Fluorescent / photoacoustic dual-mode diagnosis and treatment probe for detecting and photodynamically removing senescent cells, and preparation method and application of fluorescent / photoacoustic dual-mode diagnosis and treatment probe

Abstract

The invention discloses a fluorescent / photoacoustic dual-mode diagnosis and treatment probe for detection and photodynamic removal of senescent cells, a preparation method and application, the probe takes a sulfur-substituted hemicyanine dye as a parent structure, a novel bromo-glycoside ligand is covalently coupled, and a blue-green solid is obtained after gradient purification. The probe system shows a remarkable optical response characteristic on beta-galactosidase (beta-Gal) and ideal singlet oxygen (1O2) generation efficiency; an optical signal and a photodynamic therapy effect of the probe can be specifically activated by beta-galactosidase (beta-Gal) overexpressed in cancerous cells and senescent cells under a light excitation condition; the result is successfully verified in a palbociclib-induced senescence melanoma model. According to the probe disclosed by the invention, collaborative regulation and control of dual-mode visual monitoring and precise photodynamic therapy of beta-Gal enzyme activity are realized for the first time, a novel tool is provided for researching a molecular mechanism of cell aging and developing a diagnosis and treatment strategy of aging-related diseases, and the probe has a remarkable application value in the fields of anti-tumor treatment and degenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of imaging probe detection and treatment, and specifically relates to a design, preparation method and application of a fluorescence / photoacoustic dual-mode diagnostic and therapeutic probe for detecting and photodynamically eliminating senescent cells. Background Art

[0002] Cell senescence is a process in which cells permanently stop dividing and enter an irreversible growth arrest state, mainly caused by factors such as oxidative stress, telomere dysfunction, DNA damage, and oncogene activation. Its key physiological role is to inhibit the proliferation of damaged or stressed cells and trigger tissue repair to maintain the homeostasis of organisms. However, the abnormal accumulation of senescent cells can lead to the release of chemokines, matrix remodeling agents, growth factors, and inflammatory cytokines, which may disrupt normal tissue function and exacerbate the aging process. The elimination of senescent cells has the potential to reverse degenerative changes and improve the long-term therapeutic effects of aging-related diseases and cancer. Nevertheless, most senescent cell elimination drugs tested in preclinical studies, including piperine, dasatinib, Bcl2 family inhibitors ABT-737 and ABT-263, face challenges of drug resistance and potential side effects. In addition, existing cell senescence detection methods, such as X-gal staining assay, DNA damage detection, telomere length measurement, and senescence-associated secretory phenotype (SASP) detection, are still not suitable for real-time monitoring of the degree of senescence in living systems. Therefore, there is an urgent need to develop highly selective and sensitive methods for targeting senescent cells, which is crucial for achieving accurate diagnosis and timely intervention of aging-related diseases and cancer.

[0003] A diagnostic and therapeutic probe is a reagent with both diagnostic and therapeutic functions, capable of detecting and treating diseases in a single system. When an activatable diagnostic and therapeutic probe interacts with molecular targets in a complex biological system, it undergoes an intrinsic signal change and simultaneously activates a therapeutic effect, which can improve the therapeutic effect and reduce side effects on normal organs and tissues. Senescence-associated β-galactosidase (SA-β-gal) is an endogenous lysosomal enzyme that is widely regarded as a standard marker of cellular senescence due to its elevated levels and abnormal accumulation in senescent cells. Due to the advantages of photodynamic therapy (PDT), such as low side effects, low invasiveness, and high spatiotemporal selectivity, it has been used in β-galactosidase-activatable diagnostic and therapeutic probes. The image-guided therapy function of these probes is achieved by modulating photosensitizers with activatable fluorescence and PDT effects, such as meso-4-pyridyl BODIPY. However, the absorption and emission wavelengths of meso-4-pyridyl BODIPY-derived probes are limited to the visible region (λex / em = 550 / 567 nm), indicating that the probe may be affected by background interference, weak tissue penetration ability, and ionizing radiation to organisms. Therefore, the development of novel image-guided diagnostic and therapeutic probes is of great significance for in vivo photodynamic senescent cell clearance.

[0004] As an imaging technique that detects ultrasound rather than photons, photoacoustic (PA) imaging can effectively reduce the effects of light absorption, reflection, and scattering, enabling non-invasive visualization of bioactive molecules in deep tissues (up to 7 cm) with high three-dimensional spatial resolution (sub-millimeter level). However, PA imaging still cannot perform whole-body imaging and lacks resolution and sensitivity at the single-cell level. The NIRF / PA dual-modal imaging technique combines the advantages of single imaging techniques while overcoming their respective limitations, which can significantly improve the accuracy of in vivo detection. Therefore, the development of fluorescent / photoacoustic dual-modal diagnostic and therapeutic probes for detecting and photodynamically clearing senescent cells is of great significance. Summary of the Invention

[0005] In view of the problems of insufficient sensitivity in detecting senescent cells and limited targeting of photodynamic therapy in the prior art, the present invention provides a photodynamic diagnostic and therapeutic probe (named ISH-Gal) based on fluorescence / photoacoustic dual-modal imaging, its preparation method, and biomedical applications. The probe uses a sulfur-substituted hemicyanine dye as the parent structure, covalently couples a novel bromoglycoside ligand, and obtains a blue-green solid after gradient purification. In vitro experiments confirm that the probe system exhibits significant optical response characteristics and ideal singlet oxygen ( 1O2) generation efficiency. It should be noted that the optical signal of this probe and the photodynamic therapy effect can be specifically activated by overexpressed β-galactosidase (β-Gal) in cancer cells and senescent cells under light excitation conditions. Animal model experiments further showed that in the senescent melanoma model induced by palbociclib, the photodynamic therapy system guided by near-infrared fluorescence / photoacoustic (NIRF / PA) dual-modal imaging achieved the photodynamic clearance of senescent cells through precise localization by dual-modal imaging. This probe provides a new tool for the precise diagnosis and targeted therapy of senescence-related diseases and has important application value in the fields of anti-tumor therapy, organ senescence intervention, etc. The ISH-Gal probe system has the following innovations: 1) For the first time, the coordinated regulation of dual-mode visualization monitoring of β-Gal enzyme activity and precise photodynamic therapy is realized; 2) A dynamic diagnosis and treatment integrated platform for senescence-related pathological models is established. This technical solution provides a new tool for studying the molecular mechanism of cell senescence and developing diagnosis and treatment strategies for senescence-related diseases, and has significant application value in the fields of anti-tumor therapy and degenerative diseases.

