) NADH, (6) cysteine (7) Homo-cysteine (8) L-Glutathione, (9) H ₂ O ₂ , (10) hypochlorite ion (OCl ^- ), (11) hydroxyl ions (OH ^- ). All spectra were recorded in 30% EtOH in PBS, pH 7.4, 37° C. Excitation and emission wavelengths were 709 nm and 733 nm, respectively.
10 shows the fluorescence on-off switching mechanism of dyes between acidic and basic pH.
11 shows the chemical structure of the probe NIR-GA at different acidic pH values.
12 shows 3D reconstructed confocal microscopy images of fibroblasts and Hih7 cells co-labeled with 20 μM NIR and DPAI (nuclear staining). Images were acquired using a confocal fluorescence microscope at 60X magnification.
13 is a magnified confocal microscopy image of Huh7 cells co-labeled with 20 μM NIR-GA and 5 μM of each organelle marker, showing co-localization of probe NIR-GA in lysosomes. Images were acquired using a confocal fluorescence microscope at 60X magnification. Data are expressed as mean±SD (n=3).
Fig. 14 shows fluorescence microscopy images of normal cells (fibroblasts) and liver cancer cells (HepG2 and Huh7) incubated with 20 μM Changsha dye as a result of intracellular uptake of Changsha dye. The excitation and emission wavelengths were 709 nm and 733 nm, respectively. Data are expressed as mean±SD (n=3).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

experimental method

Materials, methods and apparatus

All reagents and solvents were purchased from Sigma Aldrich, Alfa Aesar, TCI Korea and Merck and used without further purification. Column chromatography purification was performed using silica gel 60 (70-230 mesh) as a stationary phase. Analytical thin-layer chromatography (TLC) was performed using 60 silica gel. ¹ H and ¹³ C NMR spectra were recorded on a Bruker 500 NMR instrument. Chemical shift (δ is reported in ppm and coupling constants in Hz. Mass spectra were recorded with an IonSpecHiRes ESI Shimadzu LC/MS-2020 mass spectrometer. All fluorescence and UV-Vis spectra were obtained from RF-5301PC and S-3100. Each record was recorded in the spectrophotometer.Prepared stock solution of NIR-GA (5mM) in pure DMSO.For solution study, dilute the stock solution with PBS (pH 7.4, 37°C) containing EtOH (30%). to make the final working solution concentration of 5 μM.The total volume of the solution was fixed at 3.0 mL.In the experiment, the excitation wavelength was 709 nm, all excitation and emission slit widths were 5 nm, and the emission was collected at 733 nm. To monitor the fluorescence change, first, a working solution of NIR-GA (5 μM) was incubated at 37 °C for 5 min under different pH values, and the corresponding fluorescence spectra were recorded.

Cell Culture and Confocal Microscopy Imaging

Two different hepatocyte cell lines (HepG2 and Huh7, Korea Cell Line Bank) and human dermal fibroblast cell lines (HDFn, Invitrogen) were used in the experiments. HepG2, Huh7 and fibroblast cell lines (each cell number is 1×10 ⁵ cells) were seeded in 35 mm confocal dishes (glass bottom dish, SPL Life Sciences, Korea) and fetal bovine serum (FBS, Cell Signaling Technology, Danvers, Massachusetts, USA, 10%) and penicillin/streptomycin (P/S, Hyclone, 1%) supplemented with high-glucose Dulbecco's modified Eagle's medium (DMEM, GE Hyclone, Logan, UT, USA) at 37 °C, 5% CO Incubated in _{2 atmospheres.} When cells reached 75-80% confluence, they were treated with NIR-GA at various concentrations (0, 5, 20, 40 μM). After incubation for 15 h, cells were washed with PBS, fixed with paraformaldehyde (PFA, 4%), and stored at 48 °C until further analysis. Confocal fluorescence images were obtained using a confocal laser scanning microscope (Carl Zeiss LSM 700 Exciter, Germany). The excitation wavelength was 639 nm at 5 mW and the emission was collected using a long-pass filter of 700-1000 nm. Images were analyzed using Zen imaging software (ZEISS, Oberko, Germany).

