A fluorescent / photoacoustic molecular probe for imaging tumor aging and its application

Abstract

The present invention discloses a fluorescent / photoacoustic molecular probe for imaging tumor aging and its application, wherein the fluorescent / photoacoustic molecular probe for imaging tumor aging is abbreviated as Gal-HCy-Biotin, and its structural formula is as follows: The fluorescence and photoacoustic signals of Gal-HCy-Biotin are "off". When Gal-HCy-Biotin is targeted and enters tumor cells, it can effectively accumulate in the cells. The β-gal highly expressed in the lysosomes of aging tumor cells will specifically recognize and hydrolyze the galactose residues of Gal-HCy-Biotin, quickly release free HCy-Biotin, and turn on the near-infrared fluorescence / photoacoustic signal through the intramolecular charge transfer process for imaging tumor aging. The fluorescent / photoacoustic molecular probe Gal-HCy without targeting function is used as a control probe.

Description

Technical Field

[0001] The invention belongs to the field of biological probes, and in particular relates to a fluorescent / photoacoustic molecular probe for imaging tumor aging and an application thereof. Background Art

[0002] Cellular senescence is a permanent blockage of cell cycle progression that can be caused by a variety of reasons, including excessive proliferation, telomere dysfunction, oxidative stress, DNA damage, and oncogenic activation. β-Galactosidase (β-gal) is an endogenous enzyme produced by lysosomes and is responsible for removing galactose residues from various protein and non-protein substrates. The activity of β-Gal is directly related to cellular senescence. It is overexpressed in many aging-related diseases, such as atherosclerosis, cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, and Alzheimer's disease. It is currently the most widely used biomarker for detecting cellular senescence.

[0003] So far, researchers have developed a variety of fluorescent probes for visualizing the activity of β-gal in senescent cells in vitro and in vivo. According to the Journal of the American Chemical Society (J.Am.Chem.Soc.2020,142,18005), Tian et al. designed a β-Gal-responsive photochromic fluorescent probe for locating β-Gal in senescent cells. By using super-resolution microscopy, the fluorescent probe can accurately locate the subcellular distribution of β-Gal. The German Journal of Applied Chemistry (Angew.Chem.Int.Ed.,2020,59,15152) reported a nanoprobe loaded with Nile blue dye and covered with galactose, which can release Nile blue dye in the specific presence of β-Gal. In both cell and mouse aging models, the nanoprobe showed excellent visualization ability to image senescent cells. In recent years, molecular probes for detecting and imaging β-gal in senescent cells are booming, and it is still necessary to continue to develop some excellent molecular probes that can monitor the activity of β-gal in senescent cells in real time in vivo.

[0004] Optical imaging has the advantages of high sensitivity, non-invasiveness, and low cost, but it has low spatial resolution and poor depth penetration. Photoacoustic imaging has high spatial resolution and deep tissue penetration, but the sensitivity needs to be further improved. Therefore, fluorescence / photoacoustic multimodal imaging can not only achieve highly sensitive functional imaging, but also provide high-resolution structural histological information, providing more comprehensive information for in vivo research, and will have broader application prospects in scientific research and medical clinics. However, there is currently no fluorescent / photoacoustic molecular probe for imaging the activity of β-gal in aging tumors, so we developed a tumor-targeted fluorescent / photoacoustic molecular probe to image tumor aging by monitoring the activity of β-gal. Summary of the invention

[0005] The present invention aims to provide a fluorescent / photoacoustic molecular probe for imaging tumor aging and its application. The fluorescent / photoacoustic molecular probe Gal-HCy-Biotin of the present invention can successfully monitor the activity of β-gal in the process of tumor aging. The fluorescent / photoacoustic molecular probe Gal-HCy without targeting function is used as a control probe. In vitro experiments show that both molecular probes have good sensitivity and selectivity. Since the catalytic efficiency of β-gal to Gal-HCy-Biotin is higher than that of Gal-HCy, Gal-HCy-Biotin has a lower detection limit than Gal-HCy. The in vivo imaging results further confirm that the overexpressed β-gal in the process of tumor aging can successfully turn on the fluorescence / photoacoustic signal of the probe. Both molecular probes can be used for in vivo imaging of tumor aging, and the molecular probe Gal-HCy-Biotin that can target tumors has better detection performance than the control probe Gal-HCy.

