CN112592386A - Red light mediated nucleic acid anchoring type fluorescent probe and preparation method and application thereof - Google Patents
Red light mediated nucleic acid anchoring type fluorescent probe and preparation method and application thereof Download PDFInfo
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- CN112592386A CN112592386A CN202011263517.1A CN202011263517A CN112592386A CN 112592386 A CN112592386 A CN 112592386A CN 202011263517 A CN202011263517 A CN 202011263517A CN 112592386 A CN112592386 A CN 112592386A
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/64—Cyclic peptides containing only normal peptide links
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- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0076—PDT with expanded (metallo)porphyrins, i.e. having more than 20 ring atoms, e.g. texaphyrins, sapphyrins, hexaphyrins, pentaphyrins, porphocyanines
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- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
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Abstract
The invention discloses a red light mediated nucleic acid anchoring type fluorescent probe and a preparation method and application thereof, wherein the fluorescent probe has the capability of carrying out cross-linking reaction with RNA in cytoplasm under the mediation of singlet oxygen, and realizes the imaging of a tumor tissue in a long window period; meanwhile, the phenomenon of serious apoptosis of tumor cells is found after the RNA is crosslinked, and the diagnosis and treatment of the tumor are integrated.
Description
Technical Field
The invention belongs to the technical field of small molecule fluorescent probe biological imaging and tumor treatment, and particularly relates to a novel anchoring type molecular probe, a preparation method thereof and application of the probe in multi-modal imaging and tumor treatment.
Background
It is known that cancer is one of the most major diseases threatening human life and health, and it is a great obstacle to the healthy development of the economic society. According to data released by the national cancer center, the incidence rate of tumors in China increases year by year, and the trend of the tumors is younger, so that the development of materials and new technologies for tumor diagnosis and treatment is urgent. In recent years, researchers have designed various materials for tumor imaging and therapy, however, the materials often cannot be enriched in tumor tissues for a long time, thereby greatly reducing the bioavailability of the materials. Therefore, the development of a novel probe aiming at overcoming the high metabolism of tumor tissues has obvious clinical significance. Biological cross-linking reaction means that a compound can chemically react with macromolecules in an organism to form covalent bonds under the stimulation of certain exogenous conditions.
Disclosure of Invention
In order to overcome the problems of the existing materials and the prior art, the invention constructs a novel anchoring molecular probe, and long-time living body fluorescence, photoacoustic imaging and tumor treatment are carried out by utilizing the advantages of the anchoring molecular probe, such as crosslinking capability group, better biocompatibility, active targeting integrin and near infrared emission.
The invention adopts the following technical scheme:
a novel red light mediated nucleic acid anchoring type fluorescent probe has the following chemical structural formula:
the preparation method of the red light mediated nucleic acid anchoring type fluorescent probe comprises the following steps:
(1) carrying out deprotection on tert-butyloxycarbonyl-fluorenylmethyloxycarbonyl-lysine, and then reacting with 3- (2-furan) propionic acid to obtain a compound 1;
(2) activating carboxyl by the compound 1, reacting with cyclopeptide cRGD to obtain a compound 2, and deprotecting to obtain a compound 3;
(3) and reacting the compound 3 with Cy7 SE to obtain the red light mediated nucleic acid anchoring type fluorescent probe.
The preparation method of the red light mediated nucleic acid anchoring type fluorescent probe specifically comprises the following steps:
(1) reacting tert-butyloxycarbonyl-fluorenylmethoxycarbonyl-lysine with 3- (2-furan) propionic acid after deprotection in a dichloromethane solution containing trifluoroacetic acid to obtain a compound 1;
(2) activating carboxyl by using N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and reacting the activated carboxyl with cyclopeptide cRGD to obtain a compound 2;
(3) deprotecting the compound 2 in N, N-dimethylformamide solution containing piperidine to obtain a compound 3;
(4) and reacting the compound 3 with Cy7 SE to obtain the red light mediated nucleic acid anchoring type fluorescent probe f-CR.
