CN111999504B

CN111999504B - Mucin 1 and sialylglycosyl dual fluorescence imaging method and application thereof

Info

Publication number: CN111999504B
Application number: CN202010795818.2A
Authority: CN
Inventors: 尹健; 王晓丽; 胡静; 叶雨霏
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-08-10
Filing date: 2020-08-10
Publication date: 2021-09-28
Anticipated expiration: 2040-08-10
Also published as: CN111999504A

Abstract

The invention discloses a mucin 1 and a sialylglycosyl dual fluorescence imaging method and application thereof, belonging to the technical field of fluorescence imaging and the field of biomedicine. The invention successfully constructs two sialic acid-gold nanostar probes Sia-GNSs and a mucin 1-quantum dot probe MUC1-QDs with high specificity and biocompatibility. Through the combined use of Sia-GNSs and MUC1-QDs, MUC1 in MCF-7 cells can be specifically recognized. The nano probe prepared by the invention can realize double fluorescence imaging and relative quantification of MUC1 protein skeleton and Sia glycosyl thereof in vitro, in vivo and clinical breast cancer patient tissue samples. The present invention provides a simple and effective platform with great potential in clinical cancer diagnosis.

Description

Mucin 1 and sialylglycosyl dual fluorescence imaging method and application thereof

Technical Field

The invention relates to a mucin 1 and a sialylglycosyl dual fluorescence imaging method and application thereof, belonging to the technical field of fluorescence imaging and the field of biomedicine.

Background

Cancer is a major cause of disease burden, and cancer detection and imaging for clinical use is extremely important for early cancer diagnosis, monitoring recurrence, and drug discovery and development. Clinical diagnosis is usually performed by combining various biochemical and imaging techniques, such as enzyme linked immunosorbent assay (ELISA), hematoxylin-eosin (H & E) staining, Computed Tomography (CT), or Magnetic Resonance Imaging (MRI), which complicates the analysis, resulting in lack of specificity. Detection of protein biomarkers can be used for identification and monitoring of cancer. Mucin 1(MUC1) is a highly glycosylated type I transmembrane O-glycoprotein that is abnormally expressed on the surface of a variety of tumor cells. The MUC1 molecular protein skeleton consists of two parts, namely a protein skeleton and a sugar chain, which are connected through an O-glycosidic bond. Currently, several optical imaging techniques (e.g., fluorescence, MRI and surface enhanced raman scattering) targeting MUC1 have been used for cancer detection. Since single biomarker detection is not sufficient for clinical diagnosis, multiple diagnostic methods are urgently needed. Tumor-associated MUC1 is often sialylated prematurely, and the glycan chain of MUC1 in normal cells is simpler and shorter in structure than the glycan chain of MUC 1. Sialic acid (Sia) generally modifies the termini of surface glycans, and studies have shown that sialylation is involved in malignant transformation and tumorigenesis. To better understand the glycosylation process of proteins, researchers have developed several protein-specific glycoimaging strategies, including Fluorescence Resonance Energy Transfer (FRET), chemical covalent recognition, and metabolic labeling, among others.

Disclosure of Invention

The invention provides a detection tool for double imaging of MUC1 based on fluorescence energy resonance transfer (FRET), which can simultaneously detect MUC1 skeleton and glycosylation thereof, thereby carrying out accurate cancer detection and diagnosis.

The invention relates to a nano probe composition for quickly detecting MUC1 and Sia glycosyl thereof, which can realize specific target recognition and fluorescence imaging, wherein the nano probe comprises a sialic acid-gold nanostar probe (Sia-GNSs) and a mucin 1-quantum dot probe (MUC 1-QDs).

In one embodiment, the sialic acid-venomous probe (Sia-GNSs) is prepared by immobilizing DNA1 and polyethylene glycol on the surface of venomous (GNSs) through disulfide bonds, base-complementary pairing short complementary single-stranded DNA2 with sialic acid aptamer, and coupling with thiol-modified PEG; the DNA1 is linked to a sialic acid (Sia) aptamer labeled with the near-infrared dye Cy5 by base complementary pairing; the sialic acid aptamer can specifically recognize sialic acid on mucin 1(MUC 1).

In one embodiment, the nucleotide sequence of the DNA1 is: 5'-AGGCAATCGATATAGTCACGGACTAGGCCCAGCGTC-3' -SH.

In one embodiment, the nucleotide sequence of the DNA2 is: 5'-GACGCAAGTCTGAAA-3' are provided.

In one embodiment, the mucin 1-quantum dot probe (MUC1-QDs) is formed by quantum dots sequentially mixed with MUC1 aptamer, NH₂PEG coupling synthesis, in which MUC1 aptamer (MUC1-Apt) capable of specifically recognizing with MUC1 protein skeleton and polyethylene glycol are fixed on the surface of the quantum dot through amido bond.

