A method for detecting tumor cells using a fluorescent tracer.

Abstract

The present invention discloses the use of a near-infrared fluorescent molecule of pemetrexed targeting the α-folate receptor as a fluorescent tracer, the structural formula of which is as follows: [C9] TIFF2025534729000010.tif71170Such actively targeted near-infrared fluorescent molecules have high affinity for alpha-type folate receptors and low affinity for beta-type folate receptors, effectively distinguishing tumors from inflammation. Furthermore, they have the advantages of high water solubility and high fluorescence quantum yield, and have great potential for development in the fields of tumor surgery navigation imaging and medical cell labeling using near-infrared fluorescence imaging.

Description

Technical Field

[0001] The present invention relates to the use of a novel pemetrexed near-infrared fluorescence tracer targeting the α-type folate receptor to distinguish between non-small cell lung cancer with high expression of the α-type folate receptor and inflammation, and relates to fields such as near-infrared surgical navigation fluorescence molecules and cell labeling imaging.

Background Art

[0002] In recent years, near-infrared (NIR) imaging has been widely applied in clinical tumor imaging. Indocyanine green (ICG) is currently the only fluorescent agent for human medical imaging and clinical diagnosis clinically approved by the China National Food and Drug Administration (CFDA) and the US Food and Drug Administration (FDA). ICG is a hydrophilic tricarbocyanine dye with a molecular weight of 776. The maximum absorption peak in an aqueous solution is located at 785 nm, and the maximum emission peak is located at 810 - 820 nm. According to reports, ICG can provide minimally invasive imaging of tumors and metastatic lymph nodes including real-time imaging during surgery. Generally, ICG can bind to albumin in plasma to form nanoparticles with a size of 4 - 6 nm and is considered to be concentrated in tumors by the enhanced permeability and retention (EPR) effect. However, since ICG is not a receptor-specific tracer, it does not preferentially accumulate in tumor tissues. It has been confirmed that ICG accumulates not only in tumors but also in other highly permeable tissues such as inflammatory tissues. According to research, ICG also shows fluorescence in the inflammatory tissues around tumors, which is the main limitation of the application of ICG. To improve this situation, it is necessary to design a near-infrared fluorescence tracer with active targeting properties.

[0003] The targeted delivery of fluorescent probes to tumor cancer cells by utilizing the specific binding properties of folic acid and folate receptors has attracted widespread attention from researchers both domestically and internationally. However, folate receptors have four subtypes: FRα, FRβ, FRγ, and FRδ. In cancer tissue, mainly two subtypes, FRα and FRβ, are expressed. FRα is overexpressed mainly in malignant tumors of epithelial tissue such as ovarian cancer, lung cancer, and uterine cancer, while FRβ is overexpressed in the placenta, monocytes, and macrophages. Furthermore, these two subtypes can be expressed simultaneously in the same organism. Patients with malignant tumors may also have inflammatory diseases, and activated macrophages accumulate at the site of inflammation, leading to high expression of FRβ. Since folic acid and folate complexes exhibit similar affinity to these two subtypes of folate receptors, they cannot selectively identify tumor cells or inflammatory sites. Therefore, improving the specificity of tumor targeting by denaturing folic acid and folate complexes has significant practical applications.

[0004] Studies have revealed that pemetrexed exhibits significant differences in affinity for FRα and FRβ. By utilizing this property, a targeted labeling system that efficiently mediates FRα can be obtained by selecting a pemetrexed derivative as a target group for tumor cancer cells that highly express the folate receptor α subtype. Compared to conventional targeted labeling systems, this system allows for more efficient delivery to FRα-expressing tumor cancer cells while simultaneously reducing interference with the targeting of the labeling system by FRβ-subtype-expressing inflammatory cells. This is of significant importance for the rapid and highly efficient detection of tumor cells. [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to provide a pemetrexed near-infrared fluorescent molecule that targets the α-folate receptor as a fluorescent tracer, thereby enabling rapid and efficient detection of tumor cells. [Means for solving the problem]

[0006] To achieve the above objective, the present invention employs the following technical means.

[0007] The application is as a fluorescent tracer for a pemetrexed near-infrared fluorescent molecule that targets the α-type folate receptor, and the structural formula of the pemetrexed near-infrared fluorescent molecule is as follows. [ka]

[0008] The aforementioned fluorescent tracer effectively distinguishes tumors from inflammatory tissues by utilizing differences in specificity for different folate receptor subtypes.

[0009] The aforementioned fluorescent tracer can distinguish between non-small cell lung cancer with high α-folate receptor expression and inflammation.

