TARGETING VEHICLES, COMPOSITIONS and USES THEREOF

Abstract

A targeted vector comprising an extracellular vesicle wherein an anti-dopamine transporter antibody is bound to a transmembrane protein, the extracellular vesicle being secreted by a cell genetically transfected with a vector gene, and at least a portion of the vector gene comprising SEQ ID No: 1. The targeted vector provided by this invention can load drugs to cross the blood-brain barrier and achieve specific binding to dopamine-producing neurons, thereby modulating the secretion of Parkinson's disease marker proteins and producing excellent effects in delaying the progression of Parkinson's disease.

Description

Technical Field

[0001] A carrier, particularly a targeted carrier, a medical preparation and its uses. Prior Technology

[0002] Parkinson's disease (PD) is a common degenerative disease of the central nervous system (CNS) in the brain. Symptoms typically appear slowly over time, affecting the motor nervous system, and the disease is incurable. Traditionally, treatment involves administering large doses of dopamine (L-DOPA) to slow disease progression. However, with neuronal degeneration, loss of neurons, and the blockage of the blood-brain barrier, dopamine therapy gradually becomes ineffective. Furthermore, the indiscriminate administration of large doses of dopamine to the central nervous system inevitably affects the physiological mechanisms of other brain regions. Therefore, developing other technologies to help alleviate Parkinson's disease is a pressing goal in this field. Summary of the Invention

[0003] In order to develop technologies that can alleviate Parkinson's disease, the present invention provides a targeted vector comprising an extracellular vesicle wherein a dopamine transporter antibody is bound to a membrane-penetrating protein, the protein sequence of which includes SEQ ID NO:4.

[0004] The extracellular vesicles are secreted by the gene-transfected HEK-293 cell line, and at least a portion of a gene sequence of the gene-transfected HEK-293 cell line contains SEQ ID No:1.

[0005] The targeted carrier provided by the present invention can be further loaded with a drug as needed, thereby producing a biocompatible medical preparation.

[0006] The drug includes, but is not limited to, gene fragments (DNA, RNA), protein sequences, chemical agents, etc.

[0007] The targeted carrier provided by this invention can carry drugs across the blood-brain barrier and can specifically bind to dopamine nerve cells, thereby achieving excellent therapeutic effects in regulating the secretion of Parkinson's disease marker proteins and delaying the progression of Parkinson's disease.

[0008] The present invention also provides a medical preparation comprising the aforementioned targeted carrier, wherein the targeted carrier is loaded with curcumin.

[0009] Medical preparations that incorporate curcumin into targeted carriers can effectively reduce the accumulation of synuclein and the expression of inflammatory response-related proteins (INF-γ, IL-1β, IL-6, TNF-α), enhance the expression of Parkinson's protein, DJ-1 protein, TH protein, and neural regeneration-related proteins (BDNF, NGF, VEGF, IL-10, CNTF), and maintain the activity of neural stem cells (nesin and Ki67 protein) in the striatum brain region. This can not only delay the symptoms of Parkinson's disease, but also achieve the effects of dopamine-induced neural cell regeneration and differentiation, showing great potential for the treatment of Parkinson's disease. Simple Explanation of the Diagram