The action principle of the nano probe composition is as follows: when irradiated with near infrared light (NIR), Sia-GNSs generate heat due to absorption of near infrared light, causing DNA2 to unwind and the Cy 5-labeled Sia aptamers can then specifically bind Sia at the desired region and simultaneously separate from the GNSs nanoprobes. The MUC1-QDs nanoprobe can be specifically combined with MUC1 protein skeleton through MUC1 aptamer. Only when MUC1-QDs and Cy5 labeled Sia aptamers are bound to the same MUC1 molecule (with a distance of less than 10nm), the QDs can excite the fluorescence of Cy5 by FRET effect. The fluorescence intensity of FRET-induced Cy5 may reflect MUC 1-specific sialylation. By imaging the fluorescence of QDs and Cy5, the dual detection of MUC1 protein skeleton and Sia glycosyl thereof can be realized.

The second aspect of the present invention is to provide a method for preparing the nanoprobe.

In one embodiment, the Sia-GNSs probes are prepared as follows:

(1) synthesizing the gold nano star GNSs by adopting a seed crystal growth method;

(2) coupling the GNSs synthesized in the step (1) and the sulfhydryl-modified DNA1 to form GNS-2;

(3) generating GNS-3 by the base complementary pairing principle of the GNS-2 prepared in the step (2) and a Cy 5-labeled Sia aptamer (Cy5-Sia Apt);

(4) coupling the GNS-3 prepared in the step (3) with DNA2 to form GNS-4;

(5) coupling the GNS-4 prepared in the step (4) with PEG modified by sulfydryl to obtain a Sia-GNSs probe;

the DNA1 and DNA2 are single stranded nucleic acids complementary to Sia aptamers.

In one embodiment, the nucleotide sequence of the Sia aptamer is 5 '-Cy 5-GACGCUGGGCCUAGUCCGUGACUAUAUCGAUUGCCUUUUCAGACUUGCGUC-3'; the MUC1 aptamer has the following structure: 5' -NH₂-C₆-GCAGTTGATCCTTTGGATACCCTGG-3’。

In one embodiment, the nucleotide sequence of the DNA1 is 5'-AGGCAATCGATATAGTCACGGACTAGGCCCAGCGTC-3' -SH.

In one embodiment, the nucleotide sequence of the DNA2 is 5'-GACGCAAGTCTGAAA-3'.

In one embodiment, the MUC1-QDs probe is prepared as follows:

(1) coupling of CdSe @ ZnS QDs and amino-modified MUC1 aptamers in the presence of EDC and NHS to form QD-2;

(2) and (2) coupling the QD-2 prepared in the step (1) with PEG modified by amino in an environment containing EDC and NHS to obtain a quantum dot probe MUC 1-QDs.

The third purpose of the invention is to provide the application of the nano-probe composition in preparing a breast cancer diagnosis tool.

In one embodiment, the diagnostic tool includes, but is not limited to, a test kit.

In one embodiment, the assays include in vitro assays, in vivo assays, and clinical tissue sample assays.

In one embodiment, the detection is performed by co-incubating the tumor tissue slices to be detected in an environment containing the nanoprobe composition, and then irradiating with NIR for 2min under 980nm, 5mW/cm²And fluorescence imaging of QDs and FRET-induced Cy5 with laser confocal to reflect the levels of MUC1 protein backbone and its sialic acid, respectively.

The fourth purpose of the invention is to provide a tumor screening kit.

In one embodiment, the tumor is selected from the group consisting of: lung cancer, liver cancer, stomach cancer, esophagus cancer, intestinal cancer, nasopharyngeal carcinoma, breast cancer, lymph cancer, kidney cancer, pancreatic cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, gallbladder cancer, lip cancer, melanoma, tongue cancer, larynx cancer, leukemia, prostate cancer, brain cancer, squamous cell cancer, skin cancer, hemangioma, lipoma, thyroid cancer, lung cancer, glioma, cervical cancer or a plurality of the concurrent diseases.

Has the advantages that:

(1) compared with the prior art, the nanoprobe has dual fluorescence imaging characteristics activated by NIR, under the irradiation of near infrared light (NIR), Sia aptamers marked by Cy5 on the Sia-GNSs probe can target and recognize Sia on MUC1, and MUC1 aptamers on the MUC1-QDs probe can target and recognize MUC1 protein skeletons. Since MUC1 is closely related to the occurrence, development and metastasis of tumors, and MUC1 is abnormally highly expressed in 70% of solid tumors, the fluorescence imaging of MUC1 has important significance for the diagnosis, treatment effect and detection prognosis of tumors.

(2) The excitation wavelength of the probe QDs designed by the invention is 448nm, the emission wavelength range is 605-.

(3) The nano probe has better stability and lower cytotoxicity.

