Nano-probe for detecting expression of epidermal growth factor receptor of head and neck squamous carcinoma cells and preparation method of nano-probe

Abstract

The invention relates to the technical field of medical detection. The invention aims to provide a nano-probe for detecting the expression of an epidermal growth factor receptor of head and neck squamous carcinoma cells. The nano-probe is a nano-particle with a core-shell structure, the nano particle takes a nano Au particle as a core, and 4-mercaptobenzoic acid (4-MBA) is adsorbed on the surface of the Au core as a Raman signal internal reference molecule; according to the nanoparticle, SiO2 is used as a shell, and PEG and EGFR antibodies are linked to the SiO2 shell. According to the nanoprobe, head and neck squamous cell cancer cell and tissue imaging is researched by adopting a surface enhanced Raman spectroscopy technology, tumor cell and tissue EGFR expression images are drawn, and quantitative analysis and research are carried out. A rapid, visual and accurate new tool is provided for clinically detecting the squamous cell carcinoma of the head and neck and the EGFR expression condition of the squamous cell carcinoma of the head and neck.

Description

Technical Field

[0001] The present invention relates to the field of medical detection, and in particular to a nanoprobe for detecting epidermal growth factor receptor expression in head and neck squamous cell carcinoma cells and a preparation method thereof. Background Art

[0002] Head and neck squamous cell carcinoma (HNSCC) ranks sixth in incidence among all cancers worldwide, with an estimated 890,000 new cases and over 450,000 deaths each year. The prognosis of HNSCC varies depending on the stage at diagnosis: for 70%-90% of patients with early-stage HNSCC, surgery alone and radiotherapy alone can significantly improve long-term survival. However, locally advanced HNSCC carries a high risk of local recurrence and distant metastasis, resulting in a poor prognosis (5-year overall survival rate <50%). Approximately 60% of patients are in advanced stages (III to IVB) at diagnosis, of which approximately 10% have already developed distant metastases, and an additional 20%-30% are expected to develop distant metastases during the course of the disease. Therefore, timely detection and screening of HNSCC are of great significance.

[0003] Epidermal growth factor (EGFR) is a proto-oncogene α activated on the cell membrane by transforming growth factor to promote cell proliferation in cancer tissues. EGFR has the strongest prognostic significance among various markers found in HNSCC. EGFR expression in HNSCC tissue is significantly higher than in normal mucosal and muscle tissues, and EGFR expression in HNSCC cells is also significantly higher than in normal fibroblasts. EGFR stimulation can enhance cancer processes, including increased cell division, neovascularization, invasion / metastasis, and cell apoptosis escape. EGFR detection in HNSCC can play a variety of roles in cancer diagnosis, such as early detection, prognostic assessment, and treatment planning. Currently, the detection of EGFR expression in tumor tissues usually relies on several common laboratory methods, most of which require cumbersome processing steps and experimental processes, are often time-consuming and labor-intensive, and each has its own shortcomings.

[0004] Surface-enhanced Raman spectroscopy (SERS) is a new Raman spectroscopy technique based on electromagnetic field enhancement and charge transfer enhancement. It not only shares the advantages of Raman spectroscopy but also boasts higher sensitivity than conventional Raman spectroscopy, even capable of detecting single-molecule structures. In medical research, it is widely used to detect biochemical changes in disease and for differential diagnosis. Research has shown that SERS can successfully differentiate between tumor and normal tissue, achieving high accuracy, specificity, and sensitivity. The principle of SERS electromagnetic field enhancement and charge transfer enhancement is to add nanoscale probes to the sample. When the component to be measured and the nanoprobes accumulate with each other, the local electromagnetic field is enhanced. The Raman scattering intensity is proportional to the square of the optical field strength of the molecule. The enhanced local electromagnetic field greatly increases the probability of Raman scattering by molecules adsorbed on the probe surface, thereby enhancing the Raman intensity of the sample to be measured and exponentially amplifying weak Raman signals. The SERS peak is narrower than the fluorescence peak, and there is no photobleaching or quenching phenomenon, which significantly improves its signal-to-noise ratio. The probe material itself has no obvious Raman signal and will not interfere with the sample signal. It can reflect information such as changes in substance content and structural changes of molecules and groups. SERS offers great prospects for simplified and sensitive detection of biomolecular interactions. Compared with the detection methods mentioned above, it has many advantages.

