Anti-dat antibodies and compositions thereof

Abstract

To provide anti-DAT antibodies and compositions thereof for slowing the progression of Parkinson's disease.SOLUTION: Anti-DAT antibodies are formed by transcription and translation of gene fragments containing specific sequences. The anti-DAT antibody provided by the present invention is used for labeling a dopamine transporter. The anti-DAT antibody provided by the present invention is capable of forming a composition that crosses the blood-brain barrier, is capable of specifically binding to dopaminergic neurons, and is capable of specifically binding to cells in the striatum The use of reducing the deposition of α - synuclein protein in the brain region is achieved, and the excellent effect of delaying the progress of Parkinson's disease is exerted.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to antibodies, and in particular to anti-DAT antibodies and compositions thereof. [Background technology]

[0002] Parkinson's disease (PD) is a common disorder characterized by degeneration of the central nervous system (CNS) in the brain. Symptoms of the disease typically appear gradually over time and affect the motor nervous system, but no cure exists. Traditionally, high-dose administration of dopamine (L-dopa) has been used to slow the progression of the disease. However, the therapeutic effect of dopamine gradually declines due to neuronal degeneration, neuronal loss, and the blood-brain barrier. Furthermore, after the central nervous system in the cerebral cortex is uniformly processed by high-dose dopamine, it is difficult to avoid the drug affecting the physiological mechanisms of the central nervous system in other brain regions. Summary of the Invention [Problem to be solved by the invention]

[0003] In view of this, the development of new technologies for inhibiting the progression of Parkinson's disease has become an urgent issue in related fields. Therefore, an object of the present invention is to provide an anti-DAT antibody and a composition thereof for slowing the progression of Parkinson's disease. [Means for solving the problem]

[0004] In order to develop a related technology that can slow the progression of Parkinson's disease, the present invention provides an anti-DAT antibody formed by transcription and translation of a gene fragment comprising SEQ ID No:2 and used to label the dopamine transporter.

[0005] The anti-DAT antibodies provided by the present invention can optionally be formed into compositions containing the target gene SEQ ID No:3, in which any portion of the target gene SEQ ID No:3 is inserted into the gene fragment SEQ ID No:2.

[0006] The anti-DAT antibodies provided by the present invention can further form compositions containing extracellular vesicles in which the anti-DAT antibodies are bound to transmembrane proteins, if necessary.

[0007] In the present invention, the extracellular vesicles are secreted by cells into which a vector gene has been transfected, and at least a portion of the vector gene includes SEQ ID No: 2 or SEQ ID No: 4.

[0008] In the present invention, the dopamine transporter antibody is formed in the extracellular loop between the third and fourth transmembrane domains of the transmembrane protein.

[0009] In the present invention, the transmembrane protein is CD63, and the cells are HEK-293 cells.

[0010] In the present invention, the extracellular vesicles are loaded with drugs, which include, but are not limited to, gene fragments (DNA, RNA), protein sequences, or chemical agents.

[0011] The anti-DAT antibodies provided by the present invention can carry drugs and pass through the blood-brain barrier, specifically bind to dopamine neurons, and regulate the secretion of Parkinson's disease marker proteins and reduce the deposition of α-synuclein protein in the striatum, a brain region where dopamine neurons are degenerated or damaged, thereby demonstrating excellent therapeutic effects in slowing the progression of Parkinson's disease. [Effects of the Invention]

[0012] The anti-DAT antibodies and compositions thereof provided by the present invention are effective in slowing the progression of the symptoms of Parkinson's disease. [Brief explanation of the drawings]