[0006] To solve the above technical problems, the basic concept of the technical solution adopted in the present invention is:

[0007] On the one hand, the present invention provides a fluorescence / photoacoustic dual-modal diagnostic and therapeutic probe for detecting and photodynamically clearing senescent cells. The probe is named ISH-Gal, and its chemical structural formula is shown in Formula I:

[0008]

[0009] On the other hand, the present invention provides a preparation method of a fluorescence / photoacoustic dual-modal diagnostic and therapeutic probe for detecting and photodynamically clearing senescent cells. The preparation process is as follows:

[0010] Bromoglycoside (640 mg, 1.2 mmol), dye ISH-OH (160 mg, 0.31 mmol), anhydrous acetonitrile and cesium carbonate (400 mg, 1.2 mmol) are mixed and stirred at 80 °C for 16 h. After vacuum concentration, the obtained crude product is dissolved in anhydrous ethanol, 1 M NaOCH3 is added, the mixture is reacted at 0 °C, and after monitoring the reaction by TLC, it is vacuum concentrated. Dichloromethane and methanol are used as eluents (volume ratio 10:1), and the residue obtained by purification by column chromatography is a blue-green solid product named ISH-Gal.

[0011] Furthermore, it specifically includes the following steps:

[0012] Step 1, synthesis of compound 2:

[0013] Dissolve ISH-OH (160 mg, 0.31 mmol) and Cs2CO3 (400 mg, 1.2 mmol) in dry CH3CN (10 mL). The mixture was refluxed and stirred at 80 °C for 10 minutes, then a solution of bromoglycoside (640 mg, 1.2 mmol) in dry CH3CN (10 mL) was added to the mixture. The reaction mixture was stirred overnight. Subsequently, the solvent was evaporated under reduced pressure, and the crude product was directly used in the next reaction without further purification;

[0014] Step 2, Synthesis of ISH-Gal:

[0015] Dissolve compound 2 (152 mg, 0.16 mmol) in a solution in absolute ethanol. Add 1 M sodium methoxide (NaOCH3) at 0 °C. The reaction was monitored by thin-layer chromatography (TLC) after 20 minutes. Evaporate the solvent in vacuo. The residue was purified by column chromatography to obtain the product ISH-Gal.

[0016] Among them, the preparation process of ISH-OH:

[0017] Step 11, Preparation of compound 1:

[0018] Dissolve 6-methoxy-2,3-dihydro-1H-xanthene-4-carbaldehyde (0.19 g, 0.74 mmol) and 5-iodo-1,2,3,3-tetramethyl-3H-indol-1-ium (0.33 g, 1.1 mmol) in absolute ethanol, then add piperidine to the solution. The system was heated to reflux under nitrogen protection and reacted overnight. Finally, the solvent was evaporated under reduced pressure, and the obtained crude compound 1 was directly used in the next reaction without further purification;

[0019] Step 2, Preparation of hemicyanine dye ISH-OH:

[0020] Dissolve compound 1 (400 mg, 0.74 mmol) in anhydrous dichloromethane, then add BBr3 (710 μL, 7.4 mmol) at 0 °C, stir the reaction at room temperature for 16 h, and add saturated sodium bicarbonate solution (30 mL) to terminate the reaction. Then extract the aqueous layer with dichloromethane, combine the organic layers, dry over anhydrous sodium sulfate, and then concentrate. The residue was purified by silica gel column chromatography (DCM / CH3OH = 10:1) to obtain the dye ISH-OH, whose chemical structural formula is shown in Formula II:

[0021]

[0022] Formula II.

[0023] Another aspect of the present invention provides an application of a fluorescence / photoacoustic dual-modal diagnostic and therapeutic probe for detecting and photodynamically eliminating senescent cells in detecting and photodynamically eliminating senescent cells at the cellular level.

[0024] Another aspect of the present invention provides an application of a fluorescence / photoacoustic dual-modal diagnostic and therapeutic probe for detecting and photodynamically eliminating senescent cells in diagnosing and treating senescent melanoma at the in vivo level.

[0025] The fluorescence / photoacoustic dual-modal diagnostic and therapeutic probe for detecting and photodynamically eliminating senescent cells of the present invention is composed of a β-D-galactopyranoside moiety as an identification unit and a hemicyanine dye (ISH-OH) as a photosensitizer. After reacting with β-galactosidase, the glycoside moiety of the probe ISH-Gal is specifically cleaved and removed, and then spontaneous 1,6-elimination occurs, resulting in the release of ISH-OH, thereby activating the NIRF / PA signal. Under the irradiation of a 660 nm laser lamp, singlet oxygen can be released, thus realizing the imaging and treatment of senescent tumors. Research shows that ISH-Gal exhibits excellent optical responses and ideal 1 O2 generation efficiency in vitro for β-gal detection. Further cell studies show that the optical signals and photodynamic therapeutic effects of ISH-Gal can be specifically activated by overexpressed β-Gal in cancer cells and senescent cells under light irradiation. ISH-Gal has been successfully applied to a palbociclib-induced senescent melanoma tumor model, and photodynamic elimination of senescent cells has been achieved through accurate localization by NIRF / PA dual-modal imaging.