[0005] This nanoprobe uses Au as a core, 4-MBA as a Raman signal internal reference molecule adsorbed on the Au core, and a SiO2 shell as a nanoparticle. PEG and EGFR antibodies are linked to the SiO2 shell. The nanoprobe is used to detect EGFR expression in HNSCC cells and tissues, create real-time two-dimensional images of EGFR expression in cells and tissues, and perform quantitative analysis. This provides a rapid, visual, and convenient new method for clinical detection of HNSCC cells and tissues, and also provides a theoretical basis for noninvasive, real-time detection and quantitative analysis of EGFR expression. Summary of the Invention

[0006] The purpose of the present invention is to provide a nanoprobe for detecting the expression of epidermal growth factor receptor in head and neck squamous cell carcinoma cells. The nanoprobe is capped with 4-MBA, SiO2, EGFR antibody and PEG to form a core-shell structure with better SERS activity, stability and biocompatibility.

[0007] To achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a nanoprobe for detecting the expression of epidermal growth factor receptor in head and neck squamous cell carcinoma cells, which is a nanoparticle with a core-shell structure;

[0008] The nanoparticles are nano-Au particles as cores, and 4-MBA is adsorbed on the surface of the Au core as a Raman signal internal reference molecule;

[0009] The nanoparticles use SiO2 as a shell, and PEG and EGFR antibodies are linked to the SiO2 shell.

[0010] The beneficial effects of the present invention are concentrated in:

[0011] 1. With Au nanoparticles as the core, 4-MBA was selected as the internal reference signal molecule. 4-MBA is a molecule with high Raman activity. The thiol group in its molecular structure can bind to the gold nanoparticles through the Au-S bond, thereby determining the distribution of the nanoprobe in head and neck squamous cell carcinoma tumor cells.

[0012] 2. Using SiO2 as the shell layer of the core-shell structure can reduce the volume conductivity and increase the suspension stability of gold nanoparticles without interfering with surface redox reactions. It can also regulate the position and intensity of surface plasmon absorption bands. Furthermore, because SiO2 is optically transparent, chemical reactions on the surface of gold nanoparticles can be studied spectroscopically. Furthermore, SiO2 can hydrate with water molecules in water, producing hydroxyl groups, which can form stable chemical bonds with functional groups on polymer chains. The shell structure formed by SiO2 can also enhance its affinity for organic polymers through mechanisms such as electrostatic interactions and adsorption layer mediation, resulting in an organic adsorption layer coating the surface.

[0013] 3. Introducing EGFR antibodies as a link between the probe and the tumor, and by activating the carboxyl group of PEG, the EGFR antibodies are bound to the surface of the core-shell nanoprobe to form a "sandwich" structure. This can be used to create SERS images of EGFR expression in head and neck squamous cell carcinoma tumor tissue, thereby more intuitively and conveniently displaying EGFR expression in tumor tissue, providing a rapid, non-invasive, and convenient visualization imaging technology for head and neck squamous cell carcinoma tumor cells and tumor tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of the formation of the nanoprobe core-shell structure of the present invention;

[0015] Figure 2 This is a statistical graph showing that EGFR expression in head and neck squamous cell carcinoma tissue is significantly higher than that in normal tissue;

[0016] Figure 3 Nanoprobe scanning electron microscopy and Raman spectroscopy images;

[0017] Figure 4 Light microscopy images, SERS images, and EGFR immunofluorescence images of SCC4, Tca8113 squamous cell carcinoma cells and Fibroblast cells;

[0018] Figure 5 Surface-enhanced Raman spectroscopy images of excised tissue and tumor tissue from nude mice. DETAILED DESCRIPTION

[0019] like Figure 1 As shown, a nanoprobe for detecting epidermal growth factor receptor expression in head and neck squamous cell carcinoma is a nanoparticle with a core-shell structure; the nanoparticle uses nano-Au particles as the core, and 4-MBA is adsorbed on the surface of the Au core as a Raman signal internal reference molecule; the nanoparticle uses SiO2 as the shell, and PEG and EGFR antibodies are linked to the SiO2 shell. Figure 1 The overall molding process and the final structural morphology of the product are schematically illustrated in the figure. To achieve better biocompatibility and SERS activity, the nanoparticle size is ≤100 nm, and the EGFR mutation-specific antibodies selected are E746-A750 and L858R.