[0013] [Figure 1] 1 is a step flow chart of a preferred embodiment of the targeted carrier provided by the present invention. [Figure 2A] FIG. 1 shows the results of protein expression in a first preferred embodiment of the targeted carrier provided by the present invention. [Figure 2B] FIG. 1 shows the results of protein expression in a first preferred embodiment of the targeted carrier provided by the present invention. [Figure 3A] FIG. 1 is a transmission electron microscope (TEM) image of a preferred embodiment of a targeted carrier provided by the present invention. [Figure 3B] FIG. 1 is a diagram showing the results of Western blot analysis of the first preferred embodiment of the targeted carrier provided by the present invention. [Figure 4A] 3A and 3B are electron microscope TEM images of the first and second preferred embodiments of the targeted carriers provided by the present invention after loading a drug thereon. [Figure 4B] 3A and 3B are electron microscope TEM images of the first and second preferred embodiments of the targeted carriers provided by the present invention after loading a drug thereon. [Figure 5A] FIG. 10 shows the results of flow cytometry analysis after loading drugs in the first and second preferred embodiments of the targeted carriers provided by the present invention. [Figure 5B] FIG. 10 shows the results of flow cytometry analysis after loading drugs in the first and second preferred embodiments of the targeted carriers provided by the present invention. [Figure 6A] FIG. 1 is a schematic diagram of the blood-brain barrier cell culture model provided by the present invention. [Figure 6B] FIG. 1 shows the results of immunofluorescence staining in Experiment 1 provided by the present invention. [Figure 7A]FIG. 1 is a graph showing the results of measuring intracellular reactive oxygen species (ROS) concentrations in Experiment 2 provided by the present invention. [Figure 7B] FIG. 1 shows the results of Western blot analysis in Experiment 2 provided by the present invention. [Figure 7C] FIG. 10 is a graph showing the results of measuring cell death rates in Experiment 2 provided by the present invention. [Figure 8] 1 is a distribution diagram of the in vivo targeting carrier in the Parkinson's disease animal model provided by the present invention. [Figure 9] FIG. 10 is a graph showing the results of a rolling roller running test in an animal model of Parkinson's disease treated with the first preferred embodiment of the targeted carrier provided by the present invention. [Figure 10A] FIG. 1 is a graph showing the results of an open space trajectory analysis test in an animal model of Parkinson's disease treated with the first preferred embodiment of the targeted carrier provided by the present invention. [Figure 10B] FIG. 1 is a graph showing the results of an open space trajectory analysis test in an animal model of Parkinson's disease treated with the first preferred embodiment of the targeted carrier provided by the present invention. [Figure 10C] FIG. 1 is a graph showing the results of an open space trajectory analysis test in an animal model of Parkinson's disease treated with the first preferred embodiment of the targeted carrier provided by the present invention. [Figure 11A] FIG. 1 shows brain section and blood analysis results of an animal model of Parkinson's disease treated with a first preferred embodiment of the targeted carrier provided by the present invention. [Figure 11B] FIG. 1 shows brain section and blood analysis results of an animal model of Parkinson's disease treated with a first preferred embodiment of the targeted carrier provided by the present invention. [Figure 11C] FIG. 1 shows brain section and blood analysis results of an animal model of Parkinson's disease treated with a first preferred embodiment of the targeted carrier provided by the present invention. [Figure 11D] FIG. 1 is a diagram showing the results of brain slice analysis in an animal model of Parkinson's disease treated with a first preferred embodiment of the targeted carrier provided by the present invention. [Figure 12A]FIG. 10 shows the results of a rotary roller running test and an open space trajectory analysis test in an animal model of Parkinson's disease treated with the second preferred embodiment of the targeted carrier provided by the present invention. [Figure 12B] FIG. 10 shows the results of a rotary roller running test and an open space trajectory analysis test in an animal model of Parkinson's disease treated with the second preferred embodiment of the targeted carrier provided by the present invention. [Figure 12C] FIG. 10 is a diagram showing the results of brain slice analysis in an animal model of Parkinson's disease treated with a second preferred embodiment of the targeted carrier provided by the present invention. [Figure 13A] FIG. 1 shows the protein expression results of neurons (PD) differentiated from induced pluripotent stem cells (iPSCs) of a Parkinson's disease patient, after treatment with a first preferred embodiment of the targeting carrier provided by the present invention. [Figure 13B] FIG. 1 shows the protein expression results of neurons (PD) differentiated from induced pluripotent stem cells (iPSCs) of a Parkinson's disease patient, after treatment with a first preferred embodiment of the targeting carrier provided by the present invention. [Figure 14A] FIG. 10 is a graph showing the results of measuring intracellular and extracellular exosome protein concentrations in Experiment 7 provided by the present invention. [Figure 14B] FIG. 10 shows the results of Western blot analysis in Experiment 7 of the present invention. [Figure 15A] FIG. 10 shows the results of a rotary roller running test and an open space trajectory analysis test in an animal model of Parkinson's disease treated with the third preferred embodiment of the targeted carrier provided by the present invention. [Figure 15B] FIG. 10 shows the results of a rotary roller running test and an open space trajectory analysis test in an animal model of Parkinson's disease treated with the third preferred embodiment of the targeted carrier provided by the present invention. [Figure 15C] FIG. 10 is a surface analysis of protein expression in brain regions of a Parkinson's disease animal model treated with the third preferred embodiment of the targeted carrier provided by the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0014] The pathological features of Parkinson's disease (PD) are caused by the degeneration and death of dopamine neurons projecting from the substantia nigra to the striatum. The expression level of the dopamine transporter (DAT) in dopamine neurons in the striatum can be used as an indicator of the progression of Parkinson's disease.

[0015] See Figure 1. In order to achieve a more specific therapeutic effect, in the present invention, a mother cell line was modified by genetic engineering techniques to express a dopamine transporter antibody (hereinafter abbreviated as anti-DAT) on the surface of extracellular vesicles secreted by the mother cell line, and targeted extracellular vesicles (hereinafter referred to as "DATEV carriers") were designed. This allows the DATEV carriers to specifically bind to dopaminergic neurons expressing DAT via the anti-DAT, effectively promoting uptake by dopaminergic neurons and enabling normal cells to avoid the binding reaction of the extracellular vesicles.

[0016] The anti-DAT is preferably expressed on a transmembrane protein (e.g., CD63, CD81, and CD9) on the DATEV carrier, and in this example, the dopamine transporter antibody is expressed on the CD63 transmembrane protein.

[0017] In this embodiment, the steps of preparing the DATEV carrier include: In step S1, a dopamine transporter antibody plasmid is constructed. A gene fragment of a dopamine transporter antibody is prepared, and the nucleic acid sequence of the dopamine transporter antibody is inserted into the target gene of the transmembrane protein using genetic engineering techniques. This results in the gene fragment and the target gene being linked to form a vector gene. Hereinafter, the present invention will be described using the target gene SEQ ID No: 3 of the CD63 transmembrane protein expressed in the extracellular vesicles as an example.

[0018] The gene fragment comprises SEQ ID No: 1 or SEQ ID No: 2.

[0019] The gene fragment is inserted into the gene sequence corresponding to the extracellular loop between the third and fourth transmembrane domains of the transmembrane protein.

[0020] In one embodiment, the gene fragment SEQ ID No: 1 or SEQ ID No: 2 is inserted into the target gene SEQ ID No: 3 or SEQ ID No: 5 to form the vector gene.

[0021] In one embodiment, the gene fragment SEQ ID No:2 is inserted into the target gene SEQ ID No:3, and then the vector gene SEQ ID No:4 is formed.

[0022] In one embodiment, the gene fragment SEQ ID No:1 is inserted into the target gene SEQ ID No:5, and then the vector gene SEQ ID No:6 is formed.

[0023] Subsequently, the vector gene SEQ ID No: 4 is integrated into a plasmid using gene cloning technology. In this example, the plasmid is pEXO plasmid (Addgene, Watertown, MA, USA).

[0024] In step S2, the vector gene is introduced into the mother cell line and cultured. The plasmid containing the vector gene SEQ ID No. 4 is transfected into the mother cell line using transfection technology to form a DAT cell line. This DAT cell line highly expresses the anti-DAT antibody. Transfection techniques include electroporation, cell squeezing, ultrasound, and chemical transfection. In this example, lipofection (Lipofectamine 3000, L3000015, Invitrogen, Waltham, MA, USA) was used.

[0025] The selection of the mother cell line is not particularly limited and can be made according to the cell characteristics of the mother cell line. For example, the human embryonic kidney cell line 293 (HEK-293) cell line (hereinafter referred to as the HEK-293 cell line) has high transfection efficiency and rapid growth rate, making it easy to culture and capable of obtaining a large number of cells and extracellular vesicles secreted by them in a short period of time. Alternatively, mesenchymal stem cells (MSCs) contain abundant growth factors and anti-inflammatory factors, and the extracellular vesicles secreted by them can also contain abundant growth factors and anti-inflammatory factors, thereby providing a direct therapeutic effect.

