In-vivo selective probe for mammalian nerve cells and application thereof

Abstract

The invention relates to the technical field of nerve cell marking and regulation and control, in particular to a nerve cell living body selective protein modification probe capable of penetrating a blood brain barrier and application of the nerve cell living body selective protein modification probe. The protein body part specifically targets a living glutamic acid energetic neuron cell membrane, the protein body part and / or a modified part thereof can regulate and control the function of the glutamic acid energetic neuron, and a coding sequence of the protein body part comprises a sequence as shown in SEQ ID NO: 1 or a sequence having at least 85% identity with the SEQ ID NO: 1. The protein modified probe provided by the invention provides a new feasible thought for developing in-vivo nerve cell selective markers and regulation tools which are non-gene transfection of blood brain barrier penetration, are high in biological safety, are more stable and are easy to obtain and have nerve cell specific type selectivity.

Description

Technical Field

[0001] The present application relates to the technical field of neural cell labeling and regulation, and in particular to a neural cell living selective protein modification probe capable of penetrating the blood-brain barrier and its application. Background Art

[0002] Since the beginning of the 21st century, countries around the world have successively launched large-scale scientific projects related to brain science, revealing the mysteries of brain function through the development of forward-looking and innovative neurotechnologies. Among them, the development of neural markers and neuromodulation technologies at the in vivo level is a cutting-edge cross-cutting hotspot and key issue at this stage, providing a huge boost to the study of neurophysiological and pathological processes such as cell interactions, neuronal cell transmission, and signal tracking in complex neural networks. In recent years, with the continuous development of brain science, the research on neurochemical imaging and regulation has put forward the requirement of selective regulation using non-transfection and blood-brain barrier permeability, aiming to more accurately describe and regulate the functions of specific brain regions and specific types of neurons in physiological and pathological environments under the premise of high biosafety.

[0003] Currently, neural labeling and regulation mainly rely on research strategies using viral transfection, small molecule overall regulation, or chemical carrier modification: on the one hand, chemical molecular drugs that can penetrate the blood-brain barrier can be administered as a whole to interfere with cellular function throughout the brain and body, or by modifying blood-brain barrier-related receptor binding proteins (such as transferrin, growth hormone analogs, etc.) on chemical carriers in order to break through the blood-brain barrier and thus manipulate neural activity; on the other hand, they rely on viral vectors to directly manipulate genes through viral transfection to change the molecular composition and function of nerve cells. However, these methods are limited by the extremely low permeability of the blood-brain barrier, the difficulty in achieving efficient labeling and precise regulation of nerve cells, or the biosafety concerns and ethical restrictions brought about by gene transfection, making them difficult to apply to large mammals or even humans.

[0004] Recently, a series of exogenous field intervention strategies based on invasiveness (in situ injection, component implantation, etc.) have been developed. For example, nanoparticles that are sensitive to light, electricity, magnetism, heat, etc. are constructed, and the chemical molecules loaded in the nanoparticles are released through in situ brain injection and field regulation, thereby regulating the neural function of the specific brain area injected. In addition, chemically modified electrodes can also be implanted in the brain, and physiologically active molecules can be controllably synthesized by changing the applied voltage to achieve neural function intervention. At the same time, the strategy of exogenous field intervention can be associated with gene transfection to increase the expression of related receptors (such as heat-sensitive receptors) on the nerve cell membranes in specific brain areas, thereby achieving targeted and precise regulation of nerve cells. Although the above-mentioned methods can directly intervene in neural activity without considering the limitations of the blood-brain barrier, they still require invasive operations of physical implantation of brain tissue and genetic manipulation to ensure the limitations of selective labeling of nerve cells, and face a series of challenges such as biosafety concerns and limited application scenarios.

[0005] In view of this, it is very necessary to develop non-genetic transfection tools that can penetrate the blood-brain barrier, have high biosafety, are more stable and easy to obtain, and have selective labeling and regulation of living neurons with specific neuronal cell types. Summary of the Invention

[0006] The present application solves at least one of the problems of the related art from the following aspects.

[0007] To this end, an embodiment of the present application provides a method for targeted regulation of glutamatergic neuron function using a protein modification probe, wherein the protein modification probe comprises a protein body portion, wherein the protein body portion specifically targets the cell membrane of a living glutamatergic neuron, wherein the protein body portion comprises a structural domain capable of responding to a first exogenous stimulus, wherein the structural domain mediates the production of a first regulatory molecule in response to the first exogenous stimulus for regulating the function of the glutamatergic neuron, and the coding sequence of the protein body portion comprises the sequence shown in SEQ ID NO: 1 or a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with SEQ ID NO: 1.

[0008] In some embodiments, the domain comprises a flavin mononucleotide, the first exogenous stimulus comprises light, and the first regulatory molecule comprises reactive oxygen species.

[0009] In some embodiments, the protein modification probe penetrates the blood-brain barrier and contacts the cell membrane of the living glutamatergic neurons, thereby regulating the function of the glutamatergic neurons.

[0010] In some embodiments, the protein modification probe further comprises a modifying portion comprising a second regulatory molecule capable of regulating the function of the glutamatergic neurons. In some embodiments, the modifying portion is covalently or non-covalently linked to the protein body portion. In some embodiments, the modifying portion is connected to the protein body portion via a linker. In some embodiments, the linker is capable of releasing the modifying portion in response to the environment surrounding the living glutamatergic neurons.

[0011] In some embodiments, the second regulatory molecule is selected from: small molecule drugs, dyes, peptides, antibodies, plasmid DNA, nucleic acids, liposomes, phage particles, superparamagnetic particles, fluorescent dyes, nanoparticles, viruses, quantum dots or magnetic resonance imaging contrast agents.

[0012] In some embodiments, the second regulatory molecule is a molecule capable of responding to a second external stimulus, which may optionally include light, sound, heat, electricity, and magnetism.

[0013] In some embodiments, the second exogenous stimulus is light, and the second regulatory molecule is selected from one or more of the following photosensitizers: indocyanine green, chlorin e6, methylene blue, acridine orange, pyropheophytin-a, and isomers or analogs thereof.

[0014] In some embodiments, the second exogenous stimulus is sound, and the second regulatory molecule is selected from one or more of the following sonosensitizers: 5-aminolevulinic acid, protoporphyrin IX, hematoporphyrin, rose bengal, curcumin, titanium nanoparticles, dihydrochlorin e6, pheobromide-a, ATX-S10 photoporphyrin, photoporphyrin II, DCPH-P-Na(I), NPe6, polyhydroxyfullerene, hypocrellin-B, ZnPcS2P2, methylene blue, sodium chloroporphyrin and its isomers or analogs.

[0015] In some embodiments, the method further comprises providing the first and / or second exogenous stimulus to the brain after administering the protein modification probe.

[0016] In some embodiments, the first and / or second exogenous stimulation is provided by implanting an exogenous stimuli-responsive element into the brain, the exogenous stimuli-responsive element optionally comprising an optical fiber and an electrode.

