Application of GRP94 protein as a drug delivery target in the construction of drug carriers for Parkinson's disease

Abstract

This invention discloses the application of GRP94 protein as a drug delivery target in the construction of drug carriers for Parkinson's disease. GRP94 is highly upregulated on the cell membranes of dopaminergic neurons in the substantia nigra of Parkinson's disease cells, while its expression is extremely low on the surface of normal brain cells. The invention also discloses the application of GRP94 as a target in a targeted delivery system for anti-Parkinson's disease drugs. By targeting GRP94, the delivered anti-Parkinson's drug can efficiently penetrate the blood-brain barrier to reach the brain parenchyma, avoiding normal brain cells and specifically targeting dopaminergic neurons in the substantia nigra of Parkinson's disease cells, thus achieving targeted drug delivery for Parkinson's disease. This invention applies GRP94 as a target in the construction of drug carriers for Parkinson's disease and in the preparation of targeted delivery systems for anti-Parkinson's disease drugs. Compared to other target receptors that cross the blood-brain barrier for targeted brain delivery, targeting GRP94 more specifically targets dopaminergic neurons in the substantia nigra, avoiding drug accumulation and neurotoxicity in normal intracranial regions.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of GRP94 protein as a drug delivery target in the construction of drug carriers for Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease in the world. Its main pathological feature is the death of dopaminergic neurons in the substantia nigra pars compacta, and its main clinical feature is motor dysfunction. Furthermore, when clinical symptoms of Parkinson's disease appear, at least 50% of the dopaminergic neurons in the substantia nigra have died, and the dopamine content in the striatum is reduced by more than 70-80%.

[0003] The blood-brain barrier (BBB) ​​is a barrier between blood plasma and brain cells, composed of densely connected brain microvascular endothelial cells, pericytes, astrocytes, and the basement membrane. Its main function is to selectively allow substances to pass through, preventing potentially harmful substances from entering the brain while allowing nutrients and signaling molecules to pass normally, thereby maintaining brain homeostasis. Currently, the main cross-BBB transport mechanisms used in the treatment of Parkinson's disease include passive transport and diffusion, carrier-mediated transport, and receptor-mediated transport. Passive transport and diffusion and carrier-mediated transport require sophisticated drug molecular structures, while receptor-mediated transport is highly efficient and specific, suitable for large molecule drugs, and delivers drugs efficiently.

[0004] Current research indicates that brain-mediated BBB receptors used to treat Parkinson's disease include transferrin receptor (TfR), low-density lipoprotein receptor-associated protein 1 (LRP1), and nicotinic acetylcholine receptor (α7-nAChR). However, brain delivery via these receptors carries certain risks. These BBB-penetrating receptors are also expressed in normal brain parenchyma regions, such as normal neurons, astrocytes, and microglia, where LRP1 and α7-nAChR are widely expressed. This lack of targeting to lesion neurons leads to unnecessary drug accumulation and neurotoxicity in normal intracranial regions. Therefore, there is an urgent need to discover novel drug delivery targets expressed only on the blood-brain barrier and the cell membranes of diseased cells, capable of penetrating the blood-brain barrier to effectively bypass normal brain cells and reach the lesion area within the brain parenchyma.

[0005] Glucose-regulated protein 94 (GRP94) is expressed on the membranes of brain microvascular endothelial cells. Outer membrane protein A (OmpA) on the surface of nanoparticles encapsulated by bacterial outer membrane vesicles can efficiently cross the blood-brain barrier to reach the brain parenchyma for drug delivery by targeting GRP94. One of the common characteristics of Parkinson's disease is endoplasmic reticulum stress, which upregulates GRP94 expression in dopaminergic neurons and increases GRP94 expression on the cell membrane. Targeting GRP94 can further target dopaminergic neurons and prevent uptake by other normal brain cells, such as astrocytes, microglia, and neurons.

