Nano fluorescent probe for detecting activity of FAPalpha and preparation method of nano fluorescent probe

Abstract

The invention discloses a nano fluorescent probe for detecting FAPalpha activity and a preparation method of the nano fluorescent probe. The preparation method comprises the following steps: triggering a CBT-Cys click reaction by using a prepared probe precursor Cys (StBu)-Gly-Pro-Ala-His-Lys (IR780)-CBT through a reducing agent to generate a cyclic dimer NIR-CBT-Dimer, and further carrying out self-assembly to form a nano fluorescent probe NIR-CBT-NPs; according to the prepared nano fluorescent probe, the imaging time for detecting the activity of the FAPalpha is prolonged, and the detection time is long; compared with the prior art, the method has the advantages that the penetration depth and the signal-to-noise ratio of fluorescence imaging are enhanced, in addition, the half-life period for monitoring the activity of the FAPalpha is prolonged, and the degradation and metabolism time of the FAPalpha in vivo is slowed down.

Description

Technical Field

[0001] The present invention relates to a nano-fluorescent probe and a preparation method thereof; in particular, it relates to a nano-fluorescent probe for detecting fibroblast activation protein α (FAPα) and a preparation method thereof. Background Art

[0002] Fibroblast activation protein α, namely FAPα, is up-regulated in a variety of human malignant tumors (especially pancreatic cancer and breast cancer), and can affect tumor growth through multiple mechanisms, including promoting proliferation, invasion, angiogenesis, immunosuppression, and drug resistance; therefore, it is of great significance to visualize the activity of FAPα in vivo, which helps to reveal the mechanism of tumorigenesis and development and achieve early diagnosis of tumors.

[0003] Existing various imaging techniques are used to monitor the activity of FAPα in physiological processes, including radiological imaging (such as positron emission tomography, magnetic resonance imaging) and optical imaging (such as fluorescence, chemiluminescence, and bioluminescence imaging), etc.; however, radiological imaging usually involves ionizing radiation, often accompanied by high costs and complex operation requirements.

[0004] Generally, near-infrared fluorescent probes can be mainly divided into two categories according to their signal modes: "always-on" probes and "turn-on" probes; traditional "always-on" probes amplify signals through passive or active targeting, but often result in a low signal-to-noise ratio; in contrast, "turn-on" probes maintain the fluorescence "off" state when not activated by the target molecule, and once activated, they can provide a higher signal-to-noise ratio for biological imaging; however, currently, almost all "turn-on" fluorescent probes for FAPα detection are small molecules, which have a short half-life, are easily degraded and metabolized rapidly in vivo, and are difficult to achieve continuous monitoring of the activity of FAPα. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide a nano-fluorescent probe that can persistently monitor FAPα, and another object of the present invention is to provide a preparation method of a nano-fluorescent probe that can persistently monitor FAPα.

[0006] Technical Solution: A preparation method of a nano-fluorescent probe for detecting the activity of FAPα according to the present invention, the prepared probe precursor Cys(StBu)-Gly-Pro-Ala-His-Lys(IR780)-CBT (NIR-CBT) triggers a CBT-Cys click reaction through a reducing agent to generate a cyclic dimer NIR-CBT-Dimer, and further self-assembles to form a fluorescence-quenched nano-fluorescent probe NIR-CBT-NPs;

[0007] Wherein, the molecular structure of the dimer NIR-CBT-Dimer is shown in the following formula:

[0008]

[0009] Furthermore, a preparation method of a nano-fluorescent probe for detecting FAPα activity is as follows:

[0010] (1) Dissolve IR780 in an organic solvent, then add 3-mercaptopropionic acid and triethylamine, react at room temperature under the protection of an inert gas, and obtain IR780-COOH after purification by high performance liquid chromatography, denoted as P1;

[0011] (2) Dissolve Fmoc-K(Boc)-OH (lysine derivative) in an organic solvent, and add N-methylmorpholine, isobutyl chloroformate and CBT to the above solution step by step under the protection of an inert gas. After stirring and reacting in an ice-water bath, restore to room temperature for reaction, and obtain Fmoc-K(Boc)-CBT after purification by high performance liquid chromatography, denoted as P2;

[0012] (3) Prepare an acid cleavage solution to remove the protecting group of the side-chain amino group on P2, and obtain Fmoc-K-CBT after purification by high performance liquid chromatography, denoted as P3;

