Targeted nuclide probe for realizing visualization of inner ear spiral ganglion neurons

Abstract

The invention provides a targeted nuclide probe for realizing visualization of inner ear spiral ganglion neurons. For severe and extremely severe hearing loss patients, artificial cochlea implantation, as a common and effective hearing recovery means, can generate electrical activity and form auditory perception by directly stimulating the SGN. However, the curative effect after cochlear implantation is significantly affected by the number of survival SGNs, and the existing hearing function and imaging examination methods cannot display the integrity of the inner ear neurons. Therefore, a technical means capable of directly and effectively evaluating the integrity of the SGN in vivo is lacked. According to the invention, the radionuclide tracer agent is combined with the specific inner ear marker, so that the in-vivo key protein expression level is detected, the auditory function of the inner ear is reflected, and the problem that the integrity of the cochlea spiral ganglion cannot be accurately and directly evaluated in the prior art is solved.

Description

Technical Field:

[0001] The present invention relates to the field of biomedical technologies, and particularly to a targeted radionuclide probe for realizing in vivo visualization of inner ear spiral ganglion neurons 177 Lu-DOTA-anti-VGLUT1. Background Art:

[0002] Hearing loss is one of the most common sensory disorders, affecting more than 5% of the global population, that is, approximately 430 million people suffer from disabling hearing loss. So far, more than 220,000 people globally have received cochlear implants, which are widely regarded as the only treatment option for severe to profound sensorineural hearing loss (SNHL) in children and adults. The efficacy of cochlear implants (CI) is related to multiple factors such as the cause of deafness, cochlear development, cochlear nerve and auditory pathway integrity, and speech rehabilitation. Among them, the function of the cochlear nerve and auditory pathway is one of the most important factors affecting the prognosis of CI, and it is also the focus of preoperative evaluation of CI.

[0003] Multiple factors such as genetics, aging, exposure to ototoxic drugs, and noise can all lead to hair cell (HC) death and deafness. Cochlear implants bypass the damaged HCs and directly stimulate the remaining spiral ganglion neurons (SGN) to generate electrical activity and form auditory perception. Therefore, the number of surviving SGNs is a key factor affecting the efficacy of CI. Currently, preoperative evaluation of cochlear implant implantation usually includes audiological evaluation and imaging studies. The main purposes are to determine whether the patient's hearing loss meets the criteria for CI implantation, and secondly, whether the anatomical structure is suitable for implantation. However, effectively evaluating the number of surviving SGNs in the cochlea remains a major challenge. Some studies have shown that the development of targeted contrast agents can play a key role in determining the cause of hearing loss and guiding treatment.

[0004] Immuno-positron emission tomography (immunoPET) combines a highly target-specific antibody with a radionuclide through a coupling agent to generate PET images with high sensitivity and spatial resolution. In the era of molecular targeted therapy and cancer immunotherapy, the development of molecular imaging probes based on antibodies, antibody fragments, and nanobodies can help visualize the heterogeneous expression of tumor antigens, evaluate the pharmacokinetics of therapeutic antibodies in vivo, and predict the efficacy of treatment effects, thereby achieving non-invasive and precise screening. PD-1 / PD-L1, HER-2, CD4+ / CD8+ targeted radionuclide antibody probes have been translated into clinical molecular imaging and will provide important reference values.

[0005] For patients with severe and profound hearing loss, cochlear implantation, as a commonly used and effective means of restoring hearing, can directly stimulate spiral ganglion neurons (SGNs) to generate electrical activity and form auditory perception. However, the therapeutic effect after cochlear implantation is significantly affected by the number of surviving SGNs, and existing hearing function and imaging examination methods cannot show the integrity of inner ear neurons. Therefore, there is a lack of a technical means to directly and effectively evaluate the integrity of SGNs in vivo.