[0006] However, it has some shortcomings. For example, Parkinson's disease, as a neurodegenerative disease, requires long-term medication, and the use of bacterial outer membrane vesicles for long-term drug delivery may pose safety concerns. Furthermore, the preparation of nanoparticles encapsulated by bacterial outer membrane vesicles requires advanced technology and is costly. OmpA, as a large protein molecule, has a high molecular weight, making its synthesis, purification, and transformation difficult. Therefore, selecting a portion of the amino acid sequence from the OmpA protein to construct the GRP94-targeting peptide N, which is then modified onto the carrier surface, solves the safety, technical requirements, and cost issues. Therefore, developing a novel drug delivery strategy targeting Parkinson's disease and related aspects using the GRP94 protein as the target is of paramount importance. Summary of the Invention

[0007] Therefore, the purpose of this invention is to provide the application of GRP94 protein as a drug delivery target in the construction of drug carriers for Parkinson's disease, that is, to use GRP94 protein as a novel drug delivery target for dopaminergic neurons in the substantia nigra of Parkinson's disease, thereby facilitating the design, development and application of GRP94-based targeted delivery strategies for dopaminergic neurons in the substantia nigra of Parkinson's disease, so as to avoid drug accumulation and neurotoxicity in normal intracranial regions.

[0008] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution:

[0009] This invention provides the application of GRP94 protein as a drug delivery target in the construction of drug carriers for Parkinson's disease.

[0010] Furthermore, the GRP94 protein is highly upregulated on the cell membrane of dopaminergic neurons in the substantia nigra of Parkinson's disease, but expressed at extremely low levels on the surface of normal brain cells.

[0011] This invention provides the application of GRP94 protein as a drug delivery target in the preparation of targeted delivery systems for anti-Parkinson's disease drugs.

[0012] Furthermore, the targeted delivery system, by targeting the GRP94 protein, can efficiently penetrate the blood-brain barrier to reach the brain parenchyma with the anti-Parkinson's drug, avoiding normal brain cells, and specifically targeting the dopaminergic neurons in the substantia nigra of Parkinson's disease, thereby achieving targeted drug delivery for Parkinson's disease.

[0013] The present invention provides a targeted delivery system for an anti-Parkinson's disease drug, comprising at least one delivery carrier having a surface modified with a specific ligand for the GRP94 protein.

[0014] Furthermore, techniques such as chemical linkage, electrostatic adsorption, and membrane encapsulation are used to modify the surface of the delivery carrier with specific ligands of the GRP94 protein.

[0015] Furthermore, the specific ligands for the GRP94 protein include:

[0016] A GRP94-targeting polypeptide with the sequence NGPTHE;

[0017] A GRP94-targeting polypeptide with the sequence GSVEN;

[0018] Other peptides containing the GRP94-targeting peptide sequence or the GRP94-targeting peptide sequence;

[0019] A polypeptide obtained by adding one or more amino acids, removing one or more amino acids, or replacing any one or more amino acids based on the GRP94 targeting polypeptide (N) sequence or the GRP94 targeting polypeptide (G) sequence.

[0020] GRP94 antibody;

[0021] Target peptide OmpA;

[0022] Alternatively, the surface of nanoparticles may be coated with the outer membrane of GRP94-targeted E. coli K1 and E. coli DH5α.

[0023] Furthermore, the delivery carrier may be a nanocarrier, including liposome carriers, polymer carriers, nanoparticle carriers, exosomes, etc.

[0024] This invention provides the application of the above-mentioned targeted delivery system for anti-Parkinson's disease drugs in targeted drug delivery for Parkinson's disease.

[0025] This invention provides the application of GRP94 protein as a drug delivery target in the preparation of anti-Parkinson's disease drugs.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention is the first to discover that GRP94 expression is upregulated on the surface of dopaminergic neurons in the substantia nigra in Parkinson's disease cells and mouse models, while GRP94 expression is extremely low on the surface of normal brain cells. This demonstrates that GRP94 protein can serve as a novel drug delivery target for Parkinson's disease, specifically targeting dopaminergic neurons in the substantia nigra. Therefore, it is hoped that GRP94 protein can be used as a new target for the design, development, and application of targeted drug delivery strategies for Parkinson's disease.

