A hybrid supramolecular fluorescent nanoprobe system and its preparation method and application

Abstract

The present invention discloses a method for preparing a hybrid supramolecular fluorescent nanoprobe system, comprising the following steps: (1) preparing a β-cyclodextrin polymer; (2) preparing an adamantane-derivatized nucleic acid aptamer; (3) preparing an adamantane-derivatized folic acid; (4) preparing a self-assembled supramolecular fluorescent nanoprobe targeted by a nucleic acid aptamer; (5) preparing a self-assembled supramolecular fluorescent nanoprobe targeted by a folic acid; (6) mixing the nanoprobes prepared in step (4) and step (5) to obtain a hybrid supramolecular fluorescent nanoprobe system. The present invention successfully constructs two supramolecular fluorescent nanoprobes based on a self-assembly method, and the hybrid probe system composed of the two can not only effectively distinguish human lung adenocarcinoma cells, activated macrophages and normal cells, but also can successfully distinguish lung adenocarcinoma areas, inflammatory areas and normal areas in paracancerous tissue sections, providing valuable detection methods and technical support for the diagnosis of lung adenocarcinoma.

Description

Technical Field

[0001] The invention belongs to the field of nano-biomaterials, and in particular relates to a hybrid supramolecular fluorescent nano-probe system and a preparation method and application thereof. Background Art

[0002] Cancer is a major disease that is widespread throughout the world. In the 2022 global cancer ranking, lung cancer ranks first in mortality and second in incidence. Lung adenocarcinoma is a type of lung cancer. Its early diagnosis is extremely challenging and has become an active research field. For example, cancer is sometimes misdiagnosed as inflammation because the early symptoms of cancer are similar to those of chronic inflammation. In addition, during cancer treatment, accurate diagnosis of tumor boundaries can prevent tumor persistence or recurrence due to incomplete tumor resection. At present, the standard diagnostic methods for tumor detection include imaging examinations, imaging-mediated lung puncture biopsy, and histopathological examinations. Although imaging methods such as X-ray, CT, and MRI can provide effective information about the size, number, and location of lesions, they lack specificity, and the instruments and equipment are expensive. They are often effective only in the middle and late stages of the disease. They cannot confirm whether the lesions are lung cancer, and cannot effectively distinguish them from diseases such as pneumonia. Although histopathological examinations can more sensitively and accurately determine the source, nature, and range of tumor tissue, the examination results are highly subjective, experience-dependent, and face challenges such as manpower, cost, and time.

[0003] Fluorescence imaging technology has shown great potential in the sensitive and specific diagnosis of cancer. Fluorescence labeling with organic dyes has been widely used in tumor-targeted imaging and diagnosis. However, the poor photostability of organic dyes limits their application in the field of biology. In recent years, with the development of nanotechnology and the excellent properties of nanostructures themselves, such as unique signal generation and enhancement effects, anti-enzymatic properties, EPR enhancement effects, etc., a series of functionalized nanomaterials have been successfully used to enhance the photostability and fluorescence signal intensity of organic dyes, thereby effectively improving the sensitivity of tumor detection and imaging, such as silica nanoparticles, metal nanomaterials, etc.; however, they have shortcomings such as cytotoxicity and the need for complex derivatization and covalent modification, which limits their wide application.

[0004] Compared with the above-mentioned functionalized nanomaterials constructed based on covalent interactions, supramolecular nanomaterials are self-assembled through weak bond interactions of assembly primitives. The method is simple and easy, avoiding continuous multi-step synthesis and complex purification treatment, and providing the possibility of establishing a simple, sensitive, and specific labeling technology that can simultaneously distinguish pneumonia and lung cancer. In view of this, it is of great significance to establish a simple, sensitive, rapid, and specific fluorescence imaging technology that can simultaneously distinguish pneumonia and lung cancer based on the new labeling technology constructed by the self-assembly method, visualize the pathological site, and thus easily and effectively distinguish pneumonia from lung cancer. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a method for preparing a hybrid supramolecular fluorescent nanoprobe system, and two supramolecular fluorescent nanoprobes are successfully constructed based on self-assembly.

[0006] A second object of the present invention is to provide a hybrid supramolecular fluorescent nanoprobe system.

[0007] The third object of the present invention is to provide an application of a hybrid supramolecular fluorescent nanoprobe system.

[0008] One of the purposes of the present invention is achieved by the following technical solution:

[0009] A method for preparing a hybrid supramolecular fluorescent nanoprobe system comprises the following steps:

[0010] (1) preparing a β-cyclodextrin polymer: dissolving β-cyclodextrin in an alkaline solution, stirring, adding toluene, continuing to stir, and then dropping a crosslinking agent to obtain a β-cyclodextrin polymer after reaction;

[0011] Furthermore, the crosslinking agent is epichlorohydrin, the molar ratio of β-cyclodextrin to toluene and epichlorohydrin is 1:2:5.5, and in the above step (1), β-cyclodextrin is dissolved in a 15-20wt% NaOH aqueous solution, and the reaction temperature is 30-37°C;

[0012] Furthermore, step (1) also includes separating and purifying the β-cyclodextrin polymer, and the process is as follows: adding isopropanol to the crude product obtained after the reaction for pre-precipitation treatment, collecting the precipitate and dissolving it in water, adjusting the pH to neutral, dialyzing the product with a dialysis membrane with Mw=8000, changing the water every 3 hours, and dialysis for a total of 2 days, freeze-drying the dialyzed product, and storing it at -20°C for future use.