[0020] The present invention takes nano-Au particles as the core and selects 4-MBA as the internal reference signal molecule. 4-MBA is a molecule with high Raman activity. The thiol group in its molecular structure can bind to the nano-gold particles through the Au-S bond, thereby determining the distribution of the nanoprobe in head and neck squamous cell carcinoma tumor cells.

[0021] At the same time, using SiO2 as the shell layer of the core-shell structure can reduce the volume conductivity and increase the suspension stability of gold nanoparticles without interfering with surface redox reactions, while also regulating the position and intensity of surface plasmon absorption bands. Furthermore, because SiO2 is optically transparent, chemical reactions on the surface of gold nanoparticles can be studied spectroscopically. Furthermore, SiO2 can hydrate with water molecules in water, producing hydroxyl groups, which can form stable chemical bonds with functional groups on polymer chains. Furthermore, the shell structure formed by SiO2 can enhance its affinity with organic polymers through mechanisms such as electrostatic interactions and adsorption layer mediation, resulting in a layer of organic adsorption on the surface.

[0022] In addition, an EGFR antibody is introduced as a link between the probe and the tumor. By activating the carboxyl group of PEG, the EGFR antibody is bound to the surface of the core-shell nanoprobe to form a "sandwich" structure. This can be used to create SERS images of EGFR expression in head and neck squamous cell carcinoma tumor tissue, thereby more intuitively and conveniently displaying EGFR expression in tumor tissue, providing a rapid, non-invasive, and convenient visualization imaging technology for head and neck squamous cell carcinoma tumor cells and tumor tissue.

[0023] The preparation method of the nanoprobe of the present invention is as follows:

[0024] Step S1:

[0025] Heat 100 ml of a 0.01% w / v HAuCl4·H2O aqueous solution to boiling, then quickly add 1.4 ml of a 1% trisodium citrate aqueous solution, stir evenly and keep boiling. After 30 minutes, a gold nanosol with a concentration of approximately 0.03 mol / L is generated. Use a pipette to draw 100 ml of the gold nanosol into a centrifuge tube, centrifuge at 6000 rpm for 10 minutes, remove the supernatant, and resuspend in 50 ml of a 0.01% trisodium citrate aqueous solution to obtain a gold nanoparticle solution for use.

[0026] Step S2:

[0027] Take 20mL of gold nanoparticle solution and place it in a conical flask. Add 100μL of 10 - 2 mol·L -1 4-MBA ethanol solution; after stirring for 30 minutes, centrifuge at 8900 rpm for 30 minutes, and redisperse in 20 ml of deionized water. The color of the gold nanoparticle solution changes from wine red to dark blue purple;

[0028] Step S3:

[0029] Add 100 μL of 2.6 μmol·L -1 The surface of gold nanoparticles was silanized by MPTMS ethanol solution; after continuous stirring for 15 min, 20 μL of 1 mol·L -1 A NaOH solution was dissolved in 0.5 mL of deionized water and added dropwise to the silanized reaction solution; finally, 3 mL of a sodium silicate aqueous solution with a pH value between 10 and 11 was added to the solution, and the mixture was reacted at 90°C with magnetic stirring for 1.5 hours to obtain a solution of gold nanoparticles with a SiO2 shell; the solution of gold nanoparticles with a SiO2 shell was centrifuged at 7000 rpm for 10 minutes, the supernatant was removed, and the solution was redispersed in 20 mL of deionized water to form a solution of nanoparticles with a core-shell structure;

[0030] Step S4:

[0031] S4.1 distributes PEG onto the SiO2 shell of the nanoparticles via electrostatic adsorption. To activate the carboxyl groups, an excess of 50 mg / mL ethyldimethylaminopropylcarbodiimide (EDC) solution and 50 mg / mL sulfo-NHS solution are added in a 1:1 ratio and vigorously mixed at room temperature for 20 minutes. After three rounds of centrifugation, the mixture is resuspended in PBS, and the excess EDC and sulfo-NHS are separated from the suspension to obtain an intermediate with activated carboxyl groups.