The invention discloses a red light mediated probe cell anchoring method, which comprises the following steps:
(1) carrying out deprotection on tert-butyloxycarbonyl-fluorenylmethyloxycarbonyl-lysine, and then reacting with 3- (2-furan) propionic acid to obtain a compound 1;
(2) activating carboxyl by the compound 1, reacting with cyclopeptide cRGD to obtain a compound 2, and deprotecting to obtain a compound 3;
(3) reacting the compound 3 with Cy7 SE to obtain the red light mediated nucleic acid anchoring type fluorescent probe;
(4) co-incubating the red light mediated nucleic acid anchoring type fluorescent probe, methylene blue and cells to realize the anchoring of the probe to the cells; wherein, the co-incubation is carried out in a culture medium under illumination, and the preferable molar ratio of the red light mediated nucleic acid anchoring type fluorescent probe to the methylene blue is 100: 0.8-1.2, and is preferably 100: 1.
In the technical scheme, tert-butyloxycarbonyl-fluorenylmethyloxycarbonyl-lysine (Boc-Lys (Fmoc) -OH), trifluoroacetic acid and 3- (2-furan) propionic acid react in an organic solvent to obtain a compound 1; carrying out the compound 1, N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and cRGD in an organic solvent to obtain a compound 2; reacting the compound 2 with piperidine in an organic solvent to obtain a compound 3; reacting the compound 3 with Cy7 SE in an organic solvent to obtain an anchored molecular probe f-CR; the molar ratio of the tert-butyloxycarbonyl-fluorenylmethoxycarbonyl-lysine to the 3- (2-furan) propionic acid is 1: 1-1.5; preferably, the molar ratio of the reaction of the t-butyloxycarbonyl-fluorenylmethoxycarbonyl-lysine, the trifluoroacetic acid and the 3- (2-furan) propionic acid is 1: 10: 1.2, the molar ratio of the compound 1 to the N-hydroxysuccinimide, the molar ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the cRGD is 1: 1.2: 1.5: 1.1, the molar ratio of the compound C-1-4 to the piperidine is 1: 10, and the molar ratio of the compound 3 to the Cy7 SE is 1: 1.1.
The invention discloses application of the anchoring molecular probe in preparation of photoacoustic and fluorescent imaging reagents and tumor inhibition.
According to the technical scheme of the invention, the method comprises the following steps:
in the step (1), the reaction of the compound tert-butyloxycarbonyl-fluorenylmethyloxycarbonyl-lysine and trifluoroacetic acid is carried out in dichloromethane, and the molar ratio of the reaction of the tert-butyloxycarbonyl-fluorenylmethyloxycarbonyl-lysine and the trifluoroacetic acid is 1: 10; preferably, the reaction is carried out at room temperature for 0.5 h.
In the step (2), the molar ratio of the compound 1 to the cRGD is 1 to (1-1.2); preferably, compound 1 is reacted with N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in N, N-dimethylformamide in a molar ratio of 1 to N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride of 1: 1.2: 1.5; preferably, the reaction is at 0oC is reacted for 2 hours at room temperature after 0.5 hour. Then adding cRGD to react in N, N-dimethylformamide solvent containing N, N-diisopropylethylamine, wherein the molar ratio of cRGD to N, N-diisopropylethylamine is 1: 1.1: 1; preferably, the reaction is continued for 2h at room temperature.
In the step (3), the reaction of the compound 2 and piperidine is carried out in N, N-dimethylformamide solvent, and the molar ratio of the compound 2 to piperidine is 1: 10; preferably, the reaction is carried out at room temperature for 0.2 h.
In the step (4), the reaction of the compound 3 with Cy7 SE is carried out in an N, N-dimethylformamide solvent containing N, N-diisopropylethylamine; the molar ratio of the compound 3 to the Cy7 SE is 1 to (1-1.2), and preferably, the molar ratio of the compound 3, the Cy7 SE and the N, N-diisopropylethylamine is 1: 1.1: 1; preferably, the reaction is carried out at room temperature for 1 h.