[0013] (4) Dissolve P1 and P3 in an organic solvent, add an activator and an acid-binding agent, react at room temperature under the protection of an inert gas, and obtain Fmoc-K(IR780)-CBT after purification by high performance liquid chromatography, denoted as P4;

[0014] (5) Prepare a base cleavage solution to remove the protecting group of the amino group on P4, and obtain NH2-K(IR780)-CBT after purification by high performance liquid chromatography, denoted as P5;

[0015] (6) Swell 2-chlorotrityl chloride resin in an organic solvent, add Fmoc-His(Boc)-OH (histidine derivative) and an acid-binding agent to react, use a base cleavage solution to remove the protecting group of the amino group, use an activator and an acid-binding agent to couple Fmoc-Ala-OH (alanine derivative) to the polypeptide, and use a base cleavage solution to remove the protecting group of the amino group; repeat the above coupling and deprotection steps (including the thiol group of the cysteine side chain, which is protected by tert-butylthio (StBu)) to achieve the growth of the peptide chain. Finally, cut the polypeptide from the resin with an acid cleavage solution, precipitate the product with a solvent (one of petroleum ether, ether or water), discard the supernatant by freezing and centrifugation, and obtain Fmoc-C(StBu)GPAH(Boc)-OH after purification of the precipitate by high performance liquid chromatography, denoted as P6;

[0016] (7) Dissolve P5 and P6 in an organic solvent, add an activator and a base scavenger, and react at room temperature under the protection of an inert gas. After purification by high performance liquid chromatography, Fmoc-C(StBu)GPAH(Boc)K(IR780)-CBT is obtained, denoted as P7;

[0017] (8) Use an alkaline cleavage solution and an acidic cleavage solution to remove the protecting groups on P7 respectively. After purification by high performance liquid chromatography, the fluorescent probe precursor NIR-CBT is obtained, denoted as P8;

[0018] (9) Dissolve P8 in phosphate buffer solution, add a reducing agent to remove the protecting group of the side chain mercapto group on P8; adjust the pH of the solution to weakly alkaline and continue the reaction until precipitation occurs; after centrifugation of the reaction suspension, discard the supernatant, and the remaining precipitate is the final nano-fluorescent probe NIR-CBT-NPs.

[0019] Further, the organic solvent in the synthesis step is selected from one of methanol, isopropanol, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide or tetrahydrofuran.

[0020] Further, the base scavenger in the synthesis step is selected from one of triethylamine, N,N-diisopropylethylamine, 4-dimethylaminopyridine, piperidine or pyridine.

[0021] Further, the activator in the synthesis step is selected from one of O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate, 1-hydroxybenzotriazole, O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate or O-(5-chlorobenzotriazol-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate.

[0022] Further, the alkaline cleavage solution in the synthesis step is selected from one of piperidine / methanol (1-50%, v / v), piperidine / N,N-dimethylformamide (1-50%, v / v), piperidine / dichloromethane (1-50%, v / v), pyridine / dichloromethane (10-60%, v / v), pyridine / N,N-dimethylformamide (10-60%, v / v), hydrazine hydrate / N,N-dimethylformamide (1-60%, v / v) or hydrazine hydrate / methanol (1-60%, v / v).

[0023] Further, the acidic cleavage solution in the synthesis step is selected from one of trifluoroacetic acid / acetonitrile (1-99%, v / v), trifluoroacetic acid / N,N-dimethylformamide (1-99%, v / v) or trifluoroacetic acid / methanol (1-99%, v / v).

[0024] Further, the inert gas in the synthesis step is selected from one of nitrogen, argon or helium.

[0025] Furthermore, the amino acids used have 9-fluorenylmethoxycarbonyl (Fmoc) as the α-amino protecting group; the side-chain amino group of histidine used is protected by tert-butoxycarbonyl (Boc).

[0026] Furthermore, the reducing agent is selected from reduced glutathione, dithiothreitol, tris(2-carboxyethyl)phosphine, and trihydroxypropylphosphine.

[0027] Furthermore, the phosphate buffer is: PBS, pH 7.4, containing 1% DMSO.

[0028] Furthermore, steps (1)-(9) are all carried out by high-performance liquid chromatography technology, and the high-performance liquid chromatographs used are all various types of high-performance liquid chromatographs currently available on the market, such as the e2695 high-performance liquid chromatograph, etc.