[0010] Figure 1 is a block diagram of the steps in a preferred embodiment of the targeted carrier provided by the present invention. Figures 2A and 2B show the protein performance results of the first preferred embodiment of the targeting vector provided by the present invention. Figure 3A is a transmission electron microscope (TEM) image of a preferred embodiment of the targeted carrier provided by the present invention. Figure 3B shows the results of the Western ink dot method for the first preferred embodiment of the targeted carrier provided by the present invention. Figures 4A and 4B are electron microscopy (TEM) images of the first and second preferred embodiments of the targeted carrier provided by the present invention after drug loading. Figures 5A and 5B show the flow cytometry results of the first and second preferred embodiments of the targeted carrier provided by the present invention after drug loading. Figure 6A is a simulation diagram of the blood-brain barrier cell culture model provided by the present invention. Figure 6B shows the results of immunofluorescence staining in Experiment 1 provided by this invention. Figure 7A shows the intracellular reactive oxygen species concentration detection in Experiment 2 provided by this invention. Figure 7B shows the results of the Western ink dot method in Experiment 2 provided by this invention. Figure 7C shows the cell death rate results in Experiment 2 provided by this invention. Figure 8 shows the distribution of the targeted vector in the animal model of Parkinson's disease provided by this invention. Figure 9 shows the results of the roller running test of the Parkinson's disease animal model provided by the present invention after treatment with the first preferred embodiment of the targeted vector. Figures 10A to 10C show the open space trajectory analysis results of the Parkinson's disease animal model provided by this invention after treatment with the first preferred embodiment of the targeted vector. Figures 11A to 11C show brain slices and blood analysis results of the Parkinson's disease animal model provided by this invention after treatment with the first preferred embodiment of the targeted vector. Figures 12A and 12B show the results of the roller running test and open space trajectory analysis tests of the Parkinson's disease animal model provided by the present invention after treatment with the targeted carrier in the second preferred embodiment. Figure 12C shows the brain slice analysis results of the Parkinson's disease animal model provided by the present invention after treatment with the targeted vector in the second preferred embodiment. Figures 13A and 13B show the protein expression results of nerve cells (PDs) differentiated from induced pluripotent stem cells (iPSCs) of Parkinson's disease patients provided in this invention after treatment with a targeting vector in the first preferred embodiment. Figure 14A shows the detection of intracellular and extracellular vesicle protein concentrations in Experiment 7 provided by this invention. Figure 14B shows the results of the Western ink dot method in Experiment 7 provided by this invention. Figures 15A and 15B show the results of the roller running test and open space trajectory analysis tests of the Parkinson's disease animal model provided by the present invention after treatment with the targeted carrier in the third preferred embodiment. Figure 15C shows the surface analysis of brain proteins in the Parkinson's disease animal model provided by this invention after treatment with the targeted vector in the third preferred embodiment. Implementation

[0011] The known pathological features of Parkinson's disease (PD) are the degeneration and death of dopamine neurons projected from the substantia nigra to the striatum. The expression level of the dopamine transporter (DAT) in the striatum can be used to assess the disease progression of Parkinson's disease.

[0012] Please refer to Figure 1. In order to produce a specific therapeutic effect, this invention uses genetic engineering to modify a mother cell line so that the surface of an extracellular vesicle secreted by the mother cell line expresses a dopamine transporter antibody (hereinafter referred to as anti-DAT). A target extracellular vesicle (hereinafter referred to as DATEV vector) is designed so that the DATEV vector can specifically bind to dopamine neurons expressing dopamine transporter (DAT) through anti-DAT, thereby effectively promoting the phagocytic behavior of dopamine neurons and avoiding the binding reaction of normal cells to extracellular vesicles.

[0013] The dopamine transporter antibody (hereinafter referred to as anti-DAT) is preferably expressed on the membrane-penetrating proteins (such as CD63, CD81 and CD9) of the DATEV vector. In this embodiment, the dopamine transporter antibody is expressed on the CD63 membrane-penetrating protein of the DATEV vector.

[0014] In this embodiment, the fabrication steps of the DATEV carrier include:

[0015] Step S1, constructing a dopamine transporter antibody plasmid: A fragment gene expressing a dopamine transporter antibody is prepared, and the nucleic acid sequence of the dopamine transporter antibody is inserted into a target gene of the membrane-penetrating protein using genetic engineering techniques, so that the fragment gene binds to the target gene and forms a vector gene. The present invention will subsequently use SEQ ID No:3 of the target gene of the membrane-penetrating protein CD63 expressed in the extracellular vesicle as an example for illustration.

[0016] The gene fragment contains either SEQ ID No:1 or SEQ ID No:2.

[0017] Specifically, this gene fragment is inserted into the gene sequence of the transmembrane protein in the extracellular loop between the third and fourth transmembrane domains.

[0018]

[0019] In one embodiment, the fragment gene 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.

[0020] In one embodiment, the fragment gene SEQ ID No:2 is inserted into the target gene SEQ ID No:3 to form the vector gene SEQ ID No:4.

[0021] In one embodiment, the fragment gene SEQ ID No:1 is inserted into the target gene SEQ ID No:5 to form the vector gene SEQ ID No:6.

[0022] Next, the vector gene SEQ ID No:4 was recombined into a plasmid using gene recombination technology. In this embodiment, the plasmid was a pEXO plasmid (Addgene, Watertown, MA, USA).

[0023] Step S2: Transfect the vector gene into the mother cell line and culture: The plastid carrying the vector gene SEQ ID No:4 is inserted into the mother cell line using cell transfection technology to form a DAT cell line, which highly expresses the dopamine transporter antibody (anti-DAT). The cell transfection technology includes electroporation, cell extrusion, ultrasound, viral transfection, or chemical transfection. In this embodiment, liposome transfection (Lipofectamine 3000, L3000015, Invitrogen, Waltham, MA, USA) is used.