[0013] (2) Preparation of adamantane-derivatized nucleic acid aptamers: The nucleic acid aptamer primary product and adamantane amine are mixed in a solvent with DIPEA added, shaken overnight, the reaction solution is centrifuged and the precipitate is collected, and ammonia water is added to the precipitate for aminolysis overnight;

[0014] (3) Preparation of adamantane-derivatized folic acid: folic acid, adamantane, dicyclohexylcarbodiimide, N-hydroxysuccinimide and DIPEA are uniformly mixed in a solvent, and the mixture is shaken in the dark to obtain the obtained product;

[0015] (4) preparing a nucleic acid aptamer-targeted self-assembled supramolecular fluorescent nanoprobe: taking the β-cyclodextrin polymer prepared in step (1) and the adamantane-derivatized rhodamine and mixing them in a solvent, incubating them with constant temperature oscillation for a period of time, then adding the adamantane-derivatized nucleic acid aptamer prepared in step (2), and continuing to oscillate for a period of time to obtain a nucleic acid aptamer-targeted self-assembled supramolecular fluorescent nanoprobe solution;

[0016] (5) preparing a folic acid-targeted self-assembled supramolecular fluorescent nanoprobe: taking the β-cyclodextrin polymer prepared in step (1) and the adamantane-derivatized cyanine dye 5.5, mixing them in a solvent, incubating them with constant temperature oscillation for a period of time, then adding the adamantane-derivatized folic acid prepared in step (3), and continuing to oscillate for a period of time to obtain a folic acid-targeted self-assembled supramolecular fluorescent nanoprobe solution;

[0017] (6) The nanoprobes prepared in step (4) and step (5) are mixed to obtain a mixed supramolecular fluorescent nanoprobe system.

[0018] Furthermore, in the mixed supramolecular fluorescent nanoprobe system in step (6), the molar ratio of the nucleic acid aptamer-targeted self-assembled supramolecular fluorescent nanoprobe to the folic acid-targeted self-assembled supramolecular fluorescent nanoprobe is 1:1.

[0019] Furthermore, the initial product of the nucleic acid aptamer in step (2) is S11e nucleic acid aptamer, and the molar ratio of the S11e nucleic acid aptamer, adamantane amine and DIPEA is 1:1800:14000.

[0020] Furthermore, in step (2), the mixture is shaken at 30-37°C overnight, 30% aqueous ammonia is added to the precipitate, and the ammonolysis process is carried out at a constant temperature of 50-55°C. After the ammonolysis is completed, the supernatant is collected, the product is separated and purified by high performance liquid chromatography, the separated product is collected and freeze-dried, and stored at -80°C for future use.

[0021] Furthermore, in step (3), the molar ratio of folic acid, adamantane amine, dicyclohexylcarbodiimide, N-hydroxysuccinimide and DIPEA is 1:5:2:1:9, and the reaction is carried out at 30-37°C in the dark with shaking for 8-10 hours, and then the reaction solution is separated and purified by high performance liquid chromatography, and the separated product is collected, freeze-dried, and stored at -20°C for later use.

[0022] Furthermore, in step (4), the molar ratio of β-cyclodextrin polymer to adamantane-derivatized rhodamine and adamantane-derivatized nucleic acid aptamer is 5000:100:1. During the reaction, the β-cyclodextrin polymer and adamantane-derivatized rhodamine are first incubated at 25-30° C. with constant temperature shaking for 2-3 hours, and then the adamantane-derivatized nucleic acid aptamer is added and then incubated at 25-30° C. with constant temperature shaking for 2-3 hours.

[0023] Furthermore, in step (5), the molar ratio of the β-cyclodextrin polymer to the adamantane-derivatized cyanine dye 5.5 and the adamantane-derivatized folic acid is 5000:100:1. During the reaction, the β-cyclodextrin polymer and the adamantane-derivatized cyanine dye 5.5 are first incubated at 25-30° C. with constant temperature shaking for 2-3 hours, and then the adamantane-derivatized folic acid is added and the constant temperature shaking is continued at 25-30° C. for 2-3 hours.

[0024] Furthermore, the preparation process of adamantane-derivatized rhodamine in step (4) is as follows: rhodamine B isothiocyanate, adamantane, DIPEA, DMSO, and carbonate buffer are shaken and reacted overnight at 25-30° C. in the dark, and the separated product is collected after high performance liquid chromatography separation and purification, and freeze-dried and stored at -20° C. for later use, wherein the molar ratio of rhodamine B isothiocyanate, adamantane, and DIPEA is 1:10:170.

[0025] Furthermore, the preparation process of the adamantane-derivatized cyanine dye 5.5 in step (5) is as follows: Cy5.5-NHS is dissolved in DMSO, adamantane and DIPEA are added and mixed evenly, and the mixture is shaken at 35-40°C for 8-10h, and the separated product is collected after purification by high performance liquid chromatography, freeze-dried and stored at -20°C for later use, wherein the molar ratio of Cy5.5-NHS to adamantane and DIPEA is 1:7:40.

[0026] The second object of the present invention is achieved by adopting the following technical solution:

[0027] A hybrid supramolecular fluorescent nanoprobe system is prepared by the method.

[0028] The third object of the present invention is achieved by adopting the following technical solution:

[0029] The application of the above-mentioned mixed supramolecular fluorescent nanoprobe system in the preparation of reagents for detecting lung adenocarcinoma.