[0026] 2×10 8 DAT-293T cells were seeded onto a culture dish (CelCradle® benchtop bioreactor, ESCO Aster, Singapore) and cultured in 500 mL of Dulbecco's modified Eagle's medium (DMEM) containing exosome-depleted fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% antibiotics (penicillin / streptomycin / amphotericin B solution).

[0027] In step S3, the DATEV carrier is collected. After culturing DAT-293T cells for 3–4 days, the culture medium of the DAT-293T cell line is collected and filtered through a 0.22 μm filter. The medium is then concentrated and purified using a lateral flow filtration system (MAP.03-plus TFF System; Lefo Science) with a 300 kDa molecular weight cutoff. The supernatant after chromatographic elution is further concentrated using a 30 kDa molecular weight cutoff membrane. Finally, the DATEV carrier released into the medium is obtained and resuspended in phosphate buffered saline (PBS).

[0028] According to the above steps, the DATEV carrier secreted from the DAT-293T cell line transfected with the gene fragment SEQ ID No. 1 and the gene fragment SEQ ID No. 2 was identified as the first and second examples. The DATEV carriers purified in the first and second examples had KDs of 1.3 to 6.4 × 10, respectively. -9 It was shown to have a binding affinity of M.

[0029] 2A and 2B, the expression levels of CD63 and anti-DAT on the DATEV carrier of Example 1 were quantified using an antibody-antigen method and compared with those of conventional extracellular vesicles (EV carriers) secreted from conventional HEK-293 cells, confirming the difference in expression of the transmembrane proteins CD63 and anti-DAT. The conventional HEK-293 cell culture and the conventional extracellular vesicle collection methods were similar to those used for culturing DATEV-293T cells and collecting DATEV carriers.

[0030] After harvesting, DATEV carriers and EV carriers were incubated in a reaction solution containing a fluorescently labeled CD63 antibody and a fluorescently labeled DAT recombinant protein for 6–12 hours. The reaction solution was then replaced with a buffer solution (PBS-T containing 0.1% casein) and injected into each well of a detection plate in 50 μL aliquots (50 μL / well). The percentage of DATEV carriers and EV carriers bearing CD63 and / or anti-DAT on their surfaces was then measured using an automated extracellular vesicle absolute quantification analyzer (ExoCounter, JVCKENWOOD Corporation, Yokosuka, Japan). As a result, DATEV carriers derived from transfected DAT-293T cells exhibited higher expression levels of the transmembrane protein CD63 and anti-DAT than non-transfected EV carriers derived from HEK-293 cells. The CD63 and anti-DAT expression rates in the DATEV carriers of Example 2 reached 94% and 91%, respectively (not shown).

[0031] The present invention further provides a third embodiment, which differs from the second embodiment in that mesenchymal stem cells (MSCs) were used as the parent cell line and transfected with lentivirus to produce DAT-MSCs. The DAT-MSCs were cultured in 20 mL of medium containing fetal bovine serum (FBS). The FBS medium contained 8 μg / mL polybrene (PB), 50 mg / mL protamine sulfate (PS), and 1100 mg / mL polyethylene glycol-polypropylene glycol copolymer (Synperonic F108). Furthermore, the lentivirus was inoculated into the mesenchymal stem cells (MSCs) at a multiplicity of infection (MOI) of greater than 2 (number of viruses / number of cells).

[0032] In Figure 3A, transmission electron microscope (TEM) images were taken. In all three examples, the DATEV carriers obtained in Examples 1, 2, and 3 expressed dopamine transporter antibodies (anti-DAT) on their surfaces, but the particle size and shape remained unchanged compared to standard EV carriers. In Figure 3B, Western blotting was used to confirm the difference in protein expression levels between the DATEV carriers and EV carriers. The results showed that the expression levels of transmembrane proteins (especially CD63 and CD81) and anti-DAT protein in the DATEV carriers were significantly higher than in the EV carriers. This confirms that the DATEV carriers secreted by the DAT-293T cell line, which was genetically transfected to express anti-DAT protein through the aforementioned steps of the present invention, truly contain anti-DAT protein.

[0033] The targeted carriers of the present invention can be loaded with drugs as needed and can be manufactured into biocompatible pharmaceutical preparations, including but not limited to gene fragments (DNA, RNA), protein sequences, chemical agents, etc.

[0034] Curcumin has excellent pharmacological properties and has been proven to be effective in wound healing, anti-cancer, anti-COVID-19, and immune system regulation. Furthermore, numerous studies have shown that curcumin suppresses the expression of neurodegeneration-related factors (phosphorylated tau protein and β-amyloid precursor protein), demonstrating its potential in the treatment of Parkinson's disease.

[0035] Brain-derived neurotrophic factor (BDNF) is an important physiologically active protein present in the brain that regulates the survival, growth, and remodeling of nerve cells. Numerous studies have shown that BDNF deficiency is involved in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.

[0036] In order to confirm that the targeted carrier provided by the present invention has targeting, loading, and release functions, the present invention further encapsulated (encapsulated) curcumin and messenger RNA (messenger ribonucleic acid, mRNA) mRNA-BDNF-CY3 for BDNF (brain-derived neurotrophic factor) expression into the DATEV carrier to form a curcumin-loaded DATEV carrier (hereinafter referred to as Cur@DATEV) and a mRNA-BDNF-CY3-loaded DATEV carrier (hereinafter referred to as BDNF@DATEV), and observed the effects of these Cur@DATEV and BDNF@DATEV on the treatment of Parkinson's disease.

[0037] See Figures 4A and 4B. In Example 1, curcumin was encapsulated in the DATEV carrier and the EV carrier, respectively, to form a curcumin-loaded DATEV carrier (Cur@datev) and a curcumin-loaded EV carrier. Cell and animal experiments were then conducted to confirm the specificity and functionality of the DATEV carrier and whether curcumin is effective in treating Parkinson's disease. In Example 2, mRNA-BDNF-CY3 was encapsulated in the DATEV carrier to form a DATEV carrier loaded with mRNA-BDNF-CY3 (BDNF@DATEV). For comparison, BDNF@EV, loaded with mRNA-BDNF-CY3 in the EV carrier, was used to confirm the specificity, functionality, and effectiveness of the DATEV carrier in treating Parkinson's disease due to reduced BDNF.

