A fluorescent / photoacoustic molecular probe targeting tumor and application thereof

Abstract

This invention discloses a fluorescent / photoacoustic molecular probe targeting tumors and its applications. The fluorescent / photoacoustic molecular probe, abbreviated as Biotin-DEVD-HCy, has the following structural formula: Under the action of the Biotin targeting group, the molecular probe binds to the biotin receptor highly expressed on the surface of cancer cells, allowing it to enter and accumulate within the cancer cells. Subsequently, Biotin-DEVD-HCy is slowly cleaved by Caspase-3 overexpressed in apoptotic cancer cells, generating a hemicyanine fluorophore derivative containing free amino groups. Due to the intramolecular charge transfer process, this fluorophore emits a strong near-infrared fluorescence / photoacoustic signal, which can be used for fluorescence / photoacoustic imaging of tumor apoptosis, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to a fluorescent / photoacoustic molecular probe for targeting tumors and its applications. Background Technology

[0002] Apoptosis is a programmed cell death that occurs during cell development or under the influence of certain factors, through the regulation of intracellular genes and their products. It is commonly involved in various processes, such as physiological cell turnover, embryogenesis, and the development of the immune system. Furthermore, with the development of various serious diseases, such as cancer, atherosclerosis, autoimmune diseases, and Alzheimer's disease, the apoptosis program can become disordered. The caspase family consists of caspase-specific proteases that play a crucial role in controlling inflammation and cell death. Caspase-3 is a key protease in the apoptosis process, and its mediated protein cleavage plays a vital role in the molecular mechanisms of apoptosis. Therefore, monitoring caspase-3 activity can be used to observe the dynamic process of apoptosis and perform real-time imaging of the apoptosis process, which is of great significance for the early diagnosis and progression of various diseases.

[0003] In recent years, researchers have developed various molecular probes based on inorganic or organic structures for fluorescence, magnetic resonance, photoacoustic, and radionuclide imaging of Caspase-3 in vitro and in vivo. According to a report in *Analytical Chemistry* (Anal. Chem., 2021, 93, 2045), Zhao et al. designed a molecular probe based on cell-permeable peptides that generates ratiometric fluorescence signals in response to Caspase-3. This ratiometric fluorescent probe can image Caspase-3 with high sensitivity in vitro to predict cell apoptosis, but its limited fluorescence penetration depth prevents its use in vivo imaging. *Angewandte Chemie* (Angew. Chem., 2019, 131, 4940) reported that Ye et al. developed a photoacoustic molecular probe for imaging Caspase-3 in chemotherapy-induced tumor apoptosis. Utilizing the high spatial resolution and penetration depth of photoacoustic imaging, this photoacoustic molecular probe can be used to analyze the distribution of apoptosis signals throughout tumor tissue, facilitating early, real-time evaluation of in vivo tumor treatment efficacy. Despite significant progress in molecular probes for detecting and imaging Caspase-3, there is still a need to develop superior molecular probes that can monitor Caspase-3 activity in vivo in real time.

[0004] Multimodal imaging technology is rapidly developing, providing more accurate images and more comprehensive information for in vivo studies by combining two or more imaging modalities. Optical imaging has advantages such as high sensitivity, non-invasiveness, and low cost. However, it has low spatial resolution and poor depth penetration. Photoacoustic imaging, a non-invasive biomedical imaging technique, has high spatial resolution and deep tissue penetration capabilities; however, its sensitivity still needs further improvement. Fluorescence / photoacoustic multimodal imaging can achieve both highly sensitive functional imaging and high-resolution structural and histological information, ultimately achieving a perfect unity of functional and structural tissue imaging, and will have broader application prospects in scientific research and clinical medicine. To date, researchers have only reported one photoacoustic / magnetic resonance molecule and one fluorescence / photoacoustic bimodal molecular probe; a Caspase-3 bimodal imaging molecular probe for imaging tumor apoptosis is still lacking. Therefore, we have developed a tumor-targeting fluorescence / photoacoustic molecular probe to image tumor apoptosis by monitoring Caspase-3 activity. Summary of the Invention

[0005] This invention addresses the shortcomings of the prior art by providing a fluorescent / photoacoustic molecular probe for targeting tumors and its applications. The molecular probe of this invention can be used for fluorescence / photoacoustic imaging of tumor apoptosis, and has promising application prospects.