[0030] Compared with the prior art, the beneficial effect of the present invention is that the present invention provides a method for preparing a hybrid supramolecular fluorescent nanoprobe system, with β-cyclodextrin polymer as the main molecule and adamantane-derivatized dye as the guest molecule, combined with the specific recognition of tumor cells by nucleic acid aptamers and the properties of folate receptors on the surface of activated macrophages during inflammatory response targeted by folic acid, and successfully constructed two supramolecular fluorescent nanoprobes based on self-assembly: nucleic acid aptamer-targeted self-assembled supramolecular fluorescent nanoprobes and folic acid-targeted self-assembled supramolecular fluorescent nanoprobes. The hybrid probe system composed of the two can not only effectively distinguish human lung adenocarcinoma cells, activated macrophages and normal cells, but also can successfully distinguish lung adenocarcinoma areas, inflammatory areas and normal areas in adjacent tissue sections. The new labeling technology constructed based on the self-assembly method can provide valuable detection methods and technical support for the diagnosis of lung adenocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the synthetic route of Ad-Aptamer;

[0032] Figure 2 The DLS and SEM characterization results of SSNP-Aptamer and SSNP-Folate, wherein (a) and (f) are the DLS and SEM characterization results of poly-β-CD, (b) and (d) are the DLS images of poly-β-CD / Ad-R and poly-β-CD / Ad-C, (c) and (g) are the DLS and SEM characterization results of SSNP-Aptamer, and (e) and (h) are the DLS and SEM characterization results of SSNP-Folate;

[0033] Figure 3 The gel electrophoresis images of SNP-Aptamer and SSNP-Folate probes, where "+" indicates the addition of the corresponding component; (a) is the electrophoresis image of each component of SSNP-Aptamer, and (b) is the electrophoresis image of each component of SSNP-Folate;

[0034] Figure 4 In vitro fluorescence spectra of SNP-Aptamer and SSNP-Folate probes;

[0035] Figure 5 The fluorescence intensity of SSNP-Aptamer and SSNP-Folate in different solutions changes with time;

[0036] Figure 6The effects of different concentrations of SSNP-Aptamer and SSNP-Folate on cell viability, (a) is the viability of A549 cells after being treated with SSNP-Aptamer, (b) is the viability of A549 cells after being treated with SSNP-Folate;

[0037] Figure 7 Confocal images of the SSNP-Aptamer and SSNP-Folate probe systems recognizing different cells.

[0038] Figure 8 The experimental principle diagram of the application of SSNP-Aptamer and SSNP-Folate probe systems;

[0039] Fig. 9 This is a graph showing the experimental results of the SSNP-Aptamer and SSNP-Folate mixed probe system for distinguishing lung adenocarcinoma adjacent tissue (TAT) and normal lung tissue (Lung). DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0041] Example 1

[0042] A method for preparing a hybrid supramolecular fluorescent nanoprobe system comprises the following steps:

[0043] (1) Preparation of β-cyclodextrin polymer (poly-β-CD): 10 g of β-CD was dissolved in 15 mL of 15 wt% NaOH aqueous solution, the reaction temperature was maintained at 35° C. and stirred for 2 h, then 2 mL of toluene was added and stirred for 2 h. After the hydrophobic cavity of β-CD was fully embedded with toluene, 3.8 mL of epichlorohydrin was slowly added dropwise and reacted for 3 h to form a cross-linked polymer poly-β-CD from β-CD. The crude product obtained after the reaction was pre-precipitated with 200 mL of isopropanol, the precipitate was collected, dissolved with water, and the pH was adjusted to neutral. The product was dialyzed with a dialysis membrane with Mw=8000 to remove oligomers and uncross-linked monomers. The water was changed every 3 hours for a total of 2 days. The dialyzed product was freeze-dried and stored at -20° C. for later use.

[0044] The product was characterized by gel permeation chromatography (GPC) to determine the molecular weight of the polymer. The GPC results showed that the number average molecular weight of poly-β-CD was 94417 Da and the dispersion was 1.45, indicating that the molecular weight distribution of the polymer was relatively concentrated.

[0045] (2) Preparation of adamantane-derivatized nucleic acid aptamers (Ad-Aptamer): Synthesis route: Figure 1 As shown: 10OD of S11e nucleic acid aptamer (Aptamer), 5mg of adamantane amine (Ad-NH2), and 50μLDIPEA were placed in 1mL of anhydrous DMSO, shaken at 37°C overnight, the mixed solution was centrifuged at 6000rpm for 3min, the supernatant was discarded, the precipitate was collected and washed three times with DMSO, 1mL of 30% ammonia water was added to the washed precipitate, and the precipitate was placed in a 55°C constant temperature water bath for aminolysis overnight, the supernatant was collected and placed in a centrifuge tube, and separated and purified by HPLC, and the separation conditions are shown in Table 1. The separated and purified product was freeze-dried and stored at -80°C.

[0046] The molecular weight was characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOFMS). The measured molecular weight was 17512.3Da, while the theoretical molecular weight after the aptamer was modified with adamantane was 17519.4Da. The comparison between the two fully proved that the adamantane-modified aptamer had been successfully prepared;

[0047] Table 1 HPLC gradient elution conditions for separation and purification of Ad-Aptamer

[0048]

[0049] Note: Column temperature is 25℃, detection wavelength is 260nm, solvent A represents 10mmol / L triethylamine acetate, solvent B represents chromatographic acetonitrile, and the chromatographic column used is: Agilent5Hc-C18, 150mmX4.6mm.