[0017] In some embodiments, the modified portion further comprises a fluorescent reporter molecule, which is optionally a first fluorescent molecule containing an isothiocyanate group and / or a second fluorescent molecule containing a succinimide ester (NHS) group. In some embodiments, the first fluorescent molecule is fluorescein isothiocyanate or an isomer or analog thereof and / or a rhodamine molecule. In some embodiments, the second fluorescent molecule is NHS fluorescein or an isomer or analog thereof, such as Cy3-NHS, Cy5-NHS activated ester, Cy5.5-NHS activated ester, Cy7-NHS activated ester and / or Cy7.5-NHS activated ester, preferably Cy3-NHS activated ester and / or Cy5-NHS activated ester.

[0018] In some embodiments, the protein modification probe is administered orally, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradurally, sublingually, buccally, rectally, vaginally, ophthalmically, otically, nasally, transdermally, subconjunctivally, and / or intracerebrally, such as intrathecally.

[0019] In some embodiments, the glutamatergic neurons are hippocampal glutamatergic neurons.

[0020] The present application also provides an embodiment of the use of a protein-modified probe capable of penetrating the blood-brain barrier as defined in any of the above embodiments in the preparation of a drug for targeted regulation of glutamatergic neuron function. In some embodiments, the glutamatergic neurons are hippocampal glutamatergic neurons.

[0021] In some embodiments, the drug penetrates the blood-brain barrier and contacts the cell membrane of living glutamatergic neurons, thereby regulating the function of the glutamatergic neurons.

[0022] In some embodiments, the drug is administered by intravenous injection or brain injection. In some embodiments, the drug is administered based on intravenous injection, and the concentration of the protein modification probe contained in the drug is 5-10 μM. In some embodiments, the drug is administered based on brain injection, and the concentration of the protein modification probe contained in the drug is 30-50 μM, and the brain injection is optionally performed in the hippocampus, preferably in the dentate gyrus of the hippocampus or the CA3 brain region of the hippocampus.

[0023] The embodiments of the present application also provide the use of a protein-modified probe capable of penetrating the blood-brain barrier as defined in any of the above embodiments in the preparation of a drug for diagnosing or treating a nervous system disease, wherein the nervous system disease is caused by abnormal glutamatergic neuron function or can be treated by regulating the function of the glutamatergic neurons.

[0024] The embodiments of the present application achieve the following beneficial effects: Compared with traditional strategies for labeling and regulating living neural cells, the protein-modified probes proposed in the embodiments of the present application have blood-brain barrier permeability and can be administered through traditional methods such as intravenous administration, under non-gene transfection conditions, to efficiently and specifically achieve selective labeling of glutamatergic neurons in vivo. Furthermore, the probes can be associated with functional molecules that regulate glutamatergic neuron functions (e.g., drug molecules that directly bind to, inhibit, or block neuronal receptors, or functional molecules that can be associated with exogenous stimuli, etc.) based on the modified portions they contain, thereby achieving selective functional regulation of neurons in vivo. At the same time, the probes are easy to prepare and low-cost, and the main protein structure is stable. They can penetrate the blood-brain barrier to achieve neural cell labeling and regulation in a very short time, thereby showing broad application value and broad prospects in the fields of brain neurochemistry, neurophysiology, and pathology research, and brain drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 The protein main structure of Example 1 of the present application and the protein modification probe based thereon, the protein modification probe and its characterization, wherein Figure 1 Figure A is a physical image of the protein main structure and protein modified probe after purification, Figure B is an SDS-Page image of the protein main structure, and Figure C is a UV-visible spectrum of the protein main structure and protein modified probe;

[0027] Figure 2 The figure shows the imaging results of GS-FITC binding to glutamatergic neurons by brain microinjection in Example 2 of the present application;

[0028] Figure 3 The blood-brain barrier penetration results of GS-Cy5 after tail vein injection in Example 2 of the present application are shown. Figure 3 The upper part shows the time-dependent results of fluorescence imaging of brain tissue after GS-Cy5 was injected into the tail vein of mice, and the lower part shows the fluorescence imaging results of brain slices after GS-Cy5 was injected into the tail vein of mice;

[0029] Figure 4 The results of regulating nerve cell function after GS-Ce6 was injected into the tail vein in Example 2 of the present application are shown. Figure 4 The top shows the changes in the firing frequency of neurons in the hippocampus recorded by electrophysiological recording (characteristic results). Figure 4 The bottom shows the statistical results of the firing frequency of neurons in the hippocampus recorded by electrophysiological recording (number of cells n>30);

[0030] Figure 5 The following figure shows the imaging results of GS-FITC specifically binding to glutamatergic neurons after incubation of GS-FITC with brain slices in vitro in Example 2 of the present application: S1BF: primary somatosensory cortex, barrel field; S1: primary somatosensory cortex; AuV: secondary auditory cortex, ventral area; IC: internal capsule; CA: hippocampus; VPM / VPL: ventral posteromedial thalamic nucleus; DG: dentate gyrus.

[0031] Figure 6Schematic diagrams showing the generation of reactive oxygen species mediated by oxygen under light conditions by GS (a) and GS-Ce6 (b) in the examples of the present application, as well as the characterization of reactive oxygen species generation and cell activity after treating cells, are shown, where Figure c is GS and Figure d is GS-Ce6. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0033] This application is made based on the following knowledge of the inventors:

[0034] Recent advances in chemical carrier modification, optogenetics, chemogenetics, and exogenous regulation technologies have provided important avenues for intervention and regulation of neural function. For example, bio-orthogonal and field-mediated approaches can be used to label and regulate neural cell function after in situ brain injection. However, optogenetics and chemogenetics, relying on common challenges of genetic manipulation, make it difficult to expand their application and research in large mammals or humans without ethical restrictions. Furthermore, due to the extremely low permeability of the blood-brain barrier, delivery strategies for large-dose small molecule systemic intervention or blood-brain barrier perturbations are often accompanied by significant side effects and face significant biosafety challenges. To bypass the blood-brain barrier and directly intervene in neural activity, exogenous field-responsive regulation strategies have emerged. However, these strategies also face challenges in biosafety due to issues such as brain implantation, in situ injection, slow tissue diffusion of nanoparticles, poor metabolism, and the potential need for viral transfection to overexpress receptors on the cell membrane. These challenges also pose significant biosafety concerns and extremely limited application scenarios.

[0035] Biomacromolecules possess rich topological structures and surface chemistry, capable of "actively" recognizing specific chemical structures based on shape complementarity and weak bond interactions. Protein-protein chemical modification can enable corresponding biochemical functions, such as selective labeling of neural cells or blood-brain barrier penetration. For example, some peptide hormone analogs and monoclonal antibodies targeting transferrin receptors can penetrate the blood-brain barrier by interacting with transferrin receptors on the blood-brain barrier, carrying small molecules or even nanoparticles to circumvent the barrier. Although these biomacromolecules can penetrate the blood-brain barrier to varying degrees, their current applications still face numerous challenges: 1. The preparation of these proteins, especially monoclonal antibodies, is difficult and expensive; 2. Their structural instability makes chemical functionalization difficult, making it difficult to achieve neural function regulation; 3. Similar to small molecules, these proteins struggle to selectively bind to target neural cells after crossing the blood-brain barrier, lacking selectivity for neural labeling and regulation. Therefore, discovering or designing functional probes that are more stable, easily available, blood-brain barrier permeable, and capable of selectively labeling and regulating the function of neural cell types will promote the development of a new generation of targeted labeling and neural function regulation of living neurons in higher mammals that does not require genetic manipulation, and has broad application prospects in brain science research fields such as neuroimaging, neuroregulation, and diagnosis and treatment of neurological diseases.