[0029] The synthesized compounds P1-P8 have the following characteristic structures respectively:

[0030]

[0031]

[0032]

[0033] Principle of the present invention: The nano-fluorescent probe synthesized in the present invention self-assembles into nanoparticles in vitro through π-π stacking and hydrophobic interactions, and at this time the fluorescence is in the "off" state; the probe is specifically recognized and cleaves the Gly-Pro-Ala sequence on the main chain of the probe by FAPα overexpressed on the tumor cell membrane. The increased water solubility of the enzymatic cleavage product causes the probe to slowly disassemble, extending the imaging time; the fluorescence quenching effect of the dye IR780 connected to the probe side chain is interrupted. The nano-fluorescent probe NIR-CBT-NPs is specifically recognized and hydrolyzed by FAPα highly expressed on the cell membrane, and the near-infrared fluorescence "turns on", enhancing the penetration depth and signal-to-noise ratio of fluorescence imaging; the present invention has good biocompatibility and realizes sensitive and long-term monitoring of FAPα activity.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following significant features: The nano-fluorescent probe extends the imaging time for detecting FAPα activity and has a long detection time; it enhances the penetration depth and signal-to-noise ratio of fluorescence imaging. In addition, it also extends the half-life of monitoring FAPα activity and slows down the degradation and metabolism time in vivo. Description of the Drawings

[0035] Figure 1 is the mass spectrum of the pure compound P1 synthesized in Example 1 of the present invention;

[0036] Figure 2It is the mass spectrum of the pure compound P2 synthesized in Example 2 of the present invention;

[0037] Figure 3 It is the mass spectrum of the pure compound P3 synthesized in Example 2 of the present invention;

[0038] Figure 4 It is the mass spectrum of the pure compound P4 synthesized in Example 2 of the present invention;

[0039] Figure 5 It is the mass spectrum of the pure compound P5 synthesized in Example 2 of the present invention;

[0040] Figure 6 It is the mass spectrum of the pure compound P6 synthesized in Example 3 of the present invention;

[0041] Figure 7 It is the mass spectrum of the pure compound P7 synthesized in Example 3 of the present invention;

[0042] Figure 8 It is the mass spectrum of the pure compound P8 synthesized in Example 3 of the present invention;

[0043] Figure 9 It is the 1H nuclear magnetic resonance spectrum of the pure compound P8 synthesized in Example 3 of the present invention;

[0044] Figure 10 It is the 13C nuclear magnetic resonance spectrum of the pure compound P8 synthesized in Example 3 of the present invention;

[0045] Figure 11 It is the mass spectrum of the pure compound P9 synthesized in Example 3 of the present invention;

[0046] Figure 12 It is the transmission electron microscopy characterization result graph of the pure compound P9 synthesized in Example 3 of the present invention before and after being recognized and cleaved by FAPα in vitro;

[0047] Figure 13 It is the high performance liquid chromatography analysis result graph of the pure compound P8 after being reduced by TCEP and the pure compound P9 before and after being recognized and cleaved by FAPα in vitro in Example 4 of the present invention;

[0048] Figure 14 It is the fluorescence spectrum analysis result graph (excitation wavelength 780 nm) of the pure compound P8 after being reduced by TCEP and the pure compound P9 before and after being recognized and cleaved by FAPα in vitro in Example 4 of the present invention;

[0049] Figure 15 It is the fluorescence intensity analysis result graph (excitation wavelength 780 nm, emission wavelength 810 nm) of the pure compound P9 after co-incubation with different biomolecules for 12 hours in Example 4 of the present invention;

[0050] Figure 16 is the preparation flow chart of the present invention. Detailed implementation manners

[0051] The content of the present invention will be further described below in conjunction with specific embodiments.

[0052] Example 1: A nano-fluorescent probe for detecting FAPα activity according to the present invention. The prepared probe precursor Cys(StBu)-Gly-Pro-Ala-His-Lys(IR780)-CBT(NIR-CBT) triggers a CBT-Cys click reaction through a reducing agent to generate a cyclic dimer NIR-CBT-Dimer, and further self-assembles to form a fluorescence-quenched nano-fluorescent probe NIR-CBT-NPs; wherein, the molecular structure of the dimer NIR-CBT-Dimer is as shown above.