[0006] The fluorescent / photoacoustic molecular probe for imaging tumor aging of the present invention is abbreviated as Gal-HCy-Biotin, and its structural formula is as follows:

[0007]

[0008] The molecular probe Gal-HCy-Biotin of the present invention consists of three parts: (1) tetraacetyl-α-D-bromogalactose is a substrate specifically recognized by β-galactosidase (β-gal); (2) near-infrared hemicyanine dye (HCy) provides activatable fluorescence and photoacoustic signals; and (3) biotin is used to target tumor cells.

[0009] The fluorescence and photoacoustic signals of Gal-HCy-Biotin are "off". When Gal-HCy-Biotin is targeted into tumor cells, it can effectively accumulate in the cells. β-gal, which is highly expressed in the lysosomes of aging tumor cells, specifically recognizes and hydrolyzes the galactose residues of Gal-HCy-Biotin, rapidly releasing free HCy-Biotin, and turning on near-infrared fluorescence / photoacoustic signals through the intramolecular charge transfer process for imaging tumor aging. The fluorescent / photoacoustic molecular probe Gal-HCy without targeting function is used as a control probe.

[0010] The method for preparing the fluorescent / photoacoustic molecular probe for imaging tumor aging of the present invention comprises the following steps:

[0011] (1) Synthesis of compound 1: 2,3,3-trimethylbutene (3.18 g, 20.0 mmol) and 3-bromopropylamine (5.25 g, 24.0 mmol) were mixed and stirred at 100°C for 4 hours. The crude product was crystallized from acetone / methanol (5:1, V / V) to obtain a pink solid, namely compound 1.

[0012] (2) Synthesis of compound 2: A mixture of 2-chloro-1-formyl-3-(hydroxymethylene) (200 mg, 1.16 mmol compound) and compound 1 (687 mg, 3.17 mmol) was dissolved in 30 mL of 1-butanol / benzene (7:3, V / V), filtered by Dean-Stark to remove water, and the reaction solution was stirred at 75°C for 6 h. The solvent was removed under vacuum and then dissolved in ethanol. Di-tert-butyl dicarbonate (322 mg, 1.73 mmol) and TEA (242 μL, 1.73 mmol) were added. The mixture was stirred at room temperature for 3 h, and then the solvent was removed under vacuum. The residue was dissolved in ethyl acetate, washed with 1 M hydrochloric acid, water, and brine, dried over anhydrous sodium sulfate, and then the solvent was removed to obtain compound 2.

[0013] (3) Synthesis of HCy: Compound 2 (478.87 mg, 0.91 mmol) was dissolved in 3.0 mL of anhydrous acetonitrile, potassium carbonate (250.66 mg, 1.81 mmol) was added to provide an alkaline environment, and the mixture was stirred at room temperature for 10 min under a nitrogen atmosphere. 1.0 mL of anhydrous acetonitrile solution containing m-diphenol (200 mg, 1.81 mmol) was added dropwise. After the addition, the system was heated to 75°C and stirred at 75°C for 5 h. After the reaction was completed, the solvent was evaporated and purified by HPLC (the eluent was CH 3 OH / H 2 O=3:5, V / V) to obtain hemicyanine dye (HCy).

[0014] (4) Synthesis of compound 3: Hemicyanine dye (HCy) (60 mg, 0.107 mmol) and galactose (87.78 mg, 0.213 mmol) were dissolved in acetonitrile, cesium carbonate (69.55 mg, 0.213 mmol) provided an alkaline environment, sodium sulfate (30.32 mg, 0.213 mmol) was used to remove water, and the reaction was carried out at room temperature under nitrogen protection for 8-12 h. The solvent was removed by rotary evaporation, and 20% TFA / CH 2 Cl 2 It was dissolved and stirred at room temperature for 2 h. The solvent was removed by rotary evaporation to obtain compound 3 (26.49 mg, 0.035 mmol).