[0006] The fluorescent / photoacoustic molecular probe for targeting tumors in this invention, abbreviated as Biotin-DEVD-HCy, has the following structural formula:

[0007]

[0008] The structure of the fluorescent / photoacoustic molecular probe (Biotin-DEVD-HCy) of this invention consists of three parts:

[0009] (1) Targeting tumor components: Biotin;

[0010] (2) Caspase-3-specific cleavage of the polypeptide substrate: Asp-Glu-Val-Asp (DEVD);

[0011] (3) Fluorescence / photoacoustic imaging part: hemicyanine dye (HCy).

[0012] The molecular probe of this invention, under the action of the biotin targeting group, binds to the biotin receptor highly expressed on the surface of cancer cells, allowing it to enter and accumulate within the cancer cells. Subsequently, Biotin-DEVD-HCy is slowly cleaved by Caspase-3 overexpressed in apoptotic cancer cells, generating a hemicyanine fluorophore derivative containing free amino groups. Due to the intramolecular charge transfer process, this fluorophore emits a strong near-infrared fluorescence / photoacoustic signal, which can be used for fluorescence / photoacoustic imaging of tumor apoptosis, showing promising application prospects. A fluorescent / photoacoustic molecular probe without targeting function (Ac-DEVD-HCy) is used as a control probe.

[0013] The present invention provides a method for synthesizing fluorescent / photoacoustic molecular probes targeting tumors, comprising the following steps:

[0014] Step 1: Synthesis of HCY

[0015] Weigh N-Boc-3-aminophenol (80 mg, 0.2 mmol) and dissolve it in 3.0 mL of water. In CH3CN, under a nitrogen atmosphere, K2CO3 (56 mg, 0.4 mmol) was added to a three-necked flask. After stirring for 30 min, IR775 (100 mg, 0.2 mmol) was dissolved in 3.0 mL of CH3CN and added to the three-necked flask. The mixture was heated to 70 °C and reacted for 2 h. The solution was then concentrated under vacuum, and dichloromethane (1.5 mL), TFA (0.5 mL), and triisopropylsilane (400 μL) were added. After stirring for 3 h to complete the removal of Boc, the solution was dissolved in CH3CN and injected into a three-necked flask containing K2CO3 (52 mg, 0.4 mmol) under a nitrogen atmosphere. The mixture was reacted at 70 °C for 7 h. The product was purified by RP-HPLC (organic phase: CH3OH + 1% TFA; aqueous phase: H2O + 1% TFA. Organic phase to aqueous phase volume ratio = 5.5:4.5) to obtain compound HCy.

[0016] Step 2: Synthesis of Compound A

[0017] Fmoc-DEVD was synthesized using solid-phase peptide synthesis (SPPS). The synthesis was achieved using 2-chlorotrimethylol chloro resin (800 mg, 0.88 mmol) and amino acids with the corresponding Fmoc side-chain protecting groups.

[0018] FmocNH-Asp(OTBU)-Glu(OTBU)-Val-Asp(OTBU)-Resin; then Fmoc-DEVD was cleaved from the resin with 1% TFA / CH2Cl2 solution, precipitated with cold diethyl ether, and purified by RP-HPLC (organic phase: CH3CN + 1% TFA; aqueous phase: H2O + 1% TFA. Organic phase to aqueous phase volume ratio = 6:4) to give yellow compound A (150.5 mg).

[0019] Step 3: Synthesis of Compound B

[0020] Compound A (87 mg, 0.1 mmol), HATU (114 mg, 0.3 mmol), and DIPEA (26 μL, 0.2 mmol) were dissolved in a DCM / DMF mixture and reacted in an ice-water bath for 30 min. Then, HCy (58 mg, 0.15 mmol) was dissolved in DCM and added to the mixture, which was stirred overnight at room temperature. DCM and DMF were removed by rotary evaporator and vacuum dryer. The mixture was then purified by RP-HPLC (organic phase: CH3OH + 1% TFA; aqueous phase: H2O + 1% TFA. Organic phase to aqueous phase volume ratio = 7:3) to obtain compound B (90 mg).