[0036] In light of this, the inventors of this application, after extensive research and experimental verification, have proposed a protein-modified probe for in vivo selection of specific neural cells with blood-brain barrier permeability. Compared with traditional technologies, the protein-modified probe proposed in the examples of this application is easy to prepare and has strong stability, effectively reducing experimental costs and facilitating chemical functionalization. At the same time, this functional probe can directly penetrate the blood-brain barrier, rapidly achieving brain aggregation and selective labeling of neural cells. In addition, this protein-modified probe can be associated with exogenous stimulus-response strategies, such as enabling precise regulation of neural cell activity through optical fiber implantation.

[0037] In the embodiments of the present application, a protein modification probe comprises a protein body portion and a modification portion. The protein body portion specifically targets the cell membrane of living glutamatergic neurons, and the protein body portion comprises a domain capable of responding to a first exogenous stimulus, such as a coenzyme factor flavin mononucleotide (FMN) that is responsive to light stimulation, which can mediate the production of a first regulatory molecule (e.g., reactive oxygen species) to regulate glutamatergic neuron function. The coding sequence of the protein body portion (also referred to herein as the main structure) comprises the sequence shown in SEQ ID NO: 1, or a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1. The amino acid sequence of the protein body portion, SEQ ID NO: 2, and its coding sequence, SEQ ID NO: 1, are shown below.

[0038] Protein main part coding sequence SEQ ID NO: 1:

[0039]

[0040] The amino acid sequence of the protein is SEQ ID NO: 2:

[0041]

[0042] In an embodiment of the present application, the coding sequence of the protein main structure of the protein modification probe may comprise a sequence having at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99% but less than 100% identity to SEQ ID NO: 1, wherein the sequence has one or more nucleotide mutations compared to SEQ ID NO: 1.

[0043] Specifically, the coding sequence of the protein main structure of the protein modification probe proposed in the embodiments of the present application contains a mutant sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with SEQ ID NO: 1, wherein the mutation includes at least one of substitution, deletion and insertion.

[0044] In some embodiments, the protein main structure of the protein modification probe comprises a mutant sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acid mutations compared to SEQ ID NO: 2. In the embodiments of the present application, unless otherwise specified, except for the mutations indicated, the remaining amino acids of the protein modification probe or its biologically active fragment are the same as SEQ ID NO: 2. In the embodiments of the present application, the protein main structure enables the protein modification probe to penetrate the blood-brain barrier and specifically target the probe to glutamatergic neurons, and can also mediate the production of regulatory molecules in response to exogenous stimuli, thereby simply and easily achieving specific regulation of glutamatergic neurons.

[0045] In some embodiments, the protein modification probe may further comprise a modification moiety comprising a second regulatory molecule capable of regulating glutamatergic neuron function.

[0046] In the embodiments of the present application, "modifying moieties / functional molecules" are modified on the surface of the main protein structure to achieve functional regulation of specific neural cells in vivo and optional fluorescent labeling based on the modifying moieties / functional molecules. In some embodiments, the modifying moieties / functional molecules can include any suitable neural cell activity regulating molecules, in particular glutamatergic neuron activity regulating molecules, such as small molecule drugs, dyes, peptides, antibodies, plasmid DNA, nucleic acids, liposomes, phage particles, superparamagnetic particles, fluorescent dyes, nanoparticles, viruses, quantum dots or magnetic resonance imaging contrast agents. In the embodiments of the present application, the protein modified probe constructed by carrying the modifying moieties / functional molecules on the main protein body can penetrate the blood-brain barrier and contact the cell membrane of living glutamatergic neurons. The modifying moieties therebetween can regulate the function of glutamatergic neurons, thereby achieving effective regulation of glutamatergic neuron function. It is understandable that the modifying moieties modified on the main protein structure can also include other forms or types of functional molecules, as long as they have the desired regulatory function of living neural cells. This application is not intended to limit the specific type of modifying moieties.

[0047] In some embodiments, the second regulatory molecule is a molecule that is responsive to an exogenous stimulus, which may optionally include light, sound, heat, electricity, and / or magnetism. Accordingly, the second regulatory molecule may be a photosensitizer, sonosensitizer, thermosensitizer, or a combination of these properties, such as being both a photosensitizer and a sonosensitizer.

[0048] In some specific embodiments, the exogenous stimulus is light, and the second regulatory molecule is selected from one or more of the following photosensitizers: indocyanine green, dihydrochlorin e6 (Ce6), methylene blue, acridine orange, pyropheophytin-a (Ppa) and its isomers or analogs.

[0049] In other specific embodiments, the exogenous stimulus is sound, and the second regulatory molecule is selected from one or more of the following sonosensitizers: 5-aminolevulinic acid (5-ALA), protoporphyrin IX (PpIX), hematoporphyrin, rose bengal, curcumin, titanium nanoparticles, dihydrochlorin e6, pheobromide-a, ATX-S10 photoporphyrin (4-formyloximethylidene-3-hydroxy-2-vinyl-deuterio-porphynyl(IX)-6,7-dia spartic acid), photoporphyrin II, DCPH-P-Na(I), NPe6 (mono-l-aspartyl dihydrochlorin e6), polyhydroxyfullerene, hypocrellin-B, ZnPcS2P2, methylene blue, sodium chloroporphyrin and its isomers or analogs.

[0050] In an embodiment of the present application, after administering a protein-modified probe, a first and / or second exogenous stimulus corresponding to the probe itself and / or its modified portion can be provided to the brain, for example, light, sound, heat, electricity, and / or magnetism can be provided to the brain, so that the main body of the probe and the modified portion can exert a neuronal regulatory function. Taking the photosensitizer Ce6 as an example, after administering a protein-modified probe containing Ce6 as a modified portion to an individual, a corresponding light signal can be applied to the individual's brain, so that the photosensitizer Ce6 releases neuromodulator molecules, reactive oxygen species (ROS), around glutamatergic neurons under light conditions. By regulating the light intensity and illumination time, controllable synthesis of ROS can be achieved, which is used to mediate and regulate neuronal function based on changes in the environment surrounding neuronal cells.

[0051] In the embodiments of the present application, an exogenous stimulus response element is implanted into the brain to provide the first and / or second exogenous stimulation, for example, an optical fiber, an electrode, etc. is implanted into the brain to provide exogenous stimulation conditions. The present application does not limit the type of implanted element.

[0052] In the embodiment of the present application, the modified portion may further comprise a fluorescent reporter molecule for labeling glutamatergic neurons. The fluorescent molecule may be a first fluorescent molecule containing an isothiocyanate group and / or a second fluorescent molecule containing an NHS group.