[0006] Inspired by in vivo PET / SPECT imaging with radionuclide molecular probes, finding a specific membrane receptor of inner ear SGNs and synthesizing a highly targeted radionuclide probe tracer based on it for in vivo PET imaging to visualize SGNs is expected to help cochlear implant candidates evaluate the integrity of inner ear nerves before surgery and assist clinical decision-making. Summary of the Invention:

[0007] (I) Technical problems to be solved

[0008] Aiming at the above deficiencies of the prior art, the purpose of the present invention is to provide a targeted radionuclide probe for realizing in vivo visualization of inner ear spiral ganglion neurons, combining a radionuclide tracer with a specific inner ear marker to detect the expression level of key proteins in vivo, reflect the auditory function of the inner ear, and solve the problem that the prior art cannot accurately and directly evaluate the integrity of the cochlear spiral ganglion.

[0009] (II) Technical solutions

[0010] To achieve the above purpose, the present invention adopts the following technical solutions:

[0011] The present invention provides a targeted radionuclide probe for realizing visualization of inner ear spiral ganglion neurons, and the targeted radionuclide probe is 177 Lu-DOTA-anti-VGLUT1, which is formed by coupling the vesicular glutamate transporter 1 antibody anti-VGLUT1 with 177 lutetium Lu through a DOTA coupling agent. The radionuclide probe targets the specific membrane protein of inner ear SGNs and visualizes SGNs in vivo through PET imaging.

[0012] The present invention also provides a preparation method of a targeted radionuclide probe for realizing visualization of inner ear spiral ganglion neurons, including the following steps:

[0013] S1, synthesize DOTA-anti-VGLUT1:

[0014] Prepare a reaction system by mixing 2-[(4-isothiocyanatophenyl)methyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) and VGLUT1 antibody in a molar ratio of 50:1. Mix well, wrap it with tin foil to avoid light, and react with shaking at room temperature for 2 h to obtain a DOTA-anti-VGLUT1 antibody mixture. The mixture is purified by PD10, and the concentration of DOTA-anti-VGLUT1 antibody is determined by ultraviolet quantification using an enzyme-linked immunosorbent assay (ELISA) reader.

[0015] S2, 177 Labeling DOTA-anti-VGLUT1 with

[0016] Prepare an aqueous solution of DOTA-anti-VGLUT1 at 5 mg / mL, MW = 1434, and add labeling buffer and neutralization buffer. Add 0.5 ml of labeling buffer to 30 ul of the DOTA-anti-VGLUT1 system, and then add 1.5 ml 177 Lu (100 mCi / vial), place it in an incubator at 95 °C, and react for 15 minutes.

[0017] S3, Radio-TLC / Radio-HPLC analysis. After RLY > 95%, add 0.1 M Na2CO3 to the system obtained in step S2 to adjust the pH to 7.0. Pass through a 0.22 μm microporous filter membrane, add 2.0 ml of normal saline to the system, and store it at 4 °C to obtain the targeted radionuclide probe 177 Lu-DOTA-anti-VGLUT1.

[0018] In the above method, the labeling buffer Labeling buffer is: Add 390 μL of 1.0 M sodium acetate to 6 ml of 0.05 M HCl, and measure its pH value with pH test paper to be 4.0. The neutralization buffer neutralization buffer is: 0.1 M Na 2 CO 3 (106 mg in 10 mL H2O) for adjusting the pH of the product.

[0019] The present invention also provides the above-mentioned targeted radionuclide probe 177 The application of Lu-DOTA-anti-VGLUT1 in the preparation of a reagent for visual detection of spiral ganglion neurons in the inner ear in vivo.

[0020] Furthermore, the application is used as a reagent for detecting cochlear SGN-specific membrane proteins.

[0021] The present invention also provides a method for visual detection of the number of cochlear SGNs. By combining a radionuclide tracer with a specific inner ear marker, the detection of the expression level of key proteins in vivo is achieved, reflecting the auditory function of the inner ear. The radionuclide tracer is 177 Lu-DOTA-anti-VGLUT1, and the inner ear marker is an SGN-specific membrane protein.

