A Therapeutic Nucleic Acid Anchored Fluorescent Probe, Its Preparation Method and Application

Abstract

This invention discloses a diagnostic and therapeutic nucleic acid-anchored fluorescent probe, its preparation method, and its application. This fluorescent probe can spontaneously generate singlet oxygen and cross-link with intracellular RNA under the mediation of singlet oxygen, realizing long-window imaging of tumor tissue. At the same time, it was found that cross-linked RNA causes mitochondrial dysfunction and further induces severe apoptosis in tumor cells, thus realizing the integrated diagnosis and treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the technical field of small molecule fluorescent probe bioimaging and tumor therapy, specifically involving novel anchored molecular probes and their preparation methods, as well as the application of these probes in multimodal imaging and tumor therapy. Background Technology

[0002] With the significant increase in morbidity and mortality rates over the past few decades, cancer has become one of the major threats to human health and life. Molecular probes, as a core element of molecular imaging technology, play a crucial role in the precision diagnosis and treatment of cancer. Although numerous molecular imaging probes have been developed for cancer imaging and treatment, 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, 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, most activatable diagnostic and therapeutic strategies rely on a single trigger, which is often insufficient to effectively avoid normal cells and precisely ablate cancer cells, leading to potential false positive or false negative results. Among the many triggering modes, light has proven to be an attractive tool for precise activation therapy with spatiotemporal controllability, without requiring physical contact. Summary of the Invention

[0003] To overcome the problems existing in the above-mentioned materials and technologies, this invention constructs a novel anchored molecular probe that utilizes its advantages of having cross-linking groups, good biocompatibility, active targeting of integrins, and near-infrared emission to perform long-term in vivo fluorescence, photoacoustic imaging, and tumor therapy.

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

[0005] A novel diagnostic and therapeutic nucleic acid-anchored fluorescent probe has the following chemical structural formula:

[0006] .

[0007] The preparation method of the above-mentioned diagnostic and therapeutic nucleic acid-anchored fluorescent probe includes the following steps:

[0008] (1) The cyclic peptide cRGD reacts with the dye IR780 to give the compound CRI;

[0009] (2) The compound CRI reacts with activated 3-(2-furan)propionic acid to obtain the diagnostic and therapeutic nucleic acid anchoring fluorescent probe ƒ-CRI.

[0010] Specifically, the preparation method of the above-mentioned diagnostic and therapeutic nucleic acid-anchored fluorescent probe includes the following steps:

[0011] (1) Cyclic peptide cRGD and dye IR780 were reacted in organic solvent DMF at room temperature for 10-15 h to obtain compound CRI;

[0012] (2) 3-(2-furan)propionic acid was activated by N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; the compound CRI was reacted with the activated 3-(2-furan)propionic acid and N,N-diisopropylethylamine (DIPEA) for 1-5 h to obtain the diagnostic and therapeutic nucleic acid anchoring fluorescent probe ƒ-CRI.

[0013] This invention discloses a red light-mediated probe-anchored cell method, comprising the following steps: co-incubating the above-mentioned diagnostic and therapeutic nucleic acid-anchored fluorescent probe with cells to achieve probe-anchored cells; wherein, the co-incubation is carried out under light and in a culture medium (such as 1640 medium).

[0014] In the above technical solution, the cyclic peptide cRGD reacts with the dye IR780 in an organic solvent to obtain the compound CRI; the compound CRI reacts with activated 3-(2-furan)propionic acid and N,N-diisopropylethylamine (DIPEA) in an organic solvent to obtain the anchored molecular probe ƒ-CRI; the molar ratio of the cyclic peptide cRGD to the dye IR780 is 1:(1-1.2); preferably, the molar ratio of the compound CRI to 3-(2-furan)propionic acid is 1:(1-1.5).