[0026] After adopting the above technical solutions, the present invention has the following beneficial effects compared with the prior art.

[0027] (1) In the structure of the sulfur-substituted hemicyanine dye (ISH-OH) designed and synthesized by the present invention, S atoms are introduced to replace O atoms, which can not only improve the dual-modal imaging performance including absorption / emission wavelength and photoacoustic intensity, but also effectively enhance the photodynamic therapy effect.

[0028] (2) The diagnostic and therapeutic probe designed and synthesized by the present invention is based on the intramolecular charge transfer strategy of hemicyanine dye induced by the analyte. Since the hydroxyl group of hemicyanine in the probe ISH-Gal is blocked and ICT is turned off, the probe does not exhibit strong fluorescence and photoacoustic signals. When it reacts with β-galactosidase, β-galactosidase (β-gal) specifically removes the glycosidic bond, and ISH-Gal will spontaneously undergo a 1,6-elimination reaction to generate ISH-OH, producing strong fluorescence and photoacoustic signals, thus allowing NIRF / PA dual-modal imaging of β-galactosidase.

[0029] (3) The diagnostic and therapeutic probe designed and synthesized in the present invention can simultaneously detect β-galactosidase in ovarian cancer cells and senescent cells, has low cytotoxicity, good biocompatibility, and can achieve the treatment of melanoma tumor-bearing mice. It is a probe that can be used for NIRF / PA dual-mode imaging of β-galactosidase in both living cells and mouse tumor models and the treatment of melanoma tumor-bearing mice.

[0030] The following further describes in detail the specific embodiments of the present invention with reference to the accompanying drawings. Description of the Drawings

[0031] The accompanying drawings, as part of this application, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the following-described drawings are only some embodiments, and those of ordinary skill in the art can obtain other drawings based on these drawings without creative efforts. In the drawings:

[0032] Figure 1 1H NMR spectrum of the hemicyanine-like dye ISH-OH in dimethyl sulfoxide-d6 (DMSO-d6); 1 1H NMR spectrum;

[0033] Figure 2 13C NMR spectrum of the hemicyanine-like dye ISH-OH in dimethyl sulfoxide-d6 (DMSO-d6); 13 13C NMR spectrum;

[0034] Figure 3 ESI-MS spectrum of the hemicyanine-like dye ISH-OH;

[0035] Figure 4 1H NMR spectrum of the hemicyanine-like dye ISH-Gal in dimethyl sulfoxide-d6 (DMSO-d6); 1 1H NMR spectrum;

[0036] Figure 5 13C NMR spectrum of the hemicyanine-like dye ISH-Gal in dimethyl sulfoxide-d6 (DMSO-d6); 13 13C NMR spectrum;

[0037] Figure 6 ESI-MS spectrum of the hemicyanine-like dye ISH-Gal;

[0038] Figure 7For the present invention, the ultraviolet absorption, fluorescence change, photoacoustic signal change, real-time response of the probe ISH-Gal to β-galactosidase, fluorescence spectra and photoacoustic response diagrams of the probe ISH-Gal to different concentrations of β-galactosidase, high-performance liquid chromatography mechanism verification diagrams, and absorption spectra of singlet oxygen generation measured with DPBF as an indicator under different treatments of the probe ISH-Gal.

[0039] Figure 8 For the NIRF / PA imaging ability of the probe ISH-Gal of the present invention against ovarian cancer cells.

[0040] Figure 9 For the photodynamic clearance ability of the probe ISH-Gal of the present invention against ovarian cancer cells.

[0041] Figure 10 For the NIRF / PA imaging ability of the probe ISH-Gal of the present invention against senescent SK-Mel-103 cells.

[0042] Figure 11 For the photodynamic clearance ability of the probe ISH-Gal of the present invention against senescent SK-Mel-103 cells.

[0043] Figure 12 and Figure 13 For the NIRF / PA imaging performance of the probe ISH-Gal of the present invention in a senescent melanoma tumor model.

[0044] Figure 14 and Figure 15 For the photodynamic therapy ability of the probe ISH-Gal of the present invention in a senescent melanoma tumor model.

[0045] Figure 16 For the hematoxylin and eosin (H&E) staining diagrams of the main tissues and tumors of mice treated with the probe ISH-Gal of the present invention.

[0046] It should be noted that these drawings and textual descriptions are not intended to limit the scope of the concept of the present invention in any way, but to illustrate the concept of the present invention to those skilled in the art by reference to specific embodiments. Detailed implementation manners

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0048] The accumulation of senescent cells disrupts normal tissue function and promotes chronic inflammation and tumorigenesis, ultimately leading to various senescence-related dysfunctions and cancer progression. Developing effective tools for detecting and eliminating senescent cells is crucial for the accurate diagnosis and timely treatment of senescence-related diseases and cancers. However, existing diagnostic and therapeutic probes for senescent cell clearance activated by β-gal have the drawbacks of short absorption / emission wavelengths and fluorescence imaging modes. Herein, the present invention provides a β-gal-activated diagnostic and therapeutic probe (ISH-Gal) capable of simultaneously performing near-infrared fluorescence / photoacoustic (NIRF / PA) dual-modal imaging and photodynamic elimination of senescent cells. The probe contains a β-D-galactopyranoside group linked to an I-atom-modified S-substituted hemicyanine dye through a self-eliminating group, having near-infrared excitation and emission wavelengths. Studies have shown that ISH-Gal exhibits satisfactory optical responses and ideal 1 O2 generation for β-gal detection in vitro. Cellular studies further demonstrated that the optical signals and photodynamic efficacy of ISH-Gal can be specifically activated by overexpressed β-Gal in cancer cells and senescent cells under light irradiation. ISH-Gal was successfully applied to a palbociclib-induced senescent melanoma tumor model for photodynamic elimination of senescent cells guided by NIRF / PA dual-modal imaging. The probe can serve as a useful tool for studying senescence-related physiological and pathological functions and the diagnosis and treatment of senescence-related diseases.