(4) The nanoprobe has high specificity and biocompatibility, can be used for high-efficiency and reliable synchronous imaging of MUC1 and glycosylation thereof in tissue sections of in vitro, in vivo and clinical patients, and can be easily used for other cancer-related biomarkers and glycosylation detection. The present invention provides a simple and versatile platform that can track and relatively quantify protein-specific glycosylation patterns, thereby providing information about glycans for further analysis. This FRET-based imaging approach has great potential in clinical cancer diagnosis.

Drawings

FIG. 1: the particle size distribution of the nanoprobe; a, a transmission electron microscope image of the Sia-GNSs nano probe; b, transmission electron microscopy images of MUC1-QDs nanoprobes; c, a dynamic light scattering particle size distribution diagram of the Sia-GNSs nano probe; d, dynamic light scattering particle size distribution of MUC1-QDs nanoprobes.

FIG. 2: ultraviolet-visible absorption spectrum of the nanoprobe; a, ultraviolet-visible light absorption diagram of the Sia-GNSs nano probe; b, ultraviolet-visible absorption Pattern of MUC 1-QDs.

FIG. 3: fluorescence spectrum of the nanoprobe; a, a fluorescence spectrum of the Sia-GNSs nano probe; b, fluorescence spectrum of MUC 1-QDs.

FIG. 4: graph of particle size change for 7 days of incubation of nanoprobes Sia-gnss (a) and nanoprobes MUC1-qds (b) in different solvents.

FIG. 5: the cytotoxicity test result of the co-incubation of the human breast cancer cell MCF-7 and the human liver cancer cell HepG2 with the nano-probes with different concentrations; a, cytotoxicity graph of Sia-GNSs nano-probe; b, cytotoxicity profile of MUC1-QDs nanoprobes; cytotoxicity profiles of C, Sia-GNSs and MUC1-QDs mixed nanoprobes (volume/volume ═ 1: 1).

FIG. 6: the results of laser confocal experiments after the co-incubation of human breast cancer cells MCF-7 and the nano-probe for different times are shown in a scale: 50 μm.

FIG. 7: the detection result of the human breast cancer cell MCF-7 and the human liver cancer cell HepG2 after being incubated with the nano probe for 6 hours; a, laser confocal experimental results (scale bar: 50 μm); b, fluorescence intensity (n ═ 6); z-stack co-localization studies of C, MCF-7 cells.

FIG. 8: the results of laser confocal experiments after the human breast cancer cell MCF-7 is respectively incubated with Sia-GNSs and MUC1-QDs nanoprobes for 6 hours, and the scale bar: 50 μm.

FIG. 9: fluorescence imaging of MUC1 and its Sia in an MCF-7 transplantable tumor mouse model; a, in vivo imaging of MUC1 and its sialylation at different times after intravenous tail vein injection (n-5); b, fluorescence imaging of MUC1 and Sia thereof in isolated tumors of MUC1 and their vital organs 12h after injection; c, H & E staining of mouse tumor sections, scale: 150 μm; d, IHC staining of MUC1 expression in mouse tumor sections, scale bar: 150 μm; e) laser confocal experimental results after incubation of mouse tumor sections with nanoprobes for 6 hours (scale bar: 150 μm); f, fluorescence intensity (n ═ 6).

FIG. 10: imaging results of MUC1 and Sia thereof in tumor tissue sections of clinical breast cancer patients; a, results of confocal experiments with or without NIR irradiation (scale bar: 50 μm) after 6 hours incubation of tumor tissue sections from clinical breast cancer patients with Sia-GNSs and MUC 1-QDs; b, fluorescence intensity (n ═ 6); c, H & E staining of tumor tissue sections of breast cancer patients, scale: 150 μm; IHC staining of tumor tissue sections of breast cancer patients, scale bar: 150 μm.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below.

The abbreviations used in the specification and examples have the following specific meanings:

example 1 Synthesis of Sia-GNSs nanoprobes

Synthesis of gold nanostars GNSs (gold nanostars)

All the glass instruments are soaked in aqua regia, washed by double distilled water and dried for later use. The gold nano star is prepared by adopting a seed crystal growth method. The water used in the following steps was 18.2 M.OMEGA.Milli-Q ultrapure water.

The synthesis steps of the gold nano star are as follows:

(1) seed crystal synthesis: 100mL of 1mM tetrachloroauric acid trihydrate is placed in a reaction flask, heated and stirred at 100 ℃ until boiling, 15mL of 10g/L sodium citrate solution is added, and boiling is continued for 15 minutes, so that the solution changes from light yellow to wine red in color. Cooled to room temperature, filtered through a 0.22 μm filter membrane, and stored in a refrigerator at 4 ℃.

