Application of GRP94 protein as drug delivery target in construction of Parkinson's disease drug carrier

Abstract

The invention discloses an application of GRP94 protein as a drug delivery target in construction of a Parkinson's disease drug carrier, the expression of GRP94 on a Parkinson's disease nigra dopaminergic neuron cell membrane is highly up-regulated, and the expression on the surface of a normal brain cell is extremely low. The invention discloses application of GRP94 as a target spot in preparation of a targeted delivery system of an anti-Parkinson disease drug. Through targeting GRP94, the carried anti-Parkinson's disease drug can efficiently penetrate through a blood brain barrier to reach brain parenchyma, avoids normal brain cells, specifically targets Parkinson's disease nigra dopaminergic neuron cells, and realizes Parkinson's disease targeted drug delivery. According to the invention, GRP94 is used as a target to be applied to construction of a Parkinson's disease drug carrier and preparation of a targeting delivery system of an anti-Parkinson's disease drug, and compared with other target receptors which cross a blood brain barrier and are used for brain targeting delivery, the targeting GRP94 more specifically targets nigra dopaminergic neurons; and drug accumulation and neurotoxicity in normal intracranial areas are avoided.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to the application of GRP94 protein as a drug delivery target in the construction of a Parkinson's disease drug carrier. Background Art

[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, and its main clinical feature is motor dysfunction. In addition, when clinical symptoms of Parkinson's disease appear, at least 50% of dopaminergic neurons in the substantia nigra die and the dopamine content in the striatum decreases by more than 70-80%.

[0003] The blood-brain barrier (BBB) ​​is a barrier between plasma and brain cells composed of densely connected brain microvascular endothelial cells, pericytes, astrocytes and basement membranes. Its main function is to selectively permeate substances to prevent potentially harmful substances from entering the brain, while allowing nutrients and signal factors to pass normally, thereby maintaining a steady-state brain environment. At present, the main trans-blood-brain barrier transport mechanisms used in the treatment of Parkinson's disease are: passive transport and diffusion, carrier-mediated transport and receptor-mediated transport. Among them, passive transport and diffusion and carrier-mediated transport have high requirements on the molecular structure of drugs, while receptor-mediated transport has high efficiency and strong specificity, and is suitable for large molecule drugs and efficient drug delivery.

[0004] Current studies have shown that the BBB receptors that can be mediated into the brain for the treatment of Parkinson's disease include transferrin receptor (TfR), low-density lipoprotein receptor-related protein 1 (LRP1) and nicotinic acetylcholine receptor (α7-nAChR). However, there are certain risks in mediated entry into the brain through these receptors. The above receptors that can mediate penetration of the BBB are also expressed in normal brain parenchyma areas, such as normal neurons, astrocytes and microglia. There is a wide range of LRP1 and α7-nAChR expression, which makes them lack of targeting of lesion neurons and leads to unnecessary drug accumulation and neurotoxicity in normal areas of the brain. Therefore, it is urgent to discover new drug delivery targets that are only expressed on the blood-brain barrier and the cell membrane of diseased cells, which can effectively avoid normal brain cells and reach the diseased area in the brain parenchyma after penetrating the blood-brain barrier.

[0005] Glucose-regulated protein 94 (GRP94) is expressed on the membrane of brain microvascular endothelial cells. The outer membrane protein A (OmpA) on the surface of nanoparticles wrapped 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 can cause upregulation of GRP94 expression in dopaminergic neurons. At the same time, GRP94 expression on the cell membrane increases. Targeting GRP94 can further target dopaminergic neurons and prevent it from being taken up by other normal cells in the brain, such as astrocytes, microglia, and neurons.

[0006] However, it has some shortcomings. For example, Parkinson's disease is a neurodegenerative disease that requires long-term medication. There may be safety issues with bacterial outer membrane vesicles for long-term drug delivery. At the same time, the preparation of nanoparticles wrapped in bacterial outer membrane vesicles has high technical requirements and high costs. OmpA is a protein macromolecule with a high molecular weight, difficult to synthesize and purify, and difficult to transform. Therefore, some amino acid sequences on the OmpA protein are selected to form the GRP94 targeting polypeptide N modification on the carrier surface, which solves the problems of safety, technical requirements and cost. Therefore, using GRP94 protein as the target for targeted delivery of Parkinson's disease, it is of great significance to develop a new drug delivery strategy for the study of anti-Parkinson's disease and related aspects. Summary of the invention

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

[0008] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions:

[0009] The present invention provides the use of GRP94 protein as a drug delivery target in the construction of a Parkinson's disease drug carrier.