[0024] There are no restrictions on the choice of the mother cell line. It can be selected based on the cell characteristics of the mother cell line itself. For example, the human embryonic kidney cell 293 (HEK-293) cell line (hereinafter referred to as HEK-293 cell line) is highly efficient for cell transfection, and it grows rapidly and has a simple culture method, which can obtain a large number of cells and their secreted extracellular vesicles in a short time. Alternatively, mesenchymal stem cells (MSCs) are rich in growth factors and anti-inflammatory factors, and their secreted extracellular vesicles can carry rich growth factors and anti-inflammatory factors at the same time to achieve the effect of direct treatment.

[0025] Two × 10⁸ DAT-293T cells were distributed in a culture dish (CelCradle® benchtop bioreactor, ESCO Aster, Singapore) and cultured with 500 mL of Dulbecco's modified Eagle's medium (DMEM), which contained exosome-depleted FBS (Gibco, Grand Island, NY, USA) and 1% antibiotics (penicillin / streptomycin / amphotericin B solution).

[0026] Step S3, collecting the DATEV vector: After culturing DAT-293T cells for 3 to 4 days, the culture medium of the DAT-293T cell line was collected and filtered through a 0.22 μm filter membrane. Then, it was concentrated and purified using a tangential flow filtration system with a molecular weight cutoff of 300 kDa (MAP.03-plus TFF System; Lefo Science). The supernatant was then obtained by elution by chromatography and concentrated through a 30 kDa molecular weight cutoff membrane. Finally, the DATEV vector released into the culture medium was obtained and resuspended in phosphate-buffered saline (PBS).

[0027] The above steps were used to differentiate the DATEV vectors secreted by DAT-293T cell lines transfected with SEQ ID No:1 and SEQ ID No:2 of the gene fragment, respectively, using a first embodiment and a second embodiment. The DATEV vectors purified in the first and second embodiments exhibited an affinity between KD: 1.3 and 6.4 x 10⁻⁹ M.

[0028] In Figures 2A and 2B, the expression levels of CD63 and anti-DAT on the DATEV vector of the first embodiment were quantified using antibody-antigen technology, and compared with those of ordinary extracellular vesicles secreted by ordinary HEK-293 cells (hereinafter referred to as EV vectors) to confirm the difference in the expression of the transmembrane protein CD63 and anti-DAT protein between the two. The methods for culturing ordinary HEK-293 cells and collecting ordinary extracellular vesicles are the same as those for culturing DAT-293T cells and collecting DATEV vectors described above.

[0029] After collecting DATEV vectors and general extracellular vesicles, they were placed in reaction solutions containing fluorescent CD63 antibody and fluorescent DAT recombinant protein, respectively, and cultured for 6 to 12 hours. The reaction solution was then replaced with a buffer solution (PBS-T containing 0.1% casein), and 50 µL (50 µL / well) was injected into each well of a detection disc. The proportion of DATEV vectors and general extracellular vesicles containing CD63 and / or anti-DAT on the surface was then calculated using an automated extracellular vesicle absolute quantification analyzer (ExoCounter, JVCKENWOOD Corporation, Yokosuka, Japan). The results showed that the expression of the transmembrane protein CD63 and anti-DAT protein in DATEV vectors obtained from the multi-DAT-293T cell line was superior to that in general extracellular vesicles obtained from the untransfected general HEK-293 cell line. The CD63 and anti-DAT expression levels on the DATEV carrier in the second embodiment were also as high as 94% and 91%, respectively (not shown in the figure).

[0030] The present invention also provides a third embodiment, which differs from the second embodiment in that the progenitor cell line is mesenchymal stem cell (MSC), and DAT-MSC cells are formed through a lentivirus transfection process. The DAT-MSC cells are cultured in 20 mL of fetal bovine serum (FBS) containing 8 μg / mL polybrene (PB), 50 mg / mL protamine sulfate (PS), and 1100 mg / mL synthesonic F108 (F108). Further, the mesenchymal stem cells (MSC) are inoculated with the lentivirus at a multiplicity of infection (MOI) greater than 2 (virus count / cell count).

