A protein-anchored copper-responsive photoacoustic probe and a preparation method and application thereof

Abstract

The application discloses a protein anchoring type copper response photoacoustic probe and a preparation method and application thereof, the probe can be selectively chelated with copper in the mitochondria of tumor cells, so that the photoacoustic signal of the probe is activated, in-situ tracing and quantification of copper ions in the tumor are realized. Meanwhile, copper chelation realizes copper depletion in the mitochondria, causes damage to the structure and function of the mitochondria, and realizes diagnosis and treatment integration of the tumor. In addition, the probe can specifically react with protein sulfenic acid in the mitochondria to form a probe-protein conjugate, promote enrichment of the probe at the tumor site, prolong the imaging window and improve the treatment effect of copper depletion.

Description

Technical Field

[0001] This invention belongs to the technical field of small molecule photoacoustic probe bioimaging and tumor therapy. Specifically, it relates to a protein-anchored copper-responsive molecular probe and its preparation method, as well as the application of this probe in multimodal imaging and tumor therapy. Background Technology

[0002] Over the past few decades, the incidence and mortality rates of cancer have increased significantly, becoming one of the major threats to human health and life. As a core element of molecular imaging technology, molecular probes play a crucial role in the precision diagnosis and treatment of cancer. Although numerous molecular imaging probes have been developed for cancer imaging and therapy, their poor tumor specificity and potential for damaging normal tissues often prevent them from achieving precise diagnosis and treatment. Based on the characteristics of the tumor microenvironment (TME), scientists have developed various activatable molecular probes that can be triggered by endogenous substances (such as overexpressed enzymes, metals, acidic pH, highly reduced glutathione, and hypoxia) and can specifically destroy cancer cells, thereby reducing the toxic side effects of traditional chemotherapy or radiotherapy on normal tissues. However, these existing probes still have some significant drawbacks in practical applications. Insufficient specificity is one of the main problems; many probes may be activated by non-target factors in complex biological environments, leading to increased background signals and reduced detection accuracy. Furthermore, long response times are also a key challenge; some probes have slow activation processes, limiting their effectiveness in applications requiring rapid responses. Delivery and distribution control are also significant challenges; probes may be unevenly distributed within the body, affecting the consistency of treatment or testing. Therefore, new technologies are needed to effectively address these issues, thereby significantly improving probe performance. Summary of the Invention

[0003] This invention discloses a protein-anchored copper-responsive photoacoustic probe and its preparation method. The probe has the advantages of chelating groups with copper ions and cross-linking groups with protein sulfonates, good biocompatibility, active targeting of integrins and near-infrared emission, and can effectively perform photoacoustic imaging and tumor treatment.

[0004] The present invention adopts the following technical solution:

[0005] A protein-anchored copper-responsive photoacoustic probe has the following chemical structural formula:

[0006]

[0007] This invention discloses a method for preparing the above-mentioned protein-anchored copper-responsive photoacoustic probe, wherein compound 8 is reacted with compound 5 to obtain the protein-anchored copper-responsive photoacoustic probe. Preferably, the reaction is carried out in the presence of an organic solvent, preferably including N,N-dimethylformamide; the reaction time is 0.5 to 10 hours; and the molar ratio of compound 8 to compound 5 is 1:(1 to 5).

[0008] In this invention, compound 7 reacts with compound 2 to obtain compound 8; preferably, the reaction is carried out in the presence of sodium ascorbate and copper sulfate; the molar ratio of compound 7, compound 2, sodium ascorbate and copper sulfate is 1:(0.8-1.2):(0.05-0.2):(0.05-0.1).

[0009] In this invention, N-Boc-ethylenediamine reacts with 2-chloromethylpyridine hydrochloride, and then the protecting group is removed by trifluoroacetic acid to obtain compound 5 (DPA); preferably, N-Boc-ethylenediamine reacts with 2-chloromethylpyridine hydrochloride in a small molecule alcohol to obtain compound 4; compound 4 is then deprotected by trifluoroacetic acid in an organic solvent to obtain compound 5.

[0010] This invention discloses the application of the above-mentioned protein-anchored copper-responsive photoacoustic probe in the preparation of reagents for chelating copper ions and / or cross-linking proteins. The probe has a copper ion chelating group and a protein sulfenic acid cross-linking group. Copper is an essential cofactor for enzymes mediating a series of basic cellular functions, including mitochondrial respiration, antioxidant defense, and the biosynthesis of hormones, neurotransmitters, and pigments. Imbalances in copper storage can induce oxidative stress and cytotoxicity. Therefore, the probe of this invention can be effectively used for photoacoustic imaging and tumor therapy.

[0011] This invention discloses the application of the aforementioned protein-anchored copper-responsive photoacoustic probe in imaging or the preparation of imaging agents; preferably, the imaging includes fluorescence imaging and / or photoacoustic imaging. Specifically, this invention discloses the application of the aforementioned protein-anchored copper-responsive photoacoustic probe in fluorescence imaging and photoacoustic imaging; or in the preparation of fluorescence imaging agents and photoacoustic imaging agents.

[0012] This invention discloses the application of the above-mentioned protein-anchored copper-responsive photoacoustic probe in the preparation of drugs; preferably, the drugs are diagnostic and / or therapeutic drugs.

[0013] In the above technical solutions, the imaging agent or drug is applied to tumors.

[0014] This invention discloses an imaging agent or drug, including the above-mentioned protein-anchored copper-responsive photoacoustic probe; it may also include other drug components.

[0015] This invention discloses the application of the above-mentioned protein-anchored copper-responsive photoacoustic probe in improving the retention time of the probe in tumor tissue or inhibiting tumors, or in the preparation of reagents for increasing the retention time of tumor tissue or inhibiting tumors.