[0038] In this example, the DATEV carrier and the EV carrier were encapsulated with curcumin and mRNA-BDNF-CY3 by ultrasonication and electroporation, respectively.

[0039] The steps of encapsulating curcumin in DATEV carriers and EV carriers by ultrasonic treatment include: The DATEV carrier and EV carrier were mixed with human serum albumin at a weight ratio ranging from 1:0.1 to 1:2 to form a mixture. This mixture was then sonicated for 1 to 10 cycles (30 seconds on / 30 seconds off) with 2 minutes of cooling on ice between cycles. The mixture was then filtered through a first filter membrane with a molecular weight cutoff of 100 kDa. Curcumin was added to the filtered mixture, and the ratio of human serum albumin, DATEV carrier / EV carrier, and curcumin was adjusted to 1:1:1 (μg), followed by six more cycles of sonication under the same conditions. The mixture was then filtered twice through a second filter membrane with a molecular weight cutoff of 30 kDa, resuspended in PBS buffer, and used for subsequent experiments.

[0040] The step of encapsulating the mRNA-BDNF-CY3 in the DATEV carrier and the EV carrier by electroporation includes the following steps: The DATEV / EV carrier and mRNA-BDNF-CY3 were mixed, and the mixture was electroporated under the following conditions: voltage 100-250 V, pulse time 50-300 μs, interpulse time 500-1500 μs, and cycle discharge 4-6 times. The mixture was then incubated in a cell incubator for 30-90 minutes, centrifuged, and resuspended in PBS buffer solution for subsequent experiments.

[0041] Transmission electron microscope (TEM) images (Figure 4A) show that compared to a general EV carrier loaded with curcumin, the overall size and shape of Cur@DATEV, formed by encapsulating curcumin in DATEV carriers, are not affected by the effects of the curcumin on the EV carriers. Similarly, Figure 4B shows no difference in size or shape between BDNF@DATEV and BDNF@EV, demonstrating that the DATEV carriers with the dopamine transporter antibody (anti-DAT) bound to their surface do not affect their drug encapsulation capacity.

[0042] Figures 5A and 5B are flow cytometric detections showing the ratio of the DATEV carrier forming Cur@DATEV loaded with curcumin and BDNF@DATEV loaded with mRNA-BDNF-CY3 through the steps of encapsulating curcumin and mRNA-BDNF-CY3 provided by the present invention in Examples 1 and 2. The flow cytometry identified Cur@DATEV by the fluorescent reaction of curcumin itself, and detected Cur@DATEV and BDNF@DATEV by the CY3 fluorescent reaction of mRNA-BDNF-CY3. After system calculation conversion, the results in the Q2 quadrant of Figures 5A and 5B confirmed that after the curcumin encapsulation step of the DATEV carrier of the first embodiment, approximately 88.3% of the carrier successfully encapsulated curcumin (i.e., Cur@DATEV was formed), and after the mRNA-BDNF-CY3 encapsulation step of the DATEV carrier of the second embodiment, approximately 69.2% of the carrier successfully encapsulated mRNA-BDNF-CY3 (i.e., BDNF@DATEV was formed).

[0043] 6A, to demonstrate the in vivo applicability of the DATEV carrier and / or Cur@DATEV, a blood-brain barrier cell culture model 10 is provided to simulate the in vivo blood-brain barrier environment and ensure that the drug carrier can cross the blood-brain barrier, enter the brain, and specifically bind to a Parkinson's disease model cell line. The Parkinson's disease model cell line is formed by culturing and differentiating a human neuroblastoma cell line (hereinafter referred to as the SH-SY5Y cell line). Due to its inherent DAT protein expression characteristics, the SH-SY5Y cell line has become a commonly used cell model in medical research related to Parkinson's disease.

[0044] SH-SY5Y cells were cultured in a 1:1 mixture of Minimum Essential Medium (MEM, Invitrogen) and Ham's F-12 Nutrient Mix (F-12, Thermo Fisher Scientific). The mixture was supplemented with 10% fetal bovine serum (FBS, US-sourced HyClone, GE), 1% sodium pyruvate (Thermo Fisher Scientific), 1% GlutaMAX™ supplement, and 1% penicillin-streptomycin (Thermo Fisher Scientific). The cells were cultured at 37°C in a 5% CO2, humidity-controlled environment. When the cells reach a distribution occupying 70-80% of the cell plate area through culture, they can be induced to differentiate into the Parkinson's disease model cell line. The method for inducing neuronal differentiation involves first treating SH-SY5Y cells with 50 μM retinoic acid (RA) for two days, followed by changing the differentiation medium every day for the following five days, in which the differentiation medium is the mixed medium containing 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA).

[0045] The blood-brain barrier cell culture model 10 includes a first medium 11 and a second medium 12, and the first medium 11 and the second medium 12 are in communication with each other via a plurality of through-holes 13. The first medium 11 and the second medium 12 are filled with medium A, and the Parkinson's disease model cell line 50 is cultured at the bottom of the first medium 11. Endothelium 20, pericytes 30, and astrocytes 40 are sequentially placed at the bottom of the second medium 12 adjacent to the plurality of through-holes 13, and the electrical resistance of the blood-brain barrier cell culture model 10 is 2144 Ω.cm. 2 Thus, an inhibitory environment at the blood-brain barrier is effectively established. In subsequent experiments, the EV carrier, the DATEV carrier, Cur@DATEV, or Cur@EV is administered sequentially to the second medium 12 of the blood-brain barrier cell culture model 10 as needed, and experimental analysis is performed on the Parkinson's disease model cell line 50 in the first medium 11 to confirm the efficacy of the drug vector's penetration from the second medium 12 to the first medium 11 and its therapeutic effect.