[0050] (3) Preparation of adamantane-derivatized folic acid (Ad-Folate): 13.2 mg of folic acid (Folate), 22.7 mg of adamantane amine (Ad-NH2), 12.4 mg of dicyclohexylcarbodiimide (DCC), 3.5 mg of N-hydroxysuccinimide (NHS) and 50 μL of DIPEA were placed in 1 mL of anhydrous DMSO and mixed thoroughly. The mixture was reacted at 37°C in the dark for 8 hours. The mixture was separated and purified by HPLC. The separation conditions are shown in Table 2. The separated product was collected and freeze-dried, stored at -20°C for later use, and characterized by mass spectrometry. The molecular weight of its molecular ion peak was measured to be 575.3 Da, while its theoretical molecular weight was 575.6 Da, indicating that Ad-Folate has been successfully prepared;

[0051] Table 2 HPLC gradient elution conditions for separation and purification of Ad-Folate

[0052]

[0053] Note: Column temperature is 25℃, detection wavelength is 281nm, solvent A represents 10mmol / L triethylamine acetate, solvent B represents chromatographic acetonitrile, and the chromatographic column used is: Agilent5Hc-C18, 150mmX4.6mm.

[0054] (4) Preparation of aptamer-targeted self-assembled supramolecular fluorescent nanoprobe (SSNP-Aptamer):

[0055] ① Preparation of adamantane-derivatized rhodamine (Ad-R): Add 1.8 mg of rhodamine B isothiocyanate (RITC), 5 mg of adamantane amine (Ad-NH2), 400 μL of anhydrous DMSO and 100 μL of anhydrous N,N-diisopropylethylamine (DIPEA) to a 1.5 mL glass bottle, and add 400 μL of carbonate buffer (pH = 8.5); shake the reaction overnight at 25°C in the dark, and use high performance liquid chromatography (HPLC) to separate and purify the initial reaction product. The gradient elution conditions are shown in Table 3. Collect the separated products, freeze-dry them, and store them at -20°C for later use. Dissolve and dilute the separated and purified products with methanol for mass spectrometry characterization. It was found that the substance separated and purified by HPLC lost one Cl - The molecular weight of the ion peak after the loss of Ad-R is 651.2Da. - The molecular weight of Ad-R was 651.3 Da, indicating that Ad-R was successfully prepared.

[0056] Table 3 HPLC gradient elution conditions for separation and purification of Ad-R

[0057]

[0058]

[0059] Note: Column temperature is 25°C, detection wavelength is 550nm, solvent A represents filtered ultrapure water, solvent B represents chromatographic grade methanol, and the chromatographic column used is: Agilent5Hc-C18, 150mmX4.6mm.

[0060] ② Take 19.2mg of poly-β-CD prepared in step (1) and dissolve it in 1mL Milli-Q water to obtain a concentration of 15mmol / L poly-β-CD (calculated based on the concentration of β-CD monomers contained); mix Ad-R with a concentration of 300μmol / L and poly-β-CD with a concentration of 15mmol / L in an equal volume of 1:1 (concentration ratio of 1:50), place it on a 25℃ constant temperature oscillator, incubate it at 300rpm for 3 hours to obtain Ad-R / poly-β-CD, add 3μmol / L Ad-Aptamer, make the volume ratio of the three substances 1:1:1, and fully oscillate at 25℃ and 300rpm for 3 hours. 10×SSNP-Aptamer solution is obtained, and the final concentration of 1×SSNP-Aptamer solution, i.e., 10μmol / L Ad-R, 500μmol / L poly-β-CD and 100nmol / L Ad-Aptamer, is used in the in vitro characterization experiment.

[0061] (5) Preparation of folate-targeted self-assembled supramolecular fluorescent nanoprobe (SSNP-Folate):

[0062] ①Adamantane-derivatized cyanine dye 5.5 (Ad-C): Weigh 5 mg Cy5.5-NHS and fully dissolve it in 1 mL DMSO, add 7.8 mg Ad-NH2 and 50 μL DIPEA, mix well, shake at 37°C for 8 hours, and separate and purify by HPLC. The separation and purification conditions are shown in Table 3. Collect the separated product, freeze-dry it, and store it at -20°C for future use.

[0063] The separated and purified product was dissolved and diluted with methanol and characterized by mass spectrometry. The Ad-C molecular ion peak (Cl removal) was measured. - ) has a molecular weight of 716.5Da, and Ad-C removes Cl - The final theoretical molecular weight was 717.0 Da, indicating that Ad-C was successfully prepared.

[0064] Table 4 HPLC gradient elution conditions for separation and purification of Ad-C

[0065]

[0066]

[0067] Note: Column temperature is 25°C, detection wavelength is 673nm, solvent A represents filtered ultrapure water, solvent B represents chromatographic grade methanol, and the chromatographic column used is: Agilent5Hc-C18, 150mmX4.6mm.