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

[0011] The present invention provides the use of GRP94 protein as a drug delivery target in the preparation of a targeted delivery system for anti-Parkinson's disease drugs.

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

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

[0014] Furthermore, the surface of the delivery vector is modified with a specific ligand of the GRP94 protein using techniques such as chemical connection, electrostatic adsorption, and membrane wrapping.

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

[0016] A GRP94 targeting peptide having a sequence of NGPTHE;

[0017] A GRP94 targeting polypeptide having a sequence of GSVEN;

[0018] Other polypeptides comprising a GRP94 targeting polypeptide sequence or a GRP94 targeting polypeptide sequence;

[0019] A polypeptide obtained by adding one or more amino acids, or reducing one or more amino acids, or replacing any one or more amino acids in the GRP94 targeting polypeptide (N) sequence or the GRP94 targeting polypeptide (G) sequence;

[0020] GRP94 antibody;

[0021] Targeting peptide OmpA;

[0022] Or the nanoparticles were coated with the GRP94-targeted outer membrane of Escherichia coli K1 and Escherichia coli DH5ɑ.

[0023] Furthermore, the delivery carrier includes nanocarriers such as liposome carriers, polymer carriers, nanoparticle carriers, and exosomes.

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

[0025] The present invention provides the use of GRP94 protein as a drug delivery target in the preparation of an anti-Parkinson's disease drug.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention discovered for the first time that GRP94 expression on the surface of substantia nigra dopaminergic neurons is upregulated in Parkinson's disease cells and mouse models, while GRP94 expression on the surface of normal brain cells in the brain is extremely low, proving that GRP94 protein can be used as a new drug delivery target for Parkinson's disease and can specifically target substantia nigra dopaminergic neurons. Therefore, it is expected 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] The present invention uses GRP94 protein as a drug delivery target, and is applied to the construction of Parkinson's disease drug carriers and the preparation of targeted delivery systems for anti-Parkinson's disease drugs. Compared with other target receptors that cross the blood-brain barrier for brain targeted delivery, targeting GRP94 more specifically targets substantia nigra dopaminergic neurons. Since GRP94 protein is only expressed on the blood-brain barrier and the surface of diseased substantia nigra dopaminergic neurons, the delivery system of the present invention can penetrate the blood-brain barrier and accumulate in the Parkinson's disease area by targeting GRP94, thereby avoiding drug accumulation and neurotoxicity in normal intracranial areas.

[0029] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. The specific implementation of the present invention is given in detail by the following embodiments and their accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0031] Figure 1 Schematic diagram of the strategy of targeted drug delivery to substantia nigra dopaminergic neurons in Parkinson's disease by targeting GRP94 in the present invention;

[0032] Figure 2 This is a schematic diagram of the expression of GRP94 protein on different cell membranes in the brain environment and the ratio of co-localization of GRP94 protein and cell membrane characterized by confocal microscopy in Experiment 1 of the present invention;

[0033] Figure 3 This is a diagram for quantitatively characterizing the uptake of different modified fluorescent probe-carrying delivery systems on different cells in Experiment 2 of the present invention;

[0034] Figure 4 This is a schematic diagram of quantitatively characterizing the crossing of the blood-brain barrier by the IR780-loaded delivery system with different modifications in the transwell model in Experiment 3 of the present invention;

[0035] Figure 5This is a schematic diagram of the distribution of IR780-carrying delivery systems with different modifications in normal mice in Experiment 4 of the present invention using small animal imaging, wherein Figure A is a diagram showing the accumulation of nano-drug delivery systems on normal mouse organs as qualitatively characterized by small animal imaging, and Figure B is a quantitative diagram of the brain in Figure A;

[0036] Figure 6 This is a quantitative graph of the expression of GRP94 protein in the Parkinson's disease cell model PC 12 cells and its gray value characterized by Western blot in Experiment 5 of the present invention;