[0053] In some embodiments, the first fluorescent molecule containing an isothiocyanate group can be fluorescein isothiocyanate (FITC) or its isomers or analogs and / or rhodamine molecules. In some embodiments, fluorescein isothiocyanate and its isomers can be selected from one or more of the following: fluorescein isothiocyanate (FITC, molecular formula: C 21 H 11 NO5S, molecular weight: 389.39) or its isomers, i.e. 5-fluorescein isothiocyanate (5-FITC) or 6-fluorescein isothiocyanate (6-FITC). In some embodiments, the analog of fluorescein isothiocyanate can be fluorescein (C 20 H 12 O5) and / or fluorescein derivatives, such as carboxyfluorescein (FAM, molecular formula: C 21 H 12O7, molecular weight: 376.32) or its isomers, i.e. 5-FAM, 6-FAM, 5(6)-FAM or 5-FAM,SE (single isomer); 6-carboxyl-2',4,4',5',7,7'-hexachlorofluorescein succinimide ester; 6-carboxyl-4',5'-dichloro-2',7'-dimethoxyfluorescein succinimide ester; 6-carboxyl-2',4,7,7'-tetrachlorofluorescein succinimide ester, etc. In some embodiments, the rhodamine-based molecules can be selected from one or more of the following: rhodamine, rhodamine 6G (molecular formula: C 28 H 31 N2O3Cl, molecular weight: 479.01), Rhodamine 123 (Rhodamine 123, molecular formula: C 21 H 17 ClN2O3, molecular weight: 380.82), Rhodamine B (Rhodamine B, RhB, molecular formula: C 28 H 31 ClN2O3, molecular weight: 479.01). In addition, rhodamine molecules can also include rhodamine derivatives, such as tetramethylrhodamine isothiocyanate, tetramethylrhodamine-5(6)-isothiocyanate, tetramethylrhodamine-5-isothiocyanate (single compound), tetramethylrhodamine-6-isothiocyanate (single compound), rhodamine B isothiocyanate, sulforhodamine B sulfonyl chloride, sulforhodamine 101 sulfonyl chloride, Dansyl sulfonyl chloride, DABSYL sulfonyl chloride, etc.

[0054] In the embodiments of the present application, the second fluorescent molecule containing a succinimidyl ester (NHS) group can be NHS fluorescein or its isomers (such as fluorescein-succinimidyl ester) or analogs, such as rhodamine B-succinimidyl ester, Cy3-NHS activated ester, Cy5-NHS activated ester, Cy5.5-NHS activated ester, Cy7-NHS activated ester, Cy7.5-NHS activated ester, NHS-AlexaFluor 488, NHS-Alexa Fluor 594, NHS-ATTO 550. It is understood that the fluorescent molecules containing fluorescent groups in the embodiments of the present application can be coupled to the main structure of the protein, and the imaging of the labeled site can be achieved by fluorescence excitation at a later stage. It should be noted that the fluorescent molecules proposed in the embodiments of the present application only need to ensure that they contain a fluorescent group (such as an isothiocyanate group or a succinimidyl ester group) that can react with the main structure of the protein, and the present application is not intended to limit the parent structure to which the fluorescent group is connected. In addition, for different fluorophores, those skilled in the art will understand the required excitation light wavelength and perform fluorescence excitation to achieve the fluorescence imaging.

[0055] In some embodiments, the protein backbone is covalently linked to the fluorescent molecule via a primary amine group therebetween. In some embodiments, the fluorescent molecule is a first fluorescent molecule containing an isothiocyanate group, and the primary amine group is covalently linked to the isothiocyanate group of the first fluorescent molecule. In other embodiments, the fluorescent molecule is a second fluorescent molecule containing a succinimidyl ester group, and the primary amine group is covalently linked to the succinimidyl ester group of the second fluorescent molecule. In some embodiments, the primary amine group can be derived from a lysine residue in the protein backbone.

[0056] In some specific embodiments, the protein-modified probe may comprise a protein structure sequence as shown in SEQ ID NO: 2, wherein the N-terminal domain is a neuronal cell membrane binding domain and the C-terminus is fused with a six-histidine affinity fragment. The fluorescent molecules modified on the protein surface may include FITC and / or RhB molecules, which can covalently bind to lysine residues in the protein structure via isothiocyanate groups, thereby modifying the protein surface.

[0057] In some embodiments, the protein main structure and the fluorescent labeling molecule are co-incubated in an aqueous solvent. In some embodiments, the side chain primary amine groups of the lysine residues on the surface of the protein main structure are covalently linked to the isothiocyanate groups of the FITC or RhB molecules. In some embodiments, the aqueous solvent can be a 0.1M sodium bicarbonate aqueous solution with a pH of 8.5±0.5; the incubation concentration of the protein main structure can be 5-10 mg / mL; the incubation concentration of the FITC molecule can be 0.5-1 mg / mL; the co-incubation time of the protein main structure and the FITC molecule can be 1-2 hours, and the co-incubation temperature can be room temperature.

[0058] In other embodiments, the main structure of the protein is covalently linked to the fluorescent molecule through a group with other modifications therein. In some specific embodiments, the main structure of the protein may have: cysteine modification, such as alkylation, maleimide reaction, disulfide modification, etc. with sulfhydryl (-SH) as the target; lysine modification, such as succinimide ester (NHS) reaction, reductive amination, isothiocyanate reaction, etc. with amino (-NH2) as the target; N-terminal and / or C-terminal modification, such as modification on the N-terminal amino (-NH2) and / or C-terminal carboxyl (-COOH), such as NHS ester reaction, transaminase-mediated modification, EDC / NHS coupling and esterification reaction, etc.; and / or tyrosine modification, such as modification with phenolic hydroxyl (-OH) as the target. It is understood that the main structure of the protein in the embodiment of the present application may include appropriate modifications, wherein the modification or part of the modification is used to covalently link with the fluorescent molecule, or to facilitate the connection between the two. The present application does not limit the type of modification on the main structure of the protein.

[0059] In the embodiments of the present application, the protein main body and the modified part can be connected by covalent bonding or non-covalent bonding, for example, by strong chemical covalent interactions or other weak interactions. In some embodiments, covalent bonding can refer to the covalent bonding of amino acid residues of the main structure of the protein with certain groups of the modified part, such as covalent bonding with succinimide esters, maleimide esters, isothiocyanate groups, etc. in fluorescent marker molecules. In some embodiments, weak interaction connection can refer to the connection between the main structure of the protein and the modified part, for example, with the second regulatory molecule in the modified part, through hydrophilic, electrostatic interactions or hydrogen bonds. The present application does not limit the connection method between the main body of the protein and the modified part.

[0060] In the embodiments of the present application, the protein body and the modified portion can also be connected by a linker. In some specific embodiments, in response to the environment surrounding the living glutamatergic neurons, for example, the corresponding environment provided by exogenous stimulation (such as ROS-enriched environment), the linker can responsively release the modified portion (for example, by breaking), thereby achieving efficient and specific regulation of the function of the living glutamatergic neurons. This application does not limit the specific type of linker.