[0068] ② Take 19.2 mg of poly-β-CD prepared in step (1) and dissolve it in 1 mL of Milli-Q water to obtain a concentration of 15 mmol / L poly-β-CD (calculated based on the concentration of the β-CD monomer contained); mix 300 μmol / L Ad-C and 15 mmol / L poly-β-CD in an equal volume ratio of 1:1 (concentration ratio of 1:50), place on a 25°C constant temperature oscillator, incubate at 300 rpm for 3 h to obtain Ad-R / poly-β-CD, add 3 μmol / L Ad-Folate, make the volume ratio of the three substances 1:1:1, and fully oscillate at 25°C and 300 rpm for 3 h to obtain 10×SSNP-Folate solution. In the in vitro characterization experiment, a final concentration of 1×SSNP-Folate solution, i.e., 10 μmol / L Ad-C, 500 μmol / L poly-β-CD, and 100 nmol / L Ad-Folate, is used;

[0069] (6) The SSNP-Aptamer nanoprobe prepared in step (4) and the SSNP-Folate nanoprobe prepared in step (5) are mixed at equal concentrations and volumes to obtain a mixed supramolecular fluorescent nanoprobe system, that is, the molar ratio of the two probes in the mixed probe system is 1:1, which contains 5×SSNP-Aptamer (50μmol / LAd-R, 2500μmol / Lpoly-β-CD and 500nmol / LAd-Aptamer) and 5×SSNP-Folate (50μmol / LAd-C, 2500μmol / Lpoly-β-CD, 500nmol / LAd-Folate).

[0070] Example 2

[0071] Compared with Example 1, Example 2 has the following differences:

[0072] (1) In step (1), β-cyclodextrin is dissolved in an 18 wt % NaOH aqueous solution, and the reaction temperature is 30-37° C.;

[0073] (2) shaking at 30° C. overnight in step (2), and then placing in a constant temperature water bath at 50° C. for aminolysis overnight;

[0074] (3) in step (3), folic acid, adamantane amine, dicyclohexylcarbodiimide, N-hydroxysuccinimide and DIPEA were mixed evenly in anhydrous DMSO, and then reacted at 30° C. in the dark under shaking for 10 hours;

[0075] (4) In the preparation process of adamantane-derivatized rhodamine in step (4), rhodamine B isothiocyanate, adamantane, DIPEA, DMSO, and carbonate buffer are shaken and reacted overnight at 28° C. in the dark; the β-cyclodextrin polymer and adamantane-derivatized rhodamine are mixed and placed on a 28° C. constant temperature shaker and incubated at 300 rpm for 3 h. After the adamantane-derivatized nucleic acid aptamer is added, the mixture is continued to be shaken at 28° C. for 3 h.

[0076] (5) In the preparation process of the adamantane-derivatized cyanine dye 5.5 in step (5), Cy5.5-NHS is dissolved in DMSO, adamantane and DIPEA are added and mixed evenly, and the mixture is shaken at 35°C for 10 hours; the β-cyclodextrin polymer and the adamantane-derivatized cyanine dye 5.5 are placed on a constant temperature shaker at 28°C and incubated at 300 rpm for 3 hours, and the adamantane-derivatized folic acid is added and the mixture is shaken at 28°C and 300 rpm for 3 hours;

[0077] The other preparation processes of Example 2 are the same as those of Example 1.

[0078] Example 3

[0079] Compared with Example 1, Example 3 has the following differences:

[0080] (1) In step (1), β-cyclodextrin is dissolved in a 20 wt % NaOH aqueous solution, and the reaction temperature is 30-37° C.;

[0081] (2) shaking at 35° C. overnight in step (2), and then placing in a constant temperature water bath at 53° C. for aminolysis overnight;

[0082] (3) in step (3), folic acid, adamantane amine, dicyclohexylcarbodiimide, N-hydroxysuccinimide and DIPEA were mixed evenly in anhydrous DMSO, and the mixture was reacted at 35° C. in the dark under shaking for 9 hours;

[0083] (4) In the preparation process of adamantane-derivatized rhodamine in step (4), rhodamine B isothiocyanate, adamantane, DIPEA, DMSO, and carbonate buffer are shaken and reacted overnight at 30° C. in the dark; the β-cyclodextrin polymer and adamantane-derivatized rhodamine are mixed and placed on a 30° C. constant temperature oscillator and incubated at 300 rpm for 2 h. After the adamantane-derivatized nucleic acid aptamer is added, the mixture is continued to be shaken at 30° C. for 2 h.

[0084] (5) In the preparation process of the adamantane-derivatized cyanine dye 5.5 in step (5), Cy5.5-NHS is dissolved in DMSO, adamantane and DIPEA are added and mixed evenly, and the mixture is shaken at 40°C for 8 hours; the β-cyclodextrin polymer and the adamantane-derivatized cyanine dye 5.5 are placed on a 30°C constant temperature shaker and incubated at 300 rpm for 2 hours, and the adamantane-derivatized folic acid is added and the mixture is shaken at 30°C and 300 rpm for 2 hours;

[0085] The other preparation processes of Example 3 are the same as those of Example 1.

[0086] The products used in Experimental Examples 1 to 6 were all prepared in Example 1.

[0087] Experimental Example 1

[0088] The morphology and size of SSNP-Aptamer and SSNP-Folate were investigated by scanning electron microscopy (SEM) and dynamic light scattering (DLS). Figure 2 SEM showed that poly-β-CD had an irregular shape and no spherical nano-shape, while the shapes of SSNP-Aptamer and SSNP-Folate probes were nearly spherical and distributed singly, with particle sizes of approximately 96nm and 110nm; DLS analysis showed that their average hydrodynamic diameters in water were approximately 154nm and 163nm, respectively.