(2) Preparing gold nano star: and (3) after the seed crystal solution in the step (1) is synthesized, growing the gold nano-star. 100mL of 0.25mM tetrachloroauric acid trihydrate was placed in a reaction flask, and 1mL of the seed crystal solution prepared in step (1) was added thereto with stirring at room temperature. Then, 1mL of 2mM silver nitrate and 0.5mL of 100mM ascorbic acid are sequentially added, the mixture is stirred and reacts for 30 seconds at room temperature, the solution is changed into blue gray from light yellow, the mixture is centrifuged at 4500rpm for 10 minutes, the supernatant is discarded, and the mixture is dispersed in water to obtain the gold nanostar solution.

(II) Synthesis of Sia-GNSs nano probe

Gold nanostar and DNA1 were coupled to synthesize GNS-2: to 1mL (10 nM) of the gold nanostar solution prepared in example 1 was added 10. mu.L of DNA1 (nucleotide sequence 5' -AGGCAATCGATATAGTCACGGACTAGGCCCAGCGTC-C) at a concentration of 100. mu.M₆-3' -SH) and mixed evenly, and reacted for 12 hours with shaking. After centrifugation at 4500rpm for 10 minutes, the supernatant was discarded and redispersed in 1mL pure water treated with diethylpyrocarbonate (DEPC water), GNS-2 was obtained.

(1) GNS-2 and SA Apt are coupled to synthesize GNS-3: to 1mL of GNS-2 prepared in step (1), 50. mu.L of SA Apt (nucleotide sequence 5 '-Cy 5-GACGCUGGGCCUAGUCCGUGACUAUAUCGAUUGCCUUUUCAGACUUGCGUC-3') was added at a concentration of 20. mu.M, mixed well, reacted at 37 ℃ for 1 hour, and then reacted at room temperature with shaking for 12 hours. After centrifugation at 4500rpm for 10 min, the supernatant was discarded and redispersed in 1mL DEPC water to give GNS-3.

(2) Coupling of GNS-3 to DNA2 Synthesis of GNS-4: to 1mL of GNS-3 prepared in step (2), 10. mu.L of DNA2 (nucleotide sequence 5'-GACGCAAGTCTGAAA-3') was added at a concentration of 100. mu.M, mixed well, reacted at 37 ℃ for 1 hour, and then reacted at room temperature with shaking for 12 hours. After centrifugation at 4500rpm for 10 min, the supernatant was discarded and redispersed in 1mL of water to give GNS-4.

(3) GNS-4 was coupled with SH-PEG (5000) to synthesize Sia-GNSs: adding 10 μ L of thiolated polyethylene glycol (SH-PEG) with a molecular weight of 5000 and a concentration of 10mg/mL into 1mL of GNS-4 prepared in step (3), mixing, and reacting with shaking at room temperature for 12 hours. Centrifuging at 4500rpm for 10 minutes, discarding the supernatant, and dispersing in 1mL of water to obtain the Sia-GNSs nano probe.

The morphological characteristics of the nanoprobe are observed through a Transmission Electron Microscope (TEM), the particle size distribution of the nanoprobe is measured through a dynamic light scattering technology (DLS), and an ultraviolet-vis (UV-vis) spectrophotometer and a fluorescence photometer are used for respectively measuring a UV-vis absorption spectrum and a fluorescence spectrum of the nanoprobe. As shown in FIG. 1, the prepared gold nanostar probe Sia-GNSs is star-shaped and has uniform particle size distribution through TEM observation; the quantum dot probe MUC1-QDs are in a circular structure, and the particle size distribution is uniform. The particle size of the Sia-GNSs was about 76nm as measured by DLS particle size. After each modification step, the particle size of Sia-GNSs nanoprobes increased slightly, indicating that DNA, RNA and PEG were successfully modified onto the surface of the nanomaterial.

As shown in FIG. 2, the UV-Vis absorption peak of Sia-GNSs is at 710nm, and no red shift or blue shift occurs after modification. As shown in FIG. 3, the fluorescence peak of labeled Cy5 was 665nm (excitation wavelength 650nm), and the fluorescence of Cy5 modified with gold nanoparticles was quenched by GNSs, so that no fluorescence was detected by Sia-GNSs.

Example 2 Synthesis of MUC1-QDs nanoprobes

(1) Quantum dots and MUC1 aptamer were coupled to synthesize QD-2:

the carboxyl water-soluble CdSe @ ZnS quantum dots are purchased from Wuhan Jia source quantum dot technology development, Inc. To 80. mu.L of PBS containing 2. mu.M QDs, 4. mu.L of EDC at a concentration of 0mg/mL and 1.6. mu.L of NHS at a concentration of 10mg/mL were added, and the mixture was reacted at room temperature for 1 hour, followed by addition of 80. mu.L of MUC1 aptamer (sequence 5' -NH) at a concentration of 100. mu.M₂-C₆-GCAGTTGATCCTTTGGATACCCTGG-3'), mixing, and shaking to react at room temperature for 2 hours. After centrifugation at 13000rpm for 10 minutes, the resulting mixture was dispersed in 80. mu.L of PBS to obtain QD-2.