[0028] This invention utilizes the GRP94 protein as a drug delivery target in the construction of drug carriers for Parkinson's disease and the preparation of targeted delivery systems for anti-Parkinson's disease drugs. Compared to other target receptors that cross the blood-brain barrier for brain-targeted delivery, targeting GRP94 more specifically targets dopaminergic neurons in the substantia nigra. Since the GRP94 protein is expressed only on the surface of the blood-brain barrier and diseased dopaminergic neurons in the substantia nigra, the delivery system of this invention can penetrate the blood-brain barrier and accumulate in the Parkinson's disease area by targeting GRP94, thus avoiding drug accumulation and neurotoxicity in normal intracranial areas.

[0029] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the invention and to implement it according to the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Specific embodiments of the present invention are given in detail below with reference to the accompanying drawings. Attached Figure Description

[0030] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0031] Figure 1 This is a schematic diagram illustrating the strategy of the present invention for targeted drug delivery to dopaminergic neurons in the substantia nigra of Parkinson's disease by targeting GRP94.

[0032] Figure 2 This is a schematic diagram illustrating the expression of GRP94 protein on different cell membranes in the brain environment and the proportion of colocalization of GRP94 protein with the cell membrane in Experiment 1 of this invention using cofocusing microscopy.

[0033] Figure 3 This is an uptake diagram of different cells for the delivery system of fluorescently loaded probes with different modifications, used in Experiment 2 of this invention to quantitatively characterize the uptake of these probes on different cells.

[0034] Figure 4 This is a schematic diagram illustrating the quantitative characterization of the blood-brain barrier crossing of IR780-loaded delivery systems with different modifications in the transwell model in Experiment 3 of this invention.

[0035] Figure 5This is a schematic diagram of the distribution of IR780-loaded delivery systems with different modifications in organs of normal mice in Experiment 4 of this invention, characterized by small animal imaging. Figure A is an accumulation map of normal mouse organs treated by the nano-drug delivery system, which is qualitatively characterized by small animal imaging, and Figure B is a quantitative map of the brain in Figure A.

[0036] Figure 6 This is a quantitative graph showing the expression of GRP94 protein in PC 12 cells, a Parkinson's disease cell model, and its grayscale values, as determined by Western blot in Experiment 5 of this invention.

[0037] Figure 7 This invention provides a co-focusing microscopy study in Experiment 5 to characterize the expression of GRP94 protein on the cell membrane of the Parkinson's disease cell model PC 12 and to obtain a quantitative map of the colocalization of GRP94 with the cell membrane.

[0038] Figure 8 This is a quantitative graph showing the expression of GRP94 protein in different brain regions of a Parkinson's disease mouse model, as well as its grayscale values, in Experiment 6 of this invention using Western blot.

[0039] Figure 9 This is a quantitative image of the expression and grayscale values ​​of α7-nAChR in different brain regions of a Parkinson's disease mouse model, as determined by Western blot in Experiment 6 of this invention.

[0040] Figure 10 This is a schematic diagram illustrating the expression of GRP94 protein in neurons of the substantia nigra region of the brain of a Parkinson's disease mouse model using fluorescence microscopy in Experiment 7 of this invention.

[0041] Figure 11 This is a schematic diagram illustrating the expression of GRP94 protein in neurons of the cerebral cortex, hippocampus, and striatum in a Parkinson's disease mouse model during Experiment 7 of this invention using fluorescence microscopy.

[0042] Figure 12 This is a schematic diagram of the distribution of IR780-loaded delivery systems with different modifications in the substantia nigra of the brain of a Parkinson's disease model mouse, qualitatively characterized by small animal imaging in Experiment 8 of this invention. Figure A shows the accumulation of the nano-drug delivery system in the substantia nigra of the brain of a Parkinson's disease model mouse treated by the nano-drug delivery system, which is qualitatively characterized by small animal imaging. Figure B is a quantitative diagram of Figure A.