[0016] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0017] 1. This invention designs and synthesizes a novel anchoring molecular probe d-IR-DPA, which can specifically chelate endogenous copper ions in tumors, thereby activating the probe's photoacoustic properties and achieving spatiotemporal tracing of copper chelation;

[0018] 2. In this invention, the target probe specifically reacts with hyposulfonic acid, a protein in the mitochondria of tumor cells, to form a probe-protein conjugate, which prolongs the probe retention time and increases the probe enrichment effect.

[0019] 3. In this invention, the target probe chelates with copper ions in tumor cells, causing mitochondrial damage and thus exhibiting a strong ability to promote tumor cell apoptosis.

[0020] 4. In this invention, the target probe undergoes copper chelation in vivo, leading to copper depletion and significantly inhibiting tumor growth in tumor-bearing mice. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the synthesis of the protein-anchored copper-responsive photoacoustic probe in Example 1.

[0022] Figure 2 The image shows the UV absorption of probe d-IR-DPA in aqueous solution in Example 2 (a), and the image shows the UV absorption of probe d-IR-DPA in Cu. 2+ Changes in UV absorption before and after solution mixing, (c) probe d-IR-DPA and Cu 2+ Fluorescence emission images before and after solution mixing, (d) probe IR-DPA and Cu 2+ Changes in UV absorption before and after solution mixing, (e) probe d-IR-RGD and Cu 2+ Changes in UV absorption before and after solution mixing, (f) probe d-IR-DPA with different concentrations of Cu 2+ Changes in UV absorption before and after solution mixing.

[0023] Figure 3(a) UV absorption and IVIS imaging of the supernatant after dialysis of the experimental probe d-IR-DPA and the control probe IR-PDA with and without hydrogen peroxide, (c) gel electrophoresis of the BSA after the reaction, with Coomassie brilliant blue staining and IVIS fluorescence imaging for image recording, (d) confocal imaging of probe d-IR-DPA and control probe IR-DPA after 48 hours of co-incubation with MDA-MB-231 cells, and (e) gel electrophoresis image after protein extraction.

[0024] Figure 4 (a) Real-time fluorescence images of subcutaneous MDA-MB-231 tumor-bearing nude mice after intravenous injection of IR-RGD and d-IR-RGD; (b) Quantitative analysis of the fluorescence images in (a); (c) Real-time photoacoustic images of subcutaneous MDA-MB-231 tumor-bearing nude mice after intravenous injection of IR-DPA and d-IR-DPA; (d) Quantitative analysis of the photoacoustic images in (c).

[0025] Figure 5 (a) Photoacoustic images of three different tumor models at different time points after intravenous injection of d-IR-DPA; (b) PA at 680 nm at different time points after injection of d-IR-DPA. 680 / PA 800 (c) Quantification of the ratio photoacoustic signal at the tumor site 4 hours after injection of d-IR-DPA; (d) Measurement of the copper ion content in the tumor by ICP-MS of mouse tumor fragments after imaging.

[0026] Figure 6 The study investigated the cytotoxicity changes of MDA-MB-231 cells after co-incubation of the experimental group probe d-IR-DPA, the control group probe d-IR-RGD, and IR-DPA with MDA-MB-231 cells for 48 h. (a) The study of intracellular copper content in MDA-MB-231 and 3T3 cells by ICP-OES. (b) The study of cytotoxicity changes of the experimental group probe d-IR-DPA, the control group probe d-IR-RGD, and IR-DPA with MDA-MB-231 cells for 48 h. (c) The study of cytotoxicity of the experimental group probe d-IR-DPA, the control group probe d-IR-RGD, and IR-DPA with MDA-MB-231 cells for 48 h. (d) The study of cell migration ability of the experimental group probe d-IR-DPA, the control group probe d-IR-RGD, and IR-DPA with MDA-MB-231 cells for 48 h by scratch assay. (e) After incubating the experimental group probe d-IR-DPA and the control group probes d-IR-RGD and IR-DPA with MDA-MB-231 cells for 48 h, the changes in cell apoptosis were observed using the live / dead kit.

[0027] Figure 7 To analyze the mitochondrial membrane potential of MDA-MB-231 cells under different treatment groups using JC-1, (a) the subcellular distribution of the probe in MDA-MB-231 cells, (b) the effect of d-IR-DPA on the mitochondrial membrane potential using JC-1, (c) the fluorescence ratio in (b), and (d) the use of MT-G to verify the damage of the probe to the mitochondrial structure. Red represents the probe, and green represents MT-G. The higher the probe concentration, the more severe the damage, and the weaker the labeling ability of MT-G on mitochondria.

[0028] Figure 8 The following figures illustrate (a) the changes in tumor inhibition over 19 consecutive days for the experimental group probe d-IR-DPA and the control group probes d-IR and IR-DPA, respectively; (b) the size of the ex vivo tumor on day 19; (c) the tumor inhibition curve; (d) the relative tumor mass; and (e) photographs of mice in different groups at different time points during treatment. Detailed Implementation

[0029] The protein-anchored copper-responsive photoacoustic probe disclosed in this invention is a novel diagnostic and therapeutic protein-anchored copper-responsive photoacoustic probe, having the following chemical structural formula:

[0030]

[0031] This invention discloses the application of the aforementioned protein-anchored copper-responsive photoacoustic probe in real-time chelation response to endogenous copper in tumors; or the application of the aforementioned protein-anchored copper-responsive photoacoustic probe in the preparation of fluorescent imaging agents and photoacoustic imaging agents; or the application of the aforementioned protein-anchored copper-responsive photoacoustic probe in increasing the retention time of the probe in tumor tissues or inhibiting tumors; or the application of the aforementioned protein-anchored copper-responsive photoacoustic probe in the preparation of reagents for increasing the retention time of the probe in tumor tissues or inhibiting tumors; or the application of the aforementioned protein-anchored copper-responsive photoacoustic probe in the preparation of reagents that react with cytoplasm; or the application of the aforementioned protein-anchored copper-responsive photoacoustic probe in prolonging tumor cell fluorescence imaging by crosslinking with protein sulfinic acid in tumor cells; or the application of the aforementioned protein-anchored copper-responsive photoacoustic probe in quantitative analysis of copper in tumors via ratiometric photoacoustics; or the application of the aforementioned protein-anchored copper-responsive photoacoustic probe in inhibiting tumor cell growth by chelating copper ions in tumor cells.