[0049] Example 1

[0050] Preparation process of hemicyanine dye ISH-OH, referring to Route 1,

[0051]

[0052] The specific steps are as follows:

[0053] Synthesis of Compound 1:

[0054] Dissolve 6-methoxy-2,3-dihydro-1H-xanthene-4-carbaldehyde (0.19 g, 0.74 mmol) and 5-iodo-1,2,3,3-tetramethyl-3H-indol-1-ium (0.33 g, 1.1 mmol) in anhydrous ethanol, then add piperidine to the solution, and heat the system to reflux under nitrogen protection for overnight reaction. Finally, evaporate the solvent under reduced pressure, and the obtained crude Compound 1 is directly used for the next reaction without further purification.

[0055] Synthesis of Compound ISH-OH:

[0056] Compound 1 (400 mg, 0.74 mmol) was dissolved in anhydrous dichloromethane, and then BBr3 (710 μL, 7.4 mmol) was added at 0 °C. The reaction was stirred at room temperature for 16 h. Saturated sodium bicarbonate solution (30 mL) was added to terminate the reaction, and then the aqueous layer was extracted with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, and then concentrated. The residue was purified by silica gel column chromatography (DCM / CH3OH = 10:1) to obtain the dye ISH-OH in a yield of 42%.

[0057] The semi - squarylium dye ISH - OH prepared in this example was detected, and the results are as Figures 1 to 3 shown, which are respectively Figure 1 the 1H NMR spectrum of the semi - squarylium dye ISH - OH in dimethyl sulfoxide - d6 (DMSO - d6); 1 Figure 2 the 13C NMR spectrum of ISH - OH in dimethyl sulfoxide - d6 (DMSO - d6); 13 Figure 3 the ESI - MS spectrum of the semi - squarylium dye ISH - OH.

[0058] Among them, Figure 1 the hydrogen spectrum analysis is 1 1H NMR (400 MHz, DMSO - d6) δ (ppm): 8.21 (d, J = 12.0 Hz, 1H), 7.94 (s, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.52 (s, 1H), 7.33 - 7.41 (m, 4H), 7.05 - 7.12 (m, 4H), 6.90 (s, 1H), 6.70 (d, J = 14.0 Hz, 1H), 6.28 - 6.35 (m, 1H), 5.61 - 5.77 (m, 1H), 5.29 - 5.32 (m, 1H), 5.15 (s, 2H), 4.99 (s, 1H), 4.86 - 4.89 (m, 2H), 2.53 (m, 4H), 1.73 - 1.95 (m, 8H), 1.21 (s, 3H).

[0059] Figure 2 the carbon spectrum analysis is as follows: 13 13C NMR (100 MHz, DMSO - d6) δ (ppm): 178.05, 155.83, 151.01, 144.90, 142.53, 138.03, 133.71, 131.94, 131.44, 127.51, 123.67, 121.82, 116.27, 109.42, 108.33, 93.80, 57.56, 51.11, 33.57, 32.15, 27.51, 20.34.​​

[0060] Figure 3 The ESI-MS mass spectrometry analysis of it is as follows: The theoretical molecular weight of ISH-OH is 526.46; the actual detection shows that the molecular weight is 526.39.

[0061] The above results prove that the semi-cyanine dye-like ISH-OH has been successfully synthesized in this example, and its structural formula is:

[0062]

[0063] Example 2

[0064] The preparation process of the dual-mode imaging probe ISH-Gal refers to Route 2.

[0065]

[0066] The specific steps are as follows:

[0067] Synthesis of Compound 2:

[0068] Dissolve ISH-OH (160 mg, 0.31 mmol) and Cs2CO3 (400 mg, 1.2 mmol) in dry CH3CN (10 mL). The mixture is refluxed and stirred at 80 °C for 10 minutes. Then, add a solution of bromoglycoside (640 mg, 1.2 mmol) in dry CH3CN (10 mL) to the mixture, and the reaction mixture is stirred overnight. Subsequently, evaporate the solvent under reduced pressure, and the crude product is directly used for the next reaction without further purification.

[0069] Synthesis of ISH-Gal:

[0070] Dissolve Compound 2 (152 mg, 0.16 mmol) in a solution of absolute ethanol, and add 1 M sodium methoxide (NaOCH3) at 0 °C. The reaction is monitored by thin-layer chromatography (TLC) after 20 minutes. Evaporate the solvent in vacuo. The residue is purified by column chromatography to obtain the product ISH-Gal as a blue solid with a yield of 45%.

[0071] Detect the probe ISH-Gal prepared in this example, and the results are as Figures 4 to 6 shown, which are respectively: Figure 4 1H NMR spectrum of the probe ISH-Gal in dimethyl sulfoxide-d6 (DMSO-d6); 1 1H NMR spectrum; Figure 5 13C NMR spectrum of the probe ISH-Gal in dimethyl sulfoxide-d6 (DMSO-d6); 13 13C NMR spectrum; Figure 6 ESI-MS spectrum of the probe ISH-Gal.

[0072] Among them, Figure 4 the hydrogen spectrum analysis is 1 H NMR(400MHz,DMSO-d6)δ(ppm):8.19(d,J=8.4Hz,1H),7.93(d,J=9.2Hz,1H),7.63(s,1H),7.49(d,J=8.4Hz,1H),7.21-7.34(m,6H),6.98-7.01(d,3H),6.42(d,J=9.2Hz,1H),6.20(s,1H),5.74(d,1H),4.84(d,J=7.2Hz,2H),4.58-4.60(m,1H),4.32(s,2H),3.67(d,J=8.4Hz,2H),3.14-3.48(m,4H),2.64(s,4H),1.93(s,6H),1.79-1.82(m,1H),1.22(s,3H).