Competitive inhibition assay using Free GA

Huh7 and fibroblasts were seeded in 35 mm glass bottom confocal dishes at a cell density ^{of 1×10 5 cells.} After incubation for 24 hours, cells were incubated with free GA (a pharmacological inhibitor) at various concentrations (0, 5, 10 and 25 μM) for 1 hour before treatment with NIR-GA (20 μM). After an additional 24 h of incubation, cells were washed 3 times with PBS and fixed with PFA (4%), and the corresponding confocal fluorescence images were recorded.

Intracellular localization of NIR-GA (co-localization study)

To confirm the subcellular localization of NIR-GA, media containing organelle trackers (Invitrogen) Mito-Track Green, Lyso-Tracker Green and ER-Tracker Green prior to incubation of cells with NIR-GA (20 μM). was pre-treated with Huh7 cells were seeded in 35 mm confocal dishes (glass bottom dishes, SPL Life Sciences). When the cell density reached about 80% confluence, the cells were washed twice with PBS and incubated with NIR-GA (20 μM) for 15 h. Cells were again washed twice with PBS and incubated in FBS medium (2.5%) containing organelle trackers (Mito-, ER- and Lyso-tracker, 100 nM) for 60 minutes. Finally, the cells were washed twice with PBS. For NIR-GA, the excitation wavelength was 639 nm at 5 mW and the emission was collected using a long-pass filter of 700-1000 nm. For the organelle tracker, the excitation wavelength was 488 nm at 5 mW, and the emission was collected using a 500-600 nm bandpass filter.

Dose-dependent intracellular uptake of NIR-GA

HepG2, Huh7 and fibroblast cell lines (each cell number is 1×10 ⁵ cells) were seeded in a 35 mm glass bottom confocal dish (SPL Life Science, Kyunghee, Korea) and cultured for 24 hours. Cells were then treated with NIR-GA at various concentrations (0, 5 and 20 μM) for 2 h at 37° C., _{under CO 2} (5%). Fluorescence images were visualized with a confocal laser scanning microscope.

Cell viability assay (MTT)

To determine the effect of NIR-GA on cell viability, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Life Technologies, Carlsbad, CA, USA) cytotoxicity assay was performed. Cell viability was assessed using the CytoTox96 non-radioactive cytotoxicity assay kit (Promega, USA). HepG2, Huh7 and fibroblast cell lines (each number of ^{cells 1×10 5} cells) were aliquoted in a 96-well plate, and treated with various concentrations of NIR-GA for 24 hours. Then, MTT reagent (5 mgmL ^{- 1} ) was added to each well, and incubated at 37 °C for 2 hours. Absorbance at 570 nm was measured with a microplate reader (Sense, Hidex, Finland). All experiments were performed in triplicate.

compound synthesis

According to the following synthetic route, a compound of [Formula 1] (expressed as NIR-GA) according to the present invention was synthesized.

[synthetic route]

Synthesis of compound 3

Compound 3 (Changsha dye) was synthesized according to the procedure reported in the conventional literature (Anal. Chem. 2016, 88, 9746-9752).

Synthesis of compound 4

Glycyrrhetic acid (GA, 0.2 g, 0.43 mmol), EDC (0.1 g, 0.52 mmol), 1-hydroxy-1H-benzotriazole (HOBt; 0.07 g, 0.52 mmol) and 4 in DMF (10 mL) A mixture of -dimethylaminopyridine (DMAP, 0.06 g, 0.52 mmol) was stirred at room temperature under nitrogen for 1 hour. Then, 17-Azido-3,6,9,12,15-pentaoxaheptadecan-1-amine (0.16 g, 0.52 mmol) was added and stirred overnight. After completion of the reaction, DMF was evaporated to obtain a crude product, which was purified by column chromatography using MeOH (5%) in _{CH 2} Cl _{2 to obtain an azide derivative in 72% yield.}