[0061] In the embodiments of the present application, the protein-modified probe can be administered by oral administration, subcutaneous injection, peritoneal administration, intravenous injection, intramuscular injection, intradural injection, sublingual administration, buccal administration, rectal administration, vaginal injection, ocular administration, ear administration, nasal administration, transdermal administration, subconjunctival administration and / or brain injection such as intrathecal administration. In the embodiments of the present application, since the protein-modified probe has blood-brain barrier permeability, it is only necessary to enter the blood through conventional methods such as intravenous administration, digestive system or respiratory system administration to achieve accessibility to living glutamatergic neurons, thereby achieving simple and rapid drug delivery without the need for genetic manipulation, and can be effectively used for in vivo selection and functional regulation of glutamatergic neurons.

[0062] In some embodiments, the protein modified probe or a drug containing it as an active substance can be administered by intravenous injection or brain injection. In some embodiments, based on intravenous injection, the concentration of the protein modified probe or the protein modified probe contained in the drug can be 5-15 μM, for example, 5 μM, 10 μM, 15 μM, or any concentration therebetween, and the corresponding administration amount can be 200 μL, etc. In some embodiments, the contact time between the protein modified probe and the glutamatergic neurons after administration of such an amount can be 1-8 hours, preferably 2 hours. In the embodiment of the present application, the protein modified probe can be injected intravenously to achieve brain aggregation, penetrate the blood-brain barrier and bind to nerve cells, and then achieve nerve cell excitability regulation after exogenous light stimulation. The protein modified probe proposed in the embodiment of the present application is a living probe for nerve cells with blood-brain barrier permeability, which can complete the labeling of brain nerve cells by intravenous injection, and promote the diagnosis and treatment research based on living neurochemical regulation and nervous system diseases.

[0063] In other embodiments, based on brain injection, the concentration of the protein-modifying probe or the protein-modifying probe contained in the drug can be 30-50 μM, for example, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, or any concentration therebetween, and the corresponding administration volume can be 3 μL, etc. In some embodiments, after administration of such an amount, the contact time between the protein-modifying probe and the glutamatergic neurons can be 1-4 hours, preferably 1 hour.

[0064] It is understood that the dosage can be determined based on the type of drug using the protein modification probe as an active ingredient and various related factors (such as the disease to be treated, the route of administration, the patient's age, gender, and weight, and the severity of the disease). Specifically, the preferred dosage can be increased or decreased based on the route of administration, the severity of the disease, gender, weight, age, etc., and therefore, it is not intended to limit the scope of the dosage of the present invention by any method.

[0065] In the examples of the present application, "living body" refers to a living animal subject / individual.

[0066] In the embodiments of the present application, the "subject / individual" can be a mammal, such as a human or a non-human primate, or can be a mouse, rat, dog, cat, horse or cow.

[0067] In the embodiments of the present application, "glutamatergic neurons" may be hippocampal glutamatergic neurons, for example, glutamatergic neurons in the dentate gyrus or CA3 region of the hippocampus, such as mouse glutamatergic neurons. It is understood that glutamatergic neurons in these locations are relatively concentrated, thereby effectively achieving in vivo selection and functional regulation of glutamatergic neurons.

[0068] The protein-modified probes proposed in the examples of the present application, the methods for regulating the function of glutamatergic neurons using the probes, and the drugs prepared therefrom can penetrate the blood-brain barrier and bind to the cell membranes of nerve cells to exert regulatory functions. According to the physicochemical properties of the modified functional molecules, the protein-modified probes can be used as fluorescent probes for in vivo imaging of specific neuronal cells in the brain on the one hand, and can be used as stimulus-responsive neural function regulatory elements on the other hand, in conjunction with exogenous means such as optical fibers to achieve neural excitability regulation. The imaging and regulatory processes in which protein-modified probes participate do not require genetic manipulation, have high biosafety, and have a wide range of applications. At the same time, as non-monoclonal antibody membrane-labeled probes, the protein-modified probes have many advantages, such as stability and availability, selectivity for neural cell types, short processing time, high labeling efficiency, and low cost. They can be effectively used in the functional regulation of mammalian nerve cells and the diagnosis and treatment of nervous system diseases.

[0069] The present invention also provides examples of a blood-brain barrier-penetrating protein-modified probe, such as described in any of the examples herein, for use in the preparation of a drug for targeted regulation of glutamatergic neuron function. In some embodiments, the drug comprises the protein-modified probe as an active ingredient, which penetrates the blood-brain barrier and contacts the cell membrane of living glutamatergic neurons, thereby regulating glutamatergic neuron function.

[0070] The present application also provides an embodiment of a use of a blood-brain barrier-permeable protein-modified probe, as described in any embodiment of the present application, in the preparation of a drug for diagnosing or treating a neurological disease, wherein the neurological disease is caused by abnormal glutamatergic neuron function or can be treated by regulating the function of the glutamatergic neurons. In the embodiment of the present application, the drug contains the protein-modified probe as an active ingredient, which penetrates the blood-brain barrier and contacts the cell membrane of living glutamatergic neurons, thereby regulating the function of the glutamatergic neurons to achieve the diagnosis and treatment of neurological diseases.

[0071] The embodiments of the present application also provide an in vitro fluorescent labeling method targeting the cell membrane of a nerve cell, comprising: contacting the protein-modified probe described in any of the above embodiments with the nerve cell to fluorescently label the cell membrane of the nerve cell.

[0072] In some embodiments, the protein modification probe can be contacted (incubated) with the neural cells at a suitable temperature and pH. In some embodiments, the contacting can be performed at 18-40° C. In other embodiments, the contacting can be performed at 25-37° C., for example, 32-37° C., such as 32° C., 34° C., or 37° C. In some embodiments, the contacting is performed at a pH of 6.0-8.5, for example, a pH of 7.2-7.4.

[0073] In some embodiments, during the contacting / incubation, the concentration of the protein modification probe can be 0.16-1 μM, for example, 0.3-0.64 μM. In some embodiments, the contacting / incubation time can be 0.5-6 hours. In some embodiments, the contacting / incubation time can be 1-4 hours.

[0074] It can be understood that the experimental conditions required for the protein-modified probes and the in vitro labeling of neural cell membranes based on the protein-modified probes proposed in the embodiments of the present application are simple and easy to control, and therefore easy to implement. In addition, compared with traditional labeling methods (such as antibody-based cell membrane labeling), the protein-modified probes and the methods based on the protein-modified probes proposed in the embodiments of the present application significantly shorten the incubation time required while ensuring accurate and effective cell membrane labeling, thereby effectively improving the experimental efficiency.

[0075] In some embodiments, the contact can be realized in a cell culture medium or an extracellular fluid, and specifically the protein modified probe can be brought into contact with a neural cell or neural cell tissue (such as a brain slice) in a cell culture medium or an extracellular fluid. In some embodiments, the cell culture medium can be a balanced salt solution, a natural cell culture medium, a synthetic cell culture medium, a serum-free cell culture medium, a cell culture medium with limited chemical composition, and the like. In some embodiments, the extracellular fluid can be tissue fluid (abbreviation for interstitial fluid), plasma (the liquid part of blood), lymph, cerebrospinal fluid, etc., wherein the extracellular fluid can be an artificially prepared extracellular fluid or a natural extracellular fluid. It is understandable that the fluorescent protein probe proposed in the embodiment of the present application can be incubated with a neural cell or neural tissue (which can be a tissue slice) in a cell culture medium or an extracellular fluid, and these cell culture media or extracellular fluids are simple and easy to obtain, and the contact (incubation) process is convenient for operation.

