Fluorescence-labeled oligonucleotide chain-loaded conjugated microporous polymer for detecting expression of Survivin gene in breast cancer

Abstract

The invention discloses a conjugated microporous polymer loaded with a fluorescence labeled oligonucleotide chain, which is used for expression detection of Survivin gene in breast cancer, and belongs to the field of biological diagnosis and disease marker detection. The monomer compound adopted by the invention is an ionic monomer, so that the polymer has good aqueous solution dispersing capacity, and the structure of the tetra (4-ethynylbenzene) methane enables the adsorption capacity of the polymer to be stronger. The surface of the polymer is modified with folic acid groups targeting tumor cells, so that the polymer is easier to target tumor tissues.

Description

Technical Field

[0001] The invention relates to a conjugated microporous polymer loaded with fluorescently labeled oligonucleotide chains for detecting the expression of Survivin gene in breast cancer, belonging to the field of biological diagnosis and disease marker detection. Background Art

[0002] The information disclosed in this background technology section is only intended to increase some understanding of the overall background of the invention, and should not be necessarily regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in this field.

[0003] Clinically, the diagnostic methods for breast cancer include breast ultrasound, mammography, magnetic resonance imaging, and pathological examination. These traditional diagnostic methods have improved the early diagnosis rate of breast cancer, enhanced the efficacy of breast cancer, and prolonged the survival of patients to a certain extent, but there are still problems such as limited detection ability, low overall treatment effectiveness, and high recurrence rate. Therefore, it is very necessary to develop effective early diagnosis methods to inhibit the development of breast cancer and reduce mortality.

[0004] Nanomaterials, due to their special physical and chemical properties, combined with traditional tumor diagnosis methods, can improve the sensitivity of diagnosis, enhance tumor treatment effects, and improve patient prognosis. They have become the research frontier and hotspot in the field of tumor diagnosis. At present, some studies have used the pore structure and high specific surface area of ​​covalent organic frameworks as receptors for loading and quenching fluorescent dyes, and used the "switch" effect of fluorescent signals to detect the expression level of corresponding tumor markers in real time. The high permeability and retention effect of tumor tissues make nanoprobes passively aggregate in tumor tissues, which makes nanoprobes show obvious advantages in early diagnosis of tumors. Summary of the invention

[0005] In view of the existing technology, the present invention aims to design a new type of microporous polymer carrier and modify the corresponding targeting group on its surface to achieve active aggregation of nanoprobes in tumor tissue and real-time imaging of breast cancer tumor markers.

[0006] The technical solution adopted by the present invention is as follows:

[0007] In the first aspect of the present invention, a conjugated microporous polymer is provided, the unit structure of which is shown in the general formula Ⅰ:

[0008] In a second aspect of the present invention, a method for preparing a conjugated microporous polymer is provided, the method comprising the following steps:

[0009] The compound tetrakis(4-ethynylphenyl)methane (TEPM) and the compound 1,1'-bis(4-aminophenyl)-[4,4'-bipyridine]-1,1'-diammonium chloride (BBDC) are dispersed in an organic solvent 1, and silicon dioxide is added to react; after the reaction is completed, the organic solvent 1 is removed; and the silicon dioxide is etched with an alkali to obtain a hollow polymer material HMON;

[0010] HMON and cysteine ​​(Cys) are dispersed in an organic solvent II, reacted under nitrogen protection, the organic solvent II is removed, washed, dried and then reacted with folic acid (FA) to obtain a microporous polymer carrier material HMON@FC, namely the conjugated microporous polymer of the present invention.

[0011] In one or some embodiments of the present invention, the usage ratio of TEPM to BBDC is (1-10):(5-20); preferably, the mass ratio is 5:13.

[0012] In one or some embodiments of the present invention, the mass ratio of TEPM to silicon dioxide is (0.5-5):(1-10); preferably, the mass ratio is 1:6.

[0013] In one or some embodiments of the present invention, after adding silicon dioxide, the reaction is carried out at 80-100° C. for 10-12 hours.

[0014] In one or some embodiments of the present invention, the mass ratio of HMON, Cys and FA is (0.5-2):(0.5-2):(1-4); preferably, the mass ratio is 1:1:2.

[0015] In one or some embodiments of the present invention, HMON and Cys are reacted at 90-100° C. for 18-32 h under nitrogen protection.

[0016] In one or some embodiments of the present invention, after drying, the reaction is carried out with FA at 10-45° C. for 18-32 hours.

[0017] In one or some embodiments of the present invention, the organic solvent 1 and the organic solvent 2 are not particularly limited and are intended to dissolve the reactants.

[0018] Preferably, the organic solvent 1 is a mixed solution of toluene and triethylamine.

[0019] Preferably, the organic solvent is toluene.

[0020] In the third aspect of the present invention, there is provided the use of the conjugated microporous polymer in the preparation of a probe loaded with fluorescently labeled oligonucleotide chains.