(2) QD-2 with an aminated polyethylene glycol (NH) of molecular weight 2000₂PEG2000) coupling to synthesize MUC 1-QDs:

adding 4. mu.L of EDC with a concentration of 10mg/mL and 1.6. mu.L of NHS with a concentration of 10mg/mL into the QD-2 prepared in step (1), reacting at room temperature for 1 hour, and adding 10. mu.L of NH with a concentration of 10mg/mL₂PEG (2000), mixing and shaking to react for 2 hours at room temperature. Ultrafiltering and centrifuging at 13000rpm for 10 min, and dispersing in 80 μ L PBS to obtain MUC 1-QDs;

the morphological characteristics of the prepared nanoprobe are observed by a Transmission Electron Microscope (TEM), the particle size distribution of the nanoprobe is measured by a dynamic light scattering technology (DLS), and an ultraviolet-vis (UV-vis) spectrophotometer and a fluorescence photometer are used for respectively measuring the UV-vis absorption spectrum and the fluorescence spectrum of the nanoprobe. As shown in FIG. 1, the quantum dot probe MUC1-QDs has a circular structure and uniform particle size distribution when observed by TEM. The particle size of MUC1-QDs was approximately 42nm as measured by DLS particle size. The particle size of MUC1-QDs nanoprobes increased slightly after each modification step, indicating that DNA, RNA and PEG were successfully modified onto the surface of the nanomaterials. As shown in FIG. 2, the UV-Vis absorption peaks of MUC1-QDs are at 385nm, respectively, and no red-shift or blue-shift occurs after modification. As shown in FIG. 3, the fluorescence peaks of QD-2 and MUC1-QDs are red shifted to 625nm compared to QDs (620nm) after binding to MUC1 aptamers, which is probably due to luminescence quenching of selective QDs and energy transfer by light scattering.

Example 3 stability test of nanoprobes

Stability is one of the most important properties of nano-carriers, and nano-particles applied to the field of biomedicine must be stably dispersed in a salt solution or a culture medium with a certain concentration range. The stability of the nanoprobes Sia-GNSs prepared in example 1 and MUC1-QDs prepared in example 2 was evaluated by diluting the nanoprobes into an aqueous solution, phosphate buffered saline (PBS, pH 7.4) and a high-glucose medium (DMEM) containing 10% fetal bovine serum, the concentration of each dilution being 0.1nM, and measuring the change in particle size within 7 days by DLS. The results are shown in fig. 4, no obvious particle size change occurs within 7 days, which indicates that the two nanoprobes have good stability.

Example 4 cytotoxicity Studies of nanoprobes

The cytotoxicity of the nanoprobe is analyzed by a cell survival rate analysis method (thiazole bromide blue tetrazole, namely MTT), and the detection principle is that MTT can be reduced into blue-purple crystalline formazan substance insoluble in an aqueous solution by succinate dehydrogenase in mitochondria of living cells, so that the detection can be carried out by a colorimetric method, and MTT cannot be reduced and developed by dead cells.

Respectively highly express human liver cancer cell MCF-7(MUC 1)Cell) and human hepatoma cell HepG2 cell (MUC1 low expression cell) suspension were diluted to a cell concentration of 5X 10⁴one/mL. And (3) taking out a sterile 96-well plate, adding 100 mu L of cell suspension into each well, adding the nanoprobes Sia-GNSs and MUC1-QDs prepared in the example 1 and the example 2 with different concentrations after the cells grow stably in the well plate for 24h at 37 ℃, and repeating 3 groups of parallel experiments for each concentration sample. After incubating the nanoprobe solutions of different concentrations with the two cells for 24h, respectively, the nanoprobe medium contained in each well was carefully removed, washed three times with sterile phosphate buffer (PBS, pH 7.4), and then 100 μ L of a PBS solution containing 0.5mg/mL MTT without phenol red added was added to each well, respectively. The 96-well plate added with the color developing agent is put into an incubator to be incubated for 4 hours, then taken out, the culture medium containing MTT is carefully sucked out, then 100 mu L of dimethyl sulfoxide is added into each well, and the mixture is mixed evenly. The absorbance values (absorption wavelength 490nm) of all the wells in the plate were then detected with a multifunctional microplate reader. The viability of the cells in each well was calculated as follows:

cell survival rate ═ a_bsample/A_bControl) x 100%;

in the formula, A_bThe samples are the absorbance values of the experimental sample set, A_bThe control is the absorbance value of the cell group that has not been exposed to the material. The results are shown in FIG. 5, the greater the concentration of the nanoprobe, the greater the toxicity to the cell, and when the concentration reaches 30nM, the better activity of the MCF-7 and HepG2 can be still maintained, the cell activity is maintained above 85%, which indicates that the cytotoxicity of the two nanoprobes is lower, and good biocompatibility is also shown.