[0043] Figure 13 This is a schematic diagram of the accumulation of different modified fluorescent probe delivery systems in the substantia nigra of the brain of a Parkinson's disease model mouse in Experiment 9 of this invention. Figure A shows the accumulation in the substantia nigra of the brain of a Parkinson's disease model mouse treated with the nano-drug delivery system as characterized by fluorescence microscopy, and Figure B is a quantitative diagram of Figure A.

[0044] Figure 14 This is a schematic diagram of the distribution of different modified IR780-loaded delivery systems in different brain regions of a Parkinson's disease model mouse in Experiment 10 of this invention, using small animal imaging to qualitatively characterize the distribution of these systems. Figure A shows the accumulation of the nano-drug delivery system in different brain regions of the Parkinson's disease model mouse treated with it, namely the cortex, hippocampus, and striatum. Figure B is a quantitative diagram of Figure A.

[0045] Figure 15 This is a schematic diagram of the accumulation of different modified fluorescent probe delivery systems in different brain regions of Parkinson's disease model mice under fluorescence microscopy in Experiment Eleven of this invention. Figure A shows the accumulation in different brain regions of Parkinson's disease model mice treated with the nano-drug delivery system under fluorescence microscopy, namely the cortex, hippocampus, and striatum. Figure B is a quantitative diagram of Figure A. Detailed Implementation

[0046] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings to provide a clearer understanding of the invention's purpose, features, and advantages. It should be understood that the embodiments shown in the drawings are not intended to limit the scope of the invention, but are merely illustrative of the essential spirit of the invention's technical solution.

[0047] In the following description, certain specific details are set forth for the purpose of illustrating various disclosed embodiments in order to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the art will recognize that embodiments may be practiced without one or more of these specific details. In other instances, well-known apparatuses, structures, and techniques associated with this application may not have been shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0048] Unless the context requires otherwise, throughout the specification and claims, the word “comprising” and its variations, such as “including” and “having”, shall be understood to have an open, inclusive meaning, that is, to be interpreted as “including, but not limited to”.

[0049] Throughout this specification, references to "an embodiment" or "an embodiment" indicate that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Therefore, the appearance of "in an embodiment" or "an embodiment" in various places throughout the specification does not necessarily refer to the same embodiment. Furthermore, a particular feature, structure, or characteristic may be combined in any manner in one or more embodiments.

[0050] The singular forms “a” and “the” used in this specification and the appended claims include plural references unless otherwise expressly stated herein. It should be noted that the term “or” is generally used to mean “and / or” unless otherwise expressly stated herein.

[0051] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0052] This invention provides the application of GRP94 protein as a drug delivery target in the construction of drug carriers for Parkinson's disease. The GRP94 protein is highly upregulated on the cell membranes of dopaminergic neurons in the substantia nigra of Parkinson's disease cells, but expressed at extremely low levels on the surface of normal brain cells.

[0053] This invention also provides the application of GRP94 protein as a drug delivery target in the preparation of targeted delivery systems for anti-Parkinson's disease drugs, thereby enabling the design of targeted delivery strategies for GRP94-targeted dopaminergic neurons in the substantia nigra of Parkinson's disease. See also Figure 1 As shown, this targeted delivery strategy can target the GRP94 protein through a targeted delivery system, thereby efficiently penetrating the blood-brain barrier to reach the brain parenchyma with the anti-Parkinson's drug, avoiding normal brain cells, and specifically targeting the dopaminergic neurons in the substantia nigra of Parkinson's disease, thus achieving targeted drug delivery for Parkinson's disease.

[0054] This invention also provides a targeted delivery system for anti-Parkinson's disease drugs. This system includes at least one delivery carrier, which is a nanocarrier including liposomes, polymers, nanoparticles, exosomes, etc. Furthermore, the surface of the delivery carrier is modified with specific ligands for the GRP94 protein through techniques such as chemical bonding, electrostatic adsorption, and membrane encapsulation.