In the step (5), cRGD reacts with Cy7 SE, and the reaction is carried out in N, N-dimethylformamide solvent containing N, N-diisopropylethylamine; the mol ratio of the compound cRGD, Cy7 SE and N, N-diisopropylethylamine is 1: 1.1: 1; preferably, the reaction is carried out at room temperature for 1 h.
In the present invention, the chemical structural formulas of compound 1, compound 2, compound 3, compound CR, and compound f-CR are as follows:
the compounds CR and f-CR of the present invention are both in the form of internal salts and are shown as conventional in the art.
The invention discloses application of the red light mediated nucleic acid anchoring type fluorescent probe in prolonging imaging time and inhibiting tumor in living body fluorescence imaging or photoacoustic imaging; or the novel anchoring type molecular probe is applied to the preparation of a long-time fluorescence imaging reagent, a photoacoustic imaging reagent or a tumor inhibition reagent; or the novel anchoring molecular probe is cross-linked with RNA in the tumor cell to prolong the application of the fluorescence imaging of the tumor cell; or the novel anchoring type molecular probe is applied to the preparation of tumor cells and the cross-linking with RNA 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 f-CR which can perform long-time living body fluorescence and photoacoustic imaging under the condition of generating singlet oxygen by red light mediation;
(2) the target probe can generate cross-linking reaction with RNA in cells under the condition of generating singlet oxygen under the mediation of red light, thereby prolonging the retention time of probe molecules in the cells.
(3) The target probe has good capacity of promoting tumor cell apoptosis after the RNA in the tumor cell is crosslinked.
(4) The target probe has the ability of inhibiting the tumor growth of tumor-bearing mice after the cross-linking reaction in vivo.
Drawings
FIG. 1 is a schematic diagram of the synthesis of the novel anchor-type molecular probe in example 1;
FIG. 2 is a graph showing the chemical structures of (a) the probes f-CR of the experimental group and the probes CR of the control group, (b) the UV absorption and fluorescence emission images of the probes f-CR of the experimental group in an aqueous solution, and (c) the TEM and particle size statistics of the probes f-CR of the experimental group in an aqueous solution in example 2; (d) a particle size distribution map;
FIG. 3 is a gel electrophoresis diagram of (b) RNA and f-CR cross-linking reaction, (c) confocal images and colocalization rate (d) of RNA Select after 6 hours incubation of the experimental group f-CR and the control group probe CR with 4T1 cells respectively, (e) fluorescence and quantification images of total RNA of cells extracted by an RNA population kit, and (f) gel electrophoresis diagram of total cytoplasmic RNA extracted by a nucleus & cytoplasmic RNA extraction kit;
FIG. 4 (a) the retention change and fluorescence intensity quantification of the probe in the cell by confocal picture observation (b) after the probe f-CR in the experimental group and the probe CR in the control group are incubated for 6 hours respectively for the MB and the 4T1 cells, (c) a schematic diagram of the animal experiment, (d) the probe f-CR in the experimental group and the probe CR in the control group are injected into the tumor at first by intratumoral injection and then by caudal vein injection, and the retention change and fluorescence intensity quantification of the probe in the tumor tissue is observed by illumination after one hour (e);
FIG. 5 (a) shows the quantification of photoacoustic signal change and photoacoustic intensity in tumor for the test probe f-CR and the control probe CR under the same test conditions (b), (c) shows the enrichment of the isolated tissue slice in tumor tissue, and (d) shows the quantification of probe in each isolated tissue (heart, liver, spleen, lung, kidney, tumor);
FIG. 6 (a) cytotoxicity Change after incubation of the test group Probe f-CR and the control group Probe CR with MB and 4T1 cells for 12 hours, respectively, (b) illumination (660 nm 50 mW/cm) after incubation of the test group Probe f-CR and the control group Probe CR with MB and 4T1 cells for 12 hours, respectively23 min) post-cytotoxicSex change, (c) the experimental probe f-CR and the control probe CR were incubated with MB and 4T1 cells for 12 h, respectively, and then light irradiation (660 nm, 50 mW/cm)23 min) observing apoptosis changes by live-dead and cell bright fields respectively, (d) transfecting GFP-mRNA into 4T1 cells after in vitro cross-linking reaction with an experimental group probe f-CR and a control group probe CR respectively, observing GFP expression by confocal observation, and observing GFP expression (e) by a flow cytometer, (f) mechanism diagram of cell apoptosis;
FIG. 7 (a) continuous 13-day change curves for tumor inhibition for experimental probe f-CR and control probe CR and blank PBS, respectively, (b) comparative size of tumors in mice on the back on day thirteen, (c) size of isolated tumors on day 13, (d) changes in Tunel, H & E, Caspase 3 were examined 48H post-treatment by immunofluorescence and immunohistochemistry, respectively.