[0015] (5) Synthesis of Compound 4: Compound 3 and biotin (12.82 mg, 0.0525 mmol) were dissolved in DMF, DIEPA (9 μL, 0.0525 mmol) was added dropwise, and then stirred at room temperature for 6 h. The product was purified by HPLC (eluent: CH 3 OH / H 2 O=3:5, V / V) to give compound 4 (32.35 mg, 0.0329 mmol).

[0016] (6) Synthesis of Gal-HCy-Biotin: Compound 4 (32.35 mg, 0.0329 mmol) was dissolved in methanol, sodium methoxide (10 mg) was added, and the mixture was stirred at room temperature for 2 h. The solvent was removed by rotary evaporation and purified by HPLC (eluent: CH 3 OH / H 2 O=3:5, V / V) to obtain compound Gal-HCy-Biotin (18.78 mg, 0.023 mmol).

[0017] The synthetic route is as follows:

[0018]

[0019] The application of the fluorescent / photoacoustic molecular probe for imaging tumor aging of the present invention is to prepare a detection reagent targeting tumors with the fluorescent / photoacoustic molecular probe.

[0020] Overexpressed β-gal during tumor senescence can successfully turn on the fluorescence / photoacoustic signal of the detection reagent, so the detection reagent can monitor the activity of β-gal in senescent cells in vivo in real time.

[0021] The detection limit of the detection reagent for β-gal is 6.8×10 -4 U / mL.

[0022] The fluorescent / photoacoustic molecular probe Gal-HCy-Biotin of the present invention can successfully monitor the activity of β-gal in the process of tumor aging. The fluorescent / photoacoustic molecular probe Gal-HCy without targeting function is used as a control probe. In vitro experiments show that both molecular probes have good sensitivity and selectivity. Since the catalytic efficiency of β-gal to Gal-HCy-Biotin is higher than that of Gal-HCy, Gal-HCy-Biotin has a lower detection limit than Gal-HCy. The in vivo imaging results further confirm that the overexpressed β-gal in the process of tumor aging can successfully turn on the fluorescence / photoacoustic signal of the probe. Both molecular probes can be used for in vivo imaging of tumor aging, and the molecular probe Gal-HCy-Biotin that can target tumors has better detection performance than the control probe Gal-HCy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the tumor-targeted fluorescent / photoacoustic molecular probe Gal-HCy-Biotin used for fluorescence / photoacoustic dual-modality imaging of β-Gal in aging tumors.

[0024] Figure 2 This is the synthetic route of Gal-HCy-Biotin.

[0025] Figure 3 This is the H NMR spectrum of Gal-HCy-Biotin.

[0026] Figure 4 This is the carbon NMR spectrum of the compound Gal-HCy-Biotin.

[0027] Figure 5 This is the mass spectrum of the compound Gal-HCy-Biotin.

[0028] Figure 6 This is the synthetic route of the compound Gal-HCy.

[0029] Figure 7 This is the carbon NMR spectrum of the compound Gal-HCy.

[0030] Figure 8 This is the carbon NMR spectrum of the compound Gal-HCy.

[0031] Fig. 9 It is the mass spectrum of compound Gal-HCy.

[0032] Fig.10 (a) Fluorescence spectra of 25 μM Gal-HCy-Biton and Gal-HCy with no or 1.2 U / mL β-gal incubated for 2 h. (b) Fluorescence intensity calibration lines fitted for 25 μM Gal-HCy-Biton and Gal-HCy in the linear range of 0 to 1.2 U / mL β-gal. Error bars represent the standard deviation of three independent measurements.

[0033] Fig.11 UV-visible spectra of 25 μM (a) Gal-HCy-Biton and (b) Gal-HCy incubated for 2 h in the absence or presence of 1.2 U / mL β-gal.

[0034] Fig.12(a) Photoacoustic spectra of 25 μM Gal-HCy-Biton and Gal-HCy with no or 1.2 U / mL β-gal incubated for 2 h. (b) Photoacoustic intensity calibration lines fitted for 25 μM Gal-HCy-Biton and Gal-HCy in the linear range of 0 to 1.2 U / mL β-gal. Error bars represent the standard deviation of three independent measurements.