Example 5 nanoprobe in vitro MUC1 fluorescence imaging

Dilution of MCF-7 cells to 8X 10⁴one/mL. Sterile confocal laser dishes were taken, 1mL of diluted cell suspension was added to each dish, and the dishes were carefully shaken to distribute the cells evenly. The well-seeded dishes were incubated in an incubator for 24 hours to reach a cell density of 70-80%, the medium in the dishes was carefully aspirated, and the cells were carefully washed 3 times with a PBS solution at pH 7.4. Add final sodium to the cuvetteMitsubes Sia-GNSs (5nM) and MUC1-QDs (50nM), and the dishes were incubated in incubators for different periods of time (2, 4, 6, 8, 12 hours). Then NIR illumination (980nm, 5 mW/cm)²And 2 minutes) and then put into an incubator to be cultured for half an hour. The sample solution in the dish was carefully aspirated and the cells were washed 3 times with PBS solution at pH 7.4. Then, 500. mu.L of a 4% paraformaldehyde-containing PBS solution was added to each dish and allowed to act at room temperature for 15 minutes to fix the cells. The paraformaldehyde solution in the dish was carefully aspirated and the cells were washed 3 times with PBS solution at pH 7.4. Then, an aqueous DAPI solution was added to cover the wells of the dish, the cells were stained for 5 to 10 minutes at room temperature, and then washed 3 times with a PBS solution having a pH of 7.4. Finally, 400. mu.L of PBS was added to maintain the cells in a wet state, and the cells were observed by scanning with a confocal laser microscope.

As a result, as shown in FIG. 6, fluorescence of QDs could be detected at each time point, indicating that MUC1-QDs nanoprobes have the ability to bind to the MUC1 protein backbone. Fluorescence of FRET-induced Cy5 was observed at the same time after 2 hours and peaked at 6 hours, so that the following cell incubation time was selected to be 6 hours.

Next, to evaluate the specificity of Sia-GNSs and MUC1-QDs nanoprobes for MUC1, HepG2 cells were selected as negative controls. Sialidases are used to hydrolyze Sia from glycoproteins. MUC1 mab was used to competitively bind to MUC 1. As shown in FIG. 7A, only fluorescence of QDs was observed in MCF-7 cells without NIR irradiation after incubating MCF-7 cells and HepG2 cells, respectively, with the two types of nanoprobes prepared in examples 1 and 2 at a final concentration of 5nM for 6 hours, suggesting that DNA2 has a stable blocking effect that prevents Sia aptamers on the nanoprobes from recognizing Sia residues (group 1). Fluorescence of QDs, FRET-induced Cy5 and Cy5 was observed on the surface of MCF-7 cells after NIR irradiation, with the fluorescence intensity of Cy5 being stronger than that of FRET-induced Cy5, since the fluorescence of Cy5 comes from MUC1 and other sialylated proteins (group 2) when excited at the normal Cy5 excitation wavelength (650 nm). Notably, the fluorescence of QDs under NIR illumination is slightly weaker than that without NIR illumination at the same time point, due to fluorescence decay caused by FRET effects. After pretreatment with sialidase, strong QDs fluorescence and slight Cy5 fluorescence were observed, but no FRET-induced Cy5 fluorescence was observed, indicating that sialidase hydrolyzed most of Sia (group 3). Only Cy5 fluorescence and negligible QDs fluorescence were observed after pretreatment with MUC1 mab, indicating that MUC1 mab binds to MUC1 competitively with the MUC1 aptamer (panel 4). No fluorescence was observed in HepG2 cells without NIR irradiation (group 5), whereas only fluorescence of Cy5 was observed in HepG2 cells under NIR irradiation (group 6), and no fluorescence of QDs and FRET-induced Cy5 was observed, indicating that our nanoprobes are specific for detection of MUC 1. The ratio of Sia to MUC1 protein in MCF-7 cells was calculated to be 0.89 by calculating the fluorescence intensity of QDs and FRET-induced Cy5 to compare the ratio of MUC1 backbone to Sia residues (fig. 7B), thus providing a sialylation analysis of specific proteins. Then, using the Z-stack technique for co-localization studies (FIG. 7C), 3,3' -octacosyloxycarbocyanine perchlorate (DiO) was used for cell membrane staining. The fluorescence of QDs and FRET-induced Cy5 is localized on the cell membrane, confirming the successful detection of the protein backbone of the membrane protein MUC1 and its sialylation. These results indicate that Sia-GNSs and MUC1-QDs nanoprobes have excellent fluorescence imaging capabilities for the MUC1 protein backbone and Sia glycosyl in living cells.