[0032] This invention discloses a method for preparing the above-mentioned protein-anchored copper-responsive photoacoustic probe, wherein compound 8 is reacted with compound 5 to obtain the protein-anchored copper-responsive photoacoustic probe. Preferably, the reaction is carried out in the presence of an organic solvent, preferably including N,N-dimethylformamide; the reaction time is 0.5 to 10 hours; and the molar ratio of compound 8 to compound 5 is 1:(1 to 5).

[0033] In this invention, compound 7 reacts with compound 2 to obtain compound 8; preferably, the reaction is carried out in the presence of sodium ascorbate and copper sulfate; the molar ratio of compound 7, compound 2, sodium ascorbate and copper sulfate is 1:(0.8-1.2):(0.05-0.2):(0.05-0.1).

[0034] In this invention, N-Boc-ethylenediamine reacts with 2-chloromethylpyridine hydrochloride, and then the protecting group is removed by trifluoroacetic acid to obtain compound 5; preferably, N-Boc-ethylenediamine reacts with 2-chloromethylpyridine hydrochloride in a small molecule alcohol to obtain compound 4; compound 4 is then deprotected by trifluoroacetic acid in an organic solvent to obtain compound 5.

[0035] The preparation method of the above-mentioned protein-anchored copper-responsive photoacoustic probe includes the following steps:

[0036] (1) 3,5-dioxanecarboxylic acid was activated by NHS and then reacted with 3-azidopropylamine to give compound 2;

[0037] (2) After N-Boc-ethylenediamine reacts with 2-chloromethylpyridine hydrochloride, the protecting group is removed by trifluoroacetic acid to obtain compound 5;

[0038] (3) Dye IR780 reacts with RGD to give compound 7;

[0039] (4) Compound 7 reacts with compound 2 to give compound d-IR;

[0040] (5) The reaction of compound d-IR-RGD with compound 5 yields the therapeutic protein-anchored copper-responsive photoacoustic probe d-IR-DPA.

[0041] Specifically, the preparation method of the above-mentioned protein-anchored copper-responsive photoacoustic probe includes the following steps.

[0042] (1) 3,5-Dioxanecarboxylic acid was activated in DMF by EDC and NHS, and then reacted with N,N-diisopropylethylamine (DIPEA) and 3-azidopropylamine for 2-5 h to give compound 2; in this reaction, the reaction of compound 3,5-dioxanecarboxylic acid with 3-azidopropylamine was carried out in the presence of EDC, NHS and DIPEA; the molar ratio of compound 3,5-dioxanecarboxylic acid, 3-azidopropylamine, EDC, NHS and DIPEA was 1:1:1.2:1.2:1; the reaction was carried out at room temperature;

[0043] (2) N-Boc-ethylenediamine and 2-chloromethylpyridine hydrochloride were reacted in methanol to give compound 4; compound 4 was deprotected in dichloromethane with trifluoroacetic acid to give compound 5; in this reaction, the reaction of N-Boc-ethylenediamine and 2-chloromethylpyridine hydrochloride was carried out in methanol; the molar ratio of N-Boc-ethylenediamine to 2-chloromethylpyridine hydrochloride was 1:2.5; the deprotection of compound 4 was carried out in a dichloromethane / trifluoroacetic acid mixed solvent;

[0044] (3) The reaction of compound IR780 with RGD yields compound 7; in this reaction, the molar ratio of cyclic peptide cRGD to dye IR780 is 1:(1-1.2); the reaction is carried out at room temperature; preferably, the reaction is carried out in the presence of sodium ascorbate and copper sulfate; the molar ratio of compound IR780, RGD, sodium ascorbate, and copper sulfate is 1:1:0.1:0.08; preferably, the RGD is azide-functionalized RGD;

[0045] (4) Compound 7 reacts with Compound 2 to give Compound 8. The reaction is carried out in the presence of sodium ascorbate and copper sulfate; the molar ratio of Compound 7, Compound 2, sodium ascorbate and copper sulfate is 1:1:0.1:0.08;

[0046] (5) Compound 8 and Compound 5 were reacted in N,N-dimethylformamide for 1-5 h to obtain the therapeutic protein-anchored copper-responsive photoacoustic probe d-IR-DPA. The molar ratio of Compound 8 to Compound 5 was 1:3.

[0047] The preparation method of the protein-anchored copper-responsive photoacoustic probes of the present invention and the chemical structural formulas of each compound are described below. Figure 1 In the compound, the coordinating anion is an iodide ion.

[0048] This invention discloses a photoacoustic imaging contrast agent, which is prepared from the above-mentioned protein-anchored copper-responsive photoacoustic probe.

[0049] This invention discloses the application of the above-mentioned protein-anchored copper-responsive photoacoustic probe in increasing the retention time of the probe in tumor tissue or inhibiting tumors, or in the preparation of reagents for increasing the retention time of the probe in tumor tissue or inhibiting tumors.

[0050] The probe of this invention is a protein-anchored copper-responsive photoacoustic probe. After entering tumor cells via a targeting peptide, it accumulates in the mitochondria and specifically cross-links with the high concentration of hyposulfonic acid protein within the mitochondria. Simultaneously, it selectively chelates with copper ions in the mitochondria, thereby activating the probe's photoacoustic properties. This also causes a copper ion depletion effect within the cell, leading to damage to mitochondrial structure and function, effectively inhibiting tumor growth.