[0035] Fig.13 Fluorescence intensity of 25 μM (a) Gal-HCy-Biton or (b) Gal-HCy acting on other biological disruptors. Error bars represent the standard deviation of three independent measurements. Photoacoustic intensity of 25 μM (c) Gal-HCy-Biton or (d) Gal-HCy acting on other biological disruptors. Error bars represent the standard deviation of three independent measurements.

[0036] Fig.14 The fluorescence intensity of β-gal in the presence of 25 μM (a) Gal-HCy-Biton or (b) Gal-HCy other biological perturbants. The error bars represent the standard deviation of three independent measurements. The photoacoustic intensity of β-gal in the presence of 25 μM (c) Gal-HCy-Biton (d) Gal-HCy other biological perturbants. The error bars represent the standard deviation of three independent measurements.

[0037] Fig.15 RP-HPLC analysis of (a) Gal-HCy-Biotin or (b) Gal-HCy in the absence and presence of β-gal (1.2 U / mL) after incubation for 2 h, wavelength: 550 nm.

[0038] Fig.16 This is the mass spectrum of the product HCy-Biotin after enzyme cleavage.

[0039] Fig.17 The mass spectrum of the product HCy after enzyme cleavage.

[0040] Fig.18 Results of nonlinear regression analysis and fitting of Michaelis-Menten model for β-gal cleavage rate V (μM / min) and different concentrations of Gal-HCy-Biton (a) or Gal-HCy (b).

[0041] Fig.19 This is the overall process of animal experiments.

[0042] Fig. 20 These are photos of tumors from mice treated with Saline and mice treated with Pal after staining with a senescence kit.

[0043] Fig.21 Ki67 staining of tumor sections from mice treated with (a) Saline and (b) Pal.

[0044] Fig. 22 (a) Dynamic time fluorescence imaging of five groups of mouse tumors after probe injection; (b) Quantitative statistical graph of tumor fluorescence intensity of Gal-HCy-Biotin under corresponding conditions in Figure (a); (c) Quantitative statistical graph of tumor fluorescence intensity of Gal-HCy under corresponding conditions in Figure (a); error bars represent the standard deviation of three independent measurements.

[0045] Fig.23 (a) Dynamic time photoacoustic imaging of five groups of mice after probe injection into tumors; (b) Quantitative statistical graph of tumor photoacoustic intensity of Gal-HCy-Biotin under corresponding conditions in Figure (a); (c) Quantitative statistical graph of tumor photoacoustic intensity of Gal-HCy under corresponding conditions in Figure (a); error bars represent the standard deviation of three independent measurements. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further analyzed and explained below through specific embodiments.

[0047] Example 1: Synthesis and Characterization of Gal-HCy-Biotin

[0048] The synthetic route of Gal-HCy-Biotin is as follows:

[0049]

[0050] The specific steps include:

[0051] (1) Synthesis of compound 1: 2,3,3-trimethylbutene (3.18 g, 20.0 mmol) and 3-bromopropylamine (5.25 g, 24.0 mmol) were mixed and stirred at 100°C for 4 hours. The crude product was crystallized from acetone / methanol (5:1, V / V) to obtain a pink solid, namely compound 1.

[0052] (2) Synthesis of compound 2: A mixture of 2-chloro-1-formyl-3-(hydroxymethylene) (200 mg, 1.16 mmol compound) and compound 1 (687 mg, 3.17 mmol) was dissolved in 30 mL of 1-butanol / benzene (7:3, V / V), filtered by Dean-Stark to remove water, and the reaction solution was stirred at 75°C for 6 h. The solvent was removed under vacuum and then dissolved in ethanol. Di-tert-butyl dicarbonate (322 mg, 1.73 mmol) and TEA (242 μL, 1.73 mmol) were added. The mixture was stirred at room temperature for 3 h, and then the solvent was removed under vacuum. The residue was dissolved in ethyl acetate, washed with 1 M hydrochloric acid, water, and brine, dried over anhydrous sodium sulfate, and then the solvent was removed to obtain compound 2.