Detailed Description
The invention will be further elucidated with reference to the drawings and specific embodiments. It should be understood that these examples are only for explaining and illustrating the technical solutions of the present invention, and are not intended to limit the scope of the present invention. In addition, unless otherwise specified, materials, reagents, instruments and the like used in the following examples are commercially available; the specific preparation method and the test method are conventional in the field.
The invention constructs and synthesizes the tumor anchoring diagnosis and treatment integration, which comprises the following steps:
the method comprises the following steps of deprotecting tert-butyloxycarbonyl-fluorenylmethoxycarbonyl-lysine in a dichloromethane solution containing trifluoroacetic acid, reacting with 3- (2-furan) propionic acid to obtain a compound 1, activating carboxyl by the compound 1 with N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, reacting with cyclopeptide cRGD to obtain a compound 2, deprotecting in an N, N-dimethylformamide solution containing piperidine to obtain a compound 3, and reacting the compound 3 with Cy7 SE to obtain the anchoring type molecular probe f-CR.
And (3) reacting the cyclic peptide cRGD with Cy7 SE to obtain a probe CR of a control group.
The method for carrying out long-time fluorescence in-vivo imaging by utilizing the novel anchoring molecular probe comprises the following steps of firstly carrying out intratumoral injection on Methylene Blue (MB), then carrying out tail vein injection on the aqueous solution of the novel anchoring molecular probe f-CR and a contrast group CR into the body of a tumor-bearing mouse, and observing in-vivo fluorescence at different time points under an anesthesia state to obtain a photoacoustic imaging effect.
The novel anchored molecular probe f-CR and the water solution of the control group CR are respectively incubated with the tumor cells and the fluorescence intensity in the tumor cells is observed at different time points.
The novel anchoring type molecular probe f-CR and the water solution of the control group CR are respectively incubated with the tumor cells for 48 hours to observe the apoptosis condition of the tumor cells.
The method for carrying out in-vivo tumor inhibition experiment by using the novel anchoring type molecular probe comprises the following steps of firstly carrying out intratumoral injection on methylene blue, then injecting the aqueous solution of the novel anchoring type molecular probe f-CR and the contrast group CR into the body of a tumor-bearing mouse through tail veins, and carrying out continuous observation and recording on the tumor inhibition condition by giving illumination after 1 h.
(1) Novel living body fluorescence imaging of tumor anchoring diagnosis and treatment integrated probe:
after intratumoral injection of MB (concentration: 0.1. mu.M, volume: 50. mu.L) for 0.5 hour, the probe f-CR of the experimental group and the probe CR of the control group obtained above were dissolved in PBS solution (concentration: 100. mu.M, volume: 200. mu.L), respectively, the probes were injected into BALB/c female mice bearing tumors (breast cancer in 4T1 mouse) by tail vein injection, and then placed in an optical imaging system of a mouse living body/IVIS Spectrum (PerkinElmer), and light (660 nm 50 mW/cm) was given to the tumor site after 1 hour 23 min), observing the imaging effect in real time, and finally calculating the fluorescence intensity of the tumor part of the mouse at different time points by using living body imaging analysis software.