[0073] Figure 5 the carbon spectrum analysis is 13 C NMR(100MHz,DMSO-d6)δ(ppm):177.51,162.61,157.55,152.43,142.54,141.28,135.28,131.89,130.04,129.82,127.01,125.17,124.28,123.46,123.16,116.65,116.59,110.16,107.33,100.69,99.69,93.37,79.23,77.46,77.02,73.66,70.11,61.06,50.88,36.29,31.25,29.03,27.61.

[0074] Figure 6 The ESI-MS mass spectrum analysis of is as follows: The theoretical molecular weight of ISH-Gal is 794.72; actual detection: the molecular weight is 794.28.

[0075] The above results prove that the probe ISH-Gal has been successfully synthesized in this example, and its structural formula is:

[0076]

[0077] Example 3

[0078] Performance analysis of the diagnostic probe ISH-Gal for detecting β-gal in vitro:

[0079] (1) Examine the ultraviolet absorption and fluorescence changes of the prepared probe ISH-Gal in response to β-galactosidase;

[0080] To study the ultraviolet absorption response and fluorescence changes of ISH-Gal to β-Gal, ISH-Gal (5 μM) was incubated with β-Gal (150 U / mL) in PBS at 37 °C for 2 hours, and the absorption spectra in the range of 450 nm to 850 nm and the fluorescence spectra in the range of 750 nm to 850 nm were recorded. The results are as Figure 7 shown in a. After incubation with β-Gal, a new absorption peak appeared at 730 nm for the probe, and the absorption was significantly red-shifted. Similarly, as Figure 7 shown in b, after incubation with β-Gal, the probe showed the maximum fluorescence intensity at 770 nm, and the signal enhancement reached 10.5 times. This indicates that the probe can sensitively detect the activity of β-Gal.

[0081] (2) Examine the photoacoustic signal changes of the prepared probe ISH-Gal in response to β-galactosidase;

[0082] To study the photoacoustic response of ISH-Gal to β-Gal, ISH-Gal (5 μM) was incubated with β-Gal (150 U / mL) in PBS at 37 °C for 2 hours. As Figure 7 shown in c, after incubation with β-Gal, the probe showed the maximum photoacoustic intensity at 730 nm, and the signal enhancement reached 9.5 times. This indicates that the probe can produce an obvious response to β-Gal.

[0083] (3) Examine the ability of the prepared probe ISH-Gal to perform real-time fluorescence response to β-galactosidase.

[0084] To study the reaction kinetics of the probe ISH-Gal to β-Gal, ISH-Gal (5 μM) was rapidly mixed with β-Gal (0 and 150 U / mL) in a PBS / DMSO solution (10 mM, pH 7.4, v / v, 4 / 1). Subsequently, at 37 °C, the fluorescence intensity at 770 nm was recorded at different time points (0, 10, 20, 30, 40, 60, 80, 90, 100, 110, 120 minutes) using an Edinburgh FLS1000-stm fluorescence spectrophotometer. The results are as Figure 7 shown in d. In the absence of β-gal, the fluorescence of the probe ISH-Gal was very weak and the fluorescence intensity remained unchanged with time. However, for the probe ISH-Gal with β-gal added, the fluorescence gradually increased with time, and the response to β-gal reached a plateau at about 90 min.

[0085] (4) Examine the fluorescence spectra and photoacoustic response maps of the prepared probe ISH-Gal against different concentrations of β-galactosidase;

[0086] To study the response of the probe ISH-Gal to different concentrations of β-galactosidase, different concentrations of β-galactosidase (0 - 150 U / mL) were added to PBS (10 mM, pH = 7.4) containing 5 μM of the probe ISH-Gal. After mixing evenly, the prepared solutions were all reacted at 37 °C for 2 h, and their fluorescence emission spectra and photoacoustic spectra were scanned respectively. As Figure 7 shown in Figures 6e and 7f, as the concentration of β-galactosidase increased (0 - 150 U / mL), the probe ISH-Gal responded with β-galactosidase to generate ISH-OH, and the fluorescence intensity at 770 nm and the photoacoustic intensity at 730 nm gradually increased. When the concentration of added β-galactosidase was 150 U / mL, the fluorescence intensity reached the maximum. This indicates that the probe completely responded with β-galactosidase at this time.

[0087] (5) Examine and verify the reaction mechanism of the prepared probe ISH-Gal against β-galactosidase;

[0088] To study the reaction mechanism of ISH-Gal against β-galactosidase, the high-performance liquid chromatography (HPLC) chromatograms of ISH-Gal (5 μM), ISH-OH (5 μM), and the product of the reaction of ISH-Gal (5 μM) with β-Gal (80 U / mL) at 37 °C for 2 h were carried out on a system with a C18 column (250 mm × 4.6 mm, 5 μm) under the following conditions: methanol / water = 85 / 15 (volume ratio); flow rate: 1.0 mL / min; detection wavelength: 700 nm. To further demonstrate the reaction mechanism, electrospray ionization mass spectrometry (ESI-MS) was introduced to study the product after the reaction of ISH-Gal (5 μM) with β-Gal (80 U / mL) at 37 °C for 2 h in the positive mode. The results are as Figure 7 shown in Figure 8g. The retention times of ISH-Gal and ISH-OH were 3.963 minutes and 8.416 minutes respectively. After co-incubation with β-Gal for 2 h, a new peak appeared at 8.416 minutes in the reaction solution. The above results well verified the proposed reaction mechanism.