[0046] Experiment 1 First, the DATEV carrier penetrates the inhibitory environment of the blood-brain barrier established by the blood-brain barrier cell culture model 10, and its uptake by the Parkinson's disease model cell line 50 is confirmed. Referring to Figure 6B, in this experiment, using the DATEV carrier of Example 1 as an example, curcumin-loaded EV carrier (Cur@EV) was introduced into the blood-brain barrier cell culture model 10 as a control group, and curcumin-loaded DATEV carrier (Cur@DATEV) was introduced as an experimental group, and the uptake effects of Cur@DATEV and Cur@EV by the Parkinson's disease model cell line were compared.

[0047] The Parkinson's disease model cell lines were incubated with Cur@DATEV and Cur@293EV for 24 hours, respectively, to confirm their uptake efficiency. The uptake of Cur@DATEV and Cur@EV by the Parkinson's disease model cell lines was compared by detecting the curcumin content in the Parkinson's disease model cell lines through the fluorescent properties of curcumin itself. Furthermore, the exosomal proteins of Cur@DATEV and Cur@EV in the Parkinson's disease model cell lines were labeled with red fluorescence using immunofluorescence staining and extracellular vesicle protein labeling technology (Protein EV Labeling Kit (Red), ExoGlow™, System Biosciences, Palo Alto, CA, USA) to confirm their uptake and entry into the Parkinson's disease model cell lines. Actin filaments (F-actin) were labeled with purple fluorescence to confirm the cytoskeleton. The cell nuclei were labeled with blue fluorescence, and curcumin was confirmed to enter the Parkinson's disease model cell line via green fluorescence reaction based on Cur@DATEV and / or Cur@EV.

[0048] The results in Figure 6B show that in the group treated with Cur@DATEV, the fluorescence expression of extracellular vesicle proteins (red fluorescence) and the fluorescence response of curcumin (green fluorescence) in the Parkinson's disease model cell line were significantly higher than in the group treated with Cur@EV, demonstrating that the DATEV carrier has the specificity to label DAT protein and can enter the Parkinson's disease model cell line through uptake.

[0049] Experiment 2 Similarly, taking the DATEV carrier of the first embodiment as an example, we will verify whether Cur@DATEV can pass through the resistance environment of the blood-brain barrier established by the blood-brain barrier cell culture model 10, be taken up by the Parkinson's disease model cell line 50, and then release curcumin within the Parkinson's disease model cell line 50 to achieve therapeutic efficacy. The blood-brain barrier cell culture model 10 is provided with the following: The group that has not undergone any additional treatment is designated as the first control group (PD). The group treated with the EV carrier was designated as the second control group (EV). The group treated with DATEV carrier was designated as the third control group (DATEV). The group treated with curcumin was designated as the fourth control group (Cur). The group treated with the addition of curcumin-loaded EV carrier was designated as the fifth control group (Cur@EV). An experimental group (Cur@datev) was treated with the addition of curcumin-loaded DATEV carrier to confirm whether or not the Parkinson's disease model cell line would release curcumin after taking up Cur@DATEV and the effectiveness of the treatment.

[0050] After 48 hours of incubation, the response mechanisms of the Parkinson's disease model cell line 50 in each group were examined. These included cell death rates, intracellular reactive oxygen species (ROS) levels, and protein expression of factors related to Parkinson's disease. Among these, alpha-synuclein (α-syn) is a common marker protein accumulated in dopaminergic neurons in Parkinson's disease patients. Parkin, DJ-1, and TH are marker proteins expressed to achieve cellular repair after dopaminergic neuronal damage.

[0051] In this experiment, the intracellular reactive oxygen species (ROS) concentration of the Parkinson's disease model cell line 50 was detected by flow cytometry analysis. The detection results in Figure 7A confirm that the intracellular reactive oxygen concentrations of the Parkinson's disease model cell line 50 in the fourth control group, fifth control group, and experimental group were significantly lower than those in the first to third control groups.

[0052] In Figure 7B, Western blotting was used to detect alpha-synuclein (α-syn), Parkin, DJ-1, and TH proteins in the Parkinson's disease model cell line 50 in each group. The results showed that after the Parkinson's disease model cell line was treated with Cur@DATEV, the expression of α-synuclein was reduced, and Parkin, DJ-1, and TH proteins were highly expressed. This indicates that the Parkinson's disease model cell line's ability to repair cells was improved after treatment with Cur@DATEV.

[0053] Referring to Figure 7C, the Parkinson's disease model cell line 50 is considered to be degenerated / pathological dopamine neurons, which have a low ability to repair themselves and are prone to death. In this experiment, the number of remaining cells in each group was normalized based on the number of remaining cells in the first control group on days 1, 3, and 7 after treatment. From day 3, the number of remaining cells of the Parkinson's disease model cell line 50 in the fourth control group, the fifth control group, and the experimental group was significantly higher than those in the first through third control groups. However, more notably, the result on day 7 showed that the number of remaining cells of the Parkinson's disease model cell line 50 in the experimental group was significantly higher than those in the fourth and fifth control groups, confirming that after the Parkinson's disease model cell line was treated with Cur@DATEV, the DATEV carrier could release curcumin intracellularly and further improve the efficacy of curcumin treatment (relative to the results of the fourth and fifth control groups).

[0054] Next, the present invention constructed an animal model of Parkinson's disease and measured behavioral changes after treatment with Cur@DATEV and BDNF@DATEV. Rats underwent roller running training to measure their motor skills in a healthy state, and an open field test was used to detect the rats' behavioral performance in a healthy state. Next, the rats underwent surgical injection of 6-hydroxydopamine (6-OHDA) into the left dorsal striatum, destroying dopamine neurons in the dorsal striatum. This resulted in the creation of an animal model of Parkinson's disease that mimicked the symptoms of Parkinson's disease. The unit volume of each injection of 6-hydroxydopamine (Oxidopamine, 6-hydroxydopamine, 6-OHDA) drug was 3.2 μL, and 11 μg of 6-hydroxydopamine (Oxidopamine, 6-hydroxydopamine, 6-OHDA) powder was dissolved in each unit volume of the 6-hydroxydopamine (Oxidopamine, 6-hydroxydopamine, 6-OHDA) drug.