[0076] It should be noted that the above explanations of the protein-modified probe embodiment are also applicable to the method of regulating glutamatergic neuron function using the probe in the above embodiment, and its pharmaceutical use, etc., and will not be repeated here.

[0077] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0078] Unless otherwise specified, the quantitative analysis experiments in the following examples were repeated at least three times, and the results were averaged.

[0079] Example 1 - Preparation of protein-modified probes targeting neuronal cell membranes

[0080] 1.1 Preparation of protein main structure

[0081] 1.1.1 Using the base sequence from base pairs 3354192 to 3358862 of the genome of cyanobacterium Synechocystis sp. PCC 6803 as a reference template (corresponding to base pairs 3354192 to 3358862 in NCBI Reference Sequence: NC_000911.1), after codon optimization in Escherichia coli, a protein expression DNA sequence (as shown in SEQ ID NO: 1) capable of transcribing and translating the main structure of the protein targeting the neuronal cell membrane was artificially synthesized;

[0082] 1.1.2 Add NdeI and XhoI restriction sites to the 5' and 3' ends of the protein expression DNA sequence (i.e., SEQ ID NO: 1), respectively. Integrate the protein expression DNA sequence into the pET-20b(+) vector through restriction endonuclease-catalyzed end cleavage reaction and DNA ligase-catalyzed sticky end ligation reaction to prepare a protein expression plasmid with a full length of 8258 base pairs;

[0083] The protein expression plasmid described in 1.1.3 was transfected into Escherichia coli BL21 (DE3) competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat. No. CB105), and the correctly connected recombinant strain was obtained through resistance screening;

[0084] The recombinant strain described in 1.1.4 was induced with 0.5 mM IPTG to express protein at 25°C for 20 hours, and then ultrasonically disrupted and centrifuged to obtain bacterial lysate;

[0085] The bacterial lysate described in 1.1.5 is loaded onto a nickel ion chelate chromatography column, where the target protein having a 6-histidine affinity tag fused to its C-terminus is affinity-bound to the column;

[0086] 1.1.6 Elute the contaminants with phosphate buffer containing 25 mM imidazole, and elute the target protein with phosphate buffer containing 100 mM imidazole;

[0087] 1.1.7 The target protein was concentrated by ultrafiltration and centrifugation, and the protein solution was replaced with 50 mM phosphate buffer (containing 150 mM NaCl) to obtain the purified target protein solution (i.e., the protein main structure GS);

[0088] 1.1.8 Quantify the concentration of the purified target protein solution and store at -80°C for later use.

[0089] The prepared protein main structure solution with the selective labeling performance of nerve cells and its SDS-Page image are shown as follows: Figure 1The results show that the main structure of the protein has extremely high purity after purification and is dispersed in the aqueous solution in the form of monomers with a molecular weight of about 180kDa. The UV-visible spectrum of the protein solution is shown in Figure 1 As shown in Section C of Figure 3, the results indicate that the Fe-S cluster and flavin mononucleotide (FMN) present in the main structure exhibit distinct UV-visible absorption peaks at 345, 417, 440, and 476 nm. Because FMN possesses photosensitivity, converting oxygen into reactive oxygen species under 470 nm illumination, the purified protein main body possesses optically regulated properties after cell labeling. Furthermore, the protein main body exhibits good structural stability and possesses multiple surface-exposed lysine and cysteine residues, making it amenable to rapid chemical functionalization.

[0090] 1.2 Preparation of protein functional probes based on protein main structure

[0091] Based on the protein main structure prepared in Example 1, it is modified with fluorescent groups or chemical functional molecules to prepare protein modification probes and function regulation tools targeting the cell membrane of neural cells. The specific steps are as follows:

[0092] 1.2.1 Disperse the purified protein bulk solution in 0.1 M sodium bicarbonate aqueous solution (pH 8.5) at a final concentration of 10 mg / mL and adjust the volume to 950 μL.

[0093] 1.2.2 Weigh 1 mg of fluorescein isothiocyanate (FITC) or N-hydroxysuccinimide ester-cyanine dye Cy5 (NHS-Cy5) and dissolve it in 50 μL of dimethyl sulfoxide;

[0094] 1.2.3 Add the FITC / Cy5 solution dropwise to the protein bulk solution under stirring and stir in the dark for 1-2 hours;

[0095] 1.2.4 Take a desalting column (PD-10, brand: Cytiva, cat. no.: 17085101) and pre-equilibrate it with 25-50 mL of 50 mM Tris-HCl solution (containing 150 mM NaCl).

[0096] 1.2.5 Purify the FITC / Cy5-labeled protein main structure using a desalting column to remove unreacted FITC molecules to obtain the purified fluorescent protein probe (GS-FITC / GS-Cy5);

[0097] 1.2.6 Concentrate GS-FITC / GS-Cy5 by ultrafiltration centrifugation and wash with the above-mentioned Tris-HCl solution until the lower layer of the ultrafiltration centrifuge tube is free of fluorescence. This is the purified GS-FITC / GS-Cy5.

[0098] 1.2.7 Quantify the protein concentration of the purified GS-FITC / GS-Cy5 solution and store it at -80°C in the dark.

[0099] It can be seen that the protein modification probe proposed in Example 1 of the present application is easy to prepare, low-cost and simple to purify; at the same time, the main structure of the protein is highly stable and can be directly modified with commercial fluorescent molecules by stirring the aqueous solution without reducing its labeling efficiency and specificity.

[0100] 1.2.8 Disperse the purified protein bulk solution in phosphate buffer (pH 8.0) at a final concentration of 10 mg / mL and adjust the volume to 980 μL.

[0101] 1.2.9 Weigh 2 mg of chlorin e6 (Ce6) and dissolve it in 20 μL of dimethyl sulfoxide.

[0102] 1.2.10 Add Ce6 solution dropwise to the protein bulk solution under stirring and stir in the dark for 1 hour.

[0103] 1.2.11 Take a desalting column (PD-10, brand: Cytiva, cat. no.: 17085101) and pre-equilibrate it with 25-50 mL of Tris-HCl solution (containing 150 mM NaCl);

[0104] 1.2.12 Purify the Ce6-labeled protein structure using a desalting column to remove unreacted Ce6 molecules, thereby obtaining the purified fluorescent protein probe (GS-Ce6);

[0105] 1.2.13 Concentrate GS-Ce6 by ultrafiltration centrifugation and wash with the above-mentioned Tris-HCl solution until the lower layer of the ultrafiltration centrifuge tube is colorless;

[0106] 1.2.14 Quantify the protein concentration of the purified GS-FITC / GS-Cy5 solution and store it at -80°C in the dark.