[0022] Furthermore, the targeted radionuclide probe 177Lu-DOTA-anti-VGLUT1 prepared in the present invention is used for in vivo PET imaging by cochlear round window injection. The radioactivity of the inner ear is detected by PET / SPECT at different time points after administration. The metabolic level of cochlear VGLUT1 is reflected by observing the magnitude of the radionuclide emission, and then the number of cochlear SGNs is visualized.

[0023] The purpose of the present invention is to design a radionuclide probe targeting the SGN-specific membrane protein of the inner ear, visualize SGNs in vivo by PET imaging, and provide a basis for the preoperative evaluation of cochlear implantation.

[0024] The present invention has verified by various means such as single-cell sequencing, immunofluorescence quantification, and RT-PCR that vesicular glutamate transporter 1 (VGLUT1) is highly expressed on the cell membrane of cochlear SGNs, but hardly expressed in other cell types of the cochlea, and can be used as a representative membrane protein marker for SGNs.

[0025] In view of the small size and deep location of the cochlea, the physical properties of lutetium-177 (Lu) make it suitable for use in this study. Its lower β-ray energy can minimize bone marrow suppression, reduce damage to surrounding tissues, and effectively concentrate the energy in the cochlea. In addition, the half-life of 177Lu is relatively long (6.73 days), making it not easy to decay rapidly, and maintaining its high activity in the cochlea with a smaller injection dose.

[0026] (III) Beneficial effects

[0027] The beneficial effects of the present invention are as follows:

[0028] The present invention discovers that the spiral ganglion cell membrane protein VGLUT1 is a promising representative marker for SGNs. The targeted radionuclide probe prepared by the present invention 177Lu-DOTA-anti-VGLUT1 was developed by conjugating VGLUT1 antibodies to DOTA and radiolabeling them with 177Lu. Due to its sensitivity, durability, and targeting specificity, it is able to image SGNs in vivo and visualize target antigen protein expression in the cochlea. This enables visual assessment of the relative number of SGNs in the cochlea. This strategy can predict the postoperative outcomes of CI candidates and identify beneficiaries before surgery, providing valuable guidance for clinical decision-making. Therefore, the present invention discovered the application potential and translational value of PET molecular probes in auditory nerve research and pioneered a new approach to nuclear medicine neuroimaging. Description of the drawings:

[0029] Figure 1 Figure 1 is the identification and screening of the spiral ganglion-specific membrane protein VGLUT1. The bubble plot in A shows the expression pattern of Slc17a7 in various cells of the cochlea. B is the tSNE plot of different cell types in the cochlea of ​​C57 / BL6 mice at P28, with SGNs in yellow. C is the tSNE plot showing that Slc17a7 is highly expressed in SGNs. D is the relative expression level of VGLUT1 mRNA in SGNs and other components of the cochlea. E is the immunofluorescence staining of different dimensions showing the expression pattern of VGLUT1 (green) in the cochlea. At P30, VGLUT1 was highly expressed in SGNs, but not in cochlear HCs. F is the quantification of VGLUT1 immunolabeling in SGNs, HCs, DCs, and PCs in wild-type cochleas (15 cells for each cell type). Abbreviations: RBC, red blood cell; SMC, smooth muscle cell; pDC, plasmacytoid dendritic cell; SGN, spiral ganglion neuron; HC, hair cell; DC: Deiter cell; PC: columnar cell.

[0030] Figure 2 This is the experimental flow chart. The VGLUT1 antibody first binds to DOTA and then 177 Lu-labeled, the synthesized radionuclide molecular probe was then injected into the mouse cochlea through the round window membrane (RWM), and finally in vivo and ex vivo PET / CT imaging was performed.