[0015] This invention discloses the application of the aforementioned therapeutic nucleic acid-anchored fluorescent probes in fluorescence imaging and photoacoustic imaging; or the application of the aforementioned therapeutic nucleic acid-anchored fluorescent probes in the preparation of fluorescence imaging agents and photoacoustic imaging agents; or the application of the aforementioned therapeutic nucleic acid-anchored fluorescent probes in increasing the retention time of probes in tumor tissues or inhibiting tumors; or the application of the aforementioned therapeutic nucleic acid-anchored fluorescent probes in the preparation of reagents for increasing the retention time of probes in tumor tissues or inhibiting tumors; or the application of the aforementioned therapeutic nucleic acid-anchored fluorescent probes in the preparation of reagents that react with cytoplasm. Alternatively, the application of the aforementioned novel anchored molecular probes in cross-linking with RNA within tumor cells to prolong tumor cell fluorescence imaging; or the application of the aforementioned novel anchored molecular probes in preparing reagents for cross-linking with RNA within tumor cells to inhibit tumor cell growth.

[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 anchored molecular probe ƒ-CRI, which can perform long-term in vivo fluorescence and photoacoustic imaging under red light-mediated singlet oxygen generation;

[0018] (2) In this invention, the target probe can undergo a cross-linking reaction with intracellular RNA under red light-mediated singlet oxygen generation, thereby prolonging the retention time of the probe molecules in the cell;

[0019] (3) The target probe in this invention has a good ability to promote tumor cell apoptosis after cross-linking RNA in tumor cells and causing mitochondrial damage;

[0020] (4) In this invention, the target probe has the ability to inhibit tumor growth in tumor-bearing mice after undergoing a cross-linking reaction in vivo. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the synthesis of the diagnostic and therapeutic nucleic acid-anchored fluorescent probe in Example 1.

[0022] Figure 2 The images show the chemical structures of probe ƒ-CRI in the experimental group and probe CRI in the control group in Example 2, (a) UV absorption and fluorescence emission images of probe ƒ-CRI and probe CRI in aqueous solution, and (c) evaluation of the ability of probe ƒ-CRI and probe CRI to generate singlet oxygen.

[0023] Figure 3 (a) Gel electrophoresis image of RNA cross-linking reaction with ƒ-CRI; (b) Confocal image and colocalization rate of ƒ-CRI in the experimental group and probe CRI in the control group after incubation with 4T1 cells for 6 hours and then incubation with RNA Select; (c, d) Fluorescence and quantitative images of total RNA extracted from cells using the total RNA kit.

[0024] Figure 4 (a) After incubating the experimental group probe ƒ-CRI and the control group probe CRI with 4T1 cells for 6 hours, they were exposed to light. Subsequently, the retention changes of the probe in the cells and the fluorescence intensity were observed by confocal imaging (b). (c) The experimental group probe ƒ-CRI and the control group probe CRI were injected into the tail vein. Four hours later, they were exposed to light to observe the retention changes of the probe in the tumor tissue and the fluorescence intensity (d). Under the same experimental conditions, the changes in photoacoustic signal of the experimental group probe ƒ-CRI and the control group probe CRI in the tumor (e) and the photoacoustic intensity were quantified (f).

[0025] Figure 5(a) Changes in cytotoxicity of 4T1 cells after co-incubation with probe ƒ-CRI in the experimental group and probe CRI in the control group for 12 h; (b) Changes in cytotoxicity of 4T1 cells after co-incubation with probe ƒ-CRI in the experimental group and probe CRI in the control group for 12 h followed by illumination (808 nm, 0.5 w / cm²). 2 Cytotoxicity changes after 3 min; (c) Experimental group probe ƒ-CRI and control group probe CRI were co-incubated with 4T1 cells for 12 h before being exposed to light (808 nm 0.5w / cm). 2 (3 min) Observe changes in cell apoptosis using live-dead and bright field methods respectively; (d) The ability to inhibit tumor cell proliferation and migration.

[0026] Figure 6 The following diagrams show the changes in tumor inhibition over 15 consecutive days for (a) the experimental group probe ƒ-CRI, the control group probe CRI, and the blank group PBS; (b) the size comparison of tumors on the back of mice on day 15; (c) the size of tumors ex vivo on day 15; (d) the tumor inhibition curve; (e) the survival cycle curve; and (f) the changes in Tunel and H&E by immunofluorescence and immunohistochemistry 48 h after treatment.