[0053] (3) Synthesis of HCy: Compound 2 (478.87 mg, 0.91 mmol) was dissolved in 3.0 mL of anhydrous acetonitrile, potassium carbonate (250.66 mg, 1.81 mmol) was added to provide an alkaline environment, and the mixture was stirred at room temperature for 10 min under a nitrogen atmosphere. 1.0 mL of anhydrous acetonitrile solution containing m-diphenol (200 mg, 1.81 mmol) was added dropwise. After the addition, the system was heated to 75°C and stirred at 75°C for 5 h. After the reaction, the solvent was evaporated and the mixture was purified by HPLC to obtain the hemicyanine dye (HCy).

[0054] (4) Synthesis of compound 3: Hemicyanine dye (HCy) (60 mg, 0.107 mmol) and galactose (87.78 mg, 0.213 mmol) were dissolved in acetonitrile, cesium carbonate (69.55 mg, 0.213 mmol) provided an alkaline environment, sodium sulfate (30.32 mg, 0.213 mmol) was used to remove water, and the reaction was carried out at room temperature under nitrogen protection for 8-12 h. The solvent was removed by rotary evaporation, and 20% TFA / CH 2 Cl 2 It was dissolved and stirred at room temperature for 2 h. The solvent was removed by rotary evaporation to obtain compound 3 (26.49 mg, 0.035 mmol).

[0055] (5) Synthesis of compound 4: Compound 3 and biotin (12.82 mg, 0.0525 mmol) were dissolved in DMF, DIEPA (9 μL, 0.0525 mmol) was added dropwise, and then stirred at room temperature for 6 h. Compound 4 (32.35 mg, 0.0329 mmol) was obtained by purification by HPLC.

[0056] (6) Synthesis of Gal-HCy-Biotin: Compound 4 (32.35 mg, 0.0329 mmol) was dissolved in methanol, sodium methoxide (10 mg) was added, and the mixture was stirred at room temperature for 2 h. The solvent was removed by rotary evaporation, and the compound Gal-HCy-Biotin (18.78 mg, 0.023 mmol) was obtained by HPLC purification. 1 H NMR (such as Figure 3 )and 13 C NMR (such as Figure 4 ) and MS (such as Figure 5 ) to confirm the molecular structure.

[0057] 1 H NMR (400 MHz, DMSO-d 6 )δ(ppm):8.57(t,J=13.7Hz,1H),7.94(t,J=5.4Hz,1H),7.66(dd,J=21.4,7.7Hz,2H),7.58-7.36(m,4H),7.14(d,J=2.1Hz,1H),7.03(dt,J=9. 3,4.6Hz,1H),6.51(d,J=15.0Hz,1H),6.33(d,J=18.4Hz,2H),4.94(d,J=7.7Hz,1H),4.36(t,J=7.2Hz,2H),4.24(dd,J=7.6,4.9Hz,1H),4.09- 4.03(m,1H),3.65(ddd,J=23.5,18.7,5.4Hz,5H),3.16(dd,J=12.4,6.5Hz,2H),3.06-2.99(m,1H),2.73-2.68(m,2H),2.64(d,J=4.5Hz,2H),2 .51(d,J=12.4Hz,1H),2.08-1.98(m,2H),1.97-1.85(m,2H),1.85-1.7 7(m,2H),1.72(d,J=11.9Hz,6H),1.59-1.39(m,4H),1.35-1.14(m,4H).

[0058] 13 C NMR (151 MHz, DMSO-d 6)δ(ppm):178.23(1C),172.93(1C),163.31(1C),160.99(1C),153.98(1C),145.78(1C),142.74(1C),141.83(1C), 129.39(1C),128.01(1C),127.68(1C),123.24(1C),116.80(1C),115.06(1C),114.54(1C),113.63(1C),104.94(1C) ),104.03(1C),101.81(1C),76.52(1C),73.76(1C),70.68(1C),68.72(1C),61.60(1C),59.76(2C),55.94(2C),51. 02(2C),44.03(1C),36.43(1C),35.74(2C),28.85(2C),28.60(2C),27.94(2C),25.75(2C),23.99(1C),20.43(1C).