[0055] The specific ligands for the GRP94 protein include: a GRP94 targeting polypeptide (N) with the sequence NGPTHE; a GRP94 targeting polypeptide (G) with the sequence GSVEN; other polypeptides containing the sequences of GRP94 targeting polypeptide (N) or GRP94 targeting polypeptide (G); polypeptides obtained by adding or removing one or more amino acids, or replacing any one or more amino acids, based on the sequences of GRP94 targeting polypeptide (N) or GRP94 targeting polypeptide (G); a GRP94 antibody; a targeting peptide OmpA; or the outer membrane of GRP94-targeting Escherichia coli K1 and Escherichia coli DH5α coated on the surface of nanoparticles.

[0056] This invention provides the application of the above-mentioned targeted delivery system for anti-Parkinson's disease drugs in targeted drug delivery for Parkinson's disease.

[0057] This invention provides the application of GRP94 protein as a drug delivery target in the preparation of anti-Parkinson's disease drugs.

[0058] The feasibility of this invention is demonstrated through the following experiments.

[0059] Experiment 1

[0060] To investigate the expression of GRP94 protein on various cell membranes in the brain environment, confocal microscopy was used to examine the proportion of GRP94 protein co-localization on different cell membranes (brain microvascular endothelial cells bEND.3, pericytes MBVP, microglia BV-2, astrocytes HA, and hippocampal neurons HT22). The experimental results are shown below. Figure 2 As shown, Figure 2 This is a schematic diagram illustrating the expression of GRP94 protein on different cell membranes as characterized by confocal microscopy. Figure 2 The numbers in the bottom right corner of each small image represent the proportions of GRP94 protein co-localization with different cell membranes.

[0061] from Figure 2 As can be seen from the confocal microscopy, GRP94 protein expression was observed on the membranes of blood-brain barrier cells (brain microvascular endothelial cells bEND.3 and pericytes MBVP), while expression was lower on the membranes of brain cells (microglia BV-2, astrocytes HA, and hippocampal neurons HT22). Therefore, this experiment demonstrates the feasibility of targeting GRP94 to penetrate the blood-brain barrier and potentially evade uptake by brain parenchymal cells.

[0062] Experiment 2

[0063] To investigate the uptake of GRP94 protein in various brain cells, flow cytometry was used to quantify the uptake of different modified fluorescent probe nanoparticles (unmodified nanoparticles (NPs), RVG-modified nanoparticles targeting the α7-nAChR peptide (RVG-NPs), and N-modified nanoparticles targeting the GRP94 peptide (N-NPs)) in different cells (brain microvascular endothelial cells bEND.3, pericytes MBVP, microglia BV-2, astrocytes HA, and hippocampal neurons HT22). Basic vectors (NPs) were prepared using (acid)-poly(ε-carbobenzoxy-L-lysine), PLGA-PLL, and surface-modified with GRP94-targeting peptide N and α7-nAChR-targeting peptide RVG to form N-NPs and RVG-NPs. Mouse brain microvascular endothelial cells bEND.3, pericytes MBVP, microglia BV-2, astrocytes HA, and hippocampal neurons HT22 were seeded in 6-well plates. NPs, N-NPs, and RVG-NPs loaded with fluorescent probes (5 μg / mL) were added, and the plates were incubated for 3 h. Cell uptake was detected by flow cytometry using the FL2 channel. Experimental results are shown below. Figure 3 As shown, Figure 3This image shows the uptake of fluorescently loaded probes with different modifications on different cells to quantitatively characterize the delivery systems.

[0064] from Figure 3 As can be seen, N-NPs, compared to RVG-NPs, showed similar or higher uptake in mouse brain microvascular endothelial cells (bEND.3) and pericytes (MBVP), but lower uptake in brain parenchymal cells. Therefore, this experiment demonstrates that targeting GRP94 can effectively cross the blood-brain barrier and evade uptake by brain parenchymal cells.

[0065] Experiment 3

[0066] To investigate the ability of GRP94 protein to cross the blood-brain barrier, mouse brain microvascular endothelial cells (bEND.3) were seeded in 12-well transwell plates and incubated for 6 hours with different modified IR780 delivery systems (NPs, RVG-NPs, and N-NPs). The concentration of IR780 was 4 μg / mL. Uptake was quantified by measuring fluorescence intensity. Results are shown below. Figure 4 As shown, Figure 4 This indicates a quantitative characterization of the crossing of the blood-brain barrier by IR780 delivery systems with different modifications in the transwell model.