[0053] As Figure 16 shown, the specific preparation steps are as follows:

[0054] The synthesis route of compound P1 is as follows: (1) Dissolve 1 mmol of IR780 in 10 mL of N,N-dimethylformamide, add 2 mmol of 3-mercaptopropionic acid and 2 mmol of triethylamine, stir overnight at room temperature under an argon atmosphere, and obtain IR780-COOH, denoted as P1, after purification by high performance liquid chromatography. The structural formula of P1 is as shown above;

[0055] Perform electrospray ionization mass spectrometry analysis on the pure compound P1 using an InfinityLab liquid chromatography-mass spectrometry instrument to obtain Figure 1 the mass spectrum as shown;

[0056] Figure 1 is the mass spectrum of the first compound P1 synthesized in this example; as Figure 1 visible, the mass spectrometry result of the first compound P1 is obsvd.ESI-MS[(M)+]: m / z 609.3;

[0057] Synthesis methods of pure compounds P2, P3, P4, and P5:

[0058] (2) Dissolve 1 mmol of Fmoc-K(Boc)-OH in 2 mL of tetrahydrofuran, place it in an ice-water bath under an argon atmosphere, add 2 mmol of N-methylmorpholine to the above reaction solution. After 3 minutes, add 1 mmol of isobutyl chloroformate, stir for 40 minutes, then add 1 mmol of CBT and 0.5 mmol of isobutyl chloroformate. The mixture continues to stir in the ice-water bath for 1 hour and react overnight at room temperature. After purification by high performance liquid chromatography, obtain Fmoc-K(Boc)-CBT, denoted as P2;

[0059] (3) Prepare a 10 mL dichloromethane solution containing 95% trifluoroacetic acid, add P2, and react at room temperature for 3 hours. After purification by high performance liquid chromatography, Fmoc-K-CBT is obtained, denoted as P3;

[0060] (4) Dissolve 1 mmol of P1 and 1 mmol of P3 in 10 mL of N,N-dimethylformamide, add 2 mmol of 1-hydroxybenzotriazole, 2 mmol of benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 3 mmol of N,N-diisopropylethylamine. Stir overnight at room temperature under an argon atmosphere. After purification by high performance liquid chromatography, Fmoc-K(IR780)-CBT is obtained, denoted as P4;

[0061] (5) Prepare a 5 mL N,N-dimethylformamide solution containing 10% piperidine, add P4 and react for 5 minutes; then add 0.6 mL of trifluoroacetic acid to neutralize piperidine. After purification by high performance liquid chromatography, NH2-K(IR780)-CBT is obtained, denoted as P5;

[0062] Electrospray ionization mass spectrometry analysis was performed on the pure compounds P2, P3, P4, P5 (the structural formulas of P2, P3, P4, P5 are as shown above) using an InfinityLab liquid chromatography-mass spectrometry instrument, and Figure 2 , Figure 3 , Figure 4 , Figure 5 the mass spectra shown were obtained;

[0063] Figure 2 is the mass spectrum of the compound P2 synthesized in this example; Figure 3 is the mass spectrum of the compound P3 synthesized in this example; Figure 4 is the mass spectrum of the compound P4 synthesized in this example; Figure 5 is the mass spectrum of the compound P5 synthesized in this example;

[0064] From Figure 2 it can be seen that the mass spectrometry result of compound P2 is obsvd.ESI-MS[(M-Boc+H)+]: m / z 526.2; from Figure 3 it can be seen that the mass spectrometry result of compound P3 is obsvd.ESI-MS[(M+H)+]: m / z 526.2; from Figure 4 it can be seen that the mass spectrometry result of compound P4 is obsvd.ESI-MS[(M)+]: m / z 1116.5; from Figure 5 it can be seen that the mass spectrometry result of compound P5 is obsvd.ESI-MS[(M)+]: m / z 894.4;

[0065] Synthesis methods of pure compounds P6, P7, P8, and P9: (6) Swell 1 mmol of 2-chlorotrityl chloride resin in 30 mL of N,N-dimethylformamide for 1 hour. Add 1.2 mmol of Fmoc-His(Boc)-OH and 3 mmol of N,N-diisopropylethylamine and react for 1.5 hours. Then, cap the reaction with methanol for 1 hour. Remove the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group using a N,N-dimethylformamide solution containing 20% piperidine for 30 minutes. Use 1-hydroxybenzotriazole, benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N-diisopropylethylamine as coupling agents to couple the next amino acid Fmoc-Ala-OH to the polypeptide. Repeat the above coupling and deprotection steps to achieve the growth of the peptide chain. Finally, wash with isopropanol and n-hexane three times each, and then cleave the polypeptide from the resin using a dichloromethane solution containing 1% trifluoroacetic acid. Precipitate the product with ether, freeze-centrifuge, and discard the supernatant. Purify the precipitate by high-performance liquid chromatography to obtain the polypeptide Fmoc-C(StBu)GPAH(Boc)-OH, denoted as P6;