[0051] The steps for synthesizing the therapeutically integrated protein-anchored copper-responsive photoacoustic probe d-IR-DPA of this invention are as follows: Cyclic peptide N3-RGD reacts with dye IR780 to obtain compound IR-RGD; compound IR-RGD then undergoes a click chemistry reaction with azide-functionalized 3,5-dioxocyclohexanecarboxylic acid to obtain compound d-IR; finally, the final probe, namely the therapeutically integrated protein-anchored copper-responsive photoacoustic probe d-IR-DPA, is obtained by reacting with the compound. IR-DPA and d-IR serve as control probes.

[0052] Aqueous solutions of protein-anchored copper-responsive molecular probe d-IR-DPA and control probes d-IR and IR-DPA were co-incubated with tumor cells for 48 hours, and the apoptosis of tumor cells was observed.

[0053] The novel protein-anchored copper-activated photoacoustic probe was used for in vivo tumor imaging and inhibition experiments. The process included the following steps: first, an aqueous solution of the probe d-IR-DPA was injected intravenously into tumor-bearing mice; photoacoustic imaging was performed at different time points after injection; or multiple injections of the probe were used to conduct tumor inhibition experiments.

[0054] (1) In vivo photoacoustic imaging: The probe was dissolved in PBS solution (concentration: 100 µM, volume: 200 µL) and injected into nude female mice bearing tumors (MDA-MB-231 human triple-negative breast cancer) via tail vein injection. Simultaneously, the small animal photoacoustic tomography imaging system was turned on. When the water temperature in the photoacoustic imaging system reached 37℃, anesthetized mice were placed in the system, and images of the tumor sites were scanned. Tumor imaging was monitored at 2, 4, 8, 12, 24, and 48 h. Finally, the photoacoustic signal intensity of the tumor sites at different time points was calculated using in vivo imaging analysis software. The obtained photoacoustic imaging data were then reconstructed and analyzed using MSOT InSight / inVision analysis software.

[0055] (2) Tumor suppression experiment: MDA-MB-231 cells (5×10⁻⁶) 6 A d-IR-DPA probe (100 µM, 200 µL) was implanted into the right back of female Nude mice. When the tumor reached 1000 mm³, it was divided into 100 mm³ pieces and transplanted into the back of the Nude mice using a puncture needle. After the transplanted tumors had fully stabilized and grown, either a d-IR-DPA probe (100 µM, 200 µL) or a control probe (100 µM, 200 µL) was injected intravenously. A second injection was given two days after the first, for a total of three treatments. After treatment, the tumor size and volume were measured in each group of mice. Fifteen days later, the mice were sacrificed, and the tumors were further dissected and weighed.

[0056] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to explain and illustrate the technical solutions in the present invention, and are not intended to limit the scope of the present invention. In addition, unless otherwise stated, the materials, reagents, instruments, etc. used in the following embodiments can be obtained by commercial means, the specific preparation operations and testing methods of the present invention are conventional techniques, and the animal experiments comply with the animal experiment regulations of Soochow University.

[0057] Example 1: The preparation method of the protein-anchored copper-responsive photoacoustic detector of the present invention, the synthesis steps are as follows: Figure 1 As shown, the coordinating anion in the compound is an iodide ion, as detailed below.

[0058] Compound 2: 20 mg (0.128 mmol) of 3,5-dioxocyclohexanecarboxylic acid, 25 mg (0.13 mmol) of 1-ethyl-3-(3-dimethylaminopropyl)carbonyldiimidazole (EDC) hydrochloride, and 15 mg (0.14 mmol) of N-hydroxysuccinimide (NHS) were dissolved in 5 mL of anhydrous DMF under stirring at 0°C, and the mixture was stirred at room temperature for 1 hour. Then, 15 mg (0.14 mmol) of 3-azidopropylamine and 10 μL of DIPEA were added to the above solution, and stirring was continued for 4 hours. The solvent was then removed under vacuum, and compound 2 (19 mg, yield 62.3%) was purified by silica gel chromatography. 1 H NMR (600 MHz, DMSO-d6) δ 5.39 (d, J = 0.7 Hz, 1H), 3.32 (s, 2H), 3.28 (s, 1H), 3.24 (s,2H), 2.72 (dd, J = 17.3, 10.6 Hz, 2H), 2.50 (dd, J = 14.5, 2.8 Hz, 2H), 1.74(s, 2H), 1.71 (d, J = 6.7 Hz, 2H).13 C NMR (151 MHz, DMSO-d6) δ 181.88,112.95, 58.03, 57.76, 53.39, 45.58, 37.95, 36.67, 31.87, 31.29. MS (MALDI-TOF) Calcd for: C 10 H 14 N4O3([M+H)) + ): 239.11, found: 238.99.

[0059] Compound DPA: N-Boc-ethylenediamine (0.4 g, 2.5 mmol), Na₂CO₃ (1.2 g, 11.3 mmol), and 2-chloromethylpyridine hydrochloride (0.9 g, 5.5 mmol) were dissolved in methanol and refluxed under a nitrogen atmosphere for 48 hours. The solvent was then removed using a rotary evaporator. The remaining reactants were dissolved in 1N NaOH aqueous solution and extracted with dichloromethane (DCM). After extraction, the organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated again to give a yellow liquid. The crude product was purified by alumina (Al₂O₃, DCM / MeOH) column chromatography to give compound 4. Compound 4 (300 mg, 0.872 mmol) was dissolved in dichloromethane, and 3 mL of trifluoroacetic acid (TFA) was added dropwise under ice bath conditions, with stirring for 3 hours, followed by neutralization with 1N NaOH aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated to give a yellow liquid, compound 5 (DPA) (187 mg, 89% yield). MS (MALDI-TOF) Calculated for: C 14 H 18 N4 ([M+H) + ): 243.15, found: 243.37.