[0021] In a fourth aspect of the present invention, a probe loaded with a fluorescently labeled oligonucleotide chain is provided, wherein the conjugated microporous polymer loads the fluorescently labeled oligonucleotide chain through hydrogen bonding interaction, π-π stacking interaction and / or electrostatic interaction.

[0022] The probe was prepared by the following method:

[0023] The conjugated microporous polymer and the fluorescently labeled antisense oligonucleotide are stirred in a buffer solution, the supernatant is separated and removed, and the fluorescently labeled oligonucleotide chain probe is loaded.

[0024] In a fifth aspect of the present invention, there is provided the use of the fluorescently labeled oligonucleotide chain probe in detecting the expression of Survivin gene in breast cancer.

[0025] Compared with the related art known to the inventor, one of the technical solutions of the present invention has the following beneficial effects:

[0026] The present invention provides a novel conjugated microporous polymer material loaded with fluorescently labeled oligonucleotide chains. Such a material can not only target tumor cells, but also has the ability to perform fluorescence imaging of Survivin mRNA in tumor cells, and can perform real-time detection of tumor disease markers.

[0027] The irregular shapes of the polymer materials in the prior art affect the uptake of cells. The polymer materials synthesized in the present invention use silicon dioxide as a template, have regular shapes, and are more easily internalized by cells.

[0028] The monomer compound used in the present invention is an ionic monomer, so that the polymer has good aqueous solution dispersibility, and the structure of tetrakis(4-ethynylphenyl)methane makes the polymer have stronger adsorption capacity.

[0029] The polymer surface is modified with folic acid groups that target tumor cells, making the polymer easier to target tumor tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0031] Figure 1 These are the scanning electron microscope and transmission electron microscope images of HMON / CF prepared in Example 1.

[0032] Figure 2 The zeta potential diagrams of HMON / CF and HMON / CF@Survivin prepared in Example 1.

[0033] Figure 3AThis is the fluorescence emission diagram of the nanoprobe in Example 2 interacting with Survivin mRNA antisense oligonucleotide.

[0034] Figure 3B This is a linear relationship diagram of the interaction between the nanoprobe in Example 2 and the Survivin mRNA antisense oligonucleotide.

[0035] Figure 4A This is the fluorescence emission diagram of the nanoprobe in Example 3 interacting with Survivin mRNA antisense oligonucleotide and other analytes.

[0036] Figure 4B This is a bar chart showing the interaction of the nanoprobe in Example 3 with Survivin mRNA antisense oligonucleotide and other analytes.

[0037] Figure 5 This is the cytotoxicity detection diagram of the nanoprobe in Example 4.

[0038] Figure 6 Schematic diagram of cell imaging of the nanoprobe in Example 5. DETAILED DESCRIPTION

[0039] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0040] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations and / or combinations thereof.

[0041] The synthetic route of the conjugated microporous polymer of the present invention is as follows:

[0042]

[0043] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0044] Example 1

[0045] 1. Disperse the compound tetrakis(4-ethynylbenzene)methane (TEPM, 50 mg) and the compound 1,1'-bis(4-aminophenyl)-[4,4'-bipyridine]-1,1'-diammonium chloride (BBDC, 130 mg) in 30 mL of a solution of toluene:triethylamine = 1:1 (v / v) and ultrasonically disperse, add 300 mg of silica, and react at 90°C for 12 hours. After the reaction is completed, remove the solvent, wash with DMF and MeOH, respectively, and dry to obtain a yellow-brown solid. Etch silica with 8% NaOH to obtain a hollow polymer material HMON.

[0046] 2. Disperse HMON (200 mg) and cysteine ​​(Cys, 200 mg) in a toluene solution (13 mL), react at 100 ° C for 24 h under nitrogen protection, remove the solvent, wash three times with DMF, and dry in an oven. Take 50 mg of the dried polymer and folic acid (FA, 100 mg) and dissolve them in 15 mL DMSO solution, react at room temperature for 24 h to obtain a microporous polymer carrier material HMON / CF. The scanning electron microscope and transmission electron microscope images are shown in the following figure. Figure 1 .

[0047] 3. HMON@FC and Survivin mRNA antisense oligonucleotide (5-TAGAGATGCGGTGGTC-3) fluorescently labeled with dye Cy5 were stirred in PBS buffer solution for 2 h, and then centrifuged to remove the supernatant to obtain the nanoprobe HMON / CF@Survivin. The zeta potential diagram is shown in Figure 2 .

[0048] Example 2

[0049] Prepare a PBS buffer solution with a pH of 7.4 and a concentration of 10mM, and use PBS to prepare a 10mg / mL nanoprobe solution, and prepare a 10μM Survivin mRNA oligonucleotide PBS buffer solution. Take the PBS buffer solution, add different gradient volumes of Survivin mRNA oligonucleotide buffer solution and a nanoprobe solution with a final concentration of 10μg / mL fluorescent probe, and place the mixed solution in a 2mL EP tube; shake at 37℃ for 30min, add the solution after the reaction to a fluorescent cuvette, and detect on a fluorescence spectrometer. As the content of Survivin mRNA antisense oligonucleotide increases, the fluorescence intensity gradually increases. Figure 3A and Figure 3B .