[0066] (7) Dissolve 1 mmol of P5 and 1 mmol of P6 in 10 mL of N,N-dimethylformamide. Add 2 mmol of 1-hydroxybenzotriazole, 2 mmol of benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 3 mmol of N,N-diisopropylethylamine. Stir overnight at room temperature under an argon atmosphere. Purify by high-performance liquid chromatography to obtain Fmoc-C(StBu)GPAH(Boc)K(IR780)-CBT, denoted as P7;

[0067] (8) Prepare a 5 mL N,N-dimethylformamide solution containing 10% piperidine and add P7 to react for 5 minutes. Subsequently, prepare a 10 mL dichloromethane solution containing 95% trifluoroacetic acid and add it to the above reaction solution. Stir at room temperature for 3 hours. Purify by high-performance liquid chromatography to obtain the fluorescent probe precursor NIR-CBT, denoted as P8;

[0068] (9) Dissolve 10 μmol of P8 in phosphate buffer (PBS, pH 7.4, containing 1% DMSO). Add 1 mmol of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and react at 37 °C for 5 hours. Then, adjust the pH to 7.4 with saturated sodium carbonate solution and continue to react for 3 hours. After centrifuging the reaction mixture, discard the supernatant. Disperse the precipitate in PBS and sonicate to obtain the final nano-fluorescent probe NIR-CBT-NPs;

[0069] Specifically, after the fluorescence probe precursor NIR-CBT was reduced by TCEP, the StBu protecting group of the Cys residue was removed; subsequently, after adjusting the pH to 7.4, the CBT and Cys at both ends of the probe underwent a click reaction, forming an intermolecular cyclic dimer NIR-CBT-Dimer; after being dispersed in PBS, the dimer self-assembled into nanoparticles, and fluorescence underwent intra- and intermolecular quenching, thus generating a fluorescence "off" nanoprobe NIR-CBT-NPs.

[0070] The electrospray ionization mass spectrometry analysis of the pure compounds P6, P7, P8, and P9 (the structural formulas of P6, P7, P8, and P9 are shown above) was performed using an InfinityLab liquid chromatography-mass spectrometry instrument, and the Figure 6 , Figure 7 , Figure 8 mass spectrometry patterns shown were obtained;

[0071] Figure 6 is the mass spectrometry pattern of the compound P6 synthesized in this example; Figure 7 is the mass spectrometry pattern of the compound P7 synthesized in this example; Figure 8 is the mass spectrometry pattern of the compound P8 synthesized in this example;

[0072] From Figure 6 it can be seen that the mass spectrometry result of the compound P6 was obsvd.ESI-MS[(M - Boc + H)+]: m / z 794.3; from Figure 7 it can be seen that the mass spectrometry result of the compound P7 was obsvd.ESI-MS[(M + H)2+ / 2]: m / z 885.1; from Figure 8 it can be seen that the mass spectrometry result of the compound P8 was obsvd.ESI-MS[(M + H)2+ / 2]: m / z 724.3.

[0073] The nuclear magnetic resonance spectra shown in Figure 9 and Figure 10 were obtained by analyzing the pure compound P8 synthesized in this example using Bruker nuclear magnetic software;