[0032] S4.2 Couple the EGFR antibody to the SiO2 shell of the nanoparticles: react the intermediate with the activated carboxyl group with the EGFR antibody at room temperature for 2 hours, and then at 4°C for 12 hours to allow the amino group in the EGFR antibody to fully react with the activated carboxyl group and connect; obtain the modified nanoprobe after purification by centrifugation and resuspend it in PBS solution for later use.

[0033] like Figure 2 As shown in Figure 2, A shows the differences in EGFR and GAPDH expression in tumor tissue T, paracancerous tissue P, and normal tissue N; B shows HE staining images of tongue squamous cell carcinoma tissue (Ta), paracancerous tissue (Pa), normal tissue (Na), EGFR immunohistochemistry images of tongue squamous cell carcinoma tissue (Tb), paracancerous tissue (Pb), and normal tissue (Nb); C shows the differences in EGFR and Actin expression in A549 cells, Tca-8113A cells, Fibroblast cells, and SSC-4 cells; D shows EGFR, DAPI, and Merge immunofluorescence images in SSC-4 cells, Fibroblast cells, and Tca-8113A cells; It can be seen that the expression of EGFR in head and neck squamous cell carcinoma tissue is significantly higher than that in normal mucosal tissue and muscle tissue, and the expression of EGFR in head and neck squamous cell carcinoma cells is also significantly higher than that in normal fibroblasts.

[0034] Nanoscale gold sol was modified with bioaffinity polymer α-mercapto-ω carboxyl polyethylene glycol (PEG) and Raman active molecule 4-MBA. EGFR antibody was combined with PEG to prepare a specific SERE probe for SERS detection of tongue squamous cell carcinoma cells. The scanning electron microscopy and Raman spectrum of the nanoprobe are shown in Figure 2. Figure 3 In the figure: (b) 1. Scanning electron microscopy and Raman spectrum of nano-gold sol. (b) 2. Scanning electron microscopy and Raman spectrum of nano-probe modified with 4-MBA, where 1073 cm -1 and 1589cm -1 The characteristic peak of 4-MBA is convenient for labeling tumor cells later. (b) 3. Scanning electron microscopy and Raman spectrum of nanoprobes modified with 4-MBA and PEG. PEG has no Raman activity and will not interfere with the Raman signal. (b) 4. Scanning electron microscopy and Raman spectrum of nanoprobes modified with MBA, PEG and EGFR antibody. -1 and 1543cm -1 The characteristic peak of EGFR antibody is obtained by using the above-mentioned polymer-modified specific nanoprobe. -1 and 1589cm -1 The two characteristic peaks of 4-MBA are used to draw Raman spectrum images of cells, such as Figure 4 As shown in the figure, the 4-MBA Raman peak in the image is set to green, and its intensity is related to the number of gold nanoparticles adsorbed or internalized by cells, while the 4-MBA peak intensity is related to the expression of EGFR on the cell surface. Therefore, surface-enhanced Raman spectroscopy technology was used to draw real-time cell imaging of EGFR expression in head and neck squamous cell carcinoma cell lines (such as Figure 4 As can be seen from the figure, the 4-MBA peak intensity of the spectral images of Tca8113 and SCC4 cells is larger, while the 4-MBA peak intensity of fibroblasts is lower, which is similar to the above-mentioned cell EGFR fluorescence immunofluorescence image, that is, squamous cell carcinoma cells highly express EGFR, while fibroblasts express it less.

[0035] The nanoprobes modified with polymer and EGFR antibody were injected into the squamous cell carcinoma and fibrosarcoma tumor tissues of nude mice. The mice were killed by cervical dislocation 24 hours after injection and the tumors were completely removed. Figure 5 a. The tumor was sliced into 10 μm thick sections using a Leica cryostat and attached to aluminum sheets. Surface-enhanced Raman spectroscopy was performed using a Nanophoton Raman-11 laser microconfocal Raman spectrometer. Figure 5 As shown in Figure b, 1. Spectral imaging of tumor tissue from the SCC4 cell line; 2. Spectral imaging of tumor tissue from the Tca8113 cell line; 3. Spectral imaging of tumor tissue from the fibrosarcoma cell line. 4-MBA-enhanced Raman spectroscopy signals are visible in squamous cell carcinoma tumor tissue (white highlights in the figure), while no significant 4-MBA-enhanced Raman signals are observed in sarcoma tumor tissue. This demonstrates that the synthesized nanoparticles possess a certain degree of selectivity and can be used for surface-enhanced Raman spectroscopy detection of squamous cell carcinoma. These results suggest that nanoscale gold particles modified with 4-MBA-PEG-EGFR antibodies can be used to screen tumor tissues with high EGFR expression, providing a theoretical basis for clinical detection of squamous cell carcinoma tumor tissues with high EGFR expression.