¹ H NMR (CDCl ₃ , 400 MHz): δ=6.28 (s, 1H), 5.68 (s, 1H), 3.66-3.55 (m, 18H), 3.39 (t, J=4.6 Hz, 2H), 3.24- 3.20 (m, 1H), 2.78 (d, J=12.0 Hz, 1H), 2.33 (s, 1H), 2.17 (d, J=12.0 Hz, 2H), 2.04-1.58 (m, 13H), 1.37 (brs) , 8H), 1.13 (s, 9H), 1.00 (s, 4H), 0.81 (s, 6H), 0.70 ppm (d, J=12.0 Hz, 1H); ¹³ C NMR (CDCl ₃ , 125 MHz): δ=200.19, 176.02, 169.44, 128.60, 78.87, 70.85, 70.82, 70.77, 70.71, 70.63, 70.36, 70.29, 70.22, 62.00, 55.14, 50.84, 48.23, 45.54, 43.75 , 43.36, 41.96, 39.33, 39.31, 37.67, 37.23, 32.94, 32.05, 32.04, 31.63, 29.63, 28.73, 28.32, 27.43, 26.67, 26.60, 23.57, 18.84, 17.66, 16.56, 15.82 ppm; MS (ESI): m/z calcd for C ₄₂ H ₇₀ N ₄ O ₈ : 758 [M+Na] ⁺ ; found: 781.

The azide derivative (0.5 g, 0.66 mmol) was then dissolved in a toluene/H ₂ O mixture (1:1, 20 mL), followed by addition of HCl (5 %, 10 mL). _{Triphenylphosphine (PPh 3} , 0.21 g, 0.79 mmol) dissolved in THF (5 mL) was slowly added dropwise to the solution. The milky suspension was stirred at room temperature overnight. After completion of the reaction, the aqueous layer was separated and washed 3-4 times with toluene. The aqueous layer was then treated with NaOH (2N) until the pH reached 10. The product was extracted with ethyl acetate, the organic layer was removed, _{dried over anhydrous NaSO 4} and concentrated under vacuum to give compound 4 in 63% yield.

NIR Synthesis of -GA

A mixture of Changsha dye (compound 3; 0.1 g, 0.18 mmol), EDC (0.04 g, 0.23 mmol), HOBt (0.03 g, 0.23 mmol) and DMAP (0.03 g, 0.23 mmol) in DMF (5 mL) under nitrogen Stirred at room temperature for 1 hour. Then amine derivative compound 4 (0.16 g, 0.22 mmol) was added and stirred overnight. After completion of the reaction, DMF was evaporated to obtain a crude product, which _{was purified by column chromatography using MeOH in CH 2} Cl ₂ (5%) to obtain NIR-GA in 68% yield.

¹ H NMR (CDCl ₃ , 400 MHz): δ=7.85-7.83 (m, 1H), 7.48-7.40 (m, 3H), 7.19-7.15 (m, 3H), 6.84 (t, J=7.6 Hz, 1H) ), 6.62 (d, J=8.0 Hz, 1H), 6.51 (brs, 1H), 6.32-6.24 (m, 3H), 5.68 (s, 1H), 5.37 (d, J=12.4 Hz, 1H), 3.62 -3.60 (m, 12H), 3.55-3.52 (m, 7H), 3.37-3.42 (m, 6H), 3.24-3.20 (m, 2H), 3.15 (s, 2H), 2.78-2.74 (m, 1H) , 2.58 (brs, 1H), 2.43 (brs, 2H), 2.32 (brs, 1H), 2.19-2.15 (m, 2H), 2.04-2.00 (m, 2H), 1.94 (d, J=10.4 Hz, 2H ), 1.85-1.81 (m, 3H), 1.74-1.70 (m, 6H) 1.66-1.58 (m, 7H), 1.36 (s, 9H), 1.25 (s, 3H), 1.17 (t, J=6.8 Hz) , 7H), 1.12-1.11 (m, 8H), 1.00 (s, 4H), 0.80-0.79 (m, 6H), 0.69 ppm (d, J=11.2 Hz, 2H); ¹³ C NMR (CDCl ₃ , 125 MHz): δ = 200.30, 200.28, 176.18, 169.59, 169.57, 168.80, 168.79, 157.90, 152.88, 157.89, 148.81, 148.07, 145.53, 139.03, 128.29.42, 131.59, 128.29, 131.90 , 127.90, 123.61, 122.98, 121.74, 120.35, 119.75, 119.46, 108.51, 105.87, 105.17, 103.60, 97.93, 92.17, 78.87, 77.62, 77.30, 76.98, 70.68, 70.33, 70.57, 70.54.23, 70.57, 70.60. , 67.03, 62.05, 55.13, 48.21, 45.63, 45.55, 44.55, 43.76, 43.37, 41.95, 39.39, 39.33, 39.30, 39.16, 37.69, 37.23, 32.94, 32.04, 31.60, 29.90, 29.63, 29.33, 28.76, 28.68, 28.47, 28. , 28.32, 27.44, 26.67, 26.61, 25.51, 23.59, 23.10, 22.39, 18.52, 17.67, 16.58, 15.84, 12.76 ppm; MS (ESI): m/z calcd for C ₇₉ H ₁₀₈ N ₄ O ₁₀ : 1272 [M+Na] ⁺ ; found: 1295.