[0031] Figure 3A, taken using a transmission electron microscope (TEM), shows that in all three embodiments—the first, second, and third—the collected DATEV vectors exhibit the appearance of the dopamine transporter antibody (anti-DAT), and their overall size and shape are not affected compared to the general EV vector. Figure 3B, using Western blotting, confirms the difference in protein expression levels between the DATEV and EV vectors. It is clearly observed that the DATEV vector shows higher expression of the membrane-penetrating proteins (especially CD63 and CD81) and anti-DAT protein compared to the EV vector. Therefore, it can be confirmed that the DATEV vector secreted by the DAT-293T cell line after gene transfection and anti-DAT protein expression, following the steps provided in this invention, carries anti-DAT protein expression.

[0032] The targeted carrier provided by this invention can be further loaded with a drug as needed, thereby creating a biocompatible medical preparation. The drug includes, but is not limited to, gene fragments (DNA, RNA), protein sequences, chemical agents, etc.

[0033] Curcumin possesses excellent medicinal properties, and has been shown to aid in wound healing, fight cancer, combat COVID-19, and regulate the function of the human immune system. Numerous studies have also demonstrated that curcumin can effectively reduce the expression of neurodegenerative factors (phosphorylation of tau and β-amiloid precursor protein), showcasing its potential in the treatment of Parkinson's disease.

[0034] Brain-derived neurotrophic factor (BDNF) is one of the important active proteins in the brain, used to regulate the survival, growth, and remodeling of neurons. A considerable body of literature has indicated a strong correlation between insufficient BDNF levels and many neurodegenerative diseases, such as Alzheimer's and Parkinson's.

[0035] To verify that the targeted vector provided by this invention possesses the functions of targeting, loading, and release, this invention further encapsulates the DATEV vector with curcumin and messenger ribonucleic acid (mRNA) mRNA-BDNF-CY3 for expressing BDNF expression, forming a DATEV vector loaded with curcumin (hereinafter referred to as Cur@DATEV) and a DATEV vector loaded with mRNA-BDNF-CY3 (hereinafter referred to as BDNF@DATEV), to observe the effects of Cur@DATEV and BDNF@DATEV on the treatment of Parkinson's disease.

[0036] Referring to Figures 4A and 4B, the DATEV and EV vectors of the first embodiment were coated with curcumin to form a curcumin-loaded DATEV vector (Cur@DATEV) and a curcumin-loaded EV vector (hereinafter referred to as Cur@EV). Subsequent cell and animal experiments were then conducted to confirm the specificity and functionality of the DATEV vector, as well as the effectiveness of curcumin in treating Parkinson's disease. In the second embodiment, the DATEV vector was coated with mRNA-BDNF-CY3 to form a mRNA-BDNF-CY3-loaded DATEV vector (BDNF@DATEV). The EV vector loaded with mRNA-BDNF-CY3 was referred to as BDNF@EV in comparison, to confirm the specificity and functionality of the DATEV vector, as well as the effectiveness of BDNF downregulation in treating Parkinson's disease.

[0037] In this embodiment, the DATEV vector and the EV vector were coated with curcumin and mRNA-BDNF-CY3 by ultrasound and electroporation, respectively.

[0038] The steps of ultrasonically encapsulating the curcumin in the DATEV and EV carriers include:

[0039] DATEV and EV vectors were mixed with human serum albumin at weight ratios ranging from 1:0.1 to 1:2 to form a mixture. The mixture was ultrasonically treated for 1-10 cycles at a 30-second on / off cycle, with the mixture cooled on ice for 2 minutes between cycles. The mixture was then filtered through a first-cell cutoff membrane with a molecular weight cutoff of 100 kDa. Curcumin was added to the filtered mixture, with the ratio of human serum albumin, DATEV / EV vectors, and curcumin being 1:1:1 (μg), and the mixture was ultrasonically treated again under the same conditions for 6 cycles. Subsequently, the mixture was filtered twice through a second-cell cutoff membrane with a molecular weight cutoff of 30 kDa and resuspended in PBS buffer for subsequent experiments.

[0040] The steps of electroporating the mRNA-BDNF-CY3 with the DATEV and EV vectors include:

[0041] Mix the DATEV / EV vector with mRNA-BDNF-CY3; then apply electroporation conditions including a voltage of 100-250V, a pulse duration of 50-300μs, a pulse interval of 500-1500μs, and 4-6 cycles; then incubate in a cell culture incubator for 30-90 min, centrifuge, and resuspend in PBS buffer for subsequent experiments.

[0042] As shown in Figure 4A using a transmission electron microscope (TEM), compared to a conventional EV carrier loaded with curcumin, the Cur@DATEV formed by coating the DATEV carrier with curcumin did not affect its overall size and shape compared to Cur@EV. Similarly, in Figure 4B, there was no difference in size and shape between BDNF@DATEV and BDNF@EV, demonstrating that the DATEV carrier with the dopamine transporter antibody (anti-DAT) bound to its surface does not affect its ability to encapsulate drugs.