(2) Novel living body photoacoustic imaging of tumor anchoring type diagnosis and treatment integrated probe:
after intratumoral injection of MB (concentration: 0.1. mu.M, volume: 50. mu.L) for 0.5 hour, the experimental probe f-CR and the control probe CR obtained above were dissolved in PBS solution (concentration: 100. mu.L), respectivelyM, volume: 200 mu L) and injecting the probe into a BALB/c female mouse with tumor (breast cancer of a 4T1 mouse) by tail vein injection, simultaneously opening a photoacoustic tomography imaging system of the mouse, placing the mouse after anesthesia when the water temperature in a water bath of a photoacoustic imager reaches 37 ℃, and scanning the tumor part image of the mouse. And 1 hour later, the tumor site was irradiated with light (660 nm, 50 mW/cm)2And 3 min), observing the imaging effect in real time, and finally calculating the fluorescence intensity of the tumor part of the mouse at different time points by using living body imaging analysis software. The obtained photoacoustic imaging data was then subjected to reconstruction analysis using MSOT InSight/inVision analysis software.
(3) Novel tumor inhibition experiment of tumor anchoring diagnosis and treatment integrated probe:
BALB/c female mice bearing bilateral dorsal tumors (breast cancer in 4T1 mice) with tumor volumes of about 20 mm3) Randomly divided into 3 groups (n ═ 5): left tumors (group 1, PBS for short) of mice injected with PBS (10 mM, 200. mu.L) in tail vein, 660 nm laser-irradiated right tumors (group 2, PBS for short +660 nm); tail vein-only CR (200. mu.M, 200. mu.L) and pre-intratumoral injection of MB (concentration: 0.1. mu.M, volume: 50. mu.L) left tumor (group 3, abbreviated CR + MB), right tumor pre-intratumorally injected MB (concentration: 0.1. mu.M, volume: 50. mu.L) and 660 nm laser-irradiated treated mice (group 4, abbreviated CR + MB +660 nm); tail vein injection of f-CR (200. mu.M, 200. mu.L) and preliminary intratumoral injection of MB (concentration: 0.1. mu.M, volume: 50. mu.L) left tumors (group 3, abbreviated as f-CR + MB), right tumors were preliminary intratumoral injection of MB (concentration: 0.1. mu.M, volume: 50. mu.L) and 660 nm laser-irradiated treated mice (group 4, abbreviated as f-CR + MB +660 nm). After treatment, mice tumor volume changes were recorded every other day and mice survival curves were plotted.
Example 1: synthesis of novel tumor anchoring diagnosis and treatment integrated probe CR and f-CR
Synthesis of compound 1: Boc-Lys (Fmoc) -OH (0.5 g, 1.07 mmol) was dissolved in dichloromethane 10 mL while adding trifluoroacetic acid 2 mL and stirring at room temperature for 10 min to remove BOC, followed by collection of product and addition of 3- (2-furan)) Propionic acid (0.2 g, 1.43 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (0.82 g, 1.43 mmol) and N-hydroxysuccinimide (0.2 g, 1.71 mmol) were stirred at room temperature for 4 h and N, N-diisopropylethylamine (5. mu.L) was added and the reaction was continued for 3 h. After the reaction was completed, it was extracted three times with ethyl acetate and then concentrated, followed by elution through a silica gel column to obtain pure product compound 1 (0.56 g, 80%). C28H30N2O6 ([M+H]+): 489.2021, found ESI-MS: m/z 489.2031;
Synthesis of Compound 2: cRGD (10 mg, 16.57. mu. mol) and 1 (19.47 mg, 33.13. mu. mol) were dissolved in DMF (5 mL) and stirred at 0 ℃ for 10 min followed by the slow addition of DIPEA (2. mu.L) simultaneously. After reacting for two hours at room temperature, collecting the product, and separating the product by using preparative HPLC to obtain a product compound 2C55H69N11O12 ([M-H]-): 1076.5190, found ESI-MS: m/z 1076.5187;
Synthesis of Compound 3: compound 2 (10 mg, 9.29. mu. mol) was dissolved in DMF/diethylamine (6 mL, 5:1, v/v) solution and reacted for 1 h at room temperature. The crude product was concentrated and the product was isolated by preparative HPLC to afford the product compound 3. C40H59N11O10 ([M-H]-): 852.4374, found ESI-MS: m/z 852.4370;
Synthesis of Compound f-CR: compound 3 (5 mg, 5.86. mu. mol), Cy7-SE (4.24 mg, 5.86. mu. mol) was dissolved in DMF (2 mL) and stirred at 0 ℃ for 10 min while dropwise adding to the reaction system, and after allowing to react for 2h at room temperature, a sample was separated by preparative HPLC to give the final product. C75H99N13O17S2 ([M-H]-): 1516.6651, found ESI-MS: m/z 1516.6664。
Synthesis of cRGD (5 mg, 8.27. mu. mol) as control Compound CR, Cy7-SE (5.99 mg, 8.27. mu. mol) was dissolved in DMF (2 mL) and DIPEA was added slowly and stirred at 0 ℃ for 10 min and then at room temperature for 2h with continued stirring to collect the crude product which was isolated by preparative HPLC to give a control product. C62H81N11O14S2 ([M-H]-): 1266.5339, found ESI-MS: m/z 1266.5321。
The reaction scheme and the chemical structural formula related to each part product are shown in figure 1.