Results and Discussion

In the present invention, a pH-responsive small molecule NIR imaging probe compound NIR-GA was synthesized in which GA was conjugated to a Changsha dye (pH-sensitive NIR fluorophore) through a polyethylene glycol linker, and NIR-GA showed that the cell viability (low demonstrated that it can be readily taken up by hepatocellular carcinoma cells without impairing cytotoxicity). In addition, in an acidic medium, NIR-GA ^{exhibits strong emission in the NIR region through H +} -triggered spirolactam ring opening, high pH selectivity and photostability, and is highly active in lysosomes among organelles. It was confirmed that it was mainly located to enable non-invasive monitoring of lysosomal pH.

NIR-GA, a compound according to the present invention, was synthesized according to the [Synthetic route] above. Briefly, NIR-GA was synthesized by coupling Changsha dye with GA-conjugated amine derivative compound 4. The presence of the synthesized compound was confirmed by mass spectrometry, ¹ H NMR/ ¹³ C NMR.

Next, the optical behavior of NIR-GA at various pH values was investigated through UV/Vis and fluorescence spectroscopy. At basic and physiological pH values (9 to 7.4), the NIR-GA probe shows no absorption band in 30% EtOH in PBS (0.1 M, pH 7.4, 37 °C). However, when the pH was gradually decreased from 7.4 to 3.0, a shoulder band was formed at 650 nm, showing a significant absorbance improvement at 709 nm (Fig. 1A). Moreover, this change was accompanied by a sharp color change from yellow to deep purple of the solution (Fig. 1A, insert). This result is due to the opening of the spirolactam ring of the Changsha dye under acidic conditions and extension of the conjugation of the p- electron π-electron cloud (see Scheme 1). Interestingly, a further decrease in the pH value below 3.0 caused a decrease in absorption, and a possible explanation for this anomalous behavior is that at very low pH the tertiary amine group of the dye may be protonated, resulting in a greater charge imbalance of the dye. Fig. 11).

[Scheme 1] Mechanism of Dye Activation in Acidic Environment

Similarly, the fluorescence behavior of NIR-GA was evaluated at various pH values. As shown in Figure 1B, the probe was non-fluorescent at basic pH, whereas a gradual enhancement of fluorescence intensity was observed as the pH value decreased from 7.5 to 3.0, and when the pH was further decreased from 3.0 to 2.0, the intensity was was kept constant. In addition, it was confirmed that the change in fluorescence intensity between pH 3.0 and pH 7.5 was reversible (Fig. 2A), suggesting the reversible sensing ability of NIR-GA. Through the time-dependent fluorescence spectrum shown in Fig. 2B, it was confirmed that the fluorescence at acidic pH increased linearly with the incubation time until it reached a maximum value after 8 min and then remained constant. In addition, NIR-GA showed high sensitivity to changes in pH, and the fluorescence spectrum was not perturbed in the presence of biologically relevant metal ions (Fig. 2C) or other analytes (Fig. 9). Next, the light stability of NIR-GA was measured, and as shown in FIG. 2D , the intensity of NIR-GA was decreased by only 5.6%, confirming that NIR-GA had excellent photostability.