[0043] Figures 5A and 5B show the ratio of Cur@DATEV loaded with curcumin to BDNF@DATEV loaded with mRNA-BDNF-CY3 in the DATEV vector formed by flow cytometry detection in the first and second embodiments, using the steps of coating curcumin and mRNA-BDNF-CY3 provided by the present invention. The flow cytometer identifies Cur@DATEV through the fluorescence reaction of curcumin itself; and detects Cur@DATEV and BDNF@DATEV through the CY3 fluorescence reaction of mRNA-BDNF-CY3. The results, calculated and converted by the system, are shown in quadrant Q2 of Figures 5A and 5B. In the first embodiment, after the curcumin-coating step, approximately 88.3% of the DATEV vector successfully coated with curcumin (i.e., forming Cur@DATEV); in the second embodiment, after the mRNA-BDNF-CY3-coating step, approximately 69.2% of the DATEV vector successfully coated with mRNA-BDNF-CY3 (i.e., forming BDNF@DATEV).

[0044] Please refer to Figure 6A. To demonstrate the effective applicability of the DATEV vector and / or Cur@DATEV in organisms, a blood-brain barrier cell culture model 10 is provided. The aim is to simulate the blood-brain barrier environment in organisms, ensuring that the drug carrier can cross the blood-brain barrier, enter the brain region, and specifically bind to a Parkinson's disease model cell line. The Parkinson's disease model cell line is formed by the differentiation of a human neuroblastoma cell line (hereinafter referred to as the SH-SY5Y cell line). Due to the DAT protein expression characteristic of the SH-SY5Y cell line, it has become a commonly used cell model in medical research related to Parkinson's disease.

[0045] 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). This mixture was supplemented with 10% fetal bovine serum (FBS, US-sourced HyClone, GE), 1% sodium pyruvate (Thermo Fisher), 1% GlutaMAX™ supplement, and 1% penicillin-streptomycin (Thermo Fisher), and cultured at 37 °C, 5% CO₂, and under controlled humidity. When the cells are cultured to the point that they occupy 70–80% of the cell disc area, they can be induced to differentiate into a Parkinson's disease model cell line. The method for inducing neural differentiation is to first treat SH-SY5Y cells with 50 μM retinoic acid (RA) for two days, and then change the differentiation culture medium daily for the next 5 days. The differentiation culture medium is a mixed culture medium containing 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA).

[0046] The blood-brain barrier cell culture model 10 includes a first culture zone 11 and a second culture zone 12, which are connected by a plurality of perforations 13. The first culture zone 11 and the second culture zone 12 are filled with a culture medium A. Parkinson's disease model cell line 50 is cultured at the bottom of the first culture zone 11, while an endothelial cell 20, a pericyte cell 30, and an astrocyte 40 are sequentially arranged at the bottom of the plurality of perforations 13 in the second culture zone 12. The resistance value of the blood-brain barrier cell culture model 10 is measured to be 2144 Ω·cm², effectively constructing the impedance environment of the blood-brain barrier. In subsequent experiments, EV vector, DATEV vector, Cur@DATEV or Cur@EV were sequentially administered to the second culture zone 12 of the blood-brain barrier cell culture model 10, and the Parkinson's disease model cell line 50 in the first culture zone 11 was used for experimental analysis to confirm the effectiveness and therapeutic effect of the drug carrier in crossing the second culture zone 12 to the first culture zone 11.

[0047] Experiment 1 First, it was confirmed that the DATEV vector could cross the impedance environment of the blood-brain barrier constructed in the blood-brain barrier cell culture model 10 and be phagocytosed by the Parkinson's disease model cell line 50. Referring to Figure 6B, this experiment used the DATEV vector of the first embodiment as an example. Curcumin-loaded EV vector (Cur@EV) was introduced into the blood-brain barrier cell culture model 10 as a control group, and curcumin-loaded DATEV vector (Cur@DATEV) was introduced as an experimental group. The effects of Cur@DATEV and Cur@EV phagocytosis by the Parkinson's disease model cell line were compared.