[0059] Example 2: Synthesis and characterization of compound Gal-HCy

[0060] The synthetic route of Gal-HCy is as follows:

[0061]

[0062] Synthesis of Gal-HCy: Compound 3 (30 mg, 0.04 mmol) was dissolved in methanol, sodium methoxide (10 mg) was added, and the mixture was stirred at room temperature for 2 h. The solvent was removed by rotary evaporation, and compound Gal-HCy was obtained by purification by HPLC. 1 HNMR (such as Figure 7 ), 13 CNMR (such as Figure 8 ) and MS (such as Fig. 9 ) to confirm the molecular structure.

[0063] 1 H NMR (400 MHz, DMSO d 6)δ(ppm):8.60(t,J=12.9Hz,1H),7.94(d,J=39.9Hz,3H),7.69(t,J=7.5Hz,2H),7.53(dd,J=15.1, 8.4Hz,3H),7.42(dd,J=14.3,7.0Hz,1H),7.15(d,J=2.0Hz,1H),7.04(dt,J=9.1,4.5Hz,1H),6.51( t,J=14.9Hz,1H),4.94(d,J=7.7Hz,1H),4.45(t,J=7.1Hz,2H),3.74–3.57(m,5H),2.95(s,2H),2.7 4–2.60(m,4H),2.02(dd,J=14.5,7.3Hz,2H),1.80(dd,J=13.2,6.9Hz,2H),1.74(d,J=11.9Hz,6H).

[0064] 13 C NMR (151 MHz, DMSO d 6 )δ(ppm):178.39(1C),160.97(1C),154.02(1C),146.09(1C),142.71(1C),141.81(1C),129.36( 1C),128.00(1C),127.62(1C),123.27(1C),116.82(1C),115.17(1C),114.78(1C),113.54(1C),1 04.91(1C),104.05(1C),101.81(1C),76.54(1C),73.78(1C),70.67(1C),68.70(1C),61.11(2C) ,51.03(2C),42.64(1C),36.84(2C),28.95(1C),28.14(2C),,26.07(1C),24.06(1C),20.42(1C).

[0065] Example 3: In vitro investigation of the response of molecular probes to β-gal

[0066] like Fig.10Figure a shows that after 25μM Gal-HCy-Biotin and Gal-HCy solution (50mM PB, pH=7.3, 2% DMSO) were incubated with 1.2U / mL β-gal at 37°C for 2h, the fluorescence signal increased significantly, indicating that β-gal can successfully release free hemicyanine dye. Specifically, the fluorescence intensity of Gal-HCy-Biotin at 710nm increased by 6.5 times, while the fluorescence intensity of Gal-HCy at 710nm increased by 5.6 times. As the concentration of β-gal gradually increased from 0 to 1.2U / mL, the fluorescence intensity of Gal-HCy-Biotin and Gal-HCy increased linearly ( Fig.10 According to the calculation formula of detection limit LOD = 3σ / k, the detection limits of Gal-HCy-Biotin and Gal-HCy for β-gal are 6.8×10 -4 U / mL and 9.8×10 -4 U / mL.

[0067] Example 4: Visible light absorption and photoacoustic properties of molecular probes

[0068] like Fig.11 As shown in Figure a, in the presence of 1.2 U / mL β-gal, the visible light absorption maximum of 25 μM Gal-HCy-Biotin red-shifted from 600 nm and 650 nm to 655 nm and 700 nm, while the visible light absorption maximum of 25 μM Gal-HCy red-shifted from 597 nm and 650 nm to 675 nm and 700 nm ( Fig.15 Specifically, the absorbance at 700 nm of Gal-HCy-Biotin increased by 15.7 times, and the absorbance at 700 nm of Gal-HCy increased by 12.4 times. Fig.12 As shown in a, the photoacoustic signal of 25 μM Gal-HCy-Biotin and Gal-HCy solution increased significantly after incubation with 1.2 U / mL β-gal at 37°C for 2 hours. Among them, the photoacoustic signal intensity of Gal-HCy-Biotin at 700 nm increased by 6.3 times, while the fluorescence intensity of Gal-HCy at 700 nm increased by 5.7 times. As the concentration of β-gal gradually increased from 0 to 1.2 U / mL, the photoacoustic signal intensity of Gal-HCy-Biotin and Gal-HCy increased linearly ( Fig.12 According to the detection limit calculation formula, the detection limits of Gal-HCy-Biotin and Gal-HCy for β-gal are 3.7×10 -3 U / mL and 7.2×10 -3 U / mL.