Next, GA-mediated cellular uptake of NIR-GA by liver cancer cell lines (HepG2 and Huh7) was evaluated using confocal fluorescence microscopy and compared with normal cell lines (fibroblasts). All cells were incubated with various concentrations of NIR-GA at 37 °C for 24 h, and the corresponding confocal microscopy images were collected. As shown in FIG. 3 , both of the liver cancer cell lines Huh7 and HepG2 cells exhibited a red fluorescence signal in which the maximum intensity was observed. In addition, as the concentration of NIR-GA increased from 5 to 20 μM, the fluorescence intensity of the corresponding cells also increased.

However, fibroblasts (normal cell line) showed only a very weak fluorescence signal at the same concentration. These results may be attributed to the activation of Changsha dye by the acidic microenvironment of cancer cells (Huh7 and HepG2). Since the fibroblast microenvironment is neutral, no activation of Changsha or a very weak fluorescence signal was observed.

A control experiment was also performed in which cells were incubated with Changsha dye without GA conjugation. As a result, none of the cell lines showed significant fluorescence intensity even at a dye concentration of 20 μM ( FIG. 14 ), suggesting the active targeting ability of NIR-GA to liver cancer cells.

In addition, inhibition assay was performed to confirm the putative GA-mediated cellular uptake (FIG. 4). Huh7 cells were pretreated with GA at various concentrations (0, 5, 10 and 25 μM) for 24 hours, and then incubated with 20 μM of NIR-GA. Corresponding confocal images showed that as the concentration of GA increased, there was a significant inhibitory effect on fluorescence intensity by competitive inhibition of the GA binding site (Figs. 4A and B). Free GA saturates GA receptors on the surface of Huh7 cells, making the binding site no longer available for NIR-GA. These results indicate that the enhanced cellular uptake of NIR-GA in liver cancer cells is due to the GA-mediated intracellular pathway.

Next, the cytotoxicity of NIR-GA to fibroblasts, Huh7 and HepG2 cells was confirmed by MTT assay. Huh7 cells were treated with NIR-GA at various concentrations (0, 10, 20, 40, 80, 160 μM). As shown in FIG. 5 , NIR-GA did not show significant cytotoxicity up to a concentration of 40 μM. However, cytotoxicity was observed at higher concentrations, ie, 80 and 160 μM, where the viability of the cells was reduced to 60% and 33%, respectively. Through these results, it was confirmed that NIR-GA exhibits excellent biocompatibility at a concentration of less than 40 μM.

Finally, a fluorescence co-localization experiment was performed to evaluate the organelle-specific localization of NIR-GA. Mito-, ER-, and Lyso-Tracker green were used as organelle markers for mitochondria, endoplasmic reticulum and lysosomes, respectively. As shown in FIG. 6 , the images of Huh7 cells incubated with 20 μM NIR-GA overlapped well with Lyto-Tracker compared to Mito and ER-Tracker, and a prominent yellow area appeared. Through these results, it was confirmed that the probe NIR-GA is mostly localized in the lysosome.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (6)

It is represented by the following [Formula 1],

It is captured inside the hepatocellular carcinoma through the GA receptors-mediated endocytic pathway,
Hepatocellular carcinoma, characterized in that it is activated at an acidic pH condition of 2 to 6 and emits fluorescence in the near-infrared region through spirolactam ring opening ^{(H + -triggered spirolactam ring opening) in the lysosome among the cell organelles of the hepatocellular carcinoma.} Specific fluorescent probe compounds. delete delete delete According to claim 1,
The hepatocellular carcinoma is HepG2 or Huh7 cells, characterized in that the hepatocellular carcinoma-specific fluorescent probe compound. A fluorescent sensor for detecting hepatocellular carcinoma comprising the fluorescent probe compound according to claim 1 .

Images