[0037] Figure 7 This is the expression of GRP94 protein on the cell membrane of PC 12 cell model of Parkinson's disease characterized by confocal microscopy in Experiment 5 of the present invention and the quantitative image of co-localization of GRP94 and cell membrane;

[0038] Figure 8 This is a quantitative graph of the expression of GRP94 protein in different brain regions of the Parkinson's disease mouse model and its grayscale value in Experiment 6 of the present invention using Western blot;

[0039] Fig. 9 This is a quantitative graph of the expression of α7-nAChR in different brain regions of the Parkinson's disease mouse model and its grayscale value in Experiment 6 of the present invention using Western blot;

[0040] Fig.10 This is a schematic diagram of the expression of GRP94 protein in neurons in the substantia nigra region of the brain of the Parkinson's disease mouse model characterized by fluorescence microscopy in Experiment 7 of the present invention;

[0041] Fig.11 This is a schematic diagram of the expression of GRP94 protein in neurons in the cerebral cortex, hippocampus, and striatum of a Parkinson's disease mouse model characterized by fluorescence microscopy in Experiment 7 of the present invention;

[0042] Fig.12 This is a schematic diagram of the distribution of differently modified IR780-carrying delivery systems in the substantia nigra of Parkinson's disease model mice in Experiment 8 of the present invention, wherein Figure A is the accumulation of nano-drug delivery systems in the substantia nigra of Parkinson's disease model mice treated with the small animal imaging method, and Figure B is a quantitative diagram of Figure A;

[0043] Fig.13 Schematic diagram of the accumulation of the delivery systems loaded with fluorescent probes with different modifications in Experiment 9 of the present invention in the substantia nigra of the brain of Parkinson's disease model mice, wherein Figure A is the accumulation of the nano drug delivery system in the substantia nigra of the brain of Parkinson's disease model mice characterized by fluorescence microscopy, and Figure B is a quantitative graph of Figure A;

[0044] Fig.14 This is a schematic diagram of the distribution of IR780-loaded delivery systems with different modifications in different brain tissues of Parkinson's disease model mice qualitatively characterized by small animal imaging in Experiment 10 of the present invention, wherein Figure A is the accumulation of different brain tissues of Parkinson's disease model mice treated with nano-drug delivery systems qualitatively characterized by small animal imaging, namely, the cortex, hippocampus, and striatum, and Figure B is a quantitative diagram of Figure A;

[0045] Fig.15 This is a schematic diagram of the accumulation of delivery systems loaded with fluorescent probes with different modifications in different brain tissues of Parkinson's disease model mice characterized by fluorescence microscopy in Experiment 11 of the present invention, wherein Figure A shows the accumulation of nano-drug delivery systems in different brain areas of Parkinson's disease model mice treated with the nano-drug delivery system characterized by fluorescence microscopy, namely the cortex, hippocampus, and striatum, and Figure B is a quantitative graph of Figure A. DETAILED DESCRIPTION

[0046] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings so that the purpose, features and advantages of the invention can be more clearly understood. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the present invention, but are only intended to illustrate the essential spirit of the technical solution of the present invention.

[0047] In the following description, certain specific details are set forth for the purpose of illustrating various disclosed embodiments to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with the present application may not be 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 "comprise" and variations such as "include" and "have" should be construed in an open, inclusive sense, ie, should be interpreted as "including, but not limited to."

[0049] References throughout the specification to "one 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. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0050] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that the term "or" is generally employed in its sense including "and / or" unless the context clearly dictates otherwise.

[0051] In addition, 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] The present invention provides the use of GRP94 protein as a drug delivery target in the construction of a Parkinson's disease drug carrier. The GRP94 protein is highly upregulated on the cell membrane of substantia nigra dopaminergic neurons in Parkinson's disease, but is extremely lowly expressed on the surface of normal brain cells.

[0053] The present invention also provides the use of GRP94 protein as a drug delivery target in the preparation of a targeted delivery system for anti-Parkinson's disease drugs, thereby designing a targeted delivery strategy for substantia nigra dopaminergic neurons of Parkinson's disease based on targeting GRP94. Figure 1 As shown, this targeted delivery strategy can target the GRP94 protein through a targeted delivery system, so that the anti-Parkinson's drug carried can efficiently penetrate the blood-brain barrier to reach the brain parenchyma, avoid normal brain cells, and specifically target the substantia nigra dopaminergic neurons of Parkinson's disease, thereby achieving targeted drug delivery for Parkinson's disease.