[0074] Figure 9 is the 1H nuclear magnetic resonance spectrum of the pure compound P8 synthesized in this example; Figure 10 is the 13C nuclear magnetic resonance spectrum of the pure compound P8 synthesized in this example; from Figure 9It can be seen that the 1H NMR (d6-DMSO, 500 MHz) of compound P8 shows δ (ppm): 10.65–10.38 (m, 1H), 8.91 (s, 1H), 8.68–8.54 (m, 5H), 8.44–8.31 (m, 2H), 8.16 (dd, J = 26.4, 20.0 Hz, 2H), 7.91 (d, J = 7.7 Hz, 2H), 7.71 (d, J = 9.0 Hz, 1H), 7.53 (d, J = 7.5 Hz, 2H), 7.36 (d, J = 6.4 Hz, 4H), 7.30 (d, J = 11.4 Hz, 1H), 7.19 (t, J = 7.3 Hz, 2H), 6.26 (d, J = 14.2 Hz, 2H), 4.59 (s, 1H), 4.48–4.20 (m, 2H), 4.11 (t, J = 7.3 Hz, 5H), 4.01 (d, J = 16.6 Hz, 1H), 3.91 (dd, J = 17.0, 4.8 Hz, 1H), 3.44 (dt, J = 32.0, 18.3 Hz, 2H), 3.12 (td, J = 12.7, 11.5, 5.9 Hz, 2H), 2.98 (s, 6H), 2.84 (s, 2H), 2.68 (s, 2H), 2.32 (t, J = 7.1 Hz, 2H), 1.73 (q, J = 7.6 Hz, 4H), 1.62 (d, J = 8.7 Hz, 12H), 1.51 (d, J = 6.1 Hz, 2H), 1.45 (s, 3H), 1.40 (d, J = 5.3 Hz, 6H), 1.24 (d, J = 3.4 Hz, 9H), 1.20–1.16 (m, 3H), 1.14 (d, J = 7.2 Hz, 2H), 0.95–0.75 (m, 6H). From Figure 10As can be seen, the carbon NMR spectrum (δ6-DMSO, 151 MHz) of compound P9 shows δ (ppm): 172.46, 171.67, 170.52, 169.85, 166.85, 165.53, 164.13, 159.24, 158.99, 155.88, 154.02, 150.40, 148.22, 145.36, 145.30, 142.82, 141.35, 140.53, 139.85, 137.14, 134.10, 133.42, 132.80, 129.72, 129.01, 127.59, 125.40, 125.28, 122.87, 121.36, 119.53, 117.92, 115.49, 113.99, 113.61, 113.20, 111.84, 111.74, 101.75, 49.29, 48.49, 45.45 (3C), 44.19, 40.88 (2C), 40.31 (2C), 29.86 (2C), 29.50, 29.31, 29.05, 28.76, 27.97 (2C), 27.95, 26.22 (7C), 24.72 (3C), 22.67 (3C), 22.13 (2C), 20.90, 11.54.

[0075] High-resolution mass spectrometry analysis was performed on pure compound P9 using high-resolution mass spectrometry, and Figure 11 the mass spectrum shown was obtained, as Figure 11 shown;

[0076] Figure 11 is the mass spectrum of compound P9 synthesized in this example; it can be seen from Figure 11 that the mass spectrometry result of compound P9 is obsvd.ESI-MS[(M + 2H)4+ / 4]: m / z 671.80432.

[0077] Detection experiment on the enzyme-responsive disassembly and fluorescence "turn-on" of the nanofluorescent probe under in vitro conditions:

[0078] In the detection experiment of this example, pure compound P9 with a concentration of 5 μmol was used. In PBS (pH 7.4, 1% DMSO), 200 ng / mL of FAPα or other biomolecules were added, and incubation was carried out at 37 °C for 12 hours for the detection experiment.

[0079] Figure 12 Figure shows the transmission electron microscopy characterization results of pure compound P9 in Example 4 before and after being recognized and cleaved by FAPα in vitro; Figure 12 The transmission electron microscopy characterization results given show that the probe appears as nanoparticles in vitro, while after cleavage by FAPα enzyme, the nanoparticles disassembled.

[0080] Figure 13 It is the high-performance liquid chromatography analysis result graph before and after the pure compound P8 in Example 4 is reduced by TCEP and the pure compound P9 is recognized and cleaved by FAPα in vitro, as shown in the figure; curve a is the chromatographic analysis result of compound P8, and its chromatographic peak a1 represents compound P8; curve b is the chromatographic analysis result obtained after compound P8 is reduced by TCEP for 8 hours, and its chromatographic peak b1 represents compound P9, proving that compound P8 has undergone a CBT-Cys click reaction to generate a cyclic dimer NIR-CBT-Dimer; curve c is the chromatographic analysis result of compound NIR-CBT-Cleaved, and its chromatographic peak c1 represents the cleaved molecule NIR-CBT-Cleaved after compound P9 is cleaved by FAPα; curve d is the chromatographic analysis result of the mixture after compound P9 is co-incubated with FAPα for 12 hours. The retention time of chromatographic peak d1 is the same as that of chromatographic peak c1 in curve c, proving that compound P9 can be specifically recognized and cleaved by FAPα in vitro to generate a cleaved molecule NIR-CBT-Cleaved with better water solubility. The retention time of chromatographic peak d2 is the same as that of chromatographic peak b1 in curve b, indicating that there is still some residue of compound P9 after being cleaved by FAPα for 12 hours.