Example 2: physicochemical properties of novel tumor anchoring diagnosis and treatment integrated probe
The control probe CR and the test probes f-CR prepared in example 1 were diluted with ultrapure water to a concentration of 10. mu.M (which was completely dissolved), and the ultraviolet-visible near-infrared spectrum and the fluorescence spectrum thereof were measured using an ultraviolet-visible near-infrared spectrophotometer and a fluorescence spectrophotometer. As shown in FIG. 2 (a) (b), the results show that the maximum absorption of probes CR and f-CR is 747 nm and the maximum emission is 789 nm; meanwhile, the control group of probes have no assembly and no nano structure, while the experimental group of probes have the assembly and the nano structure, and the size of the nano structure is larger than 7.2 +/-0.9 nm, as shown in (c) (d) of FIG. 2.
The control probe CR and the test probe f-CR prepared in example 1 were diluted with ultrapure water to a concentration of 10. mu.M, 5. mu.M custom sequence RNA (5 '-ACAUCGGGAUAGCGAAGUUGAGAGAGAGGGAG-3') was added, 10. mu.M MB was mixed, and the mixture was shaken at 4 ℃ and irradiated with light (50 mW/cm) at 660 nm 210 minutes) or not (keeping out of the light), the reaction solution is directly separated by RNA non-denaturing gel electrophoresis, as shown in FIG. 3b, the experimental group probe f-CR can be obviously labeled with RNA after adding RNA and MB and giving light, and the other groups have no obvious red fluorescence of Cy 7. The control probe CR and the test probe f-CR were added to a culture medium (Hyclone DMEM high-sugar liquid medium containing 10% FBS) at a concentration of 10. mu.M, 0.1. mu.M of MB was added thereto, and the mixture was cultured in 4T1 cells for 6 hours, followed by giving illumination at 660 nm (50 mW/cm)210 min) or not (dark), then using commercial RNA dye SYTO ™ RNaselect-green fluorescent cell stain (Thermo Fisher) to stain respectively as shown in FIG. 3d, and finding that the co-localization rate in the experimental group (f-CR + MB +660 nm) is higher than that in other groups in the co-localization experiment of the probe; then extracting total RNA in the cells by a Trizol method, and finding that the fluorescence intensity of an experimental group (f-CR + MB +660 nm) is far higher than that of other groups by fluorescence quantification as shown in figure 3 e; then through the cytoplasm&Extracting cytoplasm RNA from the total nucleus extraction kit and separating the cytoplasm RNA by using an RNA non-denaturing gelThe analysis found that the experimental group (f-CR + MB +660 nm) had significant red fluorescence, and the other groups had no significant fluorescence as shown in FIG. 3 f.