[0060] Compound 6: Synthesized and characterized according to existing methods (Journal of the American Chemical Society 2022, 144 (50), 23061-23072). 1H NMR (600 MHz, DMSO-d6) δ 8.28 (d, J =14.1 Hz, 2H), 7.64 (d, J = 7.4 Hz, 2H), 7.51-7.40 (m, 4H), 7.36-7.25 (m, 2H), 6.37 (d, J = 14.1 Hz, 2H), 4.27 (t, J = 7.3 Hz, 4H), 2.98 (s, 2H), 2.71 (t, J= 5.9 Hz, 4H), 2.50 (d, J = 1.7 Hz, 2H), 2.35 (td, J = 6.8, 2.3 Hz, 4H), 2.00- 1.89 (m, 4H), 1.69 (s, 12H), 1.23 (s, 4H). 13 C NMR (151 MHz, DMSO-d6) δ172.43, 148.18, 143.18, 142.01, 141.07, 129.62, 128.61, 126.36, 125.22,122.56, 111.30, 101.53, 83.52, 72.26, 49.06, 42.82, 29.08, 25.94, 20.33,15.30. MS (MALDI-TOF) Calcd for: C 40 H 44 ClN2 ([M] + ): 587.32, found: 587.67.

[0061] Compound 7: Compound 6 (I - The complex (13.8 mg, 0.02 mmol) and N3-RGD (14 mg, 0.02 mmol) were dissolved in 2 mL of dimethyl sulfoxide as the reaction solution. Then, an aqueous solution of copper sulfate pentahydrate (800 μL, 0.05 M) and an equal volume of sodium ascorbate aqueous solution were mixed. After sonicating for 5 minutes until the solution became an orange-yellow suspension, the mixture was added dropwise to the reaction solution and stirred at room temperature for 6 hours. After the reaction was complete, the mixture was purified and separated by preparative high-performance liquid chromatography to obtain 12.6 mL of compound 7, namely probe IR-RGD, with a yield of 46%. 1 H NMR (600 MHz, DMSO- d6) δ 8.42-8.35(m, 1H), 8.23 (dd, J = 19.6, 14.0 Hz, 2H), 8.07 (dd, J = 15.1, 12.4 Hz, 2H),7.93 (s, 1H), 7.84 (s, 1H), 7.62 (s, 4H), 7.43 (d, J = 7.9 Hz, 5H), 7.27 –7.22 (m, 2H), 6.89-6.85 (m, 2H), 6.61-6.58 (m, 2H), 6.35 (d, J = 14.2 Hz,2H), 4.59 (d, J = 6.1 Hz, 1H), 4.30-4.26 (m, 3H), 4.18 (dd, J = 13.9, 6.9 Hz,4H), 4.11 (dd, J = 14.5, 7.8 Hz, 4H), 3.22-3.17 (m, 1H), 3.06-3.00 (m, 2H),2.93 (d, J = 2.6 Hz, 2H), 2.75 (d, J = 7.4 Hz, 2H), 2.69-2.62 (m, 6H), 2.45(ddd, J = 2.9, 1.2, 0.6 Hz, 4H), 2.34 (s, 3H), 2.10-2.05 (m, 2H), 1.87 (s,2H), 1.83 (d, J = 5.4 Hz, 2H), 1.66 (d, J = 2.4 Hz, 15H), 1.55 (s, 1H), 1.41(s, 1H), 1.32-1.28 (m, 2H), 1.20-1.17 (m, 1H), 1.00-0.96 (m, 1H). 13 C NMR (151MHz, DMSO- d6) δ 172.90, 172.14, 172.08, 171.80, 171.25, 170.88, 170.06,169.68, 158.63, 158.42, 156.83, 155.89, 148.28, 145.77, 143.70, 142.88,142.19, 142.10, 141.34, 141.08, 130.13, 129.76, 128.77, 128.72, 127.42,126.59, 126.34, 125.53, 125.16, 122.70, 122.09, 115.05, 111.73, 111.25, 102.22, 101.19, 83.68, 72.37, 54.90, 54.45, 51.98, 49.28, 49.14, 49.05, 48.99, 43.62, 43.38, 42.82, 42.22, 40.51, 40.40, 36.72, 35.18, 30.71, 29.15, 28.95, 29.30, 28.60, 27.64, 27.58, 26.53, 26.00, 25.29, 22.66, 22.22, 20.51,15.43.MS (MALDI-TOF) Calcd for: C 67 H 83 ClN 13 O8 ([M+H) + ): 1233.6, found: 1233.00.