Example 6 in vitro MUC1 fluorescence imaging Using a Single fluorescent Probe

To demonstrate that the FRET-induced fluorescence signal of Cy5 is derived from the FRET effect, in vitro MUC1 fluorescence imaging was performed on both fluorescent probes alone. The specific embodiment is the same as example 5, except that MCF-7 cells were incubated with Sia-GNSs nanoprobes or MUC1-QDs fluorescent probes alone for 6 hours and then irradiated with NIR for 2 minutes. As shown in FIG. 8, only Cy5 fluorescence was observed in MCF-7 cells after incubation with Sia-GNSs, and FRET-induced fluorescence of Cy5 and QDs was not observed; at the same time, only fluorescence of QDs was observed in MCF-7 upon co-incubation with MUC 1-QDs.

Example 7 in vivo nanoprobe MUC1 fluorescence imaging

For the establishment of subcutaneous solid tumor mouse model, 4-6 weeks old female BALB/c nude mice with average weight of 18-20g are taken and connected at the right leg1X 10 seed⁷MCF-7 cells were seeded in a volume of 100. mu.L (PBS and matrigel 1:1 mix). The size of the tissue to be cancerated exceeds 60mm after about two weeks³(volume 0.5 × length of cancerous tissue × width of cancerous tissue)²) At the time, mice were divided into 3 groups (5 per group), group i: injecting Sia-GNSs and MUC1-QDs into tail vein, and adding NIR illumination; and (II) group: Sia-GNSs and MUC1-QDs are injected into tail veins without NIR illumination; group III: tail vein saline, with NIR illumination. The injection volume of nanoprobe per mouse was 100 μ L (30 nM). And (3) acquiring pictures in real time at different time points by using an In Vivo Imaging System (IVIS), and observing the enrichment condition of the fluorescent substance at the tumor part. All animal experiments were performed according to the guidelines assessed and approved by the ethics committee of south of the Yangtze university.

As a result, as shown in FIG. 9A, weak fluorescence of QDs was observed at the tumor site of the mice after 2 hours, and the fluorescence of QDs reached the maximum at 12 hours in groups I and II, indicating that MUC1-QDs nanoprobe has MUC1 skeleton detection ability. Fluorescence of FRET-induced Cy5 was only observed with a similar trend at the same position in group I, indicating that the Sia-GNSs and MUC1-QDs nanoprobes have the ability to specifically image MUC1 and Sia glycon in vivo. No fluorescence signal was observed in the control group (group III). Tumors and major organs (including heart, liver, spleen, lung and kidney) were collected after 12 hours for ex vivo imaging. As a result, as shown in FIG. 9B, significant fluorescence of QDs and FRET-induced Cy5 was observed in the tumors in group I, while no fluorescence signal was detected in other major organs. QDs fluorescence was detected only in tumors in group II, indicating that the nanoprobes specifically target tumor tissue. In addition, survival status of all mice after imaging was monitored daily, and after 30 days, mice of three groups survived and no survival difference was observed, indicating that the nanoprobes had good biosafety in vivo.

Example 8 nanoprobe tissue section MUC1 fluorescence imaging

To qualitatively analyze the detection of MUC1 by the nanoprobe in mouse tumor tissue sections and tissue sections of clinical breast cancer patients, 10 mouse tumors and 15 clinical breast cancer patients were sectioned in paraffin of 5 μm thickness and subjected to laser confocal imaging (CLSM), Immunohistochemistry (IHC) and hematoxylin and eosin staining (H & E staining), respectively.

H & E staining and IHC results of mouse tumor tissue sections are shown in fig. 9C and 9D, and laser confocal pictures of mouse tumor tissue sections are shown in fig. 9E. Fluorescence of QDs was observed on mouse tumor tissue, and detected MUC1 appeared as densely packed cell clusters, as well as in the results of H & E and IHC staining. After NIR irradiation, fluorescence of QDs and FRET-induced Cy5 could be detected simultaneously at the same site, consistent with previous in vivo experimental results. In addition, we further quantified the fluorescence of QDs and FRET-induced Cy5, with a ratio of Sia to MUC1 of 0.86 (FIG. 9F). This imaging performance shows the great potential of MUC 1-specific detection nanoprobes in breast cancer tissue diagnosis.

Based on the above experimental results, the breast cancer tissue section of the clinical patient was used to further study the feasibility of the clinical application of the nanoprobe. The study was approved by the ethical committee of clinical research in the subsidiary hospital of south of the Yangtze university and was conducted according to the International ethical guidelines for biomedical research involving the human body.