[0081] Figure 14 It is the fluorescence spectrum analysis result graph (excitation wavelength 780 nm) before and after the pure compound P8 in Example 4 is reduced by TCEP and the pure compound P9 is recognized and cleaved by FAPα in vitro, as shown in the figure; curve a is the fluorescence spectrum of the pure compound P8; curve b is the fluorescence spectrum of the pure compound P8 after being reduced by TCEP for 8 hours, and the fluorescence intensity at 810 nm is significantly reduced, proving that the fluorescence of compound P8 is significantly quenched after being reduced by TCEP; curve c is the fluorescence spectrum of the mixture after the pure compound P9 is co-incubated with FAPα for 12 hours, and the fluorescence intensity at 810 nm is increased by 11.6 times compared with curve b, indicating that compound P9 can be specifically recognized and cleaved by FAPα in vitro, the fluorescence quenching effect is interrupted, and the near-infrared fluorescence signal is enhanced; curve d is the fluorescence spectrum of the mixture after the pure compound P9 is co-incubated with FAPα and the FAPα inhibitor SP-13786 for 12 hours, and the fluorescence intensity at 810 nm is close to that of curve b, indicating that SP-13786 inhibits the cleavage activity of FAPα and the near-infrared fluorescence of compound P9 cannot be restored.

[0082] Figure 15The figure shows the analysis results of the fluorescence intensity after the pure compound P9 in Example 4 was co-incubated with different biomolecules for 12 hours (excitation wavelength: 780 nm, emission wavelength: 810 nm). The results show that after the pure compound P9 was co-incubated only with FAPα, the fluorescence intensity at 810 nm increased significantly, while the fluorescence intensity remained almost unchanged after co-incubation with other biomolecules, indicating that the pure compound P9 can be specifically recognized and cleaved by FAPα, thereby activating near-infrared fluorescence. These biomolecules include sodium ion (Na+), potassium ion (K+), calcium ion (Ca2+), cysteine (Cys), glutathione (GSH), alkaline phosphatase (ALP), carboxylesterase (CES), gamma-glutamyltransferase (GGT), tyrosinase (TYR), and caspase B (CTSB).

[0083] From the results of the examples and their detection experiments, it can be seen that the nano-fluorescent probe of the present invention can self-assemble into the morphology of nanoparticles in vitro and has an extremely low background signal. After being specifically recognized and cleaved by FAPα, the probe slowly disassembles and turns on near-infrared fluorescence. The nano-fluorescent probe of the present invention can be used to perform sensitive and persistent near-infrared fluorescence imaging of the FAPα activity at the tumor site, with a high imaging signal-to-noise ratio and a long imaging time. The present invention realizes the effective preparation of the nano-fluorescent probe and completes the detection experiment of the enzyme-responsive disassembly and fluorescence of the probe under in vitro conditions, showing its application potential in the early screening of cancer.

Claims

1. A nano-fluorescent probe for detecting FAPα activity, characterized in that, The prepared probe precursor Cys(StBu)-Gly-Pro-Ala-His-Lys(IR780)-CBT triggers the CBT-Cys click reaction through a reducing agent to generate a cyclic dimer NIR-CBT-Dimer, which self-assembles to form a nano-fluorescent probe NIR-CBT-NPs; Among them, the molecular structure of the cyclic dimer NIR-CBT-Dimer is shown in the following formula:

2. The preparation method of a nano-fluorescent probe for detecting FAPα activity according to claim 1, characterized in that, The preparation steps are as follows: (1) Dissolve IR780 in an organic solvent, then add 3-mercaptopropionic acid and triethylamine, and react at room temperature under inert gas protection. After purification by liquid chromatography, IR780-COOH is obtained, denoted as P1; (2) Dissolve the lysine derivative in another organic solvent. Under inert gas protection, add N-methylmorpholine, isobutyl chloroformate, and CBT to the above solution step by step. After stirring the reaction in an ice-water bath, restore to room temperature for reaction. After purification by liquid chromatography, Fmoc-K(Boc)-CBT is obtained, denoted as P2; (3) Prepare an acid cleavage solution to remove the protecting group of the side-chain amino group on P2. After purification by liquid chromatography, Fmoc-K-CBT is obtained, denoted as P3; (4) Dissolve P1 and P3 in another organic solvent, add an activator and a base scavenger, and react at room temperature under inert gas protection. After purification by liquid chromatography, Fmoc-K(IR780)-CBT is obtained, denoted as P4; (5) Prepare a base cleavage solution to remove the protecting group of the amino group on P4. After purification by liquid chromatography, NH2-K(IR780)-CBT is obtained, denoted as P5; (6) Swell 2-chlorotrityl chloride resin in another organic solvent, add a histidine derivative and a base scavenger for reaction, use a base cleavage solution to remove the protecting group of the amino group, use an activator and a base scavenger to couple the alanine derivative to the polypeptide, and use a base cleavage solution to remove the protecting group of the amino group; repeat the above coupling and deprotection steps; finally, cut the polypeptide from the resin with an acid cleavage solution, precipitate the product with a solvent, discard the supernatant by freezing and centrifugation, and the precipitate is purified by liquid chromatography to obtain Fmoc-C(StBu)GPAH(Boc)-OH, denoted as P6; (7) Dissolve P5 and P6 in another organic solvent, add an activator and a base scavenger, and react at room temperature under inert gas protection. After purification by liquid chromatography, Fmoc-C(StBu)GPAH(Boc)K(IR780)-CBT is obtained, denoted as P7; (8) Use a base cleavage solution and an acid cleavage solution to remove the protecting groups on P7 respectively. After purification by liquid chromatography, the fluorescent probe precursor NIR-CBT is obtained, denoted as P8; (9) Dissolve P8 in phosphate buffer, add a reducing agent to remove the protecting group of the side-chain mercapto group on P8; adjust the pH of the solution to weakly alkaline and continue the reaction until precipitation occurs; after centrifugation of the reaction suspension, discard the supernatant, and the remaining precipitate is the final nano-fluorescent probe NIR-CBT-NPs.

3. The preparation method of a nano-fluorescent probe for detecting FAPα activity according to claim 2, characterized in that, In the synthesis step, the organic solvent is selected from one of methanol, isopropanol, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide or tetrahydrofuran.

4. The preparation method of a nano-fluorescent probe for detecting FAPα activity according to claim 2, characterized in that, In the synthesis step, the acid-binding agent is selected from one of triethylamine, N,N-diisopropylethylamine, 4-dimethylaminopyridine, piperidine or pyridine.

5. The preparation method of a nano-fluorescent probe for detecting FAPα activity according to claim 2, characterized in that, In the synthesis step, the activator is selected from one of O-(7-azabenzotriazol-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate, 1-hydroxybenzotriazole, O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate or O-(5-chlorobenzotriazol-1-yl)-bis(dimethylamino)carbenium hexafluorophosphate.

6. The preparation method of a nano-fluorescent probe for detecting FAPα activity according to claim 2, characterized in that, In the synthesis step, the base cleavage solution is selected from one of piperidine / methanol, piperidine / N,N-dimethylformamide, piperidine / dichloromethane, pyridine / dichloromethane, pyridine / N,N-dimethylformamide, hydrazine hydrate / N,N-dimethylformamide or hydrazine hydrate / methanol.

7. The preparation method of a nano-fluorescent probe for detecting FAPα activity according to claim 2, wherein, In the synthesis step, the acid cleavage solution is selected from one of trifluoroacetic acid / acetonitrile, trifluoroacetic acid / N,N-dimethylformamide or trifluoroacetic acid / methanol.

8. The preparation method of a nano-fluorescent probe for detecting FAPα activity according to claim 2, characterized in that, In the synthesis step, the inert gas is selected from one of nitrogen, argon or helium.

9. The preparation method of a nano-fluorescent probe for detecting FAPα activity according to claim 2, characterized in that, All the amino acids used have 9-fluorenylmethoxycarbonyl as the α-amino protecting group; the side-chain amino groups of lysine and histidine used are protected by tert-butoxycarbonyl.

10. The preparation method of a nano-fluorescent probe for detecting FAPα activity according to claim 2, wherein, In the step (6), the solvent used to precipitate the product is selected from one of petroleum ether, ether or water; In the step (9), the reducing agent is selected from one of reduced glutathione, dithiothreitol, tris(2-carboxyethyl)phosphine or trihydroxypropylphosphine.

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