[0062] Compound d-IR-RGD: Compound 7 (15 mg, 0.012 mmol) and compound 2 (2.2 mg, 0.012 mmol) were dissolved in 1 mL of dimethyl sulfoxide. Then, an aqueous solution of copper sulfate pentahydrate (800 μL, 0.05 M) and an equal volume of sodium ascorbate aqueous solution were mixed. After sonication for 5 minutes until the solution became an orange-yellow suspension, the mixture was added dropwise to the reaction solution and stirred at room temperature for 6 hours. After the reaction was complete, the mixture was purified by preparative high-performance liquid chromatography (HPLC) to obtain 14.1 mg of compound 8, which served as the control probe d-IR, with a yield of 78%. Its molecular formula was determined by ESI-MS to be C0. 63 H 75 N 30 O 2+ The molecular ion peak is 1173.5688. 1 H NMR (600 MHz, DMSO- d6) δ 8.43 (s, 2H), 8.26 (d, J = 13.9Hz, 2H), 8.12-8.02 (m, 3H), 7.97 (d, J = 7.0 Hz, 1H), 7.92 (s, 1H), 7.87 (s,1H), 7.62 (s, 2H), 7.45-7.38 (m, 3H), 7.30-7.24 (m, 2H), 6.92 (d, J = 8.2 Hz,2H), 6.64 (d, J = 8.2 Hz, 2H), 6.34 (t, J = 14.5 Hz, 2H), 5.31 (d, J = 5.0Hz, 1H), 5.20 (s, 1H), 4.63 (dd, J = 14.6, 8.2 Hz, 1H), 4.31 (d, J = 6.6 Hz,5H), 4.21 (d, J = 6.9 Hz, 1H), 4.15 (dd, J = 14.4, 7.4 Hz, 2H), 4.04 (dd, J =14.9, 7.6 Hz, 2H), 3.96 (s, 2H), 3.66 (s, 1H), 3.33 (dd, J = 10.8, 5.8 Hz,1H), 3.27-3.17 (m, 2H), 3.06 (dd, J = 12.8, 6.3 Hz, 5H), 2.88 (s, 1H), 2.85-2.75 (m, 4H), 2.71-2.61 (m, 5H), 2.44-2.28 (m, 4H), 2.11 (s, 3H), 2.01-1.81(m, 6H), 1.73-1.62 (m, 12H), 1.45 (s, 4H), 1.35 (dd, J = 13.5, 6.3 Hz, 2H),1.28-1.15 (m, 5H), 1.10-0.95 (m, 3H), 0.84 (t, J = 6.9 Hz, 1H). 13 C NMR (151MHz, DMSO- d6) δ 172.51, 172.19, 171.85, 171.34, 170.96, 170.14, 169.75,158.95, 158.70, 158.45, 158.20, 156.88, 155.96, 148.33, 145.85, 143.39,142.22, 141.27, 130.20, 129.81, 128.77, 127.48, 126.48, 125.40, 122.71,122.50, 122.15, 115.12, 111.58, 103.48, 101.88, 65.89, 56.14, 55.20, 54.98,54.54, 52.07, 49.22, 49.08, 48.53, 47.25, 43.57, 36.81, 36.03, 35.25, MS (MALDI-TOF) Calcd for: C 77 H 97 ClN 17 O 11 ([M+H)) + ):1473.1, found:1473.3.

[0063] Compound IR-DPA: Compound 7 (15 mg, 0.012 mmol) and compound 5 (8.87 mg, 0.03 mmol) were dissolved in 1 mL of DMF. The mixture was stirred at 50 °C for 6 hours. After the reaction was complete, the mixture was purified by preparative high-performance liquid chromatography (HPLC) to obtain 9.63 mg of compound 8, which served as the control probe IR-DPA, with a yield of 75%. MS (MALDI-TOF) Calcd for: C 81 H 100 N 17 O8 ([M] + ): 1439.79, found: 1439.29.

[0064] Compound d-IR-DPA: Compound d-IR (10 mg, 0.0068 mmol) and compound 5 (6 mg, 0.02 mmol) were dissolved in 1 mL DMF. The mixture was stirred at 50 °C for 6 hours. After the reaction was complete, the mixture was purified by preparative high-performance liquid chromatography to obtain 8.32 mg of compound d-IR-DPA, with a yield of 73%. 1H NMR (600 MHz, dmso) δ 8.73-8.64 (m, 2H), 8.63-8.56 (m, 1H), 8.39 (s, 1H), 8.08 (d, J = 7.8 Hz, 3H),8.03-7.95 (m, 3H), 7.89 (ddd, J = 31.0, 11.9, 6.4 Hz, 2H), 7.77 (s, 1H), 7.70(d, J = 7.7 Hz, 2H), 7.65-7.57 (m, 2H), 7.52 (d, J = 15.3 Hz, 1H), 7.42 (dd,J = 21.8, 15.7 Hz, 3H), 7.33-7.20 (m, 2H), 7.06 (dd, J = 24.1, 16.2 Hz, 3H),6.92 (d, J = 7.2 Hz, 2H), 6.64 (d, J = 7.2 Hz, 2H), 5.77 (s, 1H), 5.32 (dd, J= 12.3, 7.7 Hz, 1H), 5.19 (s, 1H), 4.65 (dd, J = 20.5, 12.6 Hz, 10H), 4.57(s, 1H), 4.54 (s, 1H), 4.47 (s, 1H), 4.41 (d, J = 12.8 Hz, 1H), 4.36-4.25 (m,3H), 4.24-4.14 (m, 3H), 4.10 (d, J = 18.2 Hz, 3H), 4.03 (d, J = 7.2 Hz, 2H),3.93 (s, 2H), 3.84 (s, 1H), 3.72 (d, J = 19.8 Hz, 1H), 3.65 (s, 1H), 3.55-3.47 (m, 1H), 3.33 (d, J = 5.9 Hz, 1H), 3.29-3.24 (m, 1H), 3.23-3.12 (m, 9H),3.11-3.02 (m, 3H), 2.95 (t, J = 6.0 Hz, 2H), 2.88 (d, J = 1.5 Hz, 3H), 2.83-2.62 (m, 6H), 2.57-2.54 (m, 1H), 2.43 (s, 1H), 2.36 (d, J = 11.3 Hz, 1H),2.08-1.88 (m, 4H), 1.74-1.63 (m, 3H), 1.60-1.42 (m, 6H), 1.35 (d, J = 6.9 Hz, 1H), 1.31-1.19 (m, 6H), 1.04 (s, 1H), 0.84 (t, J = 6.9 Hz, 1H). 13 C NMR (151MHz, DMSO- d6 ) δ 174.83, 172.46, 172.13, 171.54, 171.16, 170.36, 169.97,164.49, 162.75, 162.38, 159.24, 159.02, 158.79, 158.56, 157.24, 156.21,155.58, 152.50, 149.44, 149.08, 148.44, 147.96, 147.72, 146.55, 146.28,143.27, 141.03, 140.17, 139.53, 139.33, 138.97, 137.94, 130.44, 130.07, 128.54, 127.76, 125.40, 124.99, 124.57, 124.44, 124.14, 123.61, 123.23, 122.86, 122.46, 122.30, 119.03, 115.37, 58.35, 57.43, 55.21, 54.79, 53.71, 52.25, 51.75, 49.44, 49.30, 49.02, 47.46, 46.98, 43.69, 37.03, 36.61, 36.21,35.54, 31.71, 31.19, 31.05, 29.61, 29.51, 29.45, 29.40, 29.26, 29.17, 29.12,29.00, 28.89, 28.65, 26.98, 26.14, 25.56, 24.58, 22.98, 22.69, 22.52, 14.35.MS (MALDI-TOF) Calcd for: C 91 H 114 N 21 O 11 ([M] + ): 1676.70.