Tissue sections from breast cancer patients (n-15) were incubated with the nanoprobes detected and visualized with CLSM. As a result, as shown in fig. 10A, strong fluorescence of QDs was observed on tumor cells, but not on normal stromal cells, confirming efficient imaging of MUC1 on tumor cells. Furthermore, after NIR irradiation, FRET-induced Cy5 fluorescence was detected on the same tumor cells, and quantitative fluorescence analysis showed a representative ratio of Sia to MUC1 of about 0.83, indicating the sialylation state of the MUC1 protein (fig. 10B). In addition, H & E and IHC staining confirmed the presence of tumor tissue and high expression of MUC1 (fig. 10C and 10D). These results demonstrate that the NIR-activated FRET-based nanoprobe of the invention has specific dual fluorescence imaging capability on MUC1 protein skeleton and Sia glycosyl, can accurately distinguish tumor tissues from normal tissues, and has huge clinical application potential.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> mucin 1 and sialylglycosyl dual fluorescence imaging method and application thereof

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<170> PatentIn version 3.3

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Claims

1. The nano probe composition for dual fluorescence imaging of mucin 1 and sialylglycosyl thereof is characterized by comprising a sialic acid-gold nano star probe and a mucin 1-quantum dot probe;

the sialic acid-gold nanostar probe is prepared by fixing a first DNA and polyethylene glycol on the surface of a gold nanostar through a disulfide bond, complementarily pairing a second DNA with a short complementary single chain and a sialic acid aptamer base, and coupling with PEG modified by sulfydryl; the first DNA is connected with sialic acid aptamer labeled by near infrared dye Cy5 through base complementary pairing; the sialic acid aptamer can specifically recognize sialic acid on mucin 1; the first and second DNAs are single-stranded nucleic acids complementary to the Sia aptamer; the nucleotide sequence of the first DNA is shown as SEQ ID NO. 1; the nucleotide sequence of the second DNA is shown as SEQ ID NO. 2; the Sia aptamer has the following structure: 5 '-Cy 5-GACGCUGGGCCUAGUCCGUGACUAUAUCGAUUGCCUUUUCAGACUUGCGUC-3';

the mucin 1-quantum dot probe is formed by sequentially mixing quantum dots with MUC1 aptamer and NH₂PEG coupling synthesis, namely fixing MUC1 aptamer and polyethylene glycol which can be specifically identified with MUC1 protein skeleton on the surface of the quantum dot through amido bond; the MUC1 aptamer has the following structure: 5' -NH₂-C₆-GCAGTTGATCCTTTGGATACCCTGG-3’。

2. The nanoprobe composition of claim 1, wherein the sialic acid-gold nanostar probe is prepared as follows:

(2) coupling the GNSs synthesized in the step (1) and the first DNA modified by sulfydryl to form GNS-2;

(3) generating GNS-3 by the base complementary pairing principle of the GNS-2 prepared in the step (2) and Cy 5-labeled Sia aptamer;

(4) coupling the GNS-3 prepared in the step (3) with a second DNA to form GNS-4;

(5) and (4) coupling the GNS-4 prepared in the step (4) with PEG modified by sulfydryl to obtain the sialic acid-gold nano star probe.

3. The nanoprobe composition of claim 1, wherein the mucin 1-quantum dot probe is prepared by the following method:

(1) coupling CdSe @ ZnS QDs and an amino-modified MUC1 aptamer in the presence of EDC and NHS to form QD-2;

4. A tumor screening kit comprising the nanoprobe composition of any one of claims 1 to 3.

5. The kit of claim 4, wherein the tumor is selected from the group consisting of: lung cancer, liver cancer, stomach cancer, esophagus cancer, intestinal cancer, nasopharyngeal carcinoma, breast cancer, lymph cancer, kidney cancer, pancreatic cancer, bladder cancer, ovarian cancer, uterine cancer, bone cancer, gallbladder cancer, lip cancer, melanoma, tongue cancer, larynx cancer, leukemia, prostate cancer, brain cancer, squamous cell cancer, skin cancer, hemangioma, lipoma, thyroid cancer, lung cancer, glioma and cervical cancer.

6. Use of the nanoprobe composition of claim 1 or 2 for the preparation of a product for the detection of MUC1 and Sia saccharides thereof.

7. The use of claim 6, wherein the product comprises a test kit.

8. The use according to claim 7, wherein the test kit is for in vitro testing.

9. The use according to claim 7, wherein the test kit is for in vivo testing.

10. The use of claim 7, wherein the test kit is for clinical tissue sample testing.

11. According to the rightThe use according to claim 6, wherein the detection is carried out by co-incubating the tumor tissue slices to be detected in an environment containing the nanoprobe composition and then irradiating with NIR for 2min under 980nm, 5mW/cm²And fluorescence imaging of QDs and FRET-induced Cy5 with laser confocal to reflect the levels of MUC1 protein backbone and its sialic acid, respectively.