[0054] The present invention also provides a targeted delivery system for anti-Parkinson's disease drugs, which includes at least one delivery carrier, which is a nanocarrier including a liposome carrier, a polymer carrier, a nanoparticle carrier, an exosome, etc. The surface of the delivery carrier is modified with a specific ligand of the GRP94 protein by chemical connection, electrostatic adsorption, membrane wrapping, etc.

[0055] The specific ligands of the GRP94 protein include: GRP94 targeting polypeptide (N), the sequence of which is NGPTHE; GRP94 targeting polypeptide (G), the sequence of which is GSVEN; other polypeptides comprising the GRP94 targeting polypeptide (N) sequence or the GRP94 targeting polypeptide (G) sequence; polypeptides obtained by adding one or several amino acids, or reducing one or several amino acids, or replacing any one or several amino acids on the basis of the GRP94 targeting polypeptide (N) sequence or the GRP94 targeting polypeptide (G) sequence; GRP94 antibodies; targeting peptide OmpA; or nanoparticles coated with the GRP94-targeting Escherichia coli K1 and Escherichia coli DH5α outer membranes.

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

[0057] The present invention provides the use of GRP94 protein as a drug delivery target in the preparation of an anti-Parkinson's disease drug.

[0058] The feasibility of the present invention is demonstrated by the following experiments.

[0059] Experiment 1

[0060] In order to investigate the expression of GRP94 protein on various cell membranes in the brain environment, the co-localization ratio of GRP94 protein on different cell membranes (brain microvascular endothelial cells bEND.3, pericytes MBVP, microglia BV-2, astrocytes HA and hippocampal neuron cells HT22) was investigated by confocal microscopy. The experimental results can be found in Figure 2 As shown, Figure 2 Schematic diagram of the expression of GRP94 protein on different cell membranes characterized by confocal microscopy. Figure 2 The numbers in the lower right corner of each small picture indicate the co-localization ratio of GRP94 protein with different cell membranes.

[0061] from Figure 2 As can be seen in the figure, confocal microscopy showed that GRP94 protein was expressed on the membrane of blood-brain barrier cells (brain microvascular endothelial cells bEND.3, pericytes MBVP), and GRP94 protein expression was low on the membrane of brain cells (microglia BV-2, astrocytes HA, hippocampal neuron cells HT22). Therefore, this experiment demonstrated the feasibility of targeted GRP94 penetrating the blood-brain barrier and the potential to escape uptake by brain parenchymal cells.

[0062] Experiment 2

[0063] In order to investigate the uptake of GRP94 protein in various cells in the brain environment, the uptake of different modified fluorescent probe-loaded nanodelivery systems (unmodified nanoparticles (NPs), α7-nAChR targeting peptide RVG-modified nanoparticles (RVG-NPs), GRP94 targeting peptide N-modified nanoparticles (N-NPs)) in different cells (brain microvascular endothelial cells bEND.3, pericytes MBVP, microglia BV-2, astrocytes HA and hippocampal neuronal cells HT22) was quantified by flow cytometry. The basic carrier (NPs) was prepared by using PLGA-PLL) and the surface was 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 inoculated in 6-well plates, and NPs, N-NPs and RVG-NPs loaded with fluorescent probes were added respectively. The concentration of fluorescent probes was 5 μg / mL, and the cells were incubated for 3 hours. The cell uptake was detected by the FL2 channel of the flow cytometer. The experimental results can be found in Figure 3 As shown, Figure 3To quantitatively characterize the uptake patterns of delivery systems loaded with fluorescent probes with different modifications on different cells.

[0064] from Figure 3 It can be seen that compared with RVG-NPs, N-NPs have similar or higher uptake in mouse brain microvascular endothelial cells bEND.3 and pericyte MBVP, and lower uptake in brain parenchymal cells. Therefore, this experiment proves that targeted GRP94 can effectively cross the blood-brain barrier and escape uptake by brain parenchymal cells.