[0065] Example 2: Physicochemical Properties of the Probe

[0066] The experimental probe d-IR-DPA prepared in Example 1 was diluted with ultrapure water to a concentration of 10 µM (completely soluble), and its ultraviolet-visible-near-infrared spectrum and fluorescence spectrum were measured using a UV-Vis-NIR spectrophotometer and a fluorescence spectrophotometer. Figure 2 The results showed that the maximum absorption of the probe d-IR-DPA was at 620 nm, and the maximum emission was at 790 nm. The control group probes IR-DPA, d-IR, and the experimental group probe d-IR-DPA were respectively reacted with 10 µM CuCl2-prepared solution. 2+ After incubation with shaking for 5 minutes, both probes d-IR-DPA and IR-DPA exhibited a redshift of maximum UV absorption from 620 nm to 680 nm (e.g., Figure 2 b、 Figure 2 c and Figure 2 As shown in d), while d-IR-RGD showed no significant change (as shown in d). Figure 2 (as shown in e), the effect of different concentrations of copper ions on the probe is described in [reference]. Figure 2 f.

[0067] IR-DPA and d-IR-DPA were diluted with ultrapure water to a concentration of 10 µM. TCEP-treated bovine serum albumin (BSA) was added, and the mixture was incubated with H2O2 (50 µM) as a variable. The mixture was then shaken at room temperature for 30 minutes, followed by centrifugation at 12000 rpm and washing three times with PBS buffer. Figure 3 As shown in a and b, UV absorption and fluorescence spectroscopy measurements revealed that the d-IR-DPA probe successfully labeled TCEP-pretreated BSA under the action of H2O2, as evidenced by a strong fluorescence signal and a UV peak at 620 nm. Further gel electrophoresis was used to determine the probe's labeling ability for BSA. Figure 3 As shown in c, in the presence of H2O2, the band of probe d-IR-DPA co-incubated with BSA showed strong fluorescence. IR-DPA and d-IR-DPA were added to the culture medium (HyClone DMEM high-glucose liquid medium containing 10% FBS) at a concentration of 10 µM and respectively added to MDA-MB-231 cells (2 × 10⁶ cells / year). 5 After culturing in the culture medium for 8 hours, the probe was washed off, and the retention of the probe in the cells was continuously observed over 48 hours. Figure 3 As shown in Figure d, the fluorescence intensity of probe d-IR-DPA was greater than that of the control group within 48 hours. IR-DPA and d-IR-DPA were added to culture medium (HyClone DMEM high-glucose liquid medium containing 10% FBS) at a concentration of 10 µM and respectively into MDA-MB-231 cells (2 × 10⁶ cells per cell line). 5After culturing in the medium for 8 hours, the probe was washed off, and the cells were cultured for another 48 hours, with the medium changed every 6 hours. After the culture was complete, proteins were extracted from MDA-MB-231 cells and subjected to electrophoresis. Following the experiment, the proteins were stained with CBB and subjected to two-zone fluorescence imaging. Figure 3 As shown in Figure e, when the protein amounts in the two groups were approximately the same, the proteins in the cells incubated with probe d-IR-DPA showed obvious fluorescent signals, confirming that the probe effectively labeled intracellular hyposulfonated proteins. The above experiments successfully demonstrated that, in the presence of protein hyposulfonates, probe d-IR-DPA can effectively label hyposulfonated proteins, forming probe-protein conjugates.

[0068] To investigate probe retention in mouse tumors, IR-RGD and d-IR-RGD were diluted with PBS to a concentration of 100 µM, and 200 µL of IR-RGD or d-IR-RGD was injected via tail vein. The metabolism of the probe within the tumor at various time points was observed using IVIS in vivo fluorescence imaging. Figure 4 As shown in Figure a, the fluorescence intensity of the experimental group (d-IR-RGD) at the tumor site was found to be significantly greater than that of the control group (IR-RGD). Quantitative data... Figure 4 b also confirms this. IR-DPA and d-IR-DPA were diluted with PBS to a concentration of 100 µM, and 200 µL of IR-DPA or d-IR-DPA were injected via tail vein. The metabolism of the probe within the tumor at each time point was observed using the MSOT photoacoustic imaging system. Figure 4 As shown in Figure c, the photoacoustic signal intensity at the tumor site was found to be significantly greater in the experimental group (d-IR-DPA) than in the control group (IR-DPA). Quantitative data... Figure 4 d also confirms this.