Control Probe CR and Experimental Probe f-CR were added to a medium (Hyclone DMEM high-sugar liquid medium containing 10% FBS) at a concentration of 10. mu.M, MB was added at 0.1. mu.M, and the mixture was cultured in 4T1 cells for 6 hours, followed by giving illumination at 660 nm (50 mW/cm)210 minutes) or not (protected from light), the retention of the material in the cells was observed, and the retention experiment in the experimental group (f-CR + MB +660 nm) was found to be much larger than that in the other groups such as FIGS. 4a and 4 b; then, in the experiment of examining the retention of the probe in the tumor of the mouse, the control probe CR and the test probe f-CR were injected into the tumor of the mouse with PBS at a concentration of 100 μ M200. mu.L at 0.5h in advance of the MB (PBS, 0.1. mu.M 50. mu.L) intratumorally, followed by administration of the tail vein injection CR or f-CR, and 1 h later with 660 nm light (50 mW/cm)23 min) or not (protected from light), the intra-tumor metabolism of the probe at each time point was observed by IVIS as shown in FIGS. 4c, 4d and 4e, and the retention time of the experimental group (f-CR + MB +660 nm) in vivo was found to be much longer than that of the other groups.
Control group Probe CR and Experimental group Probe f-CR were intratumorally injected into mouse tumors with PBS to a concentration of 100 μ M200 μ L0.5 h in advance, followed by caudal vein injection of CR or f-CR, and 660 nm illumination (50 mW/cm) 1 h later, using PBS to give an intratumoral injection of MB (PBS, 0.1 μ M50 μ L) into the mice tumors 23 minutes) or not (shielded from light), and simultaneously observing the change condition of the photoacoustic signal at each time point through a photoacoustic imaging system, performing reconstruction analysis on the photoacoustic imaging data by using MSOT InSight/INVision analysis software, and finding that an experimental group (f-CR + MB +660 nm) has a long-time photoacoustic signal compared with other groups, as shown in FIGS. 5a and 5 b; in the same experimental method as the above, after the probe is injected for 12 h, each main organ of the mouse is taken out, the tumor tissue is frozen and sliced, the graph is shown in 5c, the quantitative fluorescence intensity of the homogenate of all organ tissues is shown in 5d, and the enrichment amount of the experimental group (f-CR + MB +660 nm) in the tumor is far greater than that of other groups.
Control group Probe CR and Experimental group Probe f-CR Medium (Hyclone DMEM high sugar liquid Medium containing 10% FB)S) was diluted to a concentration of 100, 50, 20, 10, 1, 0.1. mu.M of MB was added, and after 12 h of culture in 4T1 cells, neither CR nor f-CR was found to have significant toxicity as shown in FIG. 6a, under the same experimental conditions at 660 nm (50 mW/cm) light exposure 23 min) for another 48 h, the experimental group (f-CR + MB +660 nm) was found to exhibit some cytotoxicity as shown in FIG. 6 b; meanwhile, the experimental group (f-CR + MB +660 nm) shows the capability of causing tumor cell apoptosis through the Live-dead reagent and the morphology of the cell bright field.
Using commercial GFP-mRNA 2. mu.M, control probe CR and experimental probe f-CR, the samples were diluted with PBS to a concentration of 10. mu.M, and MB was added at 0.1. mu.M, and the samples were subjected to conventional shaking at 4 ℃ while giving illumination at 660 nm (50 mW/cm)23 minutes) or not (protected from light), then each mixed solution is transfected into cells by a lipofection kit to be cultured for 24 hours, an experimental group (f-CR + MB +660 nm) shows a remarkable reduction of the expression of the GFP in the cells under a confocal picture, and an experimental group (f-CR + MB +660 nm) shows a reduction of the expression amount of the GFP by analyzing the cells through a flow cytometer is collected and is shown as figure 6 e.