[0055] To demonstrate the specific delivery of DATEV in an animal model of Parkinson's disease, we administered Cur@DATEV and Cur@EV to the animal model of Parkinson's disease via intravenous injection, using the first example described above. Prior to intravenous injection, Cur@DATEV and Cur@EV were reacted with a lipophilic fluorescent dye (XenoLight DiR, PerkinElmer) to impart fluorescent response properties. Twenty-four hours after injection, the absorption of Cur@DATEV and Cur@EV in each organ of the animal model of Parkinson's disease was confirmed by fluorescence analysis using an in vivo imaging system (IVIS Imaging System). Figure 8 shows the fluorescence values ​​measured in each organ of the animal model of Parkinson's disease, normalized to the group administered Cur@EV. It can be seen that a clear fluorescent response was observed in the cerebrum of the animal model administered Cur@DATEV. In addition to demonstrating the specificity of DATEV, this also demonstrates the ability of DATEV to cross the blood-brain barrier. In addition, it was found that DATEV was retained in the cerebrum for up to 7 days (results not shown).

[0056] Experiment 3 After confirming that Cur@DATEV can cross the blood-brain barrier in the Parkinson's disease animal model, this experiment further confirmed using Example 1 whether Cur@DATEV can release curcumin in the Parkinson's disease animal model, and whether it can change the symptoms of Parkinson's disease in the Parkinson's disease animal model, alleviate the progression of the disease, and even produce a therapeutic effect.The Parkinson's disease animal model was treated by intravenous injection every 7 days until week 5, starting from the day of surgery as week 0, and was divided into the following groups. A group that underwent a sham operation in the dorsal striatum brain region and did not undergo any drug treatment served as a healthy control group (Health). The Parkinson's disease animal model without any drug treatment was designated as the first control group (PD). The Parkinson's disease animal model given EV carrier treatment will be the second control group (EV). The Parkinson's disease animal model given DATEV vehicle treatment will be the third control group (DATEV). The Parkinson's disease animal model treated with curcumin was designated as the fourth control group (Cur). The Parkinson's disease animal model treated with curcumin-loaded EV carrier was designated as the fifth control group (Cur@EV), and the fourth and fifth control groups (Cur@EV) were treated with 1 mg of curcumin per 100 g of rat weight. The Parkinson's disease animal model treated with curcumin-loaded DATEV carrier was designated as the experimental group (Cur@datev), and this group was used to confirm the release of curcumin in the brain of the Parkinson's disease animal model and the effectiveness of the treatment after Cur@DATEV treatment.

[0057] Referring to Figure 9, starting from week 0 (W0), each group underwent a rotarod test at week 2 (W2), week 4 (W4), week 6 (W6), and week 8 (W8), respectively, to observe the performance of each group's motor parameters. The motor parameters included duration on the rotarod. The results showed that the Parkinson's disease animal models (control groups 1 to 5 and experimental groups) showed significant declines in each of the motor parameters compared to the healthy control group.

[0058] Furthermore, compared with the second (EV), third (DATEV), fourth (Cur), and fifth control groups (Cur@EV), only the motor skills of the Parkinson's disease animal models in the experimental group (Cur@datev) significantly increased with the number of treatments and the number of weeks with Cur@DATEV. Notably, after the final intravenous injection treatment of the Parkinson's disease animal models at week 5, the motor skills of the Parkinson's disease animal models in the experimental group (Cur@datev) not only did not decline but also showed a tendency to further improve at weeks 6 and 8.

[0059] Referring to Figures 10A-10C, starting from week 0, each group underwent an open field tracking test at weeks 2, 4, 6, and 8, respectively, to observe the performance of each group's motor parameters. The motor parameters in this test included speed, total distance traveled, and total rest time. Corresponding results were obtained with the rotarod test. In the experimental group treated with the curcumin-loaded DATEV carrier (Cur@datev), the motor parameters (speed, total distance traveled, and total rest time) of the Parkinson's disease animal model significantly improved with increasing weeks and treatment frequency. This indicates that treatment with the curcumin-loaded DATEV carrier not only alleviated the symptoms of Parkinson's disease (decreased motor ability) but also restored the behavioral performance of the Parkinson's disease animal model.

[0060] Experiment 4 Experiment 3 demonstrated that the behavioral ability of the Parkinson's disease animal model could be restored after treatment with the curcumin-containing DATEV carrier. Therefore, this experiment further involved collecting brain slices and blood from each of the Parkinson's disease animal models in each of the above groups for biochemical analysis.

[0061] Sections of the striatum brain region were obtained from each of the Parkinson's disease animal models in each group, and alpha-synuclein (α-syn), a marker of Parkinson's disease, TH protein, a marker of cell repair, and DAT protein, a marker of dopamine neurons, were detected through antibody-antigen reactions.

[0062] The results in Figure 11A show that in the Parkinson's disease animal model group (experimental group) treated with Cur@DATEV, not only was the accumulation of alpha-synuclein (α-syn) significantly lower in the dorsal striatum compared to control groups 1 to 5, but the expression levels of TH protein and DAT protein were also significantly higher than those of control groups 1 to 5. In addition, when comparing the experimental group (Cur@datev) with the healthy control group (Health), there were no significant differences in the expression of α-syn, TH protein, or DAT protein.

[0063] Blood samples were collected from each of the Parkinson's disease animal models in each group, and biochemical analysis was performed on a protein microarray chip using antigen labeling technology to observe the expression of neural regeneration and inflammatory response-related proteins in the blood of each of the Parkinson's disease animal models after treatment.

[0064] The nerve regeneration-related proteins include BDNF, NGF, VEGF, IL-10, and CNTF, and the inflammatory response-related proteins include INF-γ, IL-1β, IL-6, and TNF-α. The values ​​of each of the above items obtained by detection were analyzed and plotted after standardization based on the measured values ​​obtained in a healthy control group (Health).

[0065] Figure 11B shows the results of analyzing nerve regeneration-related proteins in each group of the Parkinson's disease animal model. The results show that, compared with control groups 1 to 5, the expression of nerve regeneration-related proteins in the experimental group tended to increase, with BDNF, IL-10, and CNTF being particularly prominent. It is noteworthy that the expression of the nerve regeneration-related protein BDNF in the experimental group was significantly higher than that in the healthy control group (Health).