[0069] Example 5: Selectivity of molecular probes for β-gal

[0070] like Fig.13 As shown, compared with the presence of 1.2 U / mL β-gal, in the presence of biological thiols (Cys, HCy, GSH), reactive oxygen species (H 2 O 2 , ClO - ) and common tumor markers (alkaline phosphatase (ALP), esterase, γ-glutamyl transpeptidase (GGT)), the fluorescence or photoacoustic intensity of 25μM Gal-HCy-Biotin and Gal-HCyFL can be ignored. In addition, in the mixed solution of the above-mentioned various biological interferences and β-gal, the fluorescence and photoacoustic signals of Gal-HCy-Biotin or Gal-HCy did not change significantly. Therefore, Gal-HCy-Biotin and Gal-HCy can selectively detect β-gal under physiological conditions and have good anti-interference ability.

[0071] The results of HPLC showed that ( Fig.15 ), in the presence of β-gal, the peaks of Gal-HCy-Biotin and Gal-HCy completely disappeared, and the corresponding new peaks were verified by mass spectrometry to be the products of β-gal cleavage, namely Gal-HCy and HCy ( Figure 16-17 ), which indicates that Gal-HCy-Biotin and Gal-HCy completed the cleavage process under the action of β-gal. This confirms that the enhancement of fluorescence / photoacoustic signal is indeed due to the release of free hemicyanine dye HCy by cleavage of β-gal.

[0072] Enzyme kinetic studies have found that ( Fig.18 ), the kinetic parameter turnover number (k) of β-gal to Gal-HCy-Biotin and Gal-HCy cat ) / Michaelis constant (K m ) were 4.6027 μM -1 S -1 and 3.4971 μM -1 S -1 These data indicate that the greater fluorescence / photoacoustic increase of Gal-HCy-Biotin than that of Gal-HCy is due to the higher catalytic efficiency of β-gal on Gal-HCy-Biotin than that of Gal-HCy.

[0073] Example 6: Fluorescence / photoacoustic imaging of β-gal in mouse tumors

[0074] Mouse breast cancer (4T1) cells with high expression of Biotin receptors were selected to construct a mouse tumor model ( Fig.19 5×10 5 4T1 cells were treated with drugs one week later to induce tumor senescence. The mice were randomly divided into two groups. One group of mice was gavaged with 2.5 mg of Palbociclib (Pal) every day for 7 consecutive days. The other group of mice was gavaged with saline every day for 7 consecutive days. Fig. 20 As shown in the figure, compared with the tumors of mice treated with Saline, the tumors of mice treated with Pal showed obvious blue color after staining with the aging kit. The Ki67 staining results of tumor tissues also showed that the tumors of mice treated with Pal did undergo aging ( Fig.21 ).

[0075] Then, we performed senescence imaging of Gal-HCy-Biotin and Gal-HCy in mouse tumors. Pal-treated mice were randomly divided into three groups, with 3 mice in each group. Mice in the Pal+Gal-HCy-Biotin group were injected intratumorally with Gal-HCy-Biotin (250μM, 40μL). Mice in the Pal+Gal-HCy group were injected intratumorally with Gal-HCy (250μM, 40μL).

[0076] The mice in the Gal-HCy-Biotin group were injected intratumorally with a mixture of Biotin and Gal-HCy-Biotin (40 μL, Gal-HCy-Biotin: 250 μM; Biotin: 25 mM). The mice treated with Saline were randomly divided into two groups, with 3 mice in each group. The mice in the Saline+Gal-HCy-Biotin group were injected intratumorally with Gal-HCy-Biotin (250 μM, 40 μL). The mice in the Saline+Gal-HCy group were injected intratumorally with Gal-HCy (250 μM, 40 μL).