[0107] It can be seen that the protein photosensitive probe proposed in Example 1 of the present application is also easy to prepare, low-cost and simple to purify; at the same time, the main structure of the protein is highly stable, and direct modification with chemical molecules with large conjugated structures can be achieved by stirring the aqueous solution without reducing its labeling efficiency and specificity.

[0108] Example 2 Protein-modified probes mediate the production of regulatory molecules in response to external stimuli

[0109] Based on the protein-modified probe prepared in Example 1, this example investigates the responses of the protein-modified probe body GS and the probe GS-Ce6 with the modified portion Ce6 (denoted as Ce6@GS) to light stimulation, as well as the effects of the reactive oxygen species generated in response to light stimulation on cell viability. The specific steps are as follows:

[0110] Human neuroblastoma SH-SY5Y cells were incubated with the two probes for 4 hours. After the incubation, the cells were washed three times with DPBS solution to remove residual or non-specifically bound protein probes in the solution. The cells incubated with the two probes were irradiated with white light (Ce6@GS) or 470nm blue light (GS) for 10 minutes (Ce6@GS) and 30 minutes (GS). After the illumination, the SH-SY5Y cells were treated with the ROS detection probe DCFDA (10μM) before and after illumination, and the fluorescence intensity of the cells was detected by flow cytometry to analyze the ROS level of the cells.

[0111] The results are as follows Figure 6 shown. Figure 6 Figures a and b are schematic diagrams of GS and GS-Ce6 producing reactive oxygen species in response to light-mediated oxygen, respectively, where the FMN structure in GS and Ce6 in GS-Ce6 act as active structures to mediate the reaction. Figure 6 Figures c and d show intracellular ROS levels measured by the DCFDA assay after cells were labeled with GS and GS-Ce6, respectively, and then exposed to different illumination conditions and durations. The bar graph below shows the activity of SH-SY5Y cells labeled with the corresponding proteins and then exposed to light. These results demonstrate that both GS and GS-Ce6 can generate ROS under different illumination conditions, and that this dose of ROS has no significant effect on cell viability. Further adjustments to illumination conditions can be made to generate the desired ROS, thereby altering the neuronal environment and regulating neuronal function.

[0112] Example 3 In vivo labeling and regulation of mammalian neural cells

[0113] Based on the protein-modified probe prepared in Example 1, this example verified the in vivo selective labeling and functional regulation of mammalian nerve cells. Specifically, the prepared fluorescent protein probe GS-FITC / GS-Cy5 was incubated in brain slices in vitro and microinjected or intravenously injected in situ into the brain in vivo to achieve binding to brain nerve cells.

[0114] 3.1 In vivo selective labeling based on in vitro incubation

[0115] GS-FITC was incubated with brain slices in vitro to explore the ability of GS-FITC to specifically bind to glutamatergic neurons. Figure 5 As shown. Figure 5 As shown, compared to other brain regions, neurons in the CA1, CA2, CA3, and DG regions of the hippocampus showed significantly higher levels of GS-FITC, indicating a specific interaction with GS-FITC. These regions are rich in glutamatergic neurons. These results demonstrate that GS-FITC binds specifically to glutamatergic neurons.

[0116] 3.2 In vivo selective labeling based on in situ microinjection

[0117] After the mouse's head was fixed with a stereotaxic apparatus, 3 μL of GS-FITC (10 mg / mL) solution was injected into the dentate gyrus of the hippocampus. After 0.5-2 h (1 hour was used in this embodiment), the mouse was perfused with physiological saline and 4% paraformaldehyde solution, respectively, and the mouse brain was taken out for sugar precipitation and frozen sectioning. The mouse brain slices were co-stained with immunofluorescence using commercial neuronal anti-NeuN antibody probes or glutamatergic neuron anti-VGlut-1 antibody probes (the two emit different colors of fluorescence under different excitation lights, among which the excitation light wavelength of the secondary antibody after labeling with NeuN antibody (Abcam, EPR12763) or VGlut-1 (Abcam, EPR22269) is 555 nm, and the excitation light wavelength of GS-FITC is 488 nm, so as to distinguish them). The confocal imaging results of brain slice staining are shown in the figure. Figure 2 As shown. Figure 2 It can be seen that GS-FITC has obvious and efficient labeling ability for the dentate gyrus (DG) of the hippocampus, and its fluorescence distribution is basically consistent with the immunofluorescence results of NeuN antibody or VGlut-1, and the fluorescence intensity is comparable, indicating that GS-FITC can achieve efficient and specific labeling of neurons in the hippocampus at the living level.

[0118] 3.3 In vivo selective labeling based on intravenous injection

[0119] 200 μL of GS-Cy5 solution (previously diluted with physiological saline to 1 mg / mL) was injected into the mouse via the tail vein. After circulating in the body for 0.5-8 hours (2 hours in this example), the mouse was perfused with physiological saline and 4% paraformaldehyde solution, and the mouse brain was removed for tissue fluorescence imaging analysis. Figure 3 As shown in the figure above, compared with the mouse brain injected with the same volume of Cy5 molecules or saline, the mouse brain injected with GS-Cy5 has significantly stronger fluorescence, reflecting its brain aggregation performance.

[0120] To further explore the ability of GS-Cy5 molecules to penetrate the blood-brain barrier and its selectivity in binding to nerve cells, the mouse brains after tail vein injection of GS-Cy5 were cryosectioned and confocal fluorescence imaging was performed. Figure 3 As shown in the figure below. Figure 3 As can be seen in the figure below, there are obviously lit-up neurons in the CA1, CA2, and CA3 regions of the hippocampus (marked with arrows), indicating that GS-Cy5 can penetrate the blood-brain barrier and bind to neuronal cells with good labeling selectivity.

[0121] 3.4 Regulation of neuronal function based on intravenous injection

[0122] First, the head of the mouse was fixed with a stereotaxic apparatus, and the electrophysiological array electrodes and optical fibers were implanted into the CA3 brain region of the mouse, and the spontaneous discharge of neurons (control) and the spontaneous discharge frequency under 465nm light (lighting was performed in the first ten seconds of the second minute) were recorded within 3 minutes. Next, 200-300μL GS-Ce6 (diluted to 1mg / mL with physiological saline in advance) solution was injected into the mouse through the tail vein. After circulating in the body for 0.5-8h (2h was used in this embodiment), the spontaneous discharge signals of the neurons were recorded under the conditions of 465nm light and without 465nm light (named GS-Ce6+465nm group and GS-Ce6 group, respectively). The discharge frequency of each group is as follows. Figure 4 As shown in the figure above, compared with the control group, there was no significant difference in the discharge frequency of the 465nm light group alone and the GS-Ce6 injection group alone. However, the discharge frequency of neurons irradiated with GS-Ce6+465nm light began to decrease significantly in the third minute after 10 seconds of light exposure, indicating that the trace hydrogen peroxide produced by GS-Ce6+465nm light significantly inhibited neuronal excitability (some literature reports that trace hydrogen peroxide can inhibit neuronal excitability). By increasing the number of experiments, the discharge frequency of more than 30 neurons was recorded and counted, and divided into 1min, 2min and 3min as self-control for frequency changes. The results are shown in the figure below. Figure 4 As shown in the figure below, the results showed that there was no statistically significant difference in spontaneous discharge frequency at 1, 2, and 3 minutes between the control, GS-Ce6, and 465nm groups. However, the discharge frequency of the GS-Ce6+465nm group decreased by more than 50% at 3 minutes, significantly lower than the discharge frequencies at 1 and 2 minutes. This indicates that GS-Ce6 effectively crosses the blood-brain barrier and reaches glutamatergic neurons, successfully regulating neuronal activity or function in response to external field stimulation.