[0089] (6) Examine the absorption spectra of singlet oxygen generation measured with DPBF as an indicator for the prepared probe ISH-Gal under different treatments:

[0090] To prove that the probe ISH-Gal can generate singlet oxygen after reacting with β-galactosidase, the absorption spectra were finally measured with DPBF (10 μM) as the indicator for the probe, probe ISH-Gal (5 μM), and a certain concentration of β-galactosidase (150 U / mL) under illumination with / without a 660 nm laser. The results are as Figure 7 shown in Fig. h. Only after the reaction of the probe with β-galactosidase under illumination with a 660 nm laser, the absorption intensity at 410 nm decreased significantly, proving that the dye ISH-OH can generate singlet oxygen under illumination with a 660 nm laser.

[0091] Example 4

[0092] Examine the NIRF / PA imaging and photodynamic clearance capabilities of the prepared probe ISH-Gal at the live cell level:

[0093] (1) Evaluate the NIRF / PA imaging and photodynamic clearance capabilities of the prepared probe ISH-Gal against ovarian cancer cells;

[0094] At 37 °C, SKOV-3 cells were seeded on 6 cm culture dishes in 3 ml of medium containing 10% FBS and 1% penicillin / streptomycin for 24 hours. The probe concentration for cell imaging was 10 μM. The SKOV-3 cells were incubated with 1 mL of fresh medium containing probe ISH-Gal (10 μM) for 4 h. For the control group, the cells were incubated with 1 mL of fresh medium for 4 h. In the inhibition experiment, the cells were pre-incubated with the β-galactosidase inhibitor D-(+)-galactose (100 μM) at 37 °C for 5 hours, and then incubated with ISH-Gal (10 μM) for 4 hours. The cells were washed three times with cold PBS before imaging. For cell NIRF imaging, the excitation wavelength was 640 nm, and the fluorescence signal acquisition range of probe ISH-Gal was 690 nm - 780 nm. For cell PA imaging, before imaging, the cells in each group were digested with trypsin, centrifuged, and re-dissolved in fresh medium, and then transferred to 1.5 mL centrifuge tubes and imaged with an MSOT imaging system for PA imaging. Scanning was performed from 680 nm to 850 nm at 10 nm intervals. All cell PA data were collected with an InVision 256-TF imaging system (iThera Medical GmbH), with an excitation range of 680 nm to 850 nm, a pulse length of 8 ns, and a pulse frequency of 10 Hz.

[0095] As Figure 8 a and Figure 8As shown in Fig. b, SKOV-3 cells treated with ISH-Gal alone showed very strong NIRF intensity, while cells pre-treated with the β-galactosidase inhibitor D-(+)-galactose and then incubated with the probe ISH-Gal showed relatively weak NIRF intensity. As Figure 8 Shown in the photoacoustic image of Fig. c, compared with the control group, ISH-Gal triggered a 7.5-fold increase in PA730 intensity in SKOV-3 cells, while cells treated with D-(+)-galactose showed weaker PA730 intensity, indicating that the activation of the PA signal was also due to the specific response of ISH-Gal to β-Gal. In summary, all these results demonstrated that the prepared probe ISH-Gal has the potential for NIRF / PA dual-modal imaging of β-galactosidase activity in living cells.

[0096] To evaluate the ability of the prepared probe ISH-Gal to photodynamically eliminate ovarian cancer cells, the SOSG indicator was used to detect the release of singlet oxygen. First, we selected SKOV-3 cells as the cell model and divided the cells into 4 groups: light irradiation, probe incubation, probe incubation + light irradiation, and pre-incubation with the inhibitor D-(+)-galactose + probe incubation + light irradiation. The excitation wavelength was 488 nm, and the fluorescence signal collection range was 500 nm - 550 nm.

[0097] The results are as Figure 9 Shown in Figs. a and 9b, the green fluorescence emitted by cells incubated with the probe and irradiated with light was much stronger than that of other groups because SKOV-3 cells not treated with the inhibitor expressed higher levels of β-galactosidase, activating more of the probe ISH-Gal, thus producing more of the dye ISH-OH. After irradiation with a 660 nm laser, the dye released a large amount of singlet oxygen, and SOSG is a fluorescent probe used to detect the production of singlet oxygen. It has weak blue fluorescence before reacting with singlet oxygen and emits green fluorescence after the reaction.

[0098] (2) Evaluate the NIRF / PA imaging and photodynamic elimination ability of the prepared probe ISH-Gal on senescent SK-Mel-103 cells;

[0099] To explore the NIRF / PA imaging ability of the probe ISH-Gal on senescent SK-Mel-103 cells, SK-Mel-103 cells were seeded on 6 cm culture dishes in 3 ml of medium containing 10% FBS and 1% penicillin / streptomycin for 24 hours and incubated with palbociclib (5 μM) for 7 days to establish drug-induced senescent SK-Mel-103 cells as a representative senescence model. The probe concentration for cell imaging was 10 μM. Three experimental groups were designed to study the NIRF / PA imaging ability of ISH-Gal on senescent SK-Mel-103 cells. The first and second groups incubated normal SK-Mel-103 cells and senescent SK-Mel-103 cells respectively with 1 mL of fresh medium containing the probe ISH-Gal (10 μM) for 4 h. The third group was that senescent SK-Mel-103 cells were pretreated with the β-galactosidase inhibitor D-(+)-galactose (100 μM) for 5 hours and incubated with ISH-Gal for 4 hours. For cell PA imaging, the cells in each group were digested with trypsin, centrifuged and resuspended in fresh medium before imaging, and then transferred to 1.5 mL centrifuge tubes and imaged with an MSOT imaging system. Scanning was performed from 680 nm to 850 nm at 10 nm intervals. All cell PA data were acquired using an InVision 256-TF imaging system (iThera Medical GmbH) with an excitation range from 680 nm to 850 nm, a pulse length of 8 ns, and a pulse frequency of 10 Hz.