[0050] Example 3

[0051] A PBS buffer solution with a pH of 7.4 and a concentration of 10 mM was prepared, and a 10 mg / mL nanoprobe solution was prepared with PBS, a 10 μM PBS buffer solution of Survivin mRNA oligonucleotide, and different analyte buffer solutions were prepared: Cys, Arg, Hyd, Met, Thr, Phe, His, Lys, GSH, NaCl, MnCl2, Fe3O4, CuSO4·5H2O, KCl, MgCl2, miRNA-T21, miRNA-221, mRNA-c-myc, mRNA-c-TK1. Take PBS buffer solution, put it in different EP tubes, add Survivin mRNA oligonucleotide buffer solution, different analyte buffer solutions and nanoprobe solution with a final concentration of 10 μg / mL fluorescent probe, shake the mixed solution at 37°C for 30 minutes, add the solution after reaction to the fluorescence cuvette, and detect it on the fluorescence spectrometer. Survivin mRNA antisense oligonucleotide makes the fluorescence intensity gradually increase, while other analytes basically do not cause changes in fluorescence intensity, such as Figure 4A and Figure 4B .

[0052] Example 4

[0053] Complete medium was taken and different concentrations of nanoprobe solutions were prepared with complete medium. The solutions were incubated with MCF-7 cells for 24 h, and then CCK8 was added to detect the cytotoxicity of the nanoprobes. Within the range of 0-120 μg / mL, the cell activity was maintained above 85%. Figure 5 .

[0054] Example 5

[0055] Complete culture medium was taken and used to prepare 1μM Survivin oligonucleotide solution, 50μg / mL microporous polymer material solution, 50μg / mL nanoprobe solution without targeting group, and 50μg / mL nanoprobe solution, respectively. A blank control group (Ctrl), Survivin group, HMON group, HMON@Survivin group, and HMON / CF@Survivin group were set up respectively, and incubated with MCF-7 cells for 1h, then the cell culture medium was replaced, and the cells were washed three times with PBS. The changes in fluorescence intensity were detected by laser confocal microscopy. The HMON / CF@Survivin group showed stronger blue fluorescence and red fluorescence, such as Figure 6 , indicating that the nanoprobe has good targeting ability and fluorescence imaging potential.

[0056] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A conjugated microporous polymer, characterized in that: The unit structure general formula of the conjugated microporous polymer is shown in I:

2. A method for preparing a conjugated microporous polymer, characterized in that: The method comprises the following steps: The compound tetrakis(4-ethynylphenyl)methane (TEPM) and the compound 1,1'-bis(4-aminophenyl)-[4,4'-bipyridine]-1,1'-diammonium chloride (BBDC) are dispersed in an organic solvent 1, and silicon dioxide is added to react; after the reaction is completed, the organic solvent 1 is removed; and the silicon dioxide is etched with an alkali to obtain a hollow polymer material HMON; HMON and cysteine ​​(Cys) are dispersed in an organic solvent II, reacted under nitrogen protection, the organic solvent II is removed, washed, dried and then reacted with folic acid (FA) to obtain a microporous polymer carrier material HMON@FC, which is the conjugated microporous polymer.

3. The method for preparing a conjugated microporous polymer as claimed in claim 2, characterized in that: The usage ratio of TEPM to BBDC is (1-10):(5-20); the feeding ratio of TEPM to silicon dioxide is (0.5-5):(1-10).

4. The method for preparing a conjugated microporous polymer as claimed in claim 2, characterized in that: After silica, react at 80-100°C for 10-12h.

5. The method for preparing a conjugated microporous polymer as claimed in claim 2, characterized in that: The feed ratio of HMON, Cys and FA is (0.5~2):(0.5~2):(1~4).

6. The method for preparing a conjugated microporous polymer as claimed in claim 2, characterized in that: HMON is reacted with Cys at 90-100°C for 18-32h under nitrogen protection; after drying, it is reacted with FA at 10-45°C for 18-32h.

7. Use of the conjugated microporous polymer according to claim 1 in the preparation of a probe loaded with fluorescently labeled oligonucleotide chains.

8. A fluorescently labeled oligonucleotide chain probe, characterized in that: The conjugated microporous polymer carries the fluorescent labeled oligonucleotide chain through hydrogen bond interaction, π-π stacking effect and / or electrostatic interaction.

9. The fluorescently labeled oligonucleotide chain probe according to claim 8, characterized in that: The probe is prepared by the following method: a conjugated microporous polymer and a fluorescently labeled antisense oligonucleotide are stirred in a buffer solution, the supernatant is separated and removed, and the fluorescently labeled oligonucleotide chain probe is loaded.

10. Use of the fluorescently labeled oligonucleotide chain probe according to claim 8 or 9 in detecting the expression of Survivin gene in breast cancer.

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