[0066] Figure 11C shows the analysis results of inflammatory response-related proteins in each group of the Parkinson's disease animal model. The results show that, compared with control groups 1 to 5, the expression of inflammatory response-related proteins in the experimental group showed a downward trend, particularly in INF-γ, IL-1β, IL-6, and TNF-α. It is noteworthy that the expression of TNF-α in the experimental group was significantly lower than that in the healthy control group (Health).

[0067] Figure 11D shows the comparison of neural stem cell-associated protein expression in the dorsal striatum brain sections of the Parkinson's disease animal model between the first control group (PD) and the experimental group (Cur@datev) using immunofluorescence staining. The neural stem cell-associated proteins include nestin and Ki67. The results show that the neural stem cell-associated protein expression in the Parkinson's disease animal model in the experimental group (Cur@datev) was significantly higher than that in the first control group (PD), indicating that the curcumin-loaded DATEV carrier can maintain neural stem cell activity in the dorsal striatum brain region and achieve the effects of dopamine neuron regeneration and differentiation.

[0068] Based on the above results, it can be concluded that, through the treatment with the DATEV carrier containing curcumin, curcumin can penetrate the blood-brain barrier via DATEV and be delivered to the dorsal striatum brain region, thereby reducing the secretion and accumulation of synaptic nuclear proteins, alleviating the production of inflammatory responses, as well as promoting the reduction of the expression of inflammatory response-related proteins and the enhancement of the expression of neural regeneration-related proteins. At the same time, this can maintain the activity of neural stem cells, thereby achieving the effects of regeneration and differentiation of dopamine neurons, and effectively improving the motor ability of the Parkinson's disease animal model rats.

[0069] Experiment Five This experiment uses the DATEV carrier of Example 2 as an example to verify whether BDNF@DATEV can release mRNA-BDNF-CY3 and effectively regulate BDNF expression in the Parkinson's disease model cell line 50. The Parkinson's disease animal model was treated by intravenous injection every 7 days from the day of surgery as week 0 until week 5, and was divided into the following groups: A group that underwent a sham operation in the dorsal striatum brain region and did not undergo any drug treatment served as a healthy control group (Health). The Parkinson's disease animal model without any drug treatment was used as the first control group (PD). The Parkinson's disease animal model given EV carrier treatment with loaded mRNA-BDNF-CY3 was designated as the second control group (BDNF@EV). The experimental group (BDNF@datev) was treated with DATEV carrier carrying mRNA-BDNF-CY3, and this confirmed the release of mRNA-BDNF-CY3 after BDNF@DATEV uptake by the Parkinson's disease model cell line and the efficacy of the treatment.

[0070] 12A and 12B, the Parkinson's disease animal model for each group was started at week 0 (W0), and then at week 2 (W2), week 4 (W4), week 6 (W6), week 8 (W8), and week 12 (W12), a rotarod test and an open field trajectory analysis test were performed to observe the development of motor parameters for each group. The motor parameters included duration on the rotarod and total distance traveled.

[0071] In both the rotarod test and the open field trajectory analysis test, the Parkinson's disease animal models of the first control group, the second control group, and the experimental group showed significant declines in each of the motor parameters compared to the healthy control group. However, it is noteworthy that the Parkinson's disease animal models of the experimental group showed gradual improvement over time. In particular, at week 12, the duration of the rotarod test in the experimental group improved by more than 60% compared to week 0, and the total distance traveled in the open field trajectory analysis test improved by more than 32% compared to week 0. This indicates that treatment of the experimental group with BDNF@DATEV not only alleviated the symptoms of Parkinson's disease (decreased motor ability), but also restored the behavioral ability of the Parkinson's disease animal models.

[0072] Figure 12C shows immunofluorescence staining of neural stem cell-associated proteins in dorsal striatum brain sections from the Parkinson's disease animal model. Actin filaments (F-actin) were labeled with red fluorescent dye to identify the cytoskeleton, nuclei with blue fluorescent dye, and α-synuclein (α-syn) was labeled with green fluorescent dye to identify α-syn deposition. The figure clearly shows that α-syn deposition was significantly improved in the experimental group after BDNF@DATEV treatment compared to the second control group (BDNF@EV). This result is consistent with the results of the related experiment conducted in Example 1 above.

[0073] Experiment 6 Furthermore, to prove that the DATEV carrier can produce similarly excellent effects on human cells, Figures 13A and 13B show experiments performed after differentiating induced pluripotent stem cells (iPSCs) from Parkinson's disease patients into neurons (PD). In Figure 13A, Western blotting was used to compare the high expression of alpha-synuclein (α-syn) in differentiated neurons (PD) with normal cells (Health), confirming that the neurons differentiated from Parkinson's disease patients' induced pluripotent stem cells (iPSCs) matched the protein physiological indicators of Parkinson's disease patients.

[0074] Next, the differentiated neurons were treated with Cur@DATEV, and the expression of α-synuclein (α-syn), Parkin, and DJ-1 was similarly observed on day 10. Figure 13B shows that in the group treated with Cur@DATEV, the expression of α-synuclein tended to decrease, and the expression of Parkin and DJ-1 tended to increase, consistent with the results of the previous experiments.

[0075] 14A to 14B, in order to demonstrate that DATEV can express different functions based on the cellular properties of the parent cell line itself, a Parkinson's disease model cell line 50 was similarly cultured in the blood-brain barrier cell culture model 10, and the therapeutic efficacy of the third example was demonstrated.

[0076] Experiment Seven In the blood-brain barrier cell culture model 10, the following were provided, respectively: The group without any additional treatment was designated as the first control group (PD). The group treated with the general EV carrier collected from the 293 cells was designated as the second control group (EV). The group treated with the general EV carrier collected from the stromal stem cells was designated as the third control group (MSCEV). The group treated with the DATEV carrier collected from the DAT-293 cells was designated as the fourth control group (293-DATEV). The group receiving the DATEV carrier collected from the DAT-MSC cells was designated the experimental group (MSC-DATEV). The Parkinson's disease model cell line was incubated with each of the above groups for 24 hours to confirm the endocytosis efficiency of the Parkinson's disease model cell line. After incubation for 48 hours, it was confirmed that the Parkinson's disease model cell line could take up the DATEV collected from the DAT-MSC cells and produce a therapeutic effect.