[0100] As Figure 10 shown in Figure 10 panels a and Figure 10 b, the NIRF signal observed when incubating ISH-Gal in normal SK-Mel-103 cells was negligible, while the ISH-Gal-treated senescent SK-Mel-103 cells showed a 3.5-fold enhanced NIRF signal. After adding the β-galactosidase inhibitor to senescent cells, the NIRF signal was significantly attenuated, indicating that the enhancement of the fluorescence signal in senescent cells is specific to endogenous β-galactosidase activity. As shown in

[0101] panel c, the PA signal inside senescent SK-Mel-103 cells was found to be much stronger (4.5-fold) than that in normal SK-Mel-103 cells. All these results demonstrated that the designed ISH-Gal has excellent potential for selectively imaging senescent cells.

[0101] To evaluate the ability of the prepared probe ISH-Gal to photodynamically eliminate senescent SK-Mel-103 cells, the SOSG indicator was used to detect the release of singlet oxygen, and CLSM imaging was performed on SK-Mel-103 cells treated with ISH-Gal with or without laser irradiation. The cells were divided into 4 groups: normal SK-Mel-103 cells + dark, normal SK-Mel-103 cells + light, senescent SK-Mel-103 cells + dark, and senescent SK-Mel-103 cells + light. The excitation wavelength was 488 nm, and the fluorescence signal acquisition range was 500 nm - 550 nm.

[0102] As Figure 11 shown in Figures a and 11b, the fluorescence of SOSG in the green channel was negligible in normal SK-Mel-103 cells with or without laser irradiation, and in non-laser-irradiated senescent SK-Mel-103 cells. However, in laser-irradiated senescent SK-Mel-103 cells, SOSG exhibited bright green fluorescence, confirming the satisfactory ability of ISH-Gal in photodynamic therapy for eliminating senescent cells.

[0103] Example 5

[0104] Investigate the NIRF / PA imaging performance and photodynamic therapy ability of the prepared probe ISH-Gal in a senescent melanoma tumor model:

[0105] (1) Evaluate the NIRF / PA imaging performance of the prepared probe ISH-Gal in a senescent melanoma tumor model;

[0106] First, a tumor mouse model was constructed. Female BALB / c nude mice (4 weeks old) were used for the animal tumor model. To establish an animal model of SK-Mel-103 tumors, 2×10 6SK-Mel-103 cells. After 5 days, the mice were randomly divided into two groups for imaging. One group of mice was orally administered palbociclib (100 mg / kg) daily for 10 days to induce tumor senescence. The other group was given PBS as a control. On the 11th day, the probe (100 μM, 50 μL) was injected into the mice by intratumoral injection. Subsequently, normal tumor-bearing mice and palbociclib-induced senescent melanoma-bearing mice were randomly divided into three groups and were intratumorally injected with PBS, ISH-Gal, D-galactose (500 μM), and ISH-Gal, respectively. Fluorescence images of all mice were captured on an IVIS LuminaXR small animal imaging system (Caliper, Switzerland). PA data were acquired at 10 nm intervals in the wavelength range of 680 nm to 850 nm with a scanning interval of 0.3 mm.

[0107] As Figure 12 shown in a, 12b, normal tumor-bearing mice injected with ISH-Gal showed very weak NIRF signals in the tumor region. In contrast, senescent tumor-bearing mice treated with ISH-Gal showed significant NIRF signals in the tumor region, and the enhancement of the NIRF signal was 7.6 times that of senescent tumor-bearing mice treated with PBS, which was attributed to the overexpression of β-galactosidase in senescent tumors. In addition, compared with the group treated with only ISH-Gal, the NIRF intensity of the group treated with D-galactose was significantly reduced. Similarly, the NIRF intensity gradually increased with time and reached the maximum intensity after about 5 hours ( Figure 13 a and 13b). Similarly, as Figure 12 shown in c and 12d, compared with senescent tumor-bearing mice treated with PBS, the photoacoustic (PA) signal in the senescent tumor region increased by about 6.3 times 5 hours after injection of ISH-Gal, and a significant decrease in PA intensity was observed in mice pretreated with inhibitors. These results confirmed that the activated optical signal in the senescent tumor region was attributed to the specific response of ISH-Gal to β-Gal expressed in senescent SK-Mel-103 cells.

[0108] (2) Evaluate the ability of the prepared probe ISH-Gal for photodynamic therapy in a senescent melanoma tumor model;

[0109] To explore the ability of ISH-Gal to treat senescent melanoma, after 10 days of treatment with palbociclib, mice in the senescent group and the normal group were intratumorally injected with ISH-Gal (100 μM, 50 μL) every 2 days. Laser irradiation (660 nm, 10 mW / cm 2 , 10 min) was performed 5 hours after systemic administration of ISH-Gal, and the tumor size and mouse body weight were monitored simultaneously.

[0110] As Figure 14 shown in FIGS. 14a and 14b, compared with the normal group treated with PBS, the size of the senescent tumors treated with palbociclib was slightly reduced under laser irradiation. This slight difference in tumor size was attributed to the lower growth rate of senescent tumors compared to normal tumors. Additionally, in the normal group treated with ISH-Gal, there was no significant difference in tumor size with or without laser irradiation ( Figure 15 a). As Figure 15 shown in FIGS. 15b and 15c, compared with the average tumor size of normal mice treated with ISH-Gal without laser irradiation, the tumor growth inhibition rate of the senescent group treated with ISH-Gal and laser irradiation reached 81%, indicating that laser irradiation was effective for triggering the therapeutic effect and the photodynamic therapy of our probe on senescent melanoma tumors. Additionally, the body weights of all experimental mice remained stable, indicating that these treatments had no significant side effects on the mice ( Figure 15 d).