[0021] Step 4: Synthesis of Bio-DEVD-HCy

[0022] Compound B was dissolved in 1 mL of DMF solution, and 50 μL of piperidine was added under ice-water bath conditions. After reacting for 15 min, Fmoc removal was completed. The product was then purified by RP-HPLC and dried by rotary evaporator and vacuum dryer to obtain the first-step intermediate. The first-step intermediate (101 mg, 0.1 mmol), HATU (114 mg, 0.3 mmol), and DIPEA (26 μL, 0.2 mmol) were dissolved in a dichloromethane / DCM / DMF mixture and reacted at 0 °C for 30 min. Biotin (37 mg, 0.15 mmol) was then dissolved in DCM / DMF and added to the mixture. The mixture was stirred overnight at room temperature. After removing DCM and DMF using a rotary evaporator and a vacuum dryer, the second intermediate was obtained. Dichloromethane (1.34 mL), TFA (0.66 mL), and triisopropylsilane (400 μL) were added to the second intermediate, and the mixture was stirred for 3 h to complete OTBU removal. Finally, the compound Bio-DEVD-HCy (64 mg) was purified by RP-HPLC. Each reaction step was purified by HPLC to confirm purity. 1 HNMR, 13 The molecular structure was confirmed by C NMR and mass spectrometry. Figure 3 , Figure 4 , Figure 5 ).

[0023] The synthetic route of Bio-DEVD-HCy in this invention is shown below:

[0024]

[0025] The application of the fluorescent / photoacoustic molecular probe of this invention is for the preparation of probe formulations targeting tumors. These probe formulations can reflect the tumor apoptosis process by monitoring Caspase-3 activity.

[0026] The molecular probe of this invention, under the action of the biotin targeting group, binds to the biotin receptor highly expressed on the surface of cancer cells, allowing it to enter and accumulate within the cancer cells. Subsequently, Biotin-DEVD-HCy is slowly cleaved by Caspase-3 overexpressed in apoptotic cancer cells, generating a hemicyanine fluorophore derivative containing free amino groups. Due to the intramolecular charge transfer process, this fluorophore emits a strong near-infrared fluorescence / photoacoustic signal, which can be used for fluorescence / photoacoustic imaging of tumor apoptosis, showing promising application prospects. Attached Figure Description

[0027] Figure 1 A schematic diagram of the fluorescence / photoacoustic dual-modal imaging of Caspase-3 in apoptotic tumors using the fluorescent / photoacoustic molecular probe Bio-DEVD-HCy, which targets tumors.

[0028] Figure 2 The 1H NMR spectrum of the compound Bio-DEVD-HCy is shown.

[0029] Figure 3 The image shows the carbon NMR spectrum of the compound Bio-DEVD-HCy.

[0030] Figure 4 This is the mass spectrum of the compound Bio-DEVD-HCy.

[0031] Figure 5 The 1H NMR spectrum of Ac-DEVD-HCy.

[0032] Figure 6 The NMR carbon spectrum of Ac-DEVD-HCy.

[0033] Figure 7 Mass spectrometry of Ac-DEVD-HCy.

[0034] Figure 8 (a) Fluorescence spectra of 25 μM Bio-DEVD-HCy and Ac-DEVD-HCy with and without 200 μg / mL Caspase-3 after incubation for 8 h; (b) Fluorescence intensity calibration lines fitted to the linear range of 0–8 μg / mL Caspase-3 for 25 μM Bio-DEVD-HCy and Ac-DEVD-HCy. Error bars represent the standard deviations of three independent measurements.

[0035] Figure 9The UV-Vis spectra of 25 μM (a) Bio-DEVD-HCy and (b) Ac-DEVD-HCy after incubation for 8 h with or without 200 μg / mL Caspase-3 are shown.

[0036] Figure 10 (a) Photoacoustic spectra of 25 μM Bio-DEVD-HCy and Ac-DEVD-HCy with and without 200 μg / mL Caspase-3 after incubation for 8 h. (b) Photoacoustic intensity calibration lines fitted to 25 μM Bio-DEVD-HCy and Ac-DEVD-HCy within the linear range of 0–10 μg / mL Caspase-3. Error bars represent the standard deviations of three independent measurements.

[0037] Figure 11 Fluorescence intensity of 25 μM (a) Bio-DEVD-HCy or (b) Ac-DEVD-HCy interacting with other biological interfering agents. Error bars represent the standard deviation of three independent measurements; photoacoustic intensity of 25 μM (c) Bio-DEVD-HCy or (d) Ac-DEVD-HCy interacting with other biological interfering agents. Error bars represent the standard deviation of three independent measurements.