[0031] Figure 3 This is the injury pattern of the spiral ganglion neurons at the apical, middle and basal turns of Sox2CreER Cx26-null mice (Tuj1, red) (KCNJ10, green), where A is the SGN detection diagram; B is the analysis result diagram. Compared with the control group, the SGN density at the apical, middle and basal turns of Cx26-null mice was significantly reduced.

[0032] Figure 4 A is injection 177PET / CT images of the spiral ganglion injury group and the control group at 1 h and 12 h after Lu-DOTA-anti-VGLUT1 (n = 3). Radioactive quantitative ROI analysis of the cochlea at 1 h and 12 h in B-C showed that the uptake of 177 Lu-DOTA-anti-VGLUT1 in the spiral ganglion injury group was lower than that in the control group (1 h, n = 5, P < 0.01; 12 h, control group n = 3 vs. spiral ganglion injury group n = 5, P < 0.01).

[0033] Figure 5 is the expression of VGLUT1 in Sox2CreER Cx26-null mice. A is the immunofluorescence staining of VGLUT1 (green) in the apical, middle, and basal turns of SGN in the control group and the Cx26-null group. B is the immunofluorescence quantification of VGLUT1 per unit area in the spiral ganglion of the control group and the Cx26-null group (n = 4). C shows that the number of VGLUT1-positive cells in the apical, middle, and basal turns in the Cx26-null group was significantly lower than that in the control group.

[0034] Figure 6 is the auditory brainstem response normal after mice were injected with 177 Lu-DOTA-anti-VGLUT1 through the round window and PET imaging. Specific implementation manners:

[0035] The following further describes the content of the present invention in detail in combination with specific implementation manners. It should be understood that the embodiments of the present invention are only used to illustrate the present invention rather than limit the present invention. Without departing from the technical idea of the present invention, various substitutions and changes made according to common general knowledge and customary means in the art should be included within the scope of the present invention.

[0036] Example 1

[0037] Glutamate (Glu) is the main excitatory neurotransmitter in the central nervous system. In neurons, Glu is transported into synaptic vesicles through vesicular glutamate transporters (VGLUTs). VGLUT is a key molecule responsible for transporting glutamate into synaptic vesicles in the nervous system. Among these isoforms, VGLUT1 accounts for most of the excitatory glutamatergic nerve endings and is the most reliable marker for glutamatergic nerve endings and synapses. In the cochlea, inner hair cells transmit information to SGN through ribbon synapses, where many small vesicles containing glutamate are bound. And as we verified, there is abundant membrane protein VGLUT1 expressed on the cell membrane of the spiral ganglion, which is encoded by the Slc17a7 gene. Single-cell sequencing, immunofluorescence quantification, RT-PCR and other experiments all showed that its expression in SGN was significantly higher than that in other cochlear cells (see Figure 1) Based on the specificity of VGLUT1 expression in SGN, it may be the target membrane protein required for SGN imaging.

[0038] The present invention designed and prepared a radionuclide molecular probe accordingly. 177 Lu-DOTA-anti-VGLUT1. In one embodiment, the radionuclide molecular probe was verified: (see Figure 2 )

[0039] (1) Preparation of animal model: First, an animal model suitable for cochlear nerve imaging research was established. Mutations in GJB2 (encoding Cx26) are the most common cause of non-syndromic hereditary deafness (DFNB1). The severity of deafness caused by various GJB2 mutations varies greatly and is usually accompanied by degeneration of SGN and the auditory nerve pathway. Previous studies have shown that Sox2Cre; Cx26 loxP / loxP knockout mouse models exhibit severe hearing loss and extensive HC loss, similar to the manifestations of SNHL patients who require CI candidates. In addition, targeted Cx26 knockout in Sox2 + SC results in various degrees of pathological changes in auditory primary neurons and nerve pathways, leading to unstable CI postoperative effects. There is significant SGN damage in Sox2Cre; Cx26 loxP / loxP knockout mice at 3 months, manifested as a decrease in SGN density in the apical, middle, and basal turns (see Figure 3 ). Based on these findings, the present invention selected the Sox2CreER Cx26-null mouse model to evaluate the radionuclide molecular probe 177 Lu-DOTA-anti-VGLUT1 in in vivo PET / CT imaging of cochlear SGN.