[0065] Experiment 3

[0066] In order to investigate the ability of targeted GRP94 protein to cross the blood-brain barrier, mouse brain microvascular endothelial cells bEND.3 were seeded in 12-well transwell chambers, and different modified IR780-loaded delivery systems (NPs, RVG-NPs, N-NPs) were added and incubated for 6 h. The concentration of IR780 was 4 μg / mL, and the uptake was quantified by measuring the fluorescence intensity. The experimental results can be found in Figure 4 As shown, Figure 4 Figure 5 Quantitative characterization of different modified IR780-loaded delivery systems crossing the blood-brain barrier in a transwell model.

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

[0068] Experiment 4

[0069] In order to investigate the accumulation of nanoparticles in normal mouse brains treated with different modified delivery systems, normal mice were injected with IR780-loaded NPs, RVG-NPs, and N-NPs through the tail vein. After 48 hours of injection, the mice were fixed by perfusion, and the brain tissues were taken. The accumulation of nanoparticles in normal brain tissues was observed using a small animal imaging system. The dose of IR780 was 0.75 mg / kg. The experimental results can be found in Figure 5 As shown, Figure 5 The in vivo organ distribution of IR780-loaded delivery systems with different modifications was characterized by small animal imaging, wherein Figure A is the accumulation diagram on normal mouse organs treated with the nano-drug delivery system qualitatively characterized by small animal imaging, and Figure B is the quantitative diagram of the brain in Figure A.

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

[0071] Experiment 5

[0072] In order to investigate the expression of GRP94 protein in the Parkinson's disease cell model, adrenal medullary pheochromocytoma PC12 cells were inoculated and set up a normal group and a model group. After 24 hours of cell culture, 1-methyl-4-phenylpyridinium ion (MPP+) (final concentration of 1.5mM) was added to the model group for 24 hours to induce the establishment of a Parkinson's disease cell model, and an equal volume of DMSO was added to the normal group. Western blot was used to detect the expression of GRP94 protein in the normal group and the model group. The experimental results can be found in Figure 6 and Figure 7 As shown, Figure 6 This is a quantitative graph of Western blot characterizing the expression of GRP94 protein in Parkinson's disease cell model PC12 cells and its grayscale value. Figure 7 The confocal microscope was used to characterize the expression of GRP94 protein on the cell membrane of PC12 cell model of Parkinson's disease and the quantitative map of co-localization of GRP94 protein and cell membrane.

[0073] from Figure 6 It can be seen that the expression of GRP94 protein 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, a Parkinson's disease cell model. Figure 7 As can be seen in the figure, the expression of GRP94 protein on the cell membrane is upregulated in the Parkinson's disease cell model. Therefore, this experiment shows that targeting GRP94 has the potential to further target dopaminergic neurons in Parkinson's disease.

[0074] Experiment 6

[0075] In order to investigate the expression of GRP94 protein and α7-nAChR in different brain regions of Parkinson's disease model mice, C57BL / 6J mice were randomly divided into a normal group and a model group. Starting from the first day of this experiment, the model group mice were given intraperitoneal injections of 25 mg / kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) every day for 7 days to establish a Parkinson's disease mouse model. The normal group was intraperitoneally injected with the same volume of normal saline. On the eighth day of the experiment, the mice were anesthetized, followed by cardiac perfusion, brain removal, separation of substantia nigra, cortex, hippocampus, and striatum brain tissues, extraction of brain tissue proteins by pipetting with RIPA lysis buffer, detection of sample protein concentration by the BCA method, and investigation of GRP94 protein expression by Western blot. For experimental results, see Figure 8 and Fig. 9 As shown, Figure 8 This is a quantitative graph of Western blot characterization of the expression of GRP94 protein in different brain regions (cortex, hippocampus, striatum, substantia nigra) of Parkinson's disease mouse models and its grayscale value. Fig. 9This is a quantitative graph of Western blot characterizing the expression of α7-nAChR in different brain regions (cortex, hippocampus, striatum, substantia nigra) of Parkinson's disease mouse model and its grayscale value.