[0077] The exosomal proteins of MSCEV and DATEV in the Parkinson's disease model cell lines were fluorescently labeled using immunofluorescence staining and extracellular vesicle protein labeling techniques (Protein EV Labeling Kit (Red), ExoGlow™, System Biosciences, Palo Alto, CA, USA). Flow cytometry analysis was then used to measure the uptake efficiency of MSCEV or DATEV into the Parkinson's disease model cell lines. The results for the third control group (MSCEV) and experimental group (MSC-DATEV) in Figure 16A demonstrate that Parkinson's disease model cell line 50 can more efficiently uptake extracellular vesicles secreted by MSC cells, with the uptake rate for the experimental group (MSC-DATEV) reaching over 95%.

[0078] Experiment Eight Animal experiments further confirmed that the DATEV carrier obtained by collecting DAT-MSC cells can have a direct physiological effect on the Parkinson's disease animal model. The Parkinson's disease animal model was treated by intravenous injection every 7 days from the day of surgery, counting as week 0, until week 5, and divided into the following groups: A sham operation is performed on the dorsal striatum brain region, and a group without drug treatment is used as a healthy control group (Health). The Parkinson's disease animal model without drug treatment was used as the first control group (PD). The second control group (MSCEV) was given general EV carrier treatment obtained by collecting the stromal stem cells. The experimental group (MSC-DATEV) was administered with the DATEV carrier obtained by collecting DAT-MSC cells. This group was used to confirm the physiological mechanism of the Parkinson's disease animal model after MSCEV treatment and the protein expression of Parkinson's disease-related factor α-syn, neural regeneration-related protein IL-10, and inflammatory response-related protein NF-γ in the dorsal striatum brain region.

[0079] 15A and 15B, the Parkinson's disease animal models for each group were started at week 0 (W0), and then subjected to an open field tracking test at week 2 (W2), week 4 (W4), week 6 (W6), week 8 (W8), and week 12 (W12) to observe the development of locomotion parameters for each group. The locomotion parameters included duration on the wheel and total distance traveled.

[0080] In both the wheel running test and the open field trajectory analysis test, the Parkinson's disease animal models of the first control group, the second control group, and the experimental group showed significant decreases in the motor parameters compared to the healthy control group. However, it is noteworthy that the motor parameters of the Parkinson's disease animal models of the experimental group gradually improved over time. In particular, at week 12, the duration of the wheel running test in the experimental group improved by more than 48% compared to week 0, and the total distance traveled in the open field trajectory analysis test improved by more than 17% compared to week 0. This indicates that the experimental group treated with MSCEV effectively alleviated the symptoms of Parkinson's disease (decreased motor ability) and even restored the behavioral ability of the Parkinson's disease animal models.

[0081] The results in Figure 15B confirmed that, over time, the expression of the Parkinson's disease-related factor α-syn in the dorsal striatum brain region of the Parkinson's disease animal model in the experimental group tended to decrease, while the expression of the neuroregeneration-related protein IL-10 and the inflammatory response-related protein NF-γ tended to increase. This demonstrates that extracellular vesicles secreted by DAT-MSC cells have the effect of directly regulating physiological mechanisms.

[0082] The DATEV carrier provided by the present invention can carry drugs and pass through the blood-brain barrier, achieve specific binding to dopamine neurons, and further regulate the secretion of Parkinson's disease marker proteins, thereby producing excellent therapeutic effects of delaying the progression of Parkinson's disease.

[0083] Furthermore, loading curcumin into the DATEV carrier can effectively reduce the accumulation of α-syn protein, reduce the expression of inflammatory response-related proteins (INF-γ, IL-1β, IL-6, TNF-α), increase the expression of Parkin, DJ-1, TH, and neural regeneration-related proteins (BDNF, NGF, VEGF, IL-10, CNTF), and maintain neural stem cell activity (nestin and Ki67) in the dorsal striatum. The DATEV carrier can also be loaded with BDNF mRNA, which can regulate the expression of BDNF and reduce the accumulation of α-syn protein, thereby delaying the onset of Parkinson's disease symptoms and even promoting the regeneration and differentiation of dopaminergic neurons, demonstrating its potential for the treatment of Parkinson's disease. [Explanation of symbols]

[0084] 10. Blood-brain barrier cell culture model 11 1st culture area 12 Second culture area 13 Through hole Medium A 20 endothelial cells 30 Pericite 40 Astrocytes 50 Parkinson's disease model cell lines S1 Step S2 Step S3 Step

Claims

1. An anti-DAT antibody formed by transcription and translation of a gene fragment containing SEQ ID No: 2 and used to label the dopamine transporter.

2. A composition comprising a target gene SEQ ID No: 3, wherein any part of said target gene SEQ ID No: 3 is inserted into a gene fragment SEQ ID No:

2.

3. A composition comprising extracellular vesicles in which an antibody is bound to a transmembrane protein, the extracellular vesicles being secreted by a cell transfected with a vector gene, at least a portion of which comprises SEQ ID No:

2.

4. The composition of claim 3 , wherein the dopamine transporter antibody is formed in the extracellular loop between the third and fourth transmembrane domains of the transmembrane protein.

5. A composition comprising an extracellular vesicle in which the anti-DAT antibody of claim 1 is bound to a transmembrane protein.

6. A composition comprising extracellular vesicles in which an anti-DAT antibody is bound to a transmembrane protein, the extracellular vesicles being secreted by a cell transfected with a vector gene, at least a portion of which comprises SEQ ID No:

4.

7. The composition of any one of claims 3 to 6, wherein the transmembrane protein is CD63 and the cells are HEK-293 cells.

8. The composition according to any one of claims 3 to 6, wherein the transmembrane protein is CD63 and the cells are mesenchymal stem cells.

9. The composition of any one of claims 3 to 6, wherein the extracellular vesicles are loaded with a drug, and the drug comprises a DNA or RNA gene fragment, a protein sequence, or a chemical agent.

10. The composition according to any one of claims 3 to 6, wherein the extracellular vesicles encapsulate messenger RNA used for the expression of BDNF.

11. Use of the composition of any one of claims 2 to 9 for reducing alpha-synuclein deposition in the striatum, a brain region where degeneration or lesions of dopamine neurons have occurred.

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