[0123] As can be seen from this, compared to traditional non-gene transfection type neuronal cell labeling strategy (such as monoclonal antibody and chemical functionalization delivery vector etc.), the protein modified probe in the present application embodiment can realize the aggregation of brain and the in vivo targeted labeling and functional regulation of nerve cell by non-invasive approach (intravenous injection).In addition, the cost height, functionalization difficulty of monoclonal antibody are especially difficult to break through the restriction of blood-brain barrier and arrive target spot, and this protein main structure is stable, can directly realize functionalization by chemical covalent or weak interaction, and realize brain aggregation and the in vivo labeling (1-2h) of nerve cell quickly after entering body.The synthesis of chemical functionalization delivery vector is complicated, often need to modify the structure such as transferrin to realize the penetration of blood-brain barrier, and its larger particle diameter may be accompanied by the reduction of tissue permeability and diffusivity, there is biosafety hidden danger, and as the protein modified probe of biomacromolecule, particle diameter is little, functionalization is simple, tissue diffusivity is strong, and can be degraded and metabolized by biological internal pathway completely, without biocompatibility hidden danger. In addition, this protein probe does not require genetic manipulation of the sample. Compared with the traditional viral transfection method that provides selective intervention, this protein molecular probe has greater potential and application prospects for expansion to non-human primates, and has broad application value in brain neurochemistry, neurophysiology and pathology research, diagnosis and treatment of neurological diseases and other fields.

[0124] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0125] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for targeting and regulating glutamatergic neuron function using a protein-modified probe, characterized in that: The protein modification probe comprises a protein body portion, and the protein body portion specifically targets the cell membrane of living glutamatergic neurons, wherein The protein body portion comprises a structural domain capable of responding to a first exogenous stimulus, wherein the structural domain mediates the production of a first regulatory molecule in response to the first exogenous stimulus to regulate the function of the glutamatergic neuron. The coding sequence of the protein main part comprises the sequence shown in SEQ ID NO: 1 or a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with SEQ ID NO:

1.

2. The method according to claim 1, characterized in that The domain comprises flavin mononucleotide, the first exogenous stimulus comprises light, and the first regulatory molecule comprises reactive oxygen species.

3. The method according to claim 2, characterized in that The protein-modified probe penetrates the blood-brain barrier and contacts the cell membrane of the living glutamatergic neuron, thereby regulating the function of the glutamatergic neuron.

4. The method according to claim 3, characterized in that The protein modification probe further comprises a modification portion, wherein the modification portion comprises a second regulatory molecule capable of regulating the function of the glutamatergic neurons. Optionally, the modification portion is covalently linked or non-covalently linked to the protein body. Optionally, the modification portion is connected to the protein body portion via a linker, and optionally, the linker is capable of releasing the modification portion in response to the surrounding environment of living glutamatergic neurons. Optionally, the second regulatory molecule is selected from: small molecule drugs, dyes, peptides, antibodies, plasmid DNA, nucleic acids, liposomes, phage particles, superparamagnetic particles, fluorescent dyes, nanoparticles, viruses, quantum dots or magnetic resonance imaging contrast agents.

5. The method according to claim 4, characterized in that The second regulatory molecule is a molecule that can respond to a second exogenous stimulus, and the second exogenous stimulus may optionally include light, sound, heat, electricity, and magnetism. Preferably, the second exogenous stimulus is light, and the second regulatory molecule is selected from one or more of the following photosensitizers: indocyanine green, dihydrochlorin e6, methylene blue, acridine orange, pyropheophytin-a and its isomers or analogs; Preferably, the second exogenous stimulus is sound, and the second regulatory molecule is selected from one or more of the following sonosensitizers: 5-aminolevulinic acid, protoporphyrin IX, hematoporphyrin, rose bengal, curcumin, titanium nanoparticles, dihydrochlorin e6, pheobromide-a, ATX-S10 photoporphyrin, photoporphyrin II, DCPH-P-Na(I), NPe6, polyhydroxyfullerene, hypocrellin-B, ZnPcS2P2, methylene blue, sodium chloroporphyrin and its isomers or analogs, Optionally, the method further comprises: providing the first and / or second exogenous stimulation to the brain after administering the protein modification probe, Optionally, the first and / or second exogenous stimulation is provided by implanting an exogenous stimulus-responsive element into the brain, the exogenous stimulus-responsive element optionally comprising an optical fiber and an electrode.

6. The method according to any one of claims 1 to 5, characterized in that The modification portion further comprises a fluorescent reporter molecule, which is optionally a first fluorescent molecule containing an isothiocyanate group and / or a second fluorescent molecule containing a succinimidyl ester (NHS) group. Optionally, the first fluorescent molecule is fluorescein isothiocyanate or its isomers or analogs and / or rhodamine molecules, Optionally, the second fluorescent molecule is NHS fluorescein or its isomer or analogue, preferably Cy3-NHS activated ester, Cy5-NHS activated ester, Cy5.5-NHS activated ester, Cy7-NHS activated ester and / or Cy7.5-NHS activated ester, more preferably Cy3-NHS activated ester and / or Cy5-NHS activated ester.

7. The method according to any one of claims 1 to 6, characterized in that The protein modification probe is administered orally, subcutaneously, intraperitoneally, intravenously, intramuscularly, intradurally, sublingually, buccally, rectally, vaginally, ocularly, otically, nasally, transdermally, subconjunctivally and / or intracerebrally, such as intrathecally. Optionally, the glutamatergic neurons are hippocampal glutamatergic neurons.

8. Use of the protein-modified probe capable of penetrating the blood-brain barrier as defined in any one of claims 1 to 7 in the preparation of a drug for targeted regulation of glutamatergic neuron function. Optionally, the glutamatergic neurons are hippocampal glutamatergic neurons.

9. The use according to claim 8, characterized in that The drug penetrates the blood-brain barrier and contacts the cell membrane of living glutamatergic neurons, thereby regulating the function of the glutamatergic neurons. Preferably, the drug is administered by intravenous injection or brain injection, Optionally, the drug is administered based on intravenous injection, and the concentration of the protein modification probe contained in the drug is 5-10 μM; Optionally, the drug is administered based on brain injection, wherein the concentration of the protein modification probe contained in the drug is 30-50 μM, and the brain injection is optionally performed in the hippocampus, preferably in the dentate gyrus of the hippocampus or the CA3 region of the hippocampus.

10. Use of the protein modification probe as defined in any one of claims 1 to 7 in the preparation of a drug for diagnosing or treating a nervous system disease, characterized in that: The nervous system disease is caused by abnormal function of glutamatergic neurons or can be treated by regulating the function of glutamatergic neurons.

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