[0076] from Figure 8 and Fig. 9 It can be seen that GRP94 protein expression is upregulated in the substantia nigra region, and there is no significant change in expression in other regions, while there is no significant change in α7-nAChR expression. Therefore, this experiment proves that the expression of GRP94 protein in the substantia nigra of the Parkinson's disease mouse model is abnormally elevated, 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] In order to investigate the expression of GRP94 protein in neurons in different brain regions of Parkinson's disease model mice, the expression of GRP94 protein and neurons in Parkinson's disease model mice was investigated by fluorescence microscopy. Fig.10 and Fig.11 As shown, Fig.10 To characterize the expression of GRP94 protein in neurons of the substantia nigra region of the brain of Parkinson's disease mouse models by fluorescence microscopy, Fig.11 Fluorescence microscopy was used to characterize the expression of GRP94 protein in neurons in the cerebral cortex, hippocampus, and striatum of Parkinson's disease mouse models.

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

[0080] Experiment 8

[0081] In order to investigate the distribution of different modified nanosystems in the substantia nigra of Parkinson's disease model mice, Parkinson's disease model mice were injected with IR780-loaded NPs, RVG-NPs, and N-NPs through the tail vein. After 24 hours of injection, the mice were perfused and fixed. The brain tissues were taken 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. The experimental results can be found in Fig.12 As shown, Fig.12This is a schematic diagram of the distribution of differently modified IR780-loaded delivery systems in the substantia nigra of Parkinson's disease model mice qualitatively characterized by small animal imaging, wherein Figure A shows the accumulation in the substantia nigra of Parkinson's disease model mice treated with the nanodrug delivery system qualitatively characterized by small animal imaging, and Figure B is a quantitative graph of Figure A.

[0082] from Fig.12 It can be seen that N-NPs can effectively accumulate in the substantia nigra region, which is significantly increased compared to RVG-NPs.

[0083] Experiment 9

[0084] In order to investigate the distribution of different modified nanoparticle systems in the substantia nigra of Parkinson's disease model mice at the microscopic level, the Parkinson's disease model mice were injected with fluorescent probe-loaded NPs, RVG-NPs, and N-NPs through the tail vein twice, with an interval of 12 hours. The second injection was followed by perfusion 12 hours later. The mouse brain tissue was obtained, dehydrated with sucrose, and frozen sections were cut. The distribution of nanoparticles in the substantia nigra was observed using a fluorescence microscope. The dose of fluorescent probe injected each time was 5 mg / kg. The experimental results are shown in Fig.13 As shown, Fig.13 Figure 3 shows the accumulation of differently modified fluorescent probe-loaded delivery systems in the substantia nigra of Parkinson's disease model mice. Figure A shows the accumulation of the nano-drug delivery system in the substantia nigra of Parkinson's disease model mice treated with the nano-drug delivery system as characterized by fluorescence microscopy, and Figure B is a quantitative graph of Figure A.

[0085] from Fig.13 It can be seen that N-NPs can effectively accumulate in the substantia nigra, which is significantly more than RVG-NPs. Therefore, this experiment shows that the abnormal expression of GRP94 protein in the substantia nigra in Parkinson's disease model mice can make the GRP94 targeted delivery system specifically target the substantia nigra, increase the accumulation of drugs in the substantia nigra, and increase the efficiency of drug delivery.

[0086] Experiment 10

[0087] In order to investigate the distribution of targeted GRP94 nanosystem in different brain regions of Parkinson's disease model mice, IR780-loaded NPs, RVG-NPs, and N-NPs were injected into the tail vein of Parkinson's disease model mice, and perfusion fixation was performed 24 hours after injection. The brain tissue of mice was taken, and the substantia nigra, cortex, hippocampus, and striatum brain tissue were separated. The distribution of nanoparticles in different brain tissues was observed using a small animal imaging system. The dose of IR780 was 0.75 mg / kg. The experimental results can be found in Fig.14 As shown, Fig.14Small animal imaging method was used to qualitatively characterize the distribution of IR780-loaded delivery systems in different regions of brain tissue. Figure A shows the accumulation of nano-drug delivery systems treated with the small animal imaging method in different brain regions of Parkinson's disease model mice, namely the cortex, hippocampus, and striatum. Figure B is a quantitative graph of Figure A.

[0088] from Fig.14 It can be seen that N-NPs have a significant reduction in the hippocampus compared with RVG-NPs.