Claims

1. A nanoprobe for detecting epidermal growth factor receptor expression in head and neck squamous cell carcinoma cells, characterized by: Nanoparticles with core-shell structure; The nanoparticles are nano-Au particles as cores, and 4-MBA is adsorbed on the surface of the Au core as a Raman signal internal reference molecule; The nanoparticles use SiO2 as a shell, and PEG and EGFR antibodies are linked to the SiO2 shell.

2. The nanoprobe for detecting epidermal growth factor receptor expression in head and neck squamous cell carcinoma cells according to claim 1, characterized in that: The size of the nanoparticles is ≤100 nm.

3. The nanoprobe for detecting epidermal growth factor receptor expression in head and neck squamous cell carcinoma cells according to claim 1, characterized in that: The EGFR antibodies are E746-A750 and L858R.

4. The method for preparing a nanoprobe for detecting epidermal growth factor receptor expression in head and neck squamous cell carcinoma cells according to claim 3, characterized in that: The following steps are involved: Step S1: Heat 100 ml of a 0.01% w / v HAuCl4·H2O aqueous solution to boiling, then quickly add 1.4 ml of a 1% trisodium citrate aqueous solution, stir evenly and keep boiling. After 30 minutes, a gold nanosol with a concentration of approximately 0.03 mol / L is generated. Use a pipette to draw 100 ml of the gold nanosol into a centrifuge tube, centrifuge at 6000 rpm for 10 minutes, remove the supernatant, and resuspend in 50 ml of a 0.01% trisodium citrate aqueous solution to obtain a gold nanoparticle solution for use. Step S2: Take 20mL of gold nanoparticle solution and place it in a conical flask. Add 100μL of 10 -2 mol·L -1 4-MBA ethanol solution; after stirring for 30 minutes, centrifuge at 8900 rpm for 30 minutes, and redisperse in 20 ml of deionized water. The color of the gold nanoparticle solution changes from wine red to dark blue purple; Step S3: Add 100 μL of 2.6 μmol·L -1 The surface of gold nanoparticles was silanized by MPTMS ethanol solution; after continuous stirring for 15 min, 20 μL of 1 mol·L -1 A NaOH solution was dissolved in 0.5 mL of deionized water and added dropwise to the silanized reaction solution; finally, 3 mL of a sodium silicate aqueous solution with a pH value between 10 and 11 was added to the solution, and the mixture was reacted at 90°C with magnetic stirring for 1.5 hours to obtain a solution of gold nanoparticles with a SiO2 shell; the solution of gold nanoparticles with a SiO2 shell was centrifuged at 7000 rpm for 10 minutes, the supernatant was removed, and the solution was redispersed in 20 mL of deionized water to form a solution of nanoparticles with a core-shell structure; Step S4: S4.1 distributes PEG on the SiO2 shell of nanoparticles through electrostatic adsorption; S4.2 Couple the EGFR antibody to the SiO2 shell of the nanoparticles to obtain the nanoprobe.

5. The method for preparing a nanoprobe for detecting epidermal growth factor receptor expression in head and neck squamous cell carcinoma cells according to claim 4, characterized in that: Between steps S4.1 and S4.2, in order to activate the carboxyl group, an excess of 50 mg / mL ethyldimethylaminopropylcarbodiimide (EDC) solution and 50 mg / mL sulfo-NHS solution are first added in a 1:1 ratio and vigorously mixed for 20 minutes at room temperature; after three rounds of centrifugation, the solution is resuspended in PBS solution, and then the excess EDC and sulfo-NHS are separated from the suspension to obtain an intermediate with an activated carboxyl group; in step S4.2, the intermediate with an activated carboxyl group is reacted with the EGFR antibody at room temperature for 2 hours and then at 4°C for 12 hours to allow the amino group in the EGFR antibody to fully react with the activated carboxyl group and connect; the modified nanoprobe is obtained after purification by centrifugation and resuspended in PBS solution for later use.

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