[0067] from Figure 4 As can be seen, compared with NPs and RVG-NPs, N-NPs have a stronger ability to cross the blood-brain barrier, which is conducive to achieving efficient brain-targeted delivery.

[0068] Experiment 4

[0069] To investigate the accumulation of nanoparticles in the normal mouse brain treated with different modified delivery systems, normal mice were injected intravenously via the tail vein with NPs loaded with IR780, RVG-NPs, and N-NPs, respectively. After 48 hours of injection, the mice were perfused and fixed, and mouse brain tissue was harvested. The accumulation of nanoparticles in normal brain tissue was observed using a small animal imaging system. The dose of IR780 was 0.75 mg / kg. Experimental results are shown below. Figure 5 As shown, Figure 5 To characterize the in vivo organ distribution of IR780-loaded delivery systems with different modifications using small animal imaging, Figure A shows the accumulation map on normal mouse organs treated with the nano-drug delivery system, which was qualitatively characterized by small animal imaging, and Figure B shows the quantitative map of the brain in Figure A.

[0070] from Figure 5 As can be seen, N-NPs accumulate the most in the brain tissue of mice, indicating that N-NPs have the best blood-brain barrier penetration ability.

[0071] Experiment 5

[0072] To investigate the expression of GRP94 protein in a Parkinson's disease cell model, PC12 adrenal medullary pheochromocytoma cells were seeded and divided into a normal control group and a model group. After 24 hours of cell culture, the model group was induced with 1-methyl-4-phenylpyridine ions (MPP+) (final concentration 1.5 mM) for 24 hours to establish a Parkinson's disease cell model, while the normal control group was induced with an equal volume of DMSO. Western blot analysis was performed to detect GRP94 protein expression in both groups. Results are shown below. Figure 6 and Figure 7 As shown, Figure 6 This is a Western blot diagram representing the expression of GRP94 protein in PC12 cells, a Parkinson's disease cell model, and its grayscale values. Figure 7 To characterize the expression of GRP94 protein on the cell membrane of PC12, a Parkinson's disease cell model, using confocal microscopy and to obtain a quantitative map of the colocalization of GRP94 protein with the cell membrane.

[0073] from Figure 6 As can be seen, GRP94 protein expression is upregulated in the Parkinson's disease cell model. Confocal microscopy was used to characterize the expression of GRP94 protein on the cell membrane of PC12 cells, a Parkinson's disease cell model. Figure 7 The results show that GRP94 protein expression is upregulated on the cell membrane in the Parkinson's disease cell model. Therefore, this experiment demonstrates the potential of targeting GRP94 to further target dopaminergic neurons in Parkinson's disease.

[0074] Experiment Six

[0075] To investigate the expression of GRP94 protein and α7-nAChR in different brain regions of a Parkinson's disease model mouse, C57BL / 6J mice were randomly divided into a normal control group and a model group. Starting from day 1 of the experiment, mice in the model group were intraperitoneally injected with 25 mg / kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) daily for 7 days to establish the Parkinson's disease mouse model. The normal control group received the same volume of physiological saline intraperitoneally. On day 8, the mice were anesthetized, followed by cardiac perfusion and brain harvesting. Substantia nigra, cortex, hippocampus, and striatum were isolated. Proteins were extracted from the brain tissue using RIPA lysis buffer, and protein concentration was detected by the BCA method. GRP94 protein expression was assessed by Western blot. The experimental results are shown below. Figure 8 and Figure 9 As shown, Figure 8 This is a Western blot diagram to characterize the expression and grayscale values ​​of GRP94 protein in different brain regions (cortex, hippocampus, striatum, and substantia nigra) of a Parkinson's disease mouse model. Figure 9This is a quantitative map of the expression and grayscale values ​​of α7-nAChR in different brain regions (cortex, hippocampus, striatum, and substantia nigra) of a mouse model of Parkinson's disease, as represented by Western blot.