[0111] (3) Evaluate the in vivo metabolic pathway of the prepared probe ISH-Gal;

[0112] Hematoxylin and eosin (H&E) staining analysis of major organs. Representative histological features of major organs (heart, liver, spleen, lung, kidney, tumor) were excised from normal mice or senescent melanoma mice. The excised organs and tumor tissues were fixed with 4% formaldehyde solution, embedded in paraffin, and cut into 5-μm thick sections. The sections were deparaffinized, hydrated, and stained with hematoxylin and eosin in sequence. The sections were rinsed with water and dehydrated for imaging acquisition. Fluorescent images were collected using a fluorescence microscope at a magnification of 400×.

[0113] As Figure 16 shown in FIGS. 16a and 16b, when hematoxylin-eosin (H&E) staining was performed on major organs including the heart, spleen, liver, kidney, and lung, no signs of abnormal cell structure or tissue damage were detected, indicating the low toxicity of ISH-Gal to normal organs, while there were obvious lesions in the palbociclib-induced senescent tumors treated with ISH-Gal and laser. In summary, these results demonstrated the good potential of the probe ISH-Gal for effective photodynamic therapy of senescent tumors.

[0114] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art of this patent, without departing from the scope of the technical solution of the present invention, can make some changes or modifications using the technical content prompted above to form equivalent embodiments of equivalent changes. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A fluorescence / photoacoustic dual-mode diagnostic and therapeutic probe for detecting and photodynamically removing senescent cells, characterized in that: The probe is named ISH-Gal, and its chemical structure is as follows:

2. The method for preparing the diagnostic probe according to claim 1, characterized in that: The preparation process is: Bromoglycoside, dye ISH-OH, anhydrous acetonitrile and cesium carbonate were mixed and stirred at 80°C for 16 hours. The crude product was dissolved in anhydrous ethanol after vacuum concentration, and NaOCH3 was added. The mixed solution was reacted at 0°C. The reaction was monitored by TLC and then vacuum concentrated. Dichloromethane and methanol were used as eluents, and the residue was purified by column chromatography to obtain a blue-green solid product named ISH-Gal.

3. The preparation method according to claim 2, characterized in that: The specific preparation steps are as follows: Step 1, synthesis of compound 2: ISH-OH and Cs2CO3 were dissolved in dry CH3CN, and the mixture was stirred under reflux at 80°C for 10 minutes. Then, a solution of brominated glycoside in dry CH3CN was added to the mixture, and the reaction mixture was stirred overnight. Subsequently, the solvent was evaporated under reduced pressure, and the crude product was directly used in the next reaction without further purification and was named compound 2; Step 2, synthesis of ISH-Gal: Compound 2 was dissolved in anhydrous ethanol, and 1 M sodium methoxide NaOCH3 was added at 0°C. The reaction was monitored by thin layer chromatography after 20 minutes. The solvent was evaporated in vacuo and the residue was purified by column chromatography to obtain the product ISH-Gal.

4. The preparation method according to claim 3, characterized in that: In step 1, the molar ratio of ISH-OH, Cs2CO3 and brominated glycoside is 2:5:8, wherein the concentration of ISH-OH is 0.31 mmol, the concentration of Cs2CO3 is 1.2 mmol, and the concentration of brominated glycoside is 1.2 mmol; the amount of dry CH3CN used is 10 mL; In step 2, the amount of compound 2 used is 150-160 mg, and the concentration is 0.16 mmol.

5. The preparation method according to claim 2, characterized in that: The chemical structure of ISH-OH is:

6. The preparation method according to claim 2, characterized in that: The preparation process of ISH-OH is as follows: Step 11, preparation of compound 1: 6-methoxy-2,3-dihydro-1H-xanthene-4-carbaldehyde and 5-iodo-1,2,3,3-tetramethyl-3H-indol-1-ion were dissolved in anhydrous ethanol, and then piperidine was added to the solution, and the mixture was heated to reflux under nitrogen protection, and the reaction was carried out overnight. Finally, the solvent was evaporated under reduced pressure, and the obtained crude compound 1 was directly used in the next reaction without further purification; Step 12, preparation of hemicyanine-like dye ISH-OH: Compound 1 was dissolved in anhydrous dichloromethane, and then BBr3 was added at 0°C. The reaction was stirred at room temperature for 16 h, and 30 mL of saturated sodium bicarbonate solution was added to terminate the reaction. The aqueous layer was then extracted with dichloromethane, and the organic layers were combined and dried over anhydrous sodium sulfate, then concentrated. The residue was purified by silica gel column chromatography (DCM / CH3OH=10:1) to obtain the dye ISH-OH.

7. A fluorescence / photoacoustic dual-mode diagnostic and therapeutic probe for detecting and photodynamically removing senescent cells according to claim 6, characterized in that: In step 1, the molar ratio of 6-methoxy-2,3-dihydro-1H-xanthene-4-carbaldehyde and 5-iodo-1,2,3,3-tetramethyl-3H-indole-1-ium is 19:33, wherein the concentration of 6-methoxy-2,3-dihydro-1H-xanthene-4-carbaldehyde is 0.74 mmol, and the concentration of 5-iodo-1,2,3,3-tetramethyl-3H-indole-1-ium is 1.1 mmol; In step 2, the amount of compound 1 used is 400 mg, and the concentration is 0.74 mmol; the amount of BBr3 used is 710 μL, and the concentration is 7.4 mmol.

8. The fluorescence / photoacoustic dual-mode diagnostic and therapeutic probe for detecting and photodynamically removing senescent cells according to claim 2, characterized in that: The volume ratio of dichloromethane to methanol in the eluent was 10:

1.

9. Use of the fluorescence / photoacoustic dual-mode diagnostic and therapeutic probe for detecting and photodynamically eliminating senescent cells as described in claim 1 to detect and photodynamically eliminate senescent cells at the cellular level.

10. Use of the fluorescence / photoacoustic dual-mode diagnostic and therapeutic probe for detecting and photodynamically eliminating senescent cells as claimed in claim 1 in diagnosing and treating senescent melanoma at the in vivo level.

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