[0069] IR-DPA and d-IR-DPA were diluted with PBS to a concentration of 100 µM (200 µL). Mice bearing different tumors (MDA-MB-231, MCF-7, and HepG2) were injected via tail vein with either IR-DPA or d-IR-DPA. Photoacoustic images were acquired for 4 hours using MSOT under excitation light at 680 and 800 nm. After imaging reconstruction, the probe signals in the tumor region were analyzed for Region of Interest (ROI) using the MSOT imaging system software package. The three types of tumors were removed and dissolved, and the copper ion content in the tissue was quantitatively analyzed by ICP-MS, establishing a linear relationship with the corresponding photoacoustic signals. In the in vivo imaging study, MDA-MB-231, MCF-7, and HepG2 were selected as tumor models. 200 µL of probe d-IR-DPA (100 µM concentration) was injected via tail vein, and the photoacoustic signals at 680 nm at different time points were observed. To eliminate interference from different probe concentrations, PA800 was used as an internal reference signal. Figure 5 The results showed that after probe injection, the photoacoustic signal within the tumors of the three tumor model mice increased over time, reaching a maximum at 4 hours, and then decreased. Therefore, ratiometric photoacoustic quantification was performed at the maximum time point. Figure 5 b shows that the MDA-MB-231 subcutaneous tumor model exhibited the strongest ratio photoacoustic signal, significantly higher than the non-TNBC MCF-7 tumor model. This indicates that TNBC tumors have higher copper dependence and copper content, explaining their high metastatic and invasive capabilities. Although HepG2 showed a similar photoacoustic signal at 680 nm to MDA-MB-231, its signal was stronger at 800 nm, resulting in a higher ratio signal (PA). 680 / PA 800 ) decrease, such as Figure 5 As shown in c. Further quantification of copper content within tumor tissue was performed using ICP-MS, such as... Figure 5 As shown in d, the results show the same trend as the ratio photoacoustic signal.

[0070] 3T3 and MDA-MB-231 cells were digested, centrifuged, and lysed. Intracellular copper ion content was measured using ICP-OES. Figure 6 As shown in Figure a, the copper ion content in MDA-MB-231 cells was higher than that in 3T3 cells. d-IR-RGD, IR-DPA, and d-IR-DPA were added to culture medium (HyClone DMEM high-glucose liquid medium containing 10% FBS) at a concentration of 10 µM and cultured in MDA-MB-231 and 3T3 cells for 48 hours, respectively. It was found that, compared to normal cells (3T3), they exhibited certain toxicity against triple-negative breast cancer (MDA-MB-231). Figure 6b and 6c. Simultaneously, cell migration experiments showed that the experimental group had a good ability to inhibit tumor cell proliferation and migration (see [reference needed]). Figure 6 d, and simultaneously, the Live-dead reagent showed that the experimental group (d-IR-DPA) exhibited the ability to induce tumor cell apoptosis. Figure 6 e. (****p<0.0001, t-test, n=3. ([d-IR-DPA]=[d-IR-RGD]=[IR-DPA]= 10 µM).

[0071] In the mitochondrial damage assay, d-IR-DPA was added to the culture medium (HyClone DMEM high-glucose liquid medium containing 10% FBS) at a concentration of 10 µM and cultured in MDA-MB-231 cells for 8 hours. Mitochondria were then stained using a commercial mitochondrial tracer (Mitotracker-Green). See [details omitted]. Figure 7 a) The probe d-IR-DPA was found to highly target mitochondria. Mitochondrial membrane potential was measured using the indicator JC-1. JC-1 formed J-aggregates and emitted red fluorescence in cells with high mitochondrial membrane potential, while remaining monomeric and emitting green fluorescence in cells with low mitochondrial membrane potential. Representative results of d-IR-DPA versus control treatment of MDA-MB-231 cells are shown below. Figure 7 As shown in b, the quantitative red-green fluorescence ratio of cells is shown in [reference]. Figure 7 c. MT-G was used to assess mitochondrial structural integrity after cell treatment with different concentrations of d-IR-DPA, such as... Figure 7 As shown in d, it was found that as the probe concentration increased, the ability of MT-G to stain mitochondria decreased, indicating that the mitochondrial structure was damaged.

[0072] Example 3

[0073] MDA-MB-231 cell tumor fragments were divided into 80 mm³ sections and transplanted into the right back of nude mice. After one week of fixation, the tumors grew to 100 mm³. All mice were divided into four groups (n=5). The probe d-IR-DPA (1.5 μg g) was injected via tail vein. -1 The control probe (PBS, d-IR-RGD, IR-DPA) was injected into nude mice for a total of three treatments, with two-day intervals between treatments. See [link to relevant documentation]. Figure 8 a. After the first probe injection, tumor size was measured using calipers for 19 consecutive days, and tumor changes were photographed. The results are as follows: Figure 8As shown in the results, tumors in mice in the PBS and d-IR-RGD groups exhibited exponential growth, with tumor diameters exceeding 1 cm and accompanied by ulceration. In contrast, tumors in mice in the IR-DPA and d-IR-DPA groups showed overall tumor suppression, but tumor recurrence occurred in the IR-DPA group later, while the d-IR-DPA group better delayed tumor recurrence. This indicates that probe modification of cyclohexanedione successfully promoted the copper depletion strategy and improved the therapeutic effect. (**p<0.01, ****p<0.0001, t-test, n=3. [d-IR-DPA]=[d-IR-RGD]=[IR-DPA]= 1.5 mg / g).

Claims

1. A protein-anchored copper-responsive photoacoustic probe having the following chemical structural formula: 。 2. The application of the protein-anchored copper-responsive photoacoustic probe of claim 1 in the preparation of fluorescent imaging agents and photoacoustic imaging agents.

3. The use of the protein-anchored copper-responsive photoacoustic probe of claim 1 in the preparation of reagents for chelating copper ions and / or cross-linking protein sulfenic acid.

4. An imaging agent or drug, characterized in that, Includes the protein-anchored copper-responsive photoacoustic probe of claim 1.

5. The use of the protein-anchored copper-responsive photoacoustic probe of claim 1 in the preparation of reagents that improve the retention time of probes in tumor tissues or inhibit tumors.

6. The method for preparing the protein-anchored copper-responsive photoacoustic probe according to claim 1, characterized in that, Compound 8 reacts with compound 5 to obtain the protein-anchored copper-responsive photoacoustic probe. The chemical structural formula of compound 5 is as follows: ； The chemical structural formula of compound 8 is as follows: 。

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