[0089] Experimental Example 2

[0090] Agarose gel electrophoresis was used to investigate the binding stability of adamantane-derivatized dyes to poly-β-CD: voltage 100 V, time 20 min, experimental groups and results are shown in Figure 3 As shown, Figure 3 (a) shows that the positively charged Ad-R migrates toward the negative electrode under the action of the electric field, while the bright band of Ad-R does not move when poly-β-CD is present, indicating that Ad-R as a guest molecule has been embedded in the cavity of the poly-β-CD main molecule, forming a larger and more stable Ad-R / poly-β-CD inclusion complex. It also shows that poly-β-CD has a sensitizing effect on the fluorescence intensity of the dye molecule. In addition to the obvious bands that appear in the lane of the SSNP-Aptamer obtained after the addition of Ad-Aptamer, the light bands of the corresponding nucleic acid bands are significantly weakened, indicating that part of the Ad-Aptamer has been connected to the remaining cavity of the cyclodextrin through host-guest interactions. Figure 3In (b), the fluorescence of Ad-C is almost invisible under ultraviolet excitation light (365nm) because the optimal excitation wavelength is 673nm. After adding poly-β-CD, Ad-C does not move in the swimming lane, indicating that a large molecular weight complex is obtained. Since cyclodextrin increases the fluorescence of Ad-C by about 72 times, the pores of Ad-C / poly-β-CD and SSNP-Folate show brighter bands even under ultraviolet excitation, which also indicates that Ad-C / poly-β-CD forms a stable envelope complex. Since folic acid cannot be stained on agarose, there is no band at the folic acid position.

[0091] The fluorescence spectra of the adamantane-derivatized dye at different concentrations of poly-β-CD (calculated based on the concentration of β-CD monomer) were combined and the Benesi-Hildebrand equation was used to calculate the binding constant of Ad-R to poly-β-CD to be 2.5×10 3 L / mol; the binding constant of Ad-C and poly-β-CD is 2.7×10 3 L / mol.

[0092] Since the β-CD cavity is a structure with high electron cloud density, when the dye is embedded in the β-CD cavity, its molecular mobility is reduced, maintaining a suitable microenvironment or protecting its excited state from contact with water molecules and quenchers, thereby causing the spectrum of the dye to shift or the fluorescence intensity to increase. In order to observe the fluorescence sensitization effect of poly-β-CD on fluorescent dyes, we observed the fluorescence spectrum of the probe. The results are shown in Figure 4 As shown, Figure 4 a is the fluorescence spectrum of SSNP-Aptamer, whose composition is: 100nmol / LAd-Aptamer / 10μmol / LAd-R / 500μmol / Lpoly-β-CD; Figure 4 b is the fluorescence spectrum of SSNP-Folate, whose composition is: 100nmol / LAd-Folate / 10μmol / LAd-C / 500μmol / Lpoly-β-CD. At the optimal emission wavelength, the fluorescence intensity of SSNP-Aptamer was enhanced by about 3.5 times compared with Ad-R ( Figure 4 a); The fluorescence of SSNP-Folate at the optimal emission wavelength is enhanced by about 72 times compared with Ad-C ( Figure 4 b). The above results indicate that cyclodextrin and adamantane-derivatized dyes have been successfully assembled into inclusion complexes, which have significant fluorescence sensitization effects.

[0093] Experimental Example 3

[0094] In order to demonstrate the photostability of the supramolecular fluorescent nanoprobe in a complex system, the change in the fluorescence intensity of the probe in PBS and a culture medium containing 10% FBS was investigated. The change trend of the fluorescence intensity of the probe in PBS buffer system (a) and 10% FBS culture medium (b) after continuous irradiation with UV light for different time periods is shown in the following figure. Figure 5 As shown, whether SSNP-Aptamer and SSNP-Folate are in PBS buffer or 10% FBS culture medium, the fluorescence intensity of the two probes can still be maintained at more than 90% of the initial fluorescence intensity even after continuous irradiation with a 7W handheld UV lamp for 10 hours, indicating that the probes have good photostability.

[0095] Experimental Example 4

[0096] The MTS method was used to evaluate the effect of supramolecular fluorescent nanoprobes on cell viability: the final concentrations of Ad-Aptamer or Ad-Folate were 0, 10nmol / L, 20nmol / L, 50nmol / L, 100nmol / L and 200nmol / L, respectively; the corresponding final concentrations of Ad-R or Ad-C were 0, 1μmol / L, 2μmol / L, 5μmol / L, 10μmol / L and 20μmol / L, respectively; and the corresponding concentration of poly-β-CD was added at a molar ratio of Ad-R or Ad-C to poly-β-CD of 1:50. The results are shown in Figure 2. Figure 6 As shown, the error bars represent the standard deviation of 4 independent experiments. In the experiment, different concentrations of SSNP-Aptamer and SSNP-Folate were used to incubate A549 cells for 2 hours. After two days of growth, the cell survival rate can still reach more than 85%. The above results show that the probe has low cytotoxicity and can be further used for detection in cells and tissues.