[0076] from Figure 8 and Figure 9 As can be seen, GRP94 protein expression is upregulated in the substantia nigra region, while expression in other regions shows no significant change, whereas α7-nAChR expression shows no significant change. Therefore, this experiment demonstrates that GRP94 protein expression is abnormally elevated in the substantia nigra of a Parkinson's disease mouse model, indicating that targeting GRP94 has the potential to specifically target the substantia nigra region of Parkinson's disease compared to α7-nAChR.

[0077] Experiment 7

[0078] To investigate the expression of GRP94 protein in neurons of different brain regions in a Parkinson's disease model mouse, fluorescence microscopy was used to examine the expression of GRP94 protein in neurons of the Parkinson's disease model mouse. The experimental results are shown below. Figure 10 and Figure 11 As shown, Figure 10 To characterize the expression of GRP94 protein in neurons of the substantia nigra region of the brain in a mouse model of Parkinson's disease using fluorescence microscopy. Figure 11 To characterize the expression of GRP94 protein in neurons of the cerebral cortex, hippocampus, and striatum in a mouse model of Parkinson's disease using fluorescence microscopy.

[0079] from Figure 10 and Figure 11 As can be seen from the fluorescence microscopy characterization images, GRP94 protein expression was higher in neurons of the substantia nigra region of Parkinson's disease model mice, while expression was lower in neurons of the cortex, hippocampus, and striatum. Therefore, this experiment demonstrates the feasibility of targeting substantia nigra neurons specifically by targeting GRP94, avoiding normal brain cells.

[0080] Experiment 8

[0081] To investigate the distribution of different modified nanosystems in the substantia nigra of a Parkinson's disease model mouse brain, mice were injected intravenously with IR780-loaded nanoparticles (NPs), RVG-NPs, and N-NPs, respectively. After 24 hours of injection, the mice were perfused and fixed. Brain tissue was harvested, and the substantia nigra, cortex, hippocampus, and striatum were separated. The distribution of nanoparticles in the substantia nigra was observed using a small animal imaging system. The dose of IR780 was 0.75 mg / kg. Experimental results are shown below. Figure 12 As shown, Figure 12This diagram illustrates the distribution of IR780-loaded delivery systems with different modifications in the substantia nigra of a Parkinson's disease model mouse brain, as qualitatively characterized by small animal imaging. Figure A shows the accumulation of the nano-drug delivery system in the substantia nigra of the Parkinson's disease model mouse brain treated by the nano-drug delivery system, which is qualitatively characterized by small animal imaging. Figure B is a quantitative representation of Figure A.

[0082] from Figure 12 As can be seen, N-NPs can be effectively accumulated in the substantia nigra region, and the amount is significantly increased compared to RVG-NPs.

[0083] Experiment Nine

[0084] To investigate the distribution of differently modified nanoparticle systems in the substantia nigra of a Parkinson's disease model mouse brain at the microscopic level, mice were injected twice via tail vein with fluorescent probe-loaded NPs, RVG-NPs, and N-NPs, 12 hours apart. Twelve hours after the second injection, the mice were perfused, and brain tissue was harvested, dehydrated with sucrose, frozen sections were prepared, and the distribution of nanoparticles in the substantia nigra was observed using a fluorescence microscope. The dose of the fluorescent probe was 5 mg / kg for each injection. Experimental results are shown below. Figure 13 As shown, Figure 13 The images show the accumulation of different modified fluorescent probe delivery systems in the substantia nigra of the brain of a Parkinson's disease model mouse. Figure A shows the accumulation in the substantia nigra of the brain of a Parkinson's disease model mouse treated with the nano-drug delivery system as characterized by fluorescence microscopy, and Figure B is a quantitative representation of Figure A.

[0085] from Figure 13 As can be seen, N-NPs can effectively accumulate in the substantia nigra region, showing a significant increase compared to RVG-NPs. Therefore, this experiment demonstrates that the abnormally elevated expression of GRP94 protein in the substantia nigra region in Parkinson's disease model mice can enable the GRP94 targeted delivery system to specifically target the substantia nigra region, increasing drug accumulation in the substantia nigra and enhancing drug delivery efficiency.