[0010] The aforementioned fluorescent tracer maintains high affinity for α-type folate receptors and exhibits low affinity for β-type folate receptors. [Effects of the Invention]

[0011] This invention involves preparing actively targeted near-infrared fluorescent small molecules using pemetrexed disodium and its derivatives as active target groups by a total organic synthesis method, and innovatively applying them to the imaging differentiation of tumors and inflamed tissues. Such actively targeted near-infrared fluorescent molecules maintain high affinity for α-folate receptors and low affinity for β-folate receptors, effectively differentiating tumors from inflammation. Furthermore, they possess advantages such as high water solubility and high fluorescence quantum yield, and have great potential for development in the fields of tumor surgical navigation imaging and medical cell labeling using near-infrared fluorescence imaging. [Brief explanation of the drawing]

[0012] [Figure 1] This is a synthesis flowchart for the pemetrexed near-infrared fluorescent molecule. [Figure 2]The pemetrexed near-infrared fluorescent molecule targets cells that highly express α-folate receptors. [Figure 3] The pemetrexed near-infrared fluorescent molecule targets cells that highly express β-folate receptors. [Figure 4] The pemetrexed near-infrared fluorescent molecule is a bioimaging image that distinguishes between tumors with high and low expression of α-folate receptors. [Figure 5] The pemetrexed near-infrared fluorescent molecule is a fluorescence pattern that distinguishes inflammation from non-small cell lung cancer with high α-folate receptor expression. [Figure 6] These are HE, FRα, and FRβ immunohistochemistry and fluorescence images of tumor sections. [Figure 7] These are HE, FRα, and FRβ immunohistochemistry and fluorescence images of inflammatory tissue sections. [Modes for carrying out the invention]

[0013] The present invention will be further described below with reference to the drawings.

[0014] As shown in Figure 1, first, the pemetrexed near-infrared fluorescent molecule is prepared. The preparation includes the following steps 1 to 4.

[0015] 1. Synthesis of chloro-ICG derivative matrix: (1) 4-Hydrazinobenzenesulfonic acid (1.6 g, 31.9 mmol), 3-methyl-2-butane (2.10 ml, 90 mmol), and glacial acetic acid (50 ml) were mixed and heated at 120°C for 18 hours under a nitrogen atmosphere. After precipitation in ethyl acetate, the crude product was collected by filtration as a pink solid, and the obtained product (6.5 g, 25.4 mmol) was dissolved in methanol (50 mL). Under mild conditions, this solution was added dropwise to, for example, a solution of potassium hydroxide (1.7 g, 30 mmol) and isopropanol (20 ml), and the crude mixture was filtered and washed to obtain a brown solid. yield 97%) [ka]

[0016] (2) Compound 2 (2.3 g, 8.3 mmol) and 1,4-butanethiol were added to a toluene solution under a nitrogen atmosphere and heated at 110 °C for 48 hours. The mixture was cooled to room temperature and the solvent was precipitated. Methanol (10 ml) was added to the crude mixture, stirred for 30 minutes, the crude mixture was filtered and collected, and dissolved in a 2:1 (v / v) water mixture of water (100 ml) and methanol (50 ml). The mixed solution was gradually added to acetonitrile (160 ml) from a dropping funnel. The precipitate was filtered and collected as a pink solid. ( yield 40%)

Chemical formula

[0017] (3) Compound 3 (1.5 g, 2.79 mmol), Vilsmeier-Haack reagent (0.5 g, 1.39 mmol), and sodium acetate anhydrous (0.342 g, 4.17 mmol) were added to 20 mL of anhydrous ethanol, and heated under reflux for 6 hours under a nitrogen atmosphere. After the reaction mixture was cooled to room temperature, it was filtered, washed with ethanol and methanol, and collected as a green-brown solid. ( yield 90%)

Chemical formula

[0018] 2. Synthesis of pemetrexed target drug: Pemetrexed hydrolyzed acid (1.05 g, 3.52 mmol) was dissolved in DMF, stirred until completely dissolved, HATU (2.007 g, 5.28 mmol), O-tert-butyl-L-tyrosine tert-butyl ester hydrochloride (1.161 g, 3.52 mmol), and DIEA (1.364 g, 10.56 mmol) were added to the flask in sequence, stirred until completely dissolved, reacted at room temperature for 30 min under a nitrogen atmosphere, the solution after the reaction was dropped into 0.1 N aq. HCl (1.0 L, 0.14 M), a pale yellow precipitate was formed, suction filtered, and vacuum dried to obtain 2.04 g of solid compound 5, and the yield was 95%. [ka]

[0019] Compound 5 (2.04 g, 3.34 mmol) was added to a round-bottom flask, TFA:H2O (95:510 mL) was added, and the mixture was stirred for 2 hours. The mixture was then added to methyl tert-butyl ether, the precipitate was filtered, and the mixture was vacuum-dried. 1.507 g was obtained, and the yield was 98%. [ka]