[0097] Experimental Example 5

[0098] Laser confocal imaging of different cell recognition using the SSNP-Aptamer and SSNP-Folate probe systems: In order to study the effects of SSNP-Aptamer and SSNP-Folate in distinguishing tumor cells, inflammatory cells and normal cells, a mixed probe system of SSNP-Aptamer (10μmol / LAd-R, 500μmol / Lpoly-β-CD and 100nmol / LAd-Aptamer) and SSNP-Folate (10μmol / LAd-C, 500μmol / Lpoly-β-CD, 100nmol / LAd-Folate) was incubated with cells. The pseudocolor of SSNP-Aptamer was set to green, the excitation was 552nm, and the collection band was 562nm-620nm. The pseudocolor of SSNP-Folate was set to red, the excitation was 638nm, the collection band was 650nm-750nm, the probe was incubated with cells for 10min, and the fluorescence imaging effect was observed by laser confocal imaging. The results are shown in Figure 7 As shown, among the three cells, the SSNP-Aptamer probe used to specifically target A549 lung adenocarcinoma cells showed high specific binding ability to A549 cells, A549 cells showed bright green fluorescence, BEAS-2B cells had almost no fluorescence signal, and M2 macrophages also showed bright green fluorescence, which may be because macrophages themselves belong to a type of professional phagocytes that can engulf foreign substances through phagocytosis. At the same time, the SSNP-Folate probe used to specifically target FR showed high specific binding ability to M2 macrophages, M2 macrophages showed bright red fluorescence, BEAS-2B cells showed weak red fluorescence, and A549 cells had almost no red fluorescence, indicating that M2 macrophages had the most abundant FR expression, BEAS-2B cells had low FR expression, and A549 cells had the least FR expression, which was almost negligible. The confocal imaging results of the above cells showed that the SSNP-Aptamer and SSNP-Folate probe systems can effectively distinguish tumor cells (A549), inflammatory cells (M2 macrophages) and normal cells (BEAS-2B cells).

[0099] The experimental principle of the SSNP-Aptamer and SSNP-Folate probe systems is as follows Figure 8As shown: The specific targeting molecule of SSNP-Aptamer (pseudo-color is green) is S11eAptamer, which can specifically recognize human lung adenocarcinoma cell A549; S11eAptamer is a nucleic acid sequence obtained by cell-SELEX technology screening, which has a high affinity for A549, but weak binding ability to normal lung tissue. SSNP-Folate (pseudo-color is red) uses Folate as a targeting group and can specifically recognize FR. When an inflammatory response occurs, there are a large number of activated macrophages that can express abundant FR.

[0100] SSNP-Aptamer and SSNP-Folate were used together to identify human lung adenocarcinoma cells A549, activated macrophages and normal cells: (1) When identifying A549 cells, the cells can bind to a large amount of SSNP-Aptamer under the mediation of Aptamer. However, due to the low expression level of FR on the surface of A549 cells, SSNP-Folate almost does not bind to A549 cells. At this time, the surface of A549 cells shows bright green fluorescence; (2) When identifying activated macrophages, a large amount of SSNP-Folate can be bound under the mediation of overexpressed FR on their surface. At the same time, since macrophages are professional phagocytes and have a phagocytic effect on nanoparticles, SSNP-Aptamer can also enter macrophages, which show a mixed red and green yellow fluorescence; (3) Lung adenocarcinoma originates from the bronchial mucosal epithelium or smaller bronchi. Therefore, epithelial cells around lung adenocarcinoma were selected as normal cell controls in this study. When the mixed system of the two probes is incubated with normal cells, Aptamer has a weak ability to target and bind to normal cells, and because the expression level of FR on the surface of normal lung cells is low, SSNP-Aptamer and SSNP-Folate hardly bind to normal lung cells, resulting in no obvious fluorescence signal. The above process can be used to distinguish human lung adenocarcinoma cells A549, macrophages and normal cells, and further applied to the identification of corresponding tissues, thereby constructing a simple, sensitive, specific labeling technology that can distinguish inflammation and cancer at the same time.

[0101] Experimental Example 6

[0102] The effect of SSNP-Aptamer and SSNP-Folate mixed probe system on different tissues: The tissues used in the experiment were lung adenocarcinoma paracancerous tissue (TAT) and normal lung tissue (Lung). The mixed probe system of SSNP-Aptamer (10μmol / LAd-R, 500μmol / Lpoly-β-CD and 100nmol / LAd-Aptamer) and SSNP-Folate (10μmol / LAd-C, 500μmol / Lpoly-β-CD, 100nmol / LAd-Folate) was incubated with frozen tissue sections to obtain laser confocal imaging results, which were compared with pathological HE staining and IHC staining. The results are shown in Figure 2. Fig. 9 As shown. After incubation of normal lung tissue with the mixed probe, there was almost no green fluorescence signal in the green light channel (generated by SSNP-Aptamer recognition), but a weak red fluorescence signal in the red light channel (generated by SSNP-Folate recognition). HE staining results of frozen sections of paracancerous tissue showed that the tumor tissue was distributed in clumps, with a clear boundary with the surrounding fat tissue; IHC staining results showed that positive staining areas of macrophage marker CD206 appeared in the tumor edge area (brown yellow, marked by purple arrows), indicating that there was inflammatory tissue infiltration at the tumor boundary; laser confocal imaging of TAT corresponding tissue results showed: bright green fluorescence appeared in the tumor site and inflammatory site in the green light channel; bright red fluorescence appeared in the inflammatory tissue area in the red light channel, while there was almost no red fluorescence signal in the tumor site and surrounding normal tissue, and the results of the imaging area were consistent with the pathological HE staining and IHC staining results. The above research results show that the SSNP-Aptamer and SSNP-Folate probe systems can effectively distinguish cancer tissue, inflammatory tissue and normal tissue in paracancerous tissue of lung adenocarcinoma.