[0089] Experiment 11

[0090] In order to investigate the distribution of the targeted GRP94 nanoparticle system in different brain regions of Parkinson's disease model mice at a microscopic level, the Parkinson's disease model mice were injected with fluorescent probe-loaded NPs, RVG-NPs, and N-NPs through the tail vein. The injections were repeated twice, with an interval of 12 hours. The second injection was followed by perfusion 12 hours later. The brain tissue of the mice was obtained, dehydrated with sucrose, and frozen sections were cut. The distribution of nanoparticles in different brain regions was observed using a fluorescence microscope. The dose of the fluorescent probe injected each time was 5 mg / kg. The experimental results are shown in Fig.15 As shown, Fig.15 Fluorescence microscopy was used to characterize the accumulation of differently modified fluorescent probe-loaded delivery systems in different areas of brain tissue in Parkinson's disease model mice.

[0091] from Fig.15 It can be seen that N-NPs have a significant reduction in the hippocampus compared to RVG-NPs. Therefore, this experiment proves that by targeting GRP94, it is possible to avoid normal brain areas and specifically target substantia nigra dopaminergic neurons.

[0092] The above 11 experiments showed that the abnormally increased 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, increase drug accumulation in the substantia nigra, increase drug delivery efficiency, and reduce unnecessary drug accumulation and neurotoxicity in normal areas of the brain.

[0093] The present invention discovered for the first time that GRP94 expression on the surface of substantia nigra dopaminergic neurons is upregulated in Parkinson's disease cells and mouse models, while GRP94 expression on the surface of normal brain cells in the brain is extremely low, proving that GRP94 protein can be used as a new drug delivery target for Parkinson's disease and can specifically target substantia nigra dopaminergic neurons. Therefore, it is expected 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 only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

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

2. The use according to claim 1, characterized in that: The GRP94 protein is highly upregulated on the cell membrane of dopaminergic neurons in the substantia nigra of Parkinson's disease, but is extremely lowly expressed on the surface of normal brain cells.

3. Application of GRP94 protein as a drug delivery target in the preparation of targeted delivery systems for anti-Parkinson's disease drugs.

4. The use according to claim 3, characterized in that: The targeted delivery system can efficiently penetrate the blood-brain barrier and reach the brain parenchyma by targeting the GRP94 protein, while avoiding normal brain cells and specifically targeting the substantia nigra dopaminergic neurons of Parkinson's disease, thereby achieving targeted drug delivery for Parkinson's disease.

5. A targeted delivery system for anti-Parkinson's disease drugs, characterized in that: It comprises at least one delivery vector, the surface of which is modified with a specific ligand of the GRP94 protein.

6. The targeted delivery system for anti-Parkinson's disease drugs according to claim 5, characterized in that: The surface of the delivery vector is modified with a specific ligand of the GRP94 protein using techniques including chemical connection, electrostatic adsorption, and membrane wrapping.

7. The targeted delivery system for anti-Parkinson's disease drugs according to claim 5, characterized in that: The specific ligands of the GRP94 protein include: GRP94 targeting peptide with the sequence of NGPTHE (N); a GRP94 targeting polypeptide having a sequence of GSVEN (G); Other polypeptides comprising a GRP94 targeting polypeptide (N) sequence or a GRP94 targeting polypeptide (G) sequence; A polypeptide obtained by adding one or more amino acids, or reducing one or more amino acids, or replacing any one or more amino acids in the GRP94 targeting polypeptide (N) sequence or the GRP94 targeting polypeptide (G) sequence; GRP94 antibody; Targeting peptide OmpA; Or the nanoparticles were coated with the GRP94-targeted outer membrane of Escherichia coli K1 and Escherichia coli DH5ɑ.

8. The targeted delivery system for anti-Parkinson's disease drugs according to claim 5, characterized in that: The delivery carrier is a nanocarrier including a liposome carrier, a high molecular polymer carrier, a nanoparticle carrier, and an exosome.

9. Use of the targeted delivery system for anti-Parkinson's disease drugs according to any one of claims 5 to 8 in targeted drug delivery for Parkinson's disease.

10. Application of GRP94 protein as a drug delivery target in the preparation of anti-Parkinson's disease drugs.

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