[0086] Experiment 10

[0087] To investigate the distribution of GRP94-targeted nanosystems in different brain regions of a Parkinson's disease model mouse, mice were injected intravenously with IR780-loaded nanoparticles (NPs), RVG-NPs, and N-NPs, respectively. After 24 hours of injection, the mice were perfused and fixed. Brain tissue was harvested, and the substantia nigra, cortex, hippocampus, and striatum were separated. The distribution of the nanoparticles in different brain tissues was observed using a small animal imaging system. The dose of IR780 was 0.75 mg / kg. Experimental results are shown below. Figure 14 As shown, Figure 14To qualitatively characterize the distribution of different brain tissue regions in a small animal imaging system using a delivery system carrying IR780 with different modifications, Figure A shows the accumulation in different brain regions of a Parkinson's disease model mouse treated with the nano-drug delivery system, namely the cortex, hippocampus, and striatum, as characterized by small animal imaging. Figure B is a quantitative map of Figure A.

[0088] from Figure 14 As can be seen, N-NPs are significantly reduced in the hippocampus compared to RVG-NPs.

[0089] Experiment Eleven

[0090] To investigate the distribution of the GRP94-targeted nanoparticle system in different brain regions of a Parkinson's disease model mouse at the microscopic level, mice were injected twice via tail vein with fluorescent probes NPs, RVG-NPs, and N-NPs, 12 hours apart. Twelve hours after the second injection, the mice were perfused, and brain tissue was harvested, dehydrated with sucrose, frozen sections were prepared, and the distribution of the nanoparticles in different brain regions was observed using a fluorescence microscope. The dose of the fluorescent probe was 5 mg / kg for each injection. Experimental results are shown below. Figure 15 As shown, Figure 15 To characterize the accumulation of differently modified fluorescent probe delivery systems in different brain regions of a Parkinson's disease model mouse using fluorescence microscopy.

[0091] from Figure 15 As can be seen, N-NPs are significantly reduced in the hippocampus compared to RVG-NPs. Therefore, this experiment demonstrates that targeting GRP94 can bypass normal brain regions and specifically target dopaminergic neurons in the substantia nigra.

[0092] The above 11 experiments demonstrate that the abnormal increase in GRP94 protein expression in the substantia nigra region of Parkinson's disease model mice can enable the GRP94 targeted delivery system to specifically target the substantia nigra region, increase drug accumulation in the substantia nigra, increase drug delivery efficiency, and reduce unnecessary drug accumulation and neurotoxicity in normal intracranial areas.

[0093] This invention is the first to discover that GRP94 expression is upregulated on the surface of dopaminergic neurons in the substantia nigra in Parkinson's disease cells and mouse models, while GRP94 expression is extremely low on the surface of normal brain cells. This demonstrates that GRP94 protein can serve as a novel drug delivery target for Parkinson's disease, specifically targeting dopaminergic neurons in the substantia nigra. Therefore, it is hoped that GRP94 protein can be used as a new target for the design, development, and application of targeted drug delivery strategies for Parkinson's disease.

[0094] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of a GRP94 targeting polypeptide with the sequence of NGPTHE as a specific targeting ligand in the preparation of a drug carrier for Parkinson's disease.

2. Use of a GRP94 targeting polypeptide with the sequence of NGPTHE as a specific targeting ligand in the preparation of a targeted delivery system for an anti-Parkinson's disease drug.

3. A targeted delivery system for anti-Parkinsonian drugs, characterized in that, The delivery carrier is surface-modified with a specific targeting ligand of a GRP94 targeting polypeptide with the sequence of NGPTHE.

4. The targeted delivery system of anti-Parkinson drugs according to claim 3, characterized in that, The surface of the delivery carrier is modified with a specific targeting ligand of a GRP94 protein by chemical linkage, electrostatic adsorption or membrane wrapping technology.

5. The targeted delivery system of anti-Parkinson drugs as claimed in claim 3, wherein, The delivery carrier is a nano-carrier, including a liposome carrier, a high-molecular polymer carrier, a nanoparticle carrier or an exosome.

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