[0040] (2) Preparation of radionuclide molecular probe:

[0041] 1. Synthesis of DOTA-anti-VGLUT1:

[0042] As described above, a reaction system was prepared at a molar ratio of 50:1 (SCN-DOTA:VGLUT1 antibody), quickly and thoroughly mixed, wrapped with tin foil to avoid light, and reacted with shaking at room temperature for 2 h to obtain a DOTA-anti-VGLUT1 antibody mixture. Then, the mixture was purified by PD10, and the concentration of DOTA-anti-VGLUT1 was quantitatively determined by ultraviolet spectrophotometry with an enzyme-linked immunosorbent assay reader.

[0043] 2. 177 Labeling of DOTA-anti-VGLUT1 with

[0044] 1) Take 6 mL of 0.05 M HCl and add 390 μL of 1.0 M sodium acetate to it. Measure its pH value with a pH test paper, which is about 4.0, as the labeling buffer.

[0045] 2) Prepare 0.1 M Na 2 CO 3 (106 mg in 10 mL H2O), adjust the pH of the product to be the neutralization buffer.

[0046] 3) Prepare an aqueous solution of DOTA-TATE (MW = 1434) at 5 mg / mL (100 μg for 150 mCi 177 Lulabelingper patient).

[0047] 4) Take out 0.5 mL of the labeling buffer and add the prepared DOTA-anti-VGLUT1 taken out from -20 °C to it, and label it as reaction flask 1.

[0048] 5) Add 177 Lu (100 mCi / vial) radionuclide vial with 0.5 mL of the labeling buffer, then draw 0.6 mL of the radionuclide and add it to reaction flask 1.

[0049] 6) Then add 0.5 mL of the labeling buffer to 177 the Lu vial, try to completely take out 177 Lu and add it to reaction flask 1.

[0050] 7) Add 0.5 mL of the labeling buffer to 177 the Lu vial again, cover the bottle cap and shake, try to completely take out and add it to reaction flask 1.

[0051] 8) Place it in an incubator at 95 °C and react for 15 minutes.

[0052] 9) Analyze by Radio-TLC / Radio-HPLC, RLY > 95%

[0053] 10) Add 0.1 M Na2CO3 to the system to adjust the pH to 7.0 (0.15 mL of Na 2 CO 3 ) for 1 mL of the reaction solution.

[0054] 11) Pass through a 0.22 μm microporous filter membrane, add 2.0 mL of normal saline to the system, and store it at 4 °C for later use.

[0055] 12) Radio-TLC analysis conditions:

[0056] Developing solution: ammonium acetate: methanol = 1:1; Support: ITLC-SG

[0057] 13) Radio-HPLC analysis conditions:

[0058] Instrument: Agilent 1200 series from the United States; Eclipse PLUS C18 5um

[0059] Mobile phase solution: high-purity chromatographic grade acetonitrile, 0.1% TFA ultrapure water

[0060] Mobile phase gradient: 0 - 10 min 95% H2O, 5% MeCN; 10 - 20 min 40% H2O, 60% MeCN

[0061] Flow rate: 1 mL / min Time: 20 min UV: 220 nm

[0062] 14) Obtaining the product 177 Description of Lu-DOTA-anti-VGLUT1:

[0063] Colorless transparent solution, specific activity 42.5 GBq / μmol, radiochemical purity > 95%, pH 7.0

[0064] 100 - 150 mCi / patient, Volume: 3 - 6 ml

[0065] (3) The surgical procedures and drug administration methods for mice are as follows:

[0066] 1. Anesthesia: Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (65 - 70 mg / kg, Sigma, P3761), and postoperative pain was controlled with the non-steroidal anti-inflammatory drug Meloxicam (5 mg / kg, Boehringer Ingelheim). The integrity of anesthesia was evaluated by performing noxious stimulus tests: pinching the paw; pinching the tail; and the blink response to touching the cheek and vibratory area. Sterile eye ointment was applied to the cornea.