[0048] The endocytosis efficiency of the Parkinson's disease model cell line was determined after reacting with Cur@DATEV and Cur@293EV for 24 hours, respectively. The content of curcumin in the Parkinson's disease model cell line was detected by detecting the fluorescence properties of curcumin itself to compare the phagocytosis effect of the Parkinson's disease model cell line on Cur@DATEV and Cur@EV. Simultaneously, through immunofluorescence staining and extracellular vesicle protein labeling technology (Protein EV Labeling Kit (Red), ExoGlow™, System Biosciences, Palo Alto, CA, USA), the extracellular vesicle proteins of Cur@DATEV and Cur@EV in the Parkinson's disease model cell line can be identified by red fluorescence to confirm whether Cur@DATEV and Cur@EV enter the Parkinson's disease model cell line via endocytosis; actin filaments (F-actin) can be identified by purple fluorescence to confirm the cytoskeleton; the cell nucleus is identified by blue fluorescence; and curcumin can be identified by green fluorescence to confirm whether it enters the Parkinson's disease model cell line via Cur@DATEV and / or Cur@EV.

[0049] As shown in Figure 6B, the fluorescence of the extracellular vesicle protein (red fluorescence) and the fluorescence response of curcumin (green fluorescence) in the Parkinson's disease model cell line treated with Cur@DATEV were significantly higher than those treated with Cur@EV. This confirms that the DATEV vector should have the specificity to label DAT protein and can enter the Parkinson's disease model cell line through cytophagy.

[0050] Experiment 2 Similarly, using the DATEV vector from the first embodiment as an example, it was confirmed that Cur@DATEV can cross the impedance environment of the blood-brain barrier constructed in the blood-brain barrier cell culture model 10 and be phagocytosed by the Parkinson's disease model cell line 50. Further confirmation was made regarding whether Cur@DATEV can release curcumin within the Parkinson's disease model cell line 50 and achieve therapeutic efficacy. The following were provided in the blood-brain barrier cell culture model 10: The group that received no additional treatment was used as the primary control group (PD). The group treated with EV vector was used as the second control group (EV); The groups treated with the DATEV vector were used as the third control group (DATEV). The group treated with added curcumin served as the fourth control group (Cur). The group treated with the EV carrier loaded with curcumin served as the fifth control group (Cur@EV); and The experimental group (Cur@DATEV) was treated with DATEV vector loaded with curcumin to confirm whether curcumin was released after the Parkinson's disease model cell line engulfed Cur@DATEV and the effectiveness of the treatment.

[0051] After 48 hours of reaction in each group, the response mechanisms of the Parkinson's disease model cell line 50 in each group were examined, including: mortality rate, intracellular reactive oxygen species (ROS) concentration, and protein expression of Parkinson's disease-related factors. Alpha-synuclein (α-syn) is a marker protein commonly found in dopaminergic neurons of Parkinson's patients, while Parkin protein, DJ-1 protein, and TH protein are marker proteins expressed by dopaminergic neurons after damage to achieve cell repair.

[0052] In this experiment, the intracellular reactive oxygen species (ROS) concentration of the Parkinson's disease model cell line 50 was determined by flow cytometry analysis. As shown in Figure 7A, the ROS concentration of the Parkinson's disease model cell line 50 in the fourth control group, the fifth control group, and the experimental group was significantly lower than that in the first to third control groups.

[0053] Figure 7B shows that the expression of alpha-synuclein, Parkin, DJ-1, and TH proteins in the Parkinson's disease model cell line 50 of the above groups was reduced after Cur@DATEV treatment, while Parkin, DJ-1, and TH proteins showed high expression. This indicates that the Parkinson's disease model cell line has improved cell repair ability after Cur@DATEV treatment.

[0054] Next, please refer to Figure 7C. The Parkinson's disease model cell line 50 is considered as degenerated / pathological dopamine neurons, which have low cell repair capacity and are prone to death. In this experiment, the number of remaining cells in each group was standardized on days 1, 3, and 7 after treatment, using the number of remaining cells in the first control group as a baseline. Starting from day 3, the number of remaining cells in the fourth control group, fifth control group, and experimental group was significantly higher than that in the first to third control groups. More notably, on day 7, the number of remaining cells in the experimental group was significantly higher than that in the fourth and fifth control groups, confirming that after treatment with Cur@DATEV, the DATEV vector can release curcumin intracellularly and enhance the effectiveness of curcumin treatment (compared to the results of the fourth and fifth control groups).

[0055] The present invention then constructs an animal model of Parkinson's disease to test the behavioral changes of the animal model after treatment with Cur@DATEV and BDNF@DATEV. Rats were trained to run on a roller, and their motor values ​​were measured in a healthy state. The behavioral performance of the healthy rats was also assessed