[0038] Figure 12 RP-HPLC analysis of 25 μM (a) Bio-DEVD-HCy or (b) Ac-DEVD-HCy incubated with Caspase-3 (200 μg / mL) in the absence and presence of Caspase-3, respectively, for 8 h. Wavelength: 550 nm.

[0039] Figure 13 The results of nonlinear regression analysis and fitting of the Michaelis-Menten model to the Caspase-3 cleavage rate V (μM / min) with different concentrations of Bio-DEVD-HCy (a) or Ac-DEVD-HCy (b) are presented.

[0040] Figure 14 This is the general procedure for animal experiments.

[0041] Figure 15 For (a) daily body weight changes in mice in the DOX or Saline group over 8 days, and (b) tumor volume statistics in mice in the DOX or Saline group over 8 days. *p<0.05, **p<0.01. Error bars represent the standard deviations of three independent measurements.

[0042] Figure 16 Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end marker (TUNEL) was measured in tumor tissues from DOX or Saline mice. Scale bar = 100 μm.

[0043] Figure 17 Figure (a) shows the dynamic time-lapse fluorescence imaging of tumors in five groups of mice after probe injection; (b) shows the quantitative statistical graph of tumor fluorescence intensity of Bio-DEVD-HCy under corresponding conditions in Figure (a); (c) shows the quantitative statistical graph of tumor fluorescence intensity of Ac-DEVD-HCy under corresponding conditions in Figure (a); the error bars represent the standard deviations of three independent measurements.

[0044] Figure 18 (a) Dynamic time-lapse photoacoustic imaging of tumors in five groups of mice after probe injection; (b) Quantitative statistical graph of tumor photoacoustic intensity of Bio-DEVD-HCy under corresponding conditions in Figure (a); (c) Quantitative statistical graph of tumor photoacoustic intensity of Ac-DEVD-HCy under corresponding conditions in Figure (a); Error bars represent the standard deviations of three independent measurements. Detailed Implementation

[0045] The technical solution of the present invention will be further analyzed and explained through specific embodiments below.

[0046] Example 1: Synthesis and Characterization of Bio-DEVD-HCy

[0047] 1. Synthesis of HCY

[0048] Weigh N-Boc-3-aminophenol (80 mg, 0.2 mmol) and dissolve it in 3.0 mL of CH3CN. Add this solution to a three-necked flask containing K2CO3 (56 mg, 0.4 mmol) under N2 atmosphere. After stirring for 30 min, dissolve IR775 (100 mg, 0.2 mmol) in 3.0 mL of CH3CN and add it to the three-necked flask. Heat to 70 °C and react for 2 h. Then concentrate the solution under vacuum to obtain an intermediate product. Add dichloromethane (1.5 mL), TFA (0.5 mL), and triisopropylsilane (400 μL) to the intermediate product. After stirring for 3 h to complete Boc removal, dissolve the intermediate product in CH3CN and inject it into a three-necked flask containing K2CO3 (52 mg, 0.4 mmol) under N2 atmosphere. React at 70 °C for 7 h. Purify by RP-HPLC to obtain compound HCy.

[0049] 2. Synthesis of Compound A

[0050] Fmoc-DEVD was synthesized using solid-phase peptide synthesis (SPPS). The synthesis was achieved using 2-chlorotrimethylol chloro resin (800 mg, 0.88 mmol) and amino acids with the corresponding Fmoc side-chain protecting groups.

[0051] Resin-Asp(OTBU)-Val-Glu(OTBU)-Asp(OTBU)-NHFmoc; then Fmoc-DEVD was cleaved from the resin with 1% TFA / CH2Cl2 solution, precipitated with cold diethyl ether, and purified by RP-HPLC to obtain yellow compound A (150.5 mg).

[0052] 3. Synthesis of Compound B

[0053] Fmoc-Asp(OTBU)-Glu(OTBU)-Val-Asp(OTBU), HATU and DIPEA were dissolved in a DCM / DMF mixture and reacted in an ice-water bath for 30 min. Then, compound A was dissolved in DCM and added to the mixture, and stirred overnight at room temperature. DCM and DMF were removed by rotary evaporator and vacuum dryer, and compound B was obtained by RP-HPLC purification.