[0067] 2. Skin preparation: The hair in the postauricular area was removed with a depilatory.

[0068] 3. Incision after disinfection: After anesthesia, the mice were placed in the lateral decubitus position, covered with a sterile hole towel, and the postauricular area was disinfected with a complex iodine cotton ball according to the disinfection principle. The skin was incised 1.5 cm about 1 cm from the ear root.

[0069] 4. Exposing the surgical field: Subcutaneous fat was bluntly dissected, the cervical blood vessels, muscles, and nerves were exposed and dissected, the facial nerve and the angle of the longitudinal muscle were found to determine the position of the tympanic opening, and a small hole was made and enlarged near the round window of the tympanic cavity with a drill until the stapedial artery - white ossified margin - round window shadow in the upper right were seen in sequence at the lower left of the hole.

[0070] 5. Injection: After aspirating the imaging agent using a micro syringe, inject it obliquely upwards to the right into the round window, and feel the "breaking through sensation" of the membrane rupture. Then, inject the radionuclide probe 177 Lu-DOTA-anti-VGLUT1 (2 μl) slowly and evenly.

[0071] 6. Sealing: After the injection, slowly withdraw the syringe, take a small piece of adipose tissue to seal the hole, and close the tissue layer by layer.

[0072] 7. Anesthesia recovery: Place the postoperative mice on a 37 °C constant temperature electric blanket for recovery.

[0073] (4) Imaging: After pre-anesthetizing with a 2% (v / v) isoflurane-oxygen mixture gas, place the mice on the Mediso Micro SPECT-CT scanning bed, and maintain anesthesia with a 1.5% (v / v) isoflurane-oxygen mixture gas and detect respiration for 30 min. Perform SPECT imaging at 1 h and 12 h after injection.

[0074] (5) Results: The PET imaging results showed that 177 the radioactivity of Lu-DOTA-anti-VGLUT1 was limited to the cochlea, and no radioactivity was detected in other parts of the mice. In the control group, the cochlear radioactivity was 14.18 ± 1.74% ID at 1 h after administration and 9.13 ± 0.67% ID at 12 h, which was significantly higher than that in the SGN injury group (1 h: 5.27 ± 3.09% ID; 12 h: 3.37 ± 2.23% ID) (see Figure 4 ). Over time, the cochlear radioactivity gradually decreased, indicating a stable metabolic process in the cochlea. The semi-quantitative fluorescence and positive cell analysis of VGLUT1 confirmed that the expression of VGLUT1 in the spiral ganglion of the injured group of mice was lower than that in the control group, which confirmed the PET imaging results. These results indicate that immune PET imaging can effectively evaluate the expression of VGLUT1 in the cochlea (see Figure 5 ).

[0075] (6) Toxicity and safety detection: After the surgery and PET imaging, perform auditory brainstem response tests on the mice. Anesthetize the mice with a mixture of compound anesthetic chlorpromazine hydrochloride (120 mg / kg) and chlorpromazine hydrochloride (20 mg / kg). The measurement is carried out in a special quiet room, and the mice are placed on a 37 °C constant temperature electric blanket. The recording electrode and the reference electrode are inserted subcutaneously into the skull or the ear to be measured respectively. Use the Tucker-Davis Technology (TDT) RZ6 auditory workstation to measure the hearing threshold, and use the SigGen32 software (Tucker-Davis Technologies) to amplify and record the ABR signal (seeFigure 6 )。

[0076] In summary, the present invention provides a targeted radionuclide probe for realizing in vivo visualization of inner ear spiral ganglion neurons. By combining a radionuclide tracer with a specific inner ear marker, it can detect the expression level of key proteins in vivo, reflect the auditory function of the inner ear, and solve the problem that the prior art cannot accurately and directly evaluate the integrity of the cochlear spiral ganglion.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and do not limit the protection scope of the present invention. Additionally, after reading the technical content of the present invention, those skilled in the art can make various changes, modifications or variations to the present invention, and all these equivalent forms also fall within the protection scope defined by this application.