[0027] Figure 7 This is a schematic diagram illustrating the function of the probe in this invention. Detailed Implementation

[0028] The probe of this invention is an RNA-reactive fluorescent probe. Under red light irradiation, the probe reacts with intracellular RNA, prolonging its residence time in tumor cells and enabling long-window tumor imaging. More importantly, this cross-linking reaction causes mitochondrial damage, leading to severe tumor cell apoptosis and thus inhibiting tumor growth. The invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are only for explaining and illustrating the technical solutions of this invention and are not intended to limit the scope of the invention. Furthermore, unless otherwise stated, the materials, reagents, instruments, etc., used in the following embodiments are all commercially available; the specific preparation and testing methods are conventional methods in the art.

[0029] The steps for synthesizing the therapeutically integrated nucleic acid-anchored fluorescent probe ƒ-CRI of this invention are as follows: cyclic peptide cRGD reacts with dye IR780 to obtain compound CRI; compound CRI reacts with activated 3-(2-furan)propionic acid to obtain the therapeutically integrated nucleic acid-anchored fluorescent probe ƒ-CRI. CRI serves as a control probe.

[0030] The apoptosis of tumor cells was observed after incubating the aqueous solutions of the anchored molecular probe ƒ-CRI and the control probe CRI with tumor cells for 48 h.

[0031] The method for in vivo tumor inhibition experiments using the aforementioned novel anchored molecular probe includes the following steps: First, an aqueous solution of probe ƒ-CRI is injected intravenously into tumor-bearing mice. After 4 hours, the mice are exposed to light and the tumor inhibition is continuously observed and recorded. Then, probe ƒ-CRI is replaced with control group CRI for a control experiment.

[0032] (1) In vivo fluorescence imaging: The probe was injected into BALB / c female mice bearing tumors (4T1 mouse mammary cancer) via tail vein injection, and then placed in a small animal in vivo optical imaging system / IVIS Spectrum (PerkinElmer). Four hours later, the tumor site was irradiated with light (0.5W / cm²). 2 (3 min), the imaging effect was observed in real time, and finally the fluorescence intensity of the tumor site in the mouse was calculated at different time points using in vivo imaging analysis software.

[0033] (2) In vivo photoacoustic imaging: The probe was dissolved in PBS solution (concentration: 100 μM, volume: 200 μL) and injected into BALB / c female mice bearing tumors (4T1 mouse mammary cancer) via tail vein injection. Simultaneously, the small animal photoacoustic tomography imaging system was turned on. When the water temperature in the photoacoustic imaging device's water bath reached 37℃, the anesthetized mice were placed in the bath, and images of the tumor sites were scanned. Four hours later, the tumor sites were exposed to light (808 nm, 0.5 W / cm²). 2 The imaging effect was observed in real time (3 min), and the fluorescence intensity of the tumor site in mice 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.

[0034] (3) Tumor suppression experiment: 4T1 cells (2×10 6 (Each tumor) was implanted into the left and right backs of female BALB / c mice. When the tumor size reached 50–70 mm... 3 At that time, intravenous injection f - CRI probe (100 μM, 200 μL) or control CRI probe (100 μM, 200 μL). Four hours after injection, the right tumor was placed under an 808 nm laser (0.5 W / cm²). 2The mice were irradiated for 3 minutes. BALB / c female mice bearing tumors (4T1 mouse mammary cancer) on both sides of their backs were randomly divided into three groups (n=5): Group 1 (Control) received a tail vein injection of PBS (10 mM, 200 μL) to the left tumor, and Group 2 (Control+808 nm) received 808 nm laser irradiation to the right tumor; Group 3 (CRI) received a tail vein injection of CRI (100 μM, 200 μL) to the left tumor, and Group 4 (CRI+808 nm) received 808 nm laser irradiation to the right tumor; Group 6 (ƒ-CRI+808 nm) received a tail vein injection of ƒ-CRI (100 μM, 200 μL) to the left tumor, and Group 6 (ƒ-CRI+808 nm) received 808 nm laser irradiation to the right tumor. After treatment, the tumor size and volume were measured in each group. Fifteen days later, the mice were sacrificed, and the tumors were further dissected and weighed. The animal experiments complied with the animal experiment regulations of Soochow University.

[0035] Example 1: Synthesis of control probe CRI and probe ƒ-CRI

[0036] Control group CRI synthesis: cRGD (33.5 mg, 0.06 mmol) and IR780 (13.3 mg, 0.02 mmol) were dissolved in DMF (2 mL), stirred at room temperature for 12 h, and the crude product was collected. The control group product was separated by preparative HPLC. MALDI-MS: Calcd. For C57H80N11O7S+ [M+] 1062.596, found: 1062.866. Yield 63% (15 mg).1H NMR (600 MHz, DMSO-d6) δ 8.65 (d, J = 14.1 Hz,2H), 8.27-8.19 (m, 3H),8.09 (dd, J = 8.6, 2.5 Hz, 1H), 7.89-7.83 (m, 2H), 7.79-7.77 (m, 2H), 7.69-7.67 (m, 1H), 7.57 (d, J = 7.4Hz, 2H), 7.42-7.38 (m, 3H), 7.27-7.23 (m, 2H),7.02-6.93 (m, 2H), 6.29 (d, J = 14.2 Hz, 2H), 4.83-4.79 (m, 1H), 4.65-4.62(m, 1H), 4.30-4.27 (m, 2H),4.21-4.15 (m, 6H), 3.28-3.23 (m, 2H), 3.09-3.06(m, 2H), 2.77-2.70 (m, 4H), 2.63-2.58 (m, 2H), 2.29-2.25 (m, 1H), 2.00-1.95(m, 2H), 1.88-1.85 (m, 2H),1.81-1.76 (m, 4H), 1.72-1.62 (m, 12H), 1.55-1.52(m, 2H), 1.41-1.35 (m, 4H), 0.95 (t, J = 7.4 Hz, 6H). 13C NMR (151 MHz, DMSO-d6) δ 172.86, 172.71, 172.56,171.36, 171.03, 169.93, 169.73, 157.69, 156.87,145.56, 143.27, 141.88, 133.35, 130.55, 125.72, 123.27, 112.20, 102.22,55.16, 53.69, 52.51, 49.72, 49.59,45.84, 43.86, 41.27, 39.49, 36.70, 36.03,32.78, 32.18, 29.93, 29.48, 28.66, 28.33, 27.46, 26.08, 23.32, 21.32, 12.05。

[0037] ƒ-CRI synthesis: 3-(2-furan)propionic acid (1.54 mg, 0.011 mmol), N-hydroxysuccinimide (1.38 mg, 0.012 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (2.49 mg, 0.013 mmol) were added to 5 mL of DCM and stirred at room temperature for 4 hours to obtain activated 3-(2-furan)propionic acid. The mixture was then extracted twice with an equal proportion of water and evaporated to dryness using a rotary evaporator to obtain activated 3-(2-furan)propionic acid. CRI (2.46 mg, 0.01 mmol) and activated 3-(2-furan)propionic acid were dissolved in DMF (2 mL), and 5 μL of N,N-diisopropylethylamine (DIPEA) was added. The mixture was stirred at room temperature for 4 hours, and then the sample was separated using preparative HPLC to obtain the final product. MALDI-MS: Calcd. ForC64H86N11O9S+ [M+] 1184.633, found: 1184.962. Yield 68% (8 mg).1H NMR (600 MHz,DMSO-d6) δ 8.67 (d, J = 14.0 Hz, 2H), 8.39-8.21 (m, 3H),8.18 (d, J = 8.5 Hz,1H), 8.14-8.09 (m, 1H), 7.95-7.86 (m, 2H), 7.67-7.53 (m, 3H), 7.49-7.45 (m,1H), 7.44-7.38 (m, 3H), 7.29-7.23 (m, 2H), 6.99-6.80 (m,2H), 6.37-6.25 (m,3H), 6.03 (d, J = 3.1 Hz, 1H), 4.85-4.78 (m, 1H), 4.65-4.60 (m, 1H), 4.28-4.24 (m, 1H), 4.24-4.09 (m, 6H), 3.77-3.72 (m, 2H), 3.31-3.23 (m,2H), 3.13-3.06 (m, 2H), 3.03-2.95 (m, 2H), 2.82-2.73 (m, 4H), 2.69-2.57 (m, 4H), 2.55(t, J = 5.5 Hz, 1H), 2.37-2.31 (m, 2H), 1.93-1.71 (m, 8H), 1.75-1.59(m, 12H),1.41-1.37 (m, 2H), 1.27-1.21 (m, 4H), 0.96 (t, J = 7.4 Hz, 6H).13C NMR (151MHz, DMSO-d6) δ 172.87, 172.76, 172.73, 171.65, 171.46, 171.04,169.99,169.64, 157.61, 156.82, 155.62, 145.58, 143.29, 142.19, 141.90, 133.39,129.46, 125.74, 123.32, 112.22, 111.24, 105.90, 102.23, 55.41, 53.64, 52.50,49.75, 49.61, 45.85, 43.89, 41.28, 39.21, 36.69, 35.27, 34.46, 33.04, 29.97,29.57, 28.66, 28.38, 26.61, 26.13, 24.46, 23.81, 21.35, 12.09。

[0038] The above reaction diagram and the chemical structural formulas of the products involved are as follows: Figure 1 As shown, chloride ion coordination is a conventional representation method in this field.

[0039] Example 2 Physicochemical Properties of the Probe

[0040] The control group probe CRI and the experimental group probe ƒ-CRI prepared in Example 1 were diluted with ultrapure water to a concentration of 10 μM (completely soluble), and their ultraviolet-visible-near-infrared spectrophotometer and fluorescence spectrophotometer were measured. Figure 2 (a) Figure 2 As shown in (b), the results indicate that the maximum absorption of probe CRI and ƒ-CRI is at 788 nm, and the maximum emission is at 816 nm. The control group probe CRI and the experimental group probe ƒ-CRI, under DPBF verification, are as follows: Figure 2 As shown in (c), the amount of singlet oxygen produced by the probe increases with the extension of illumination time.

[0041] CRI and ƒ-CRI were diluted with ultrapure water to a concentration of 10 μM, and 30 μM of customized sequence RNA (5`-ACAUCGGGAUAGCGAAGUUGAGAGAGAGGGAG-3`) was added. After mixing, the mixture was shaken at 4°C and exposed to 808 nm light (0.5 W / cm²). 2 (1 minute) or without light (avoid light), directly separate the reaction solution using non-denaturing RNA gel electrophoresis, see [link to documentation]. Figure 3 a. It can be observed that ƒ-CRI can clearly label RNA after adding RNA and applying light, while other groups do not show obvious red fluorescence of IR780.

[0042] CRI and ƒ-CRI were added to the culture medium (HyClone 1640 high-glucose liquid medium containing 10% FBS) at a concentration of 10 μM and then added to 4T1 cells (2 × 10⁻⁶ cells per cell line). 5 After culturing in the medium for 6 h, it was subjected to 808 nm light (0.5 W / cm²). 2 (3 minutes) or no light exposure (avoiding light), followed by staining with the singlet oxygen fluorescent probe DCFH-DA, see Figure 3 b. The CRI and ƒ-CRI illumination groups showed obvious green fluorescence, while the other groups did not.

[0043] CRI and ƒ-CRI were added to the culture medium (HyClone 1640 high-glucose liquid medium containing 10% FBS) at a concentration of 10 μM and then added to 4T1 cells (2 × 10⁻⁶ cells per cell line). 5 After culturing in the medium for 6 h, it was subjected to 808 nm light (0.5 W / cm²). 2(3 minutes) or no light exposure (avoiding light), followed by staining with the commercial RNA dye SYTORNASelect green fluorescent cell stain (Thermo Fisher), see below. Figure 3 c. Figure 3 d. The colocalization rate of the experimental group (ƒ-CRI+808 nm) was greater than that of other groups and also higher than that of the existing probe C. 75 H 99 N 13 O 17 S2 (ƒ-CR, approximately 0.63), C 72 H 90 N 15 O 16 P(ƒ-RCP, approximately 0.62); subsequently, total RNA was extracted from the cells using the Trizol method, and quantitative fluorescence analysis revealed that the fluorescence intensity of the experimental group (ƒ-CRI + 808 nm) was significantly greater than that of other groups, see [link to data]. Figure 3 e; Subsequently, cytoplasmic RNA was extracted using a cytoplasmic & nuclear total extraction kit, and non-denaturing gel analysis revealed that the experimental group (ƒ-CRI + 808 nm) exhibited significant red fluorescence, while other groups showed no obvious fluorescence. (See [link to kit]). Figure 3 f.

[0044] CRI and ƒ-CRI were added to the culture medium (HyClone 1640 high-glucose liquid medium containing 10% FBS) at a concentration of 10 μM, and then added to 4T1 cells and cultured for 6 h. The cells were then exposed to 808 nm light (0.5 W / cm²). 2 The retention of the material within cells was observed for 3 minutes (or without light) or in the dark (protected from light). The experimental group (ƒ-CRI +808 nm) showed a significantly longer retention time than the other groups. (See [reference needed]). Figure 4 a and 4b; subsequently, the retention of the probe in mouse tumors was investigated. CRI and ƒ-CRI were diluted with PBS to a concentration of 100 μM (200 μL), and CRI or ƒ-CRI was administered via tail vein injection. After 4 hours, the mice were exposed to 808 nm light (0.5 W / cm²). 2 (3 minutes) or no light exposure (avoiding light), observe the probe metabolism in the tumor at each time point using IVIS. Figure 4 c, 4d, it was found that the retention time of the experimental group (ƒ-CRI +808 nm) in vivo was much longer than that of the other groups.

[0045] CRI and ƒ-CRI were prepared with PBS to a concentration of 100 μM in 200 μL, respectively, followed by tail vein injection of CRI or ƒ-CRI. After 4 h, the patient was exposed to 808 nm light (0.5 W / cm²). 2The photoacoustic signal was examined at each time point using a photoacoustic imaging system (3 minutes) or without illumination (avoiding light). The photoacoustic imaging data were reconstructed and analyzed using MSOT InSight / inVision software. The results showed that, compared to other groups, the experimental group (ƒ-CRI +808 nm) exhibited a longer duration of photoacoustic signal. Figure 4 e and 4f.

[0046] CRI and ƒ-CRI were diluted to concentrations of 80, 40, 20, 10, 1, and 0.1 μM in RPMI 1640 medium (containing 10% FBS), respectively, and added to 4T1 cells. After culturing for 12 h, neither CRI nor ƒ-CRI showed significant toxicity. Figure 5 a, Under the same experimental conditions, with 808 nm illumination (0.5 W / cm²), 2 After culturing for another 48 hours (3 minutes), the experimental group (ƒ-CRI + 808 nm) exhibited certain cytotoxicity. (See...) Figure 5 b; Simultaneously, the morphology of the Live-dead reagent and the bright field of the cells showed that the experimental group (ƒ-CRI+808 nm) exhibited an ability to induce tumor cell apoptosis. Figure 5 c. Simultaneously, cell migration experiments showed that the experimental group (ƒ-CRI + 808 nm) had a good ability to inhibit tumor cell proliferation and migration. Figure 5 d.

[0047] In the tumor suppression experiment, CRI and ƒ-CRI were diluted with PBS to a concentration of 100 μM (200 μL), respectively, and administered via tail vein injection. Four hours later, the patients were exposed to 808 nm light (0.5 W / cm²). 2 (3 minutes) or no light exposure (avoiding light), flowchart as shown in 6a. Tumor size is then measured daily. On day 15, photographs are taken, mice are euthanized, and tumors are removed. Tumor size comparison on the mouse's back on day 15 is shown in [reference needed]. Figure 6 b, The size of the excised tumor on day 15 is shown in [the original text]. Figure 6 c. The tumors in the experimental group (ƒ-CRI +808 nm) were very small. Unexpectedly, the tumor in one mouse disappeared. In contrast, the tumors recurred on day 11 or 13 under the existing probe treatment. Figure 6 d represents the tumor inhibition curve. Figure 6 e represents the survival period curve. The survival time of all mice in the experimental group (ƒ-CRI +808 nm) was at least 15 days. (Group1: Control; Group2: Control +808 nm; Group3: CRI; Group4: CRI+808 nm; Group5: ƒ-CRI; Group6: ƒ-CRI +808 nm).

[0048] 4T1 cells (2×10) 5 (Number of mice) were inoculated into the left and right sides of Balb / c mice. CRI and ƒ-CRI were diluted with PBS to a concentration of 100 μM (200 μL), respectively. CRI or ƒ-CRI was administered via tail vein injection. Four hours later, the mice were exposed to 808 nm light (0.5 W / cm²). 2 Tumor tissue was extracted after irradiation for 3 minutes (or 48 hours without light) and stained with hematoxylin and eosin (H&E). Qualitative histological examination of the excised tumor sections revealed no significant malignant necrosis in the control group. Conversely, the tumors in the ƒ-CRI + 808 nm group showed significant and severe necrosis. TUNEL results further supported the effective inhibition of tumor cell proliferation. The experimental group (ƒ-CRI + 808 nm) exhibited good tumor inhibitory ability. Under the same conditions, mouse tumors were harvested on day 1, and H&E and Tunnel immunohistochemical and immunofluorescence analyses were performed. The experimental group (ƒ-CRI + 808 nm) probe induced apoptosis and necrosis in tumor tissue. Figure 6 f.

[0049] This invention designs and synthesizes a novel tumor-anchored, therapeutic probe capable of cross-linking with RNA in the cytoplasm under singlet oxygen-mediated interaction, enabling long-term imaging of tumor tissues. Furthermore, it was discovered that cross-linked RNA induces severe mitochondrial damage and apoptosis in tumor cells, achieving integrated diagnosis and treatment of tumors. (See [link to related document]). Figure 7 .

Claims

1. A therapeutic nucleic acid-anchored fluorescent probe, having the following chemical structural formula: 。 2. The application of the diagnostic and therapeutic nucleic acid-anchored fluorescent probe of claim 1 in the preparation of fluorescent imaging agents and photoacoustic imaging agents.

3. The method for preparing the diagnostic and therapeutic nucleic acid-anchored fluorescent probe according to claim 1, characterized in that, Includes the following steps: (1) The cyclic peptide cRGD reacts with the dye IR780 to give the compound CRI; (2) The compound CRI reacts with activated 3-(2-furan)propionic acid to obtain the diagnostic and therapeutic nucleic acid anchoring fluorescent probe.

4. The method for preparing the diagnostic and therapeutic nucleic acid-anchored fluorescent probe according to claim 3, characterized in that, In step (1), the molar ratio of cyclic peptide cRGD to dye IR780 is 1:(1-1.2); the reaction is carried out at room temperature.

5. The method for preparing the diagnostic and therapeutic nucleic acid-anchored fluorescent probe according to claim 3, characterized in that, In step (2), the molar ratio of compound CRI to 3-(2-furan)propionic acid is 1:(1 to 1.5); the reaction is carried out at room temperature.

6. The method for preparing the diagnostic and therapeutic nucleic acid-anchored fluorescent probe according to claim 3, characterized in that, In step (2), 3-(2-furan)propionic acid is activated using N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

7. A red light-mediated probe-anchored cell method, characterized in that, The procedure includes the following steps: co-incubating the diagnostic and therapeutic nucleic acid-anchored fluorescent probe of claim 1 with cells to achieve probe-anchored cell targeting.

8. The red light-mediated probe-anchored cell method according to claim 7, characterized in that, Co-incubation was carried out under light and in culture medium.

9. The use of the therapeutic nucleic acid-anchored fluorescent probe of claim 1 in the preparation of reagents that improve the retention time of the probe in tumor tissue or inhibit tumors.

10. The use of the therapeutic nucleic acid-anchored fluorescent probe of claim 1 in the preparation of reagents that react with the cytoplasm.

Images