[0020] 3. Synthesis of pemetrexed near-infrared fluorescent molecule 7: At 23°C, a solution of S0456 (2.909 g, 3.276 mmol) in water (18 mL) was added dropwise to a pH 11 Pemetrexed-Tyr (1.507 g, 3.276 mmol) trianion solution. The temperature of the reaction mixture was raised to 90°C and stirred at 90°C for 45 minutes, and the formation of compound 7 was monitored by TLC. After the formation of the product was complete, the reaction mixture was cooled to room temperature and transferred to acetone (0.5 L) under steady flow via a cannula to obtain a green precipitate. The precipitate was filtered through a sintered funnel under vacuum using an air pump and washed with acetone (3 × 500 mL). The green powdery solid was dried under high vacuum for 12 hours to quantitatively obtain compound 7 (4.34 g). [ka]

[0021] 4. In order to verify the effects of the present invention, the following verification experiments were conducted.

[0022] (1) Pemetrexed near-infrared fluorescent molecule tumor Applications to distinguishing between sores and inflammation: The pemetrexed near-infrared fluorescent molecule can specifically recognize cell lines that overexpress α-folate receptors. We selected α-folate receptor overexpression cell line H1299, low-expression cell line A549, and β-folate receptor overexpression cell line RAW264.7. Cells were cultured at 37°C in humid air with 5% CO2 and 95% air in RPMI medium containing 10% FBS and 1% diabody. A 100 nM pemetrexed near-infrared fluorescent tracer was incubated in a confocal Petri dish for 1 hour, washed with PBS, stained with DAPI, fixed with paraformaldehyde, and imaged.

[0023] The pemetrexed near-infrared fluorescent tracer was observed throughout the cytoplasm of H1299 cells but not in α-folate receptor-negative A549 cells. This confirms that the pemetrexed near-infrared fluorescent tracer is internalized by α-folate receptor-overexpressing cells and supports the idea that the pemetrexed near-infrared fluorescent tracer specifically targets α-folate receptor-overexpressing tumor cells. The absence of fluorescence detection in RAW264.7 cells indicates that the pemetrexed near-infrared fluorescent tracer does not target β-folate receptors.

[0024] (2) The pemetrexed near-infrared fluorescent molecule is used to specifically recognize non-small cell lung cancer tissue that overexpresses α-folate receptors. A 10 nmol dose of pemetrexed near-infrared fluorescent tracer was injected via tail vein into H1299 and A549 tumor-bearing mice. Two hours later, imaging effects were observed using a small animal imaging device, and the biological distribution in the tissues was analyzed. The pemetrexed near-infrared fluorescent molecule was able to specifically recognize H1299 tumor-bearing mice with high α-folate receptor expression, and a good tumor background signal ratio was observed. However, no target effect was detected in A549 tumor-bearing mice with low α-folate receptor expression.

[0025] (3) The pemetrexed near-infrared fluorescent molecule is used to differentiate inflammation from non-small cell lung cancer tissue with high α-folate receptor expression. A H1299 tumor-bearing mouse model was established. Peritonitis was induced daily by injecting persulfate into the triangular region of the peritoneum. As shown in Figure 5, the triangular region where inflammation occurred was located near the tumor tissue. The establishment of the inflammation model was verified by immunohistochemical sections. As can be seen from the immunohistochemical sections of tumor and inflammatory tissue, the brown areas indicate positive receptor expression. Alpha-folate receptors were highly expressed in the tumor, and beta-folate receptors were highly expressed in the inflammatory tissue. As shown in Figure 6, the fluorescence intensity of the non-small cell lung cancer section with high alpha-folate receptor expression was high, while as shown in Figure 7, the inflammatory tissue section with high beta-folate receptor expression showed almost no fluorescence. By utilizing the difference in specificity for different folate receptor subtypes, tumors and inflammatory tissues can be effectively distinguished.

[0026] The above description represents only preferred embodiments of the present invention, and those skilled in the art can make various improvements and modifications without departing from the principles described in the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting tumor cells using a fluorescent tracer of a pemetrexed near-infrared fluorescent molecule that targets the α-folate receptor, wherein the structural formula of the pemetrexed near-infrared fluorescent molecule is as follows: The aforementioned fluorescent tracer is a method for effectively differentiating tumors from inflammatory tissues by utilizing differences in specificity for different folate receptor subtypes. 【Transformation 8】

2. The method according to claim 1, characterized in that the fluorescent tracer can distinguish between non-small cell lung cancer with high expression of α-type folate receptor and inflammation.

3. The method according to claim 2, characterized in that the fluorescent tracer maintains high affinity for α-type folate receptors and exhibits low affinity for β-type folate receptors.

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