Tumor suppression experiments, control Probe CR and Experimental Probe f-CR were performed using PBS to a concentration of 200 μ M to 200 μ L, and MB (PBS, 0.1 μ M to 50 μ L) was injected intratumorally into mice tumors 0.5h in advance, followed by caudal vein injection of CR or f-CR, and 660 nm light (50 mW/cm) 1 h later23 min) or not (protected from light), then the tumor size was taken daily as in fig. 7a, the mice were sacrificed on day thirteen as in fig. 7b, and the tumors were taken off as in fig. 7c, and it was found that the experimental group (f-CR + MB +660 nm) had good tumor suppression ability, and the mice tumors were taken on day one under the same conditions, and subjected to H&E, immunohistochemistry and immunofluorescence analysis of Tunnel and Caspase-3 result that the probes of the experimental group (f-CR + MB +660 nm) can cause the tumor tissues to generate apoptosis and necrosis.
The anchoring type molecular probe provided by the invention is used for improving the retention time of molecules in tumor tissues and inhibiting the growth of tumors by utilizing red light mediated biological crosslinking. The intra-tumor metabolism of the probe at each time point was observed by IVIS, and the retention time of the experimental group (f-CR + MB +660 nm) in vivo was found to be much longer than that of the other groups.
Claims (10)
2. the use of the red light-mediated nucleic acid-anchored fluorescent probe of claim 1 for in vivo fluorescence, photoacoustic imaging, and tumor suppression; or the application of the red light mediated nucleic acid anchoring type fluorescent probe in the preparation of living body fluorescence, photoacoustic imaging reagents and tumor inhibition reagents according to claim 1; or the use of the red light mediated nucleic acid anchored fluorescent probe of claim 1 for increasing the retention time of the probe in tumor tissue and inhibiting tumors.
3. The method for preparing a red light-mediated nucleic acid-anchored fluorescent probe according to claim 1, comprising the steps of:
(1) carrying out deprotection on tert-butyloxycarbonyl-fluorenylmethyloxycarbonyl-lysine, and then reacting with 3- (2-furan) propionic acid to obtain a compound 1;
(2) activating carboxyl by the compound 1, reacting with cyclopeptide cRGD to obtain a compound 2, and deprotecting to obtain a compound 3;
(3) and reacting the compound 3 with Cy7 SE to obtain the red light mediated nucleic acid anchoring type fluorescent probe.
4. The method for preparing the red-light mediated nucleic acid anchored fluorescent probe according to claim 3, wherein t-butyloxycarbonyl-fluorenylmethyloxycarbonyl-lysine is deprotected in a dichloromethane solution containing trifluoroacetic acid; compound 1 activates the carboxyl group with N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
5. The method for preparing the red light-mediated nucleic acid-anchored fluorescent probe according to claim 3, wherein in the step (1), the molar ratio of tert-butyloxycarbonyl-fluorenylmethoxycarbonyl-lysine 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 red-light-mediated nucleic acid-anchored fluorescent probe according to claim 3, wherein in the step (2), the molar ratio of the compound 1 to the cRGD is 1 to (1-1.2); the reaction was carried out at room temperature.
7. The method for preparing a nucleic acid-anchored fluorescent probe mediated by red light according to claim 3, wherein in the step (3), the reaction of compound 3 with Cy7 SE is carried out in a solvent containing N, N-diisopropylethylamine; the molar ratio of the compound 3 to the Cy7 SE is 1: 1-1.2.
8. A method of red light mediated probe anchoring a cell comprising the steps of:
(1) carrying out deprotection on tert-butyloxycarbonyl-fluorenylmethyloxycarbonyl-lysine, and then reacting with 3- (2-furan) propionic acid to obtain a compound 1;
(2) activating carboxyl by the compound 1, reacting with cyclopeptide cRGD to obtain a compound 2, and deprotecting to obtain a compound 3;
(3) reacting the compound 3 with Cy7 SE to obtain the red light mediated nucleic acid anchoring type fluorescent probe;
(4) and co-incubating the red light mediated nucleic acid anchoring type fluorescent probe, methylene blue and cells to realize the anchoring of the probe to the cells.
9. The red-light mediated probe-anchored cell method of claim 8, wherein the co-incubation is performed in culture medium under light.
10. The method for anchoring cells by using the red light-mediated probe, according to claim 8, wherein the molar ratio of the red light-mediated nucleic acid anchoring type fluorescent probe to the methylene blue is 100 to (0.8-1.2).
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