[0077] like Fig. 22 As shown in Figure a, the fluorescence signals of the tumors of the three groups of mice treated with Pal increased rapidly and reached a plateau within 1 hour after the probe was injected, and then gradually weakened within the next 4 hours. The fluorescence signals of the tumors of the two groups of mice treated with Saline were very low. Fig. 22 As shown in b and c, in contrast, the fluorescence signal intensity of tumors in mice in the Pal+Gal-HCy-Biotin or Pal+Gal-HCy groups was Saline+

[0078] The fluorescence imaging results confirmed that these two molecular probes can observe the aging state of tumors by imaging β-gal, and Gal-HCy-Biotin has higher sensitivity than Gal-HCy. In addition, the fluorescence signal of the Pal+Gal-HCy-Biotin group is 1.5 times that of the Pal+Biotin+Gal-HCy-Biotin group ( Fig. 22 (b) shows that the interaction between Biotin and receptor can indeed help the probe to be enriched in tumor cells, thereby enhancing the fluorescence signal.

[0079] Fig.23 Figure a shows the photoacoustic images of mouse tumors in five groups. Similar to the trend of fluorescence imaging, the photoacoustic signal of mouse tumors in the Pal+Gal-HCy-Biotin group increased significantly and reached a maximum at 1 hour, while the photoacoustic signal of mouse tumors in the Pal+Gal-HCy group increased only slightly at 1 hour and then decreased. Quantitative analysis results showed that the photoacoustic signal intensity of the Pal+Gal-HCy-Biotin group was 3.8 times higher than that of the Saline+Gal-HCy-Biotin group, while the photoacoustic signal intensity of the Pal+Gal-HCy group was 3.8 times higher than that of the Saline+

[0080] The Gal-HCy group was 1.3 times higher ( Fig.23 bc). In addition, the photoacoustic signal of mouse tumors in the Pal+Biotin+Gal-HCy-Biotin group was 2.2 times lower than that in the Pal+Gal-HCy-Biotin group. These results confirm that Gal-HCy-Biotin and Gal-HCy can effectively image tumor senescence in mice, and that Gal-HCy-Biotin, which can target tumors, has better imaging performance than Gal-HCy.

[0081] in conclusion:

[0082] In summary, we developed a fluorescent / photoacoustic molecular probe Gal-HCy-Biotin for imaging tumor aging, which can successfully monitor the activity of β-gal during tumor aging. The fluorescent / photoacoustic molecular probe Gal-HCy without targeting function was used as a control probe. In vitro experiments showed that both molecular probes have good sensitivity and selectivity. Since the catalytic efficiency of β-gal for Gal-HCy-Biotin is higher than that of Gal-HCy, Gal-HCy-Biotin has a lower detection limit than Gal-HCy. The in vivo imaging results further confirmed that overexpressed β-gal during tumor aging can successfully turn on the fluorescence / photoacoustic signal of the probe. Both molecular probes can be used for in vivo imaging of tumor aging, and the molecular probe Gal-HCy-Biotin that can target tumors has better detection performance than the control probe Gal-HCy.

Claims

1. A fluorescent / photoacoustic molecular probe for imaging tumor senescence, referred to as Gal-HCy-Biotin, characterized in that Its structural formula is as follows: 。 2. A method for preparing the fluorescent / photoacoustic molecular probe according to claim 1, characterized in that The synthetic route is as follows: 。 3. An application of the fluorescent / photoacoustic molecular probe according to claim 1, characterized in that: The fluorescent / photoacoustic molecular probe is used to prepare a tumor-targeting detection reagent; β-gal overexpressed during tumor aging can turn on the fluorescent / photoacoustic signal of the detection reagent, so the detection reagent can monitor the activity of β-gal in aging cells in vivo in real time; the tumor is breast cancer.

4. The use according to claim 3, characterized in that: The detection limit of the detection reagent for β-gal is 6.8×10 -4 U / mL.

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