[0103] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for preparing a hybrid supramolecular fluorescent nanoprobe system, characterized in that: The following steps are involved: (1) Preparation of β-cyclodextrin polymer: dissolving β-cyclodextrin in alkaline solution, stirring, adding toluene, continuing to stir, adding a cross-linking agent dropwise, and obtaining a β-cyclodextrin polymer after reaction; (2) Preparation of adamantane-derivatized nucleic acid aptamer: the initial product of nucleic acid aptamer and adamantane amine are mixed in a solvent to which DIPEA is added, and the mixture is shaken overnight. After the reaction solution is centrifuged, the precipitate is collected, and ammonia water is added to the precipitate for aminolysis overnight. The initial product of nucleic acid aptamer is S11e nucleic acid aptamer, and the molar ratio of S11e nucleic acid aptamer, adamantane amine and DIPEA is 1:1800:14000. In step (2), the mixture is shaken overnight at 30-37°C, 30% ammonia water is added to the precipitate, and the aminolysis process is carried out at a constant temperature of 50-55°C. After the aminolysis is completed, the supernatant is collected, the product is separated and purified by high performance liquid chromatography, the separated product is collected, freeze-dried, and stored at -80°C for later use. (3) Preparation of adamantane-derivatized folic acid: folic acid, adamantane amine, dicyclohexylcarbodiimide, N-hydroxysuccinimide and DIPEA are uniformly mixed in a solvent, and the mixture is shaken in the dark to obtain the obtained product; (4) Preparing a nucleic acid aptamer-targeted self-assembled supramolecular fluorescent nanoprobe: the β-cyclodextrin polymer prepared in step (1) and adamantane-derivatized rhodamine are mixed in a solvent, incubated at a constant temperature for a period of time, and then the adamantane-derivatized nucleic acid aptamer prepared in step (2) is added, and the adamantane-derivatized nucleic acid aptamer is continued to be shaken for a period of time to obtain a nucleic acid aptamer-targeted self-assembled supramolecular fluorescent nanoprobe solution; the preparation process of the adamantane-derivatized rhodamine is as follows: rhodamine B isothiocyanate, adamantane, DIPEA, DMSO, and carbonate buffer are shaken and reacted overnight at 25-30°C in the dark, and the separated product is collected after separation and purification by high performance liquid chromatography, and then freeze-dried and stored at -20°C for future use, and the molar ratio of rhodamine B isothiocyanate, adamantane, and DIPEA is 1:10:170; (5) Preparing a folic acid-targeted self-assembled supramolecular fluorescent nanoprobe: taking the β-cyclodextrin polymer prepared in step (1) and the adamantane-derivatized cyanine dye 5.5, mixing them in a solvent, incubating them with constant temperature oscillation for a period of time, then adding the adamantane-derivatized folic acid prepared in step (3), and continuing to oscillate for a period of time to obtain a folic acid-targeted self-assembled supramolecular fluorescent nanoprobe solution; (6) The nanoprobes prepared in step (4) and step (5) are mixed to obtain a hybrid supramolecular fluorescent nanoprobe system.

2. The method for preparing the hybrid supramolecular fluorescent nanoprobe system according to claim 1, characterized in that: In step (6), the molar ratio of the nucleic acid aptamer-targeted self-assembled supramolecular fluorescent nanoprobe to the folic acid-targeted self-assembled supramolecular fluorescent nanoprobe in the mixed supramolecular fluorescent nanoprobe system is 1:

1.

3. The method for preparing the hybrid supramolecular fluorescent nanoprobe system according to claim 1, characterized in that: In step (3), the molar ratio of folic acid, adamantane amine, dicyclohexylcarbodiimide, N-hydroxysuccinimide and DIPEA is 1:5:2:1:9, and the reaction is carried out at 30-37°C in the dark with shaking for 8-10 hours, and then the reaction solution is separated and purified by high performance liquid chromatography, and the separated product is collected, freeze-dried, and stored at -20°C for later use.

4. The method for preparing the hybrid supramolecular fluorescent nanoprobe system according to claim 1, characterized in that: In step (4), the molar ratio of β-cyclodextrin polymer to adamantane-derivatized rhodamine and adamantane-derivatized nucleic acid aptamer is 5000:100:

1. During the reaction, the β-cyclodextrin polymer and adamantane-derivatized rhodamine are first incubated at 25-30° C. with constant temperature shaking for 2-3 h. After the adamantane-derivatized nucleic acid aptamer is added, the adamantane-derivatized nucleic acid aptamer is continued to be incubated at 25-30° C. with constant temperature shaking for 2-3 h.

5. The method for preparing the hybrid supramolecular fluorescent nanoprobe system according to claim 1, characterized in that: In step (5), the molar ratio of the β-cyclodextrin polymer, the adamantane-derivatized cyanine dye 5.5, and the adamantane-derivatized folic acid is 5000:100:

1. During the reaction, the β-cyclodextrin polymer and the adamantane-derivatized cyanine dye 5.5 are first incubated at 25-30° C. with constant temperature shaking for 2-3 hours. After the adamantane-derivatized folic acid is added, the adamantane-derivatized folic acid is continued to be incubated at 25-30° C. with constant temperature shaking for 2-3 hours.

6. The method for preparing the hybrid supramolecular fluorescent nanoprobe system according to claim 1, characterized in that: The preparation process of the adamantane-derivatized cyanine dye 5.5 in step (5) is as follows: Cy5.5-NHS is dissolved in DMSO, adamantane and DIPEA are added and mixed evenly, and the mixture is shaken at 35-40°C for 8-10h, and the separated product is collected after purification by high performance liquid chromatography, freeze-dried and stored at -20°C for future use. The molar ratio of Cy5.5-NHS to adamantane and DIPEA is 1:7:

40.

7. A hybrid supramolecular fluorescent nanoprobe system, characterized in that: Prepared by the method according to any one of claims 1 to 6.

8. Use of the hybrid supramolecular fluorescent nanoprobe system as claimed in claim 7 in the preparation of a reagent for detecting lung adenocarcinoma.

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