CN115998908A - Diagnosis and treatment integrated nucleic acid anchored fluorescent probe and preparation method and application thereof - Google Patents

Abstract

The invention discloses a diagnosis and treatment integrated nucleic acid anchored fluorescent probe, a preparation method and application thereof, wherein the fluorescent probe can automatically generate singlet oxygen and generate a crosslinking reaction with intracellular RNA under the mediation of the singlet oxygen, so that long-window imaging of tumor tissues is realized; meanwhile, after the cross-linked RNA is found, mitochondrial function damage further induces severe apoptosis of tumor cells, and diagnosis and treatment integration of tumors is realized.

Description

Diagnosis and treatment integrated nucleic acid anchored fluorescent probe and preparation method and application thereof

Technical Field

The invention belongs to the technical field of small molecular fluorescent probe biological imaging and tumor treatment, and particularly relates to a novel anchoring type molecular probe and a preparation method thereof, and application of the probe in multi-mode imaging and tumor treatment.

Background

With the significant increase in morbidity and mortality over the past decades, cancer has become one of the major threats to human health and life. Molecular probes are one of the key elements of molecular imaging technology, and play an important role in accurate diagnosis and treatment of cancers. Although a large number of molecular imaging probes are developed for cancer imaging and treatment, due to poor tumor specificity, normal tissues are possibly damaged, and the purpose of accurate diagnosis and treatment is often not achieved. Based on the characteristics of Tumor Microenvironment (TME), scientists develop a plurality of activatable molecular probes which can be triggered by endogenous substances (such as overexpressed enzymes, acidic pH, high-reducibility glutathione, hypoxia and the like) and can specifically destroy cancer cells, so that toxic and side effects of traditional chemotherapy or radiotherapy and the like on normal tissues are reduced. However, most activatable strategies are triggered by a single factor, and are often inadequate to effectively avoid normal cells and precisely ablate cancer cells, resulting in potentially false positive or false negative results. In many trigger modes, light has proven to be an attractive tool for precisely activating therapy with temporal and spatial controllability without physical contact.

Disclosure of Invention

In order to overcome the problems in the prior art and materials, the invention constructs a novel anchoring molecular probe, and uses the advantages of the novel anchoring molecular probe, such as crosslinking capability group, good biocompatibility, active targeting integrin and near infrared emission, to perform long-time living fluorescence, photoacoustic imaging and tumor treatment.

The invention adopts the following technical scheme:

a novel diagnosis and treatment integrated nucleic acid anchor type fluorescent probe has the following chemical structural formula:

。

the preparation method of the diagnosis and treatment integrated nucleic acid anchored fluorescent probe comprises the following steps:

(1) The cyclic peptide cRGD reacts with dye IR780 to obtain a compound CRI;

(2) The compound CRI reacts with activated 3- (2-furan) propionic acid to obtain the diagnosis and treatment integrated nucleic acid anchored fluorescent probe ƒ -CRI.

Specifically, the preparation method of the diagnosis and treatment integrated nucleic acid anchored fluorescent probe specifically comprises the following steps:

(1) The cyclic peptide cRGD reacts with dye IR780 in DMF for 10-15 h at room temperature to obtain compound CRI;

(2) Activating 3- (2-furan) propionic acid with N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; the compound CRI reacts with activated 3- (2-furan) propionic acid and N, N-Diisopropylethylamine (DIPEA) for 1-5 hours to obtain the diagnosis and treatment integrated nucleic acid anchored fluorescent probe ƒ -CRI.

The invention discloses a red light mediated probe cell anchoring method, which comprises the following steps: incubating the diagnosis and treatment integrated nucleic acid anchored fluorescent probe and the cell together to realize probe anchored cell; wherein co-incubation is performed in medium (e.g., 1640 medium) under light.

In the technical scheme, the cyclic peptide cRGD and dye IR780 react in an organic solvent to obtain a compound CRI; reacting the compound CRI with activated 3- (2-furan) propionic acid and N, N-Diisopropylethylamine (DIPEA) in an organic solvent to obtain an 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.

The invention discloses application of the diagnosis and treatment integrated nucleic acid anchored fluorescent probe in fluorescent imaging and photoacoustic imaging; or the application of the diagnosis and treatment integrated nucleic acid anchored fluorescent probe in preparing a fluorescent imaging agent and a photoacoustic imaging agent; or the application of the diagnosis and treatment integrated nucleic acid anchored fluorescent probe in improving the detention time of the probe in tumor tissues or inhibiting tumors, or the application of the diagnosis and treatment integrated nucleic acid anchored fluorescent probe in preparing a reagent for improving the detention time of the probe in tumor tissues or inhibiting tumors; or the application of the diagnosis and treatment integrated nucleic acid anchored fluorescent probe in preparing a reagent for reacting with cytoplasm. Or the novel anchoring molecular probe is applied to the fluorescent imaging of the tumor cells by crosslinking with RNA in the tumor cells; or the application of the novel anchoring molecular probe in preparing tumor cells and RNA crosslinking so as to inhibit the growth of the tumor cells.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:

(1) The invention designs and synthesizes a novel anchoring molecular probe ƒ -CRI, which can perform in-vivo fluorescence and photoacoustic imaging for a long time under the condition of generating singlet oxygen by red light mediation;

(2) The target probe can carry out crosslinking reaction on intracellular RNA under the condition of generating singlet oxygen in a red light-mediated manner so as to prolong the residence time of the probe molecule in the cell;

(3) The target probe has good capability of promoting apoptosis of tumor cells for causing mitochondrial injury after RNA crosslinking in the tumor cells;

(4) The target probe has the capability of inhibiting the tumor growth of tumor-bearing mice after the crosslinking reaction in vivo.

Drawings

FIG. 1 is a schematic diagram showing the synthesis of a nucleic acid-anchored fluorescent probe for diagnosis and treatment in example 1.

FIG. 2 is a graph of the chemical structure of (a) experimental and control group probes ƒ -CRI, (b) ultraviolet absorbance and fluorescence emission of probes ƒ -CRI and probe CRI in aqueous solution, and (c) the ability of probes ƒ -CRI and probe CRI to generate singlet oxygen, as described in example 2.

FIG. 3 shows (a) gel electrophoresis of RNA and ƒ -CRI by cross-linking reaction, and (b) fluorescence and quantification images of total RNA of cells extracted by the RNA population kit, respectively, from confocal images and co-localization ratios (c, d), (e, f) of RNA Select incubated with 4T1 cells for 6 hours, respectively, for experimental ƒ -CRI and control probe CRI.

FIG. 4 shows (a) the retention change and fluorescence intensity quantification (b) of the test group probe ƒ -CRI and the control group probe CRI and 4T1 cells in the cells after 6 hours incubation, followed by confocal images of the retention change and fluorescence intensity quantification (d) of the test group probe ƒ -CRI and the control group probe CRI in the tail vein, and four hours later the retention change and fluorescence intensity quantification (d) of the tumor tissue by the illumination observation probe; under the same experimental conditions, the experimental group probe ƒ -CRI and the control group probe CRI, the photoacoustic signal change (e) and the photoacoustic intensity quantification (f) in the tumor.

FIG. 5 shows (a) cytotoxicity changes after incubation of 12h with control probe CRI and control probe CRI in test group ƒ -CRI and 4T1 cells, (b) test groupAfter incubating the probe ƒ -CRI with the control probe CRI and 4T1 cells for 12h, light is given (808 nm 0.5 w/cm) ² 3 min) post-cytotoxicity change, (c) co-incubating experimental group probe ƒ -CRI with control group probe CRI and 4T1 cells for 12h followed by light exposure (808 nm 0.5 w/cm) ² 3 min) observing apoptosis changes with live-dead and bright cell fields, respectively; (d) ability to inhibit proliferation and migration of tumor cells.

FIG. 6 is a graph showing the continuous 15-day course of tumor inhibition for (a) experimental and control probes ƒ -CRI and blank PBS, respectively, (b) fifteenth day mouse back tumor contrast size, (c) 15 th day ex vivo tumor size, (d) tumor inhibition profile, (E) survival cycle profile, (f) 48H after treatment by immunofluorescence and immunohistochemistry, respectively, to examine Tunel, H & E changes.

FIG. 7 is a schematic diagram showing the action of the probe according to the present invention.

Detailed Description

The probe of the invention is an RNA reaction type fluorescent probe, and the probe can react with RNA in cells under the irradiation of red light, so that the detention time of the probe in tumor cells is prolonged, and long-window tumor imaging is realized; more importantly, this cross-linking reaction causes mitochondrial damage, leading to severe tumor cell apoptosis, thus inhibiting tumor growth. The invention will be further elucidated below in connection with the drawings and the specific embodiments. It should be understood that these examples are for the purpose of illustrating and explaining the technical aspects of the present invention and are not intended to limit the scope of the present invention. In addition, unless otherwise indicated, materials, reagents, instruments, and the like used in the following examples are all commercially available; the specific preparation method and the test method are conventional methods in the art.

The synthesis and diagnosis and treatment integrated nucleic acid anchor type fluorescent probe ƒ -CRI comprises the following steps: the cyclic peptide cRGD reacts with dye IR780 to obtain a compound CRI; the compound CRI reacts with activated 3- (2-furan) propionic acid to obtain the diagnosis and treatment integrated nucleic acid anchored fluorescent probe ƒ -CRI. CRI was used as control probe.

The anchorage molecular probes ƒ -CRI and the aqueous solution of the control group probe CRI are respectively incubated with tumor cells for 48 h, and then apoptosis of the tumor cells is observed.

The method for in vivo tumor inhibition experiments by using the novel anchored molecular probe comprises the following steps of firstly injecting the aqueous solution tail vein of the probe ƒ -CRI into the body of a tumor-bearing mouse, and continuously observing and recording tumor inhibition conditions by giving illumination after 4 h. Control experiments were performed with the probe ƒ -CRI replaced with the control CRI.

(1) In vivo fluorescence imaging: the probe was injected into a BALB/c female tumor-bearing (4T 1 mouse breast cancer) mouse by tail vein injection, then placed in a small animal living optical imaging system/IVIS Spectrum (Perkinelmer), and after 4 hours, light (0.5W/cm) was given to the tumor site ² 3 min), the imaging effect is observed in real time, and finally, the fluorescence intensity of the tumor part of the mouse at different time points is calculated by using in-vivo imaging analysis software.

(2) Living body photoacoustic imaging: the probe is dissolved in PBS solution (concentration: 100 mu M, volume: 200 mu L), and is injected into BALB/c female mice bearing tumor (4T 1 mouse breast cancer) in a tail vein injection mode, meanwhile, a small animal photoacoustic tomography imaging system is opened, when the water temperature in a water bath pool of a photoacoustic imager reaches 37 ℃, the anesthetized mice are put into, and tumor part images of the mice are scanned. And after 4 hours, the tumor site was irradiated with light (808 nm, 0.5W/cm) ² 3 min), the imaging effect is observed in real time, and finally, the fluorescence intensity of the tumor part of the mouse at different time points is calculated by using in-vivo imaging analysis software. The obtained photoacoustic imaging data was then subjected to reconstruction analysis using MSOT InSight/inVision analysis software.

(3) Tumor inhibition experiments: 4T1 cells (2X 10) ⁶ And) were implanted into the left and right backs of female BALB/c mice, respectively. When the tumor size reaches 50 to 70 percent 70 mm ³ When intravenous injection is performedfCRI probe (100. Mu.M, 200. Mu.L) or control CRI probe (100. Mu.M, 200. Mu.L). 4 hours after injection, right side tumors were placed on 808nm laser (0.5W/cm) ² ) Irradiating for 3 minutes. BALB/c female mice bearing tumors on the left and right bilateral backs (4T 1 mice breast cancer) were randomly divided into 3 groups (n=5): tail vein injection PBS (1)0 mM, 200 μl) of left tumor (group 1, control), 808nm laser irradiated right tumor (group 2, control+808 nm); CRI (100 μΜ,200 μΙ_) alone left tumor (group 3, CRI), right tumor 808nm laser irradiated mice (group 4, cri+808 nm); the tail vein was injected with ƒ -CRI (100. Mu.M, 200. Mu.L) left tumor (group 5, ƒ -CRI), right tumor 808nm laser irradiated mice (group 6, ƒ -CRI+808 nm). After treatment, the tumor size was then measured for each group of mice and tumor volumes were calculated. After 15 days, the mice were sacrificed and the tumors were further dissected and weighed and the animal experiments met the university of sulzhou animal experimental specifications.

Example 1 Synthesis of control probes CRI and probes ƒ -CRI

CRI synthesis of control: cRGD (33.5 mg, 0.06 mmol), IR780 (13.3 mg, 0.02 mmol) was dissolved in DMF (2 mL), stirred at room temperature for 12H, the crude product was collected and isolated by prep HPLC to give the control product MALDI-MS: calcd.for C57H80N11o7s+ [ m+ ] 1062.596, found: 1062.866. 63% yield (15 mg). 1H NMR (600 MHz, DMSO-d 6) delta 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=7.4 Hz, 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), 7.27-7.23 (m, 2H), 7.82-6.93 (m, 2H), 7.52-3.35 (m, 2H), 7.35 (m, 2H), 7.27-7.23 (m, 2H), 7.82-6.93 (m, 2H), 7.82 (3H), 4.83 (m, 2H), 4.83 (3.9-4.9 (m, 2H), 4.80-4.9 (3H), 4.80 (3H), 4.35 (3.35 (3H), 4.35 (2H), 4.35 (3.35 (2H), 2.35 (3.35 (3H), 2H), 2.35 (3.35 (2H), 2H). 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.

ƒ -CRI Synthesis: 3- (2-furoic 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 DCM and stirred at room temperature for 4 hours to give activated 3- (2-furoic acid, which was then extracted twice with an equal proportion of water and dried by a rotary evaporator to give activated 3- (2-furoic acid). CRI (2.46 mg, 0.01 mmol) and activated 3- (2-furopropionic acid) were dissolved in DMF (2 mL), 5 μ L N, N-Diisopropylethylamine (DIPEA) was added and stirred at room temperature for 4h, then the sample was separated by preparative HPLC to give the final product. MALDI-MS, calcd.for C64H86N11O9S+ [ M+ ] 1184.633, found: 1184.962. Yield 68% (8 mg). 1H NMR (600 MHz, DMSO-d 6) delta 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, 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), 6.37-6.37 (m, 2H), 6.37-6.80 (m, 2H), 6.37-6.37 (3H), 6.03 (d, J=3.1 Hz, 1H), 4.78 (m, 1H), 4.85-4.78 (m, 1H), 4.24 (m, 1.7 (3.7 (m, 1H), 4.35 (3.35 (3H), 3.35 (3.3.3H), 3.35 (3.3.7, 3H), 3.7-3 (3H), 3.7 (3.3.3 (3H), 6H) 13C NMR (151 MHz, DMSO-d 6) delta 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.

The reaction schematic and the chemical structural formula of each product are shown in figure 1, and the coordination of chloride ions is a conventional representation method in the field.

Example 2 physicochemical Properties of the Probe

The control probe CRI and the experimental probe ƒ -CRI prepared in example 1 were diluted with ultrapure water to a concentration of 10. Mu.M (completely soluble), and their ultraviolet-visible near-infrared spectrum and fluorescence spectrum were measured using an ultraviolet-visible near-infrared spectrophotometer and a fluorescence spectrophotometer, respectively. As shown in fig. 2 (a) and 2 (b), the results indicate that the maximum absorption of the probe CRI and ƒ -CRI is 788 nm and the maximum emission is 816 nm. Under the verification of DPBF, the control group probe CRI and the experimental group probe ƒ -CRI are more in singlet oxygen generated by the probes as the illumination time is prolonged, as shown in FIG. 2 (c).

Diluting CRI and ƒ -CRI with ultrapure water to a concentration of 10 μm, adding 30 μm of custom sequence RNA (5 '-ACAUCGGGAUAGCGAAGUUGAGAGAGAGGGAG-3'), mixing, shaking at 4deg.C, and irradiating with 808nm light (0.5W/cm) ² 1 minute) or without illumination (light shielding), the reaction solution was directly separated by RNA non-denaturing gel electrophoresis, see FIG. 3a, and it was found that ƒ -CRI was able to label RNA significantly after addition of RNA and administration of light, while other groups did not have significant red fluorescence of IR 780.

CRI, ƒ -CRI were added to medium (HyClone 1640 high sugar liquid medium containing 10% FBS) at a concentration of 10 μm, and added to 4T1 cells (2×10) ⁵ ) After medium culture 6h, 808nm light (0.5W/cm) ² 3 minutes) or no illumination (light-shielding) and then stained with the singlet oxygen fluorescent probe DCFH-DA, respectively, see fig. 3b, CRI, ƒ -CRI illuminated groups all exhibited a clear green fluorescence, while the remaining groups did not.

CRI, ƒ -CRI were added to medium (HyClone 1640 high sugar liquid medium containing 10% FBS) at a concentration of 10 μm, and added to 4T1 cells (2×10) ⁵ ) After medium culture 6h, 808nm light (0.5W/cm) ² 3 minutes) or no illumination (light shielding), followed by separate staining with commercial RNA dye SYTO RNASelect green fluorescent cell stain (Thermo Fisher), see FIGS. 3C, 3d, experimental groups (ƒ -CRI+808+ 808 nm) with co-localization rates greater than the other groups, and higher than that of the existing probe C ₇₅ H ₉₉ N ₁₃ O ₁₇ S ₂ (ƒ -CR, about 0.63), C ₇₂ H ₉₀ N ₁₅ O ₁₆ P (ƒ -RCP, about 0.62); the total RNA in the cells was then extracted by Trizol method and the fluorescence intensity was found to be much higher in the experimental group (ƒ -CRI+808 nm) than in the other groups by fluorescence quantification, see FIG. 3e; subsequently by fineCytoplasm and cytoplasm&The total nuclear extraction kit extracts cytoplasmic RNA and the experimental group (ƒ -CRI+808 nm) was found to have significant red fluorescence by RNA non-denaturing gel analysis, whereas the other groups did not have significant fluorescence generation, see FIG. 3f.

CRI, ƒ -CRI were added to medium (HyClone 1640 high sugar liquid medium containing 10% FBS) at a concentration of 10 μm, and cultured in 4T1 cells for 6h, respectively, followed by 808nm light (0.5W/cm) ² 3 minutes) or no light (in the absence of light) was used to observe the retention of material in cells, and the retention experiments of the experimental group (ƒ -cri+808 nm) were found to be much greater than those of the other groups, see fig. 4a and 4b; following a study of retention of the probe in mouse tumors, CRI, ƒ -CRI were measured with PBS to a concentration of 100. Mu.M 200. Mu.L, respectively, and tail vein injection of CRI or ƒ -CRI, followed by 808/nm light (0.5W/cm) after 4h ² 3 minutes) or no light (in the absence of light), the in vivo residence time of the experimental group (ƒ -cri+808 nm) was found to be much longer than the other groups by looking at the probe in-tumor metabolism at each time point by IVIS, see fig. 4c, 4 d.

CRI, ƒ -CRI 200. Mu.L with PBS to a concentration of 100. Mu.m, respectively, followed by administration of tail vein injection CRI or ƒ -CRI, followed by administration of 808nm light (0.5W/cm) after 4h ² 3 minutes) or no illumination (light shielding), and at the same time, the photoacoustic signal change condition of each time point is inspected through the photoacoustic imaging system, and the photoacoustic imaging data is subjected to reconstruction analysis by using MSOT InSight/inVision analysis software, so that compared with other groups, the experimental group (ƒ -CRI+808 nm) has a long-time photoacoustic signal, as shown in fig. 4e and 4f.

CRI, ƒ -CRI were diluted to 80, 40, 20, 10,1,0.1. Mu.M in medium (RPMI 1640 medium containing 10% FBS) and cultured in 4T1 cells for 12h, respectively, and neither CRI nor ƒ -CRI was found to be significantly toxic, as shown in FIG. 5a, under the same experimental conditions as those obtained by the administration of 808nm light (0.5W/cm ² After 3 minutes) the incubation was continued for 48 h, and the experimental group (ƒ -cri+808 nm) was found to exhibit some cytotoxicity, see fig. 5b; at the same time, the experimental group (ƒ -CRI+808-nm) shows the ability to cause apoptosis of tumor cells as seen in FIG. 5c, as seen by the Live-dead reagent and the morphology of the cell bright field. At the same time through cellsMigration experiments As can be seen, the experimental group (ƒ -CRI+808-nm) has better ability to inhibit proliferation and migration of tumor cells, as shown in FIG. 5d.

Tumor inhibition experiments, CRI, ƒ -CRI were diluted with PBS to a concentration of 100 μΜ 200 μL, respectively, and given tail vein injection CRI or ƒ -CRI, after 4h 808nm light (0.5W/cm) ² 3 minutes) or no light (light shielding), as shown in the flow chart 6a, then taking tumor size daily, taking a photograph and killing the mice on day 15 and taking the tumor off, comparing the tumor size of the back of the mice on day 15 with that of the tumor on day 15 as shown in fig. 6b, comparing the tumor size of the tumor on the ex vivo on day 15 as shown in fig. 6c, wherein the tumor of the experimental group (ƒ -cri+808 nm) is very small, unexpectedly, one mouse tumor disappears, and tumor recurrence occurs on day 11 or 13 under the existing probe treatment as a comparison; FIG. 6d shows tumor inhibition curves and FIG. 6e shows a life cycle curve, with a survival of at least 15 days for all mice in the experimental group (ƒ -CRI+808 nm). (Group 1: control; group2: control+808 nm;Group3:CRI;Group4:CRI+808 nm;Group5: ƒ -CRI; group6: ƒ -CRI+808-nm).

4T1 cells (2X 10) ⁵ And (3) inoculating to both sides of Balb/c mice, diluting CRI, ƒ -CRI with PBS to a concentration of 100 μΜ and 200 μL, administering tail vein injection CRI or ƒ -CRI, and administering 808nm light (0.5W/cm) after 4 hr ² 3 minutes) or after irradiation with no light of 48H, extracting tumor tissue, and subjecting to hematoxylin-eosin (H)&E) Dyeing. Qualitative histological examination of resected tumor sections was performed, with no apparent malignant necrosis of the control tumors. In contrast, the ƒ -CRI+808-nm group tumors showed significant severe necrosis. TUNEL results further support effective inhibition of tumor cell proliferation. The experimental group (ƒ -CRI+808-nm) has good tumor inhibition ability, and mice are taken on the first day to treat H under the same conditions&E, immunohistochemistry and immunofluorescence analysis of Tunnel, the experimental group (ƒ -CRI+808 nm) probes were able to induce apoptotic necrosis of tumor tissue, see FIG. 6f.

The invention designs and synthesizes a novel tumor anchoring type diagnosis and treatment integrated probe, has the capability of cross-linking reaction with RNA in cytoplasm under the mediation of singlet oxygen, and realizes long-window imaging of tumor tissues; meanwhile, the serious apoptosis phenomenon of the mitochondrial injury of the tumor cells is caused after the cross-linked RNA is found, so that the diagnosis and treatment integration of the tumor is realized, and the graph is shown in fig. 7.

Claims (10)

1. A diagnosis and treatment integrated nucleic acid anchored fluorescent probe has the following chemical structural formula:

。

2. the use of the diagnostic integrated nucleic acid-anchored fluorescent probe of claim 1 in fluorescence imaging and photoacoustic imaging; or the application of the diagnosis and treatment integrated nucleic acid anchored fluorescent probe in the preparation of fluorescent imaging agents and photoacoustic imaging agents.

3. The method for preparing a diagnosis and treatment integrated nucleic acid anchored fluorescent probe according to claim 1, comprising the steps of:

(1) The cyclic peptide cRGD reacts with dye IR780 to obtain a compound CRI; (2) And reacting the compound CRI with the activated 3- (2-furan) propionic acid to obtain the diagnosis and treatment integrated nucleic acid anchored fluorescent probe.

4. The method for preparing a diagnosis and treatment integrated nucleic acid anchored fluorescent probe according to claim 3, wherein in the step (1), the molar ratio of cyclic peptide cRGD to dye IR780 is 1:1-1.2; the reaction was carried out at room temperature.

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

6. The method for preparing a nucleic acid-anchored fluorescent probe for diagnosis and treatment according to claim 3, wherein in the step (2), 3- (2-furoic acid) is activated by using N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.

7. A red-mediated probe-anchored cell method comprising the steps of: incubating the diagnosis and treatment integrated nucleic acid anchored fluorescent probe and the cell according to claim 1 to realize probe anchoring of the cell.

8. The red-mediated probe-anchored cell method of claim 6, wherein the co-incubation is performed in medium under light.

9. The application of the diagnosis and treatment integrated nucleic acid anchored fluorescent probe in improving the detention time of the probe in tumor tissues or inhibiting tumors as claimed in claim 1, or the application of the diagnosis and treatment integrated nucleic acid anchored fluorescent probe in preparing a reagent for improving the detention time of the probe in tumor tissues or inhibiting tumors as claimed in claim 1.

10. The use of the nucleic acid anchored fluorescent probe for diagnosis and treatment according to claim 1 for preparing a reagent that reacts with cytoplasm.

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