Claims

1. A targeted radionuclide probe for visualizing neurons in the spiral ganglion of the inner ear, characterized in that: The targeted nuclide probe is 177 Lu-DOTA-anti-VGLUT1, composed of vesicular glutamate transporter 1 antibody anti-VGLUT1 coupled with DOTA 177 After Lu coupling, the radionuclide probe targets inner ear SGN-specific membrane proteins, and SGNs are visualized in vivo by PET imaging.

2. The method for preparing a targeted radionuclide probe for visualizing neurons of the inner ear spiral ganglion according to claim 1, characterized in that: The following steps are involved: S1, synthesis of DOTA-anti-VGLUT1: The reaction system was prepared with 2-[(4-isothiocyanatophenyl)methyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid p-SCN-Bn-DOTA and VGLUT1 antibody at a molar ratio of 50:1, mixed thoroughly, wrapped with tin foil to avoid light, and reacted at room temperature for 2 hours to obtain a DOTA-anti-VGLUT1 antibody mixture. The mixture was purified by PD10, and the DOTA-anti-VGLUT1 antibody concentration was obtained by ultraviolet quantification with an enzyme marker; S2, 177 Lu-labeled DOTA-anti-VGLUT1: Prepare 5 mg / mL DOTA-anti-VGLUT1 aqueous solution, MW = 1434, add labeling buffer and neutralization buffer; add 0.5 ml labeling buffer to 30 ul DOTA-anti-VGLUT1 system, and then add 1.5 ml 177 Lu, 100 mCi / vial, placed in an incubator at 95°C for 15 min; S3, Radio-TLC / Radio-HPLC analysis, after RLY>95%, add 0.1M Na2CO3 to the system obtained in step S2 to adjust the pH to 7.0; add 2.0ml of physiological saline to the system through a 0.22μm microporous filter membrane, store at 4°C, and obtain the targeted radionuclide probe 177 Lu-DOTA-anti-VGLUT1.

3. A method for preparing a targeted radionuclide probe for visualizing neurons in the spiral ganglion of the inner ear according to claim 2, characterized in that: The labeling buffer is: 6 ml, 0.05M HCl, to which 390 μL, 1.0M sodium acetate is added, and its pH value is 4.0 as measured by pH test paper. The neutralization buffer is: 0.1M Na2CO3, which is used to adjust the pH.

4. The nuclear probe according to any one of claims 1 to 3 177 Application of Lu-DOTA-anti-VGLUT1 in the preparation of in vivo visualization detection reagents for inner ear spiral ganglion neurons.

5. The use according to claim 4, characterized in that: The application is as a cochlear SGN-specific membrane protein detection reagent.

6. A method for visually detecting the number of cochlear SGNs, characterized in that: The radionuclide tracer is combined with a specific inner ear marker to detect the expression level of key proteins in vivo and reflect the auditory function of the inner ear. The radionuclide tracer is 177 Lu-DOTA-anti-VGLUT1, the inner ear marker is a SGN-specific membrane protein.

7. A visual detection method for the number of cochlear SGNs according to claim 6, characterized in that: In the nuclear molecular probe 177 Radioactivity was detected by PET / SPECT at different time points after Lu-DOTA-anti-VGLUT1 administration. The amount of radionuclide radiation was observed to reflect the metabolic level of cochlear VGLUT1, thereby visualizing the number of cochlear SGNs.

Images