[0054] 4. Synthesis of Bio-DEVD-HCy

[0055] Compound B was dissolved in 1 mL of DMF solution, and 50 μL of piperidine was added under ice-water bath conditions. After reacting for 15 min, Fmoc removal was completed. The mixture was then purified by RP-HPLC and dried using a rotary evaporator and vacuum dryer to obtain the first-step intermediate. The first-step intermediate, HATU, and DIPEA were dissolved in a dichloromethane / DCM / DMF mixture and reacted at 0 °C for 30 min. Biotin was then dissolved in DCM / DMF and added to the mixture. The mixture was stirred overnight at room temperature. DCM and DMF were removed using a rotary evaporator and vacuum dryer to obtain the second-step intermediate. Dichloromethane (1.34 mL), TFA (0.66 mL), and triisopropylsilane (400 μL) were added to the second-step intermediate, and the mixture was stirred for 3 h to complete OTBU removal. Finally, the compound Bio-DEVD-HCy was purified by RP-HPLC. Each reaction step was purified by HPLC to confirm purity. 1 HNMR, 13 The molecular structure was confirmed by C NMR and mass spectrometry. Figure 2 , Figure 3 , Figure 4 ).

[0056] 1H NMR spectroscopy results of Bio-DEVD-HCy: 1HNMR(600MHz, DMSO-d6) δ 8.60 (t, J = 9.7 Hz, 1H), 8.52 (t, J = 10.9 Hz, 1H), 8.13 (t, J = 9.8 Hz, 1H), 8.04 (s, 1H), 7.92 (d, J = 7.2 Hz, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.74 (dd, J = 16.5, 7.5 Hz, 1H), 7.67–7.60 (m, 1H), 7.51 (d, J = 3.4 Hz, 1H), 7.49 (d, J = 5.8 Hz, 1H), 7.43 (d, J = 6.9 Hz, 1H), 7.38–7.33 (m, 1H), 6.52 (t, J = 12.6 Hz, 1H), 6.33 (s, 1H), 5.32–5.23 (m, 1H), 4.73 (dd, J = 14.9, 7.7 Hz, 1H), 4.56–4.48 (m, 1H), 4.31–4.23 (m, 2H), 4.07 (dd, J = 7.3, 4.6 Hz, 1H), 4.00 (d, J = 20.1 Hz, 1H), 3.84 (s, 2H), 3.12 (s, 3H), 3.01 (dd, J = 11.2, 7.3 Hz, 2H), 2.83 (d, J = 15.3 Hz, 1H), 2.67 (d, J = 18.0 Hz, 4H), 2.63 (dd, J = 10.6, 5.5 Hz, 4H), 2.21–2.14 (m, 2H), 2.05 (dd, J = 17.0, 9.7 Hz, 3H), 1.95–1.89 (m, 2H), 1.81–1.77 (m, 2H), 1.71 (s, 6H), 1.44 (d, J = 6.7 Hz, 3H), 1.18 (s, 4H), 0.84 (d, J = 6.6 Hz, 3H), 0.81 (d, J = 6.5 Hz, 3H).

[0057] Carbon spectrum analysis results of Bio-DEVD-HCy: 13C NMR(150MHz,DMSO-d6)δ178.40(1C),174.59(1C),173.61(1C),172.24(2C ),171.91(1C),71.50(2C),170.68(1C),164.09(1C),153.38(1C),145.24( 1C),143.65(1C),142.79(2C),142.48(1C),142.24(1C),130.16(1C),129. 35(1C),128.64(1C),127.77(1C),123.21(1C),117.86(1C),117.76(1C),1 17.11(1C),114.45(1C),113.82(1C),105.73(1C),65.75(1C),61.34(1C), 59.43(1C),55.44(1C),51.36(1C),0.87(2C),50.18(1C),36.30(1C),35.6 3(1C),33.20(2C),31.79(1C),29.53(2C),29.25(1C),28.69(1C),27.62(1 C),27.06(2C),25.63(1C),20.39(1C),19.48(2C),19.15(1C),14.44(2C).

[0058] Example 2: Synthesis and Characterization of Ac-DEVD-HCy

[0059] The synthetic route of Ac-DEVD-HCy is shown below:

[0060]

[0061] Synthesis of Compound C: Ac-DEVD was synthesized using solid-phase peptide synthesis (SPPS). It was synthesized using 2-chlorotrimethylchloro resin (800 mg, 0.88 mmol) and amino acids with the corresponding Fmoc side-chain protecting groups.

[0062] Resin-Asp(OTBU)-Val-Glu(OTBU)-Asp(OTBU)-NHAc. Ac-DEVD was then cleaved from the resin with 1% TFA / CH2Cl2 solution, precipitated with cold diethyl ether, and purified by RP-HPLC to obtain yellow compound C.

[0063] Synthesis of compound Ac-DEVD-HCy: Compounds C, HATU, and DIPEA were dissolved in a mixture of dichloromethane (DCM) and N,N-dimethylformamide (DMF). The mixture was reacted for 30 min in an ice-water bath (0 °C). Then, HCy was dissolved in DCM and added to the mixture. The mixture was stirred overnight at room temperature. After removing DCM and DMF using a rotary evaporator and a vacuum dryer, an intermediate product was obtained. Dichloromethane (1.34 mL), TFA (0.66 mL), and triisopropylsilane (400 μL) were added to the intermediate product, and the mixture was stirred for 3 h to complete the removal of OTBU. Finally, Ac-DEVD-HCy was obtained after purification by RP-HPLC. Each reaction step was purified by HPLC to confirm purity. 1 HNMR, 13 The molecular structure was confirmed by C NMR and mass spectrometry. Figure 5 , Figure 6 , Figure 7 ).

[0064] 1H NMR spectroscopy results of Ac-DEVD-HCy: 1 H NMR (600MHz, DMSO-d6) δ10.25(d,J=10.2Hz,1H),8.63(dd,J=12.7,5.1Hz,1H),8. 56(d,J=12.7Hz,1H),8.26–8.19(m,1H),8.09(dd,J=15.7,5.9Hz,1H),8.00(dd,J= 15.3,6.3Hz,1H),7.89–7.85(m,1H),7.83–7.79(m,1H),7.70–7.67(m,1H),7.55– 7.53(m,1H),7.47(d,J=8.1Hz,1H),7.41(dd,J=8.9,4.5Hz,1H),6.61–6.56(m,1H) ,4.77(s,1H),4.55(d,J=5.1Hz,3H),4.29(d,J=16.1Hz,1H),4.10–4.05(m,1H),3 .89(s,1H),2.89(d,J=7.4Hz,1H),2.76–2.70(m,6H),2.69–2.66(m,1H),2.63(d,J =7.1Hz,1H),2.28–2.16(m,1H),1.98(ddd,J=24.6,11.9,5.6Hz,1H),1.83(s,1H), 1.77–1.74(m,1H),1.23(dd,J=31.4,10.3Hz,1H),0.89–0.87(m,1H),0.86(s,1H).

[0065] Carbon spectroscopy analysis results of Ac-DEVD-HCy:13 C NMR(150MHz,DMSO-d6)δ178.45(1C),171.50(4C),153.38(1C),142.79(2C),132.63(1C), 129.35(2C),128.64(1C),117.11(1C),114.45(1C),105.84(1C),52.12(1C),50.95(1C), 50.37(2C),49.95(1C),34.41(1C),33.20(1C),32.01(2C),31.79(1C),31.30(1C),30.93 (1C),30.81(1C),30.52(1C),29.59(1C),27.56(2C),25.63(1C),23.11(1C),18.47(2C).

[0066] Example 3: In vitro data characterization

[0067] After synthesizing and characterizing Bio-DEVD-HCy and Ac-DEVD-HCy, we investigated the fluorescence properties of the two probes in response to Caspase-3. Figure 8 As shown in Figure a, when 200 μg / mL Caspase-3 was present in the buffer solution (20 mM PIPES, 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 0.1% CHAPS, 10% Sucrose, pH = 7.4), the fluorescence intensity of 25 μM Bio-DEVD-HCy at 720 nm increased 5.5-fold after 8 h, while that of 25 μM Ac-DEVD-HCy increased 4.6-fold after 8 h. As the concentration of Caspase-3 gradually increased from 0 to 8 μg / mL, the fluorescence intensity of both 25 μM Bio-DEVD-HCy and Ac-DEVD-HCy at 720 nm increased linearly after 8 h. Figure 8 (b) Calculations showed that the limits of detection for Caspase-3 by Bio-DEVD-HCy and Ac-DEVD-HCy were 1.09 nM and 2.63 nM, respectively.

[0068] Next, we investigated the visible light absorption and photoacoustic properties of these two probes. For example... Figure 9 As shown, after incubation with 200 μg / mL Caspase-3 for 8 h, the maximum visible absorption peak of 25 μM Bio-DEVD-HCy or Ac-DEVD-HCy red-shifted from 593 nm to 675 nm. Specifically, the absorbance value at 675 nm for Bio-DEVD-HCy increased by 3.3 times, and the absorbance value at 675 nm for Ac-DEVD-HCy increased by 2.7 times. Figure 10As shown in Figure a: In the presence of 200 μg / mL Caspase-3, the photoacoustic intensity of 25 μM Bio-DEVD-HCy at 675 nm increased by 3.1 times, while that of 25 μM Ac-DEVD-HCy increased by 2.5 times. The photoacoustic image at 675 nm further confirms that the photoacoustic signal enhancement of Bio-DEVD-HCy is greater than that of Ac-DEVD-HCy. Furthermore, as the concentration of Caspase-3 gradually increased from 0 to 10 μg / mL, the photoacoustic intensity of both 25 μM Bio-DEVD-HCy and Ac-DEVD-HCy increased linearly at 670 nm after 8 h. Figure 10 (b) Calculations showed that the limits of detection for Caspase-3 were 4.38 nM and 8.90 nM for Bio-DEVD-HCy and Ac-DEVD-HCy, respectively.

[0069] We then investigated the selectivity of these two probes for other biological disruptors. For example... Figure 11 As shown, in biothiols (GSH, HCy, Cys), reactive oxygen species (H2O2, ClO) - In the presence of Caspase-3 and common tumor markers (γ-glutamyl transferase (GGT), alkaline phosphatase (ALP), and β-galactosidase (β-Gal),) the fluorescence and photoacoustic changes of Bio-DEVD-HCy and Ac-DEVD-HCy were minimal. Selectivity assays indicated that Bio-DEVD-HCy and Ac-DEVD-HCy exhibit good selectivity for Caspase-3 in the biological environment, suggesting their potential application in in vivo studies of Caspase-3.

[0070] High-performance liquid chromatography (HPLC) results showed that, in the presence of Caspase-3 for 8 hours, 48% of Bio-DEVD-HCy was cleaved by Caspase-3 to form the hemicyanine dye HCy, while only 37% of Ac-DEVD-HCy was converted to HCy. Figure 13 This confirms that the enhancement of the fluorescence / photoacoustic signal is indeed due to the release of free hemicyanine dye HCy by the cleavage of Caspase-3.

[0071] Enzyme kinetic studies revealed that Caspase-3 exhibits a significant effect on the kinetic parameter flip number (k) of Bio-DEVD-HCy and Ac-DEVD-HCy. cat ) / Michaelis constant (K m The values ​​are 0.32 × 10⁻⁶ respectively. 5 M -1 S -1 and 0.26×10 5 M -1 S -1( Figure 12 These data indicate that the greater fluorescence / photoacoustic increase of Bio-DEVD-HCy compared to Ac-DEVD-HCy is due to the higher catalytic efficiency of Caspase-3 for Bio-DEVD-HCy compared to Ac-DEVD-HCy.

[0072] Example 4: Fluorescence imaging of Caspase-3 in mouse tumors

[0073] A mouse tumor model was established using mouse breast cancer (4T1) cells that highly express the biotin receptor. Six-week-old C57BL / 6 mice were subcutaneously injected with 5 × 10⁵ cells into the left leg. 5 Four T1 cells were injected, and after one week, tumor apoptosis was induced by drug treatment. Mice were randomly divided into two groups. One group received 0.2 mg of doxorubicin (DOX) via tail vein injection every three days for a total of three injections. The other group received saline via tail vein injection every three days for a total of three injections. Figure 14 ).like Figure 15 As shown in Figure a, compared with Saline-treated mice, DOX-treated mice showed a decrease in body weight and a significant inhibition of tumor volume. Figure 15 b). TUNEL staining results of tumor tissue showed that apoptosis did indeed occur in the mouse tumors treated with DOX. Figure 16 ).

[0074] Subsequently, we performed Caspase-3 imaging of Bio-DEVD-HCy and Ac-DEVD-HCy within mouse tumors. DOX-treated mice were randomly divided into three groups of three. Mice in the DOX+Bio-DEVD-HCy group received intratumoral injections of Bio-DEVD-HCy (40 μL, 250 μM). Mice in the DOX+Ac-DEVD-HCy group received intratumoral injections of Ac-DEVD-HCy (40 μL, 250 μM). Mice in the DOX+Biotin+Bio-DEVD-HCy group received intratumoral injections of a mixture of Biotin and Bio-DEVD-HCy (40 μL, Bio-DEVD-HCy: 250 μM; Biotin: 25 mM). Saline-treated mice were randomly divided into two groups of three. Mice in the Saline+Bio-DEVD-HCy group received intratumoral injections of Bio-DEVD-HCy (40 μL, 250 μM). Mice in the Saline+Ac-DEVD-HCy group were injected intratumorally with Ac-DEVD-HCy (40 μL, 250 μM).

[0075] like Figure 17As shown in Figure a, the fluorescence signal in the tumors of mice in the DOX+Bio-DEVD-HCy and DOX+Ac-DEVD-HCy groups gradually increased within 4 hours after probe injection, and then gradually decreased. In contrast, the tumor sites of mice in the Saline+Bio-DEVD-HCy and Saline+Ac-DEVD-HCy groups showed weak fluorescence signals. Quantitative fluorescence analysis showed that the fluorescence intensity in the DOX+Bio-DEVD-HCy and DOX+Ac-DEVD-HCy groups increased by 2.5-fold and 1.6-fold, respectively, at 4 hours. Figure 17 (bc). Fluorescence imaging results confirmed that Caspase-3 overexpression during tumor apoptosis successfully activated the probe's fluorescence signal. Compared with the DOX+Bio-DEVD-HCy group, the fluorescence signal of mouse tumors in the DOX+Biotin+Bio-DEVD-HCy group was reduced by 1.6 times, indicating that Biotin can indeed help the probe target and enter tumor cells, thereby enhancing the fluorescence signal.

[0076] like Figure 18 As shown in figure a, the photoacoustic imaging trend of tumors in the five groups of mice was similar to that of fluorescence imaging. At 4 h, compared with the Saline+Bio-DEVD-HCy or Saline+Ac-DEVD-HCy groups, the DOX+Bio-DEVD-HCy or DOX+

[0077] The photoacoustic intensity of the Ac-DEVD-HCy group was increased by 4.1 times and 1.9 times, respectively. Figure 18 (bc). The photoacoustic signal of mouse tumors in the DOX+Bio-DEVD-HCy group was 3.5 times stronger than that in the DOX+Biotin+Bio-DEVD-HCy group. The photoacoustic imaging results of mouse tumors further validated that Bio-DEVD-HCy and Ac-DEVD-HCy can reflect the state of tumor apoptosis by monitoring Caspase-3 activity, and that the imaging performance of targeted Bio-DEVD-HCy is superior to that of Ac-DEVD-HCy.

[0078] in conclusion:

[0079] In summary, we developed a tumor-targeting fluorescent / photoacoustic molecular probe, Bio-DEVD-HCy, which successfully reflects tumor apoptosis by monitoring Caspase-3 activity. The non-targeting fluorescent / photoacoustic molecular probe, Ac-DEVD-HCy, was used as a control probe. In vitro experiments showed that both probes exhibited good sensitivity and selectivity. Because Caspase-3 has a higher catalytic efficiency for Bio-DEVD-HCy than Ac-DEVD-HCy, Bio-DEVD-HCy exhibits superior detection performance. In vivo imaging results further confirmed that Caspase-3 overexpression during tumor apoptosis successfully activates the probe's fluorescent / photoacoustic signal. Both probes can be used for in vivo imaging of tumor apoptosis, and the tumor-targeting probe, Bio-DEVD-HCy, is more sensitive than the control probe, Ac-DEVD-HCy.

Claims

1. A fluorescent / photoacoustic molecular probe targeting tumors, abbreviated as Biotin-DEVD-HCy, characterized in that... Its structural formula is shown below:

2. An application of the fluorescent / photoacoustic molecular probe according to claim 1, characterized in that: A probe formulation targeting tumors was prepared using the fluorescent / photoacoustic molecular probe, and the probe formulation reflects the tumor apoptosis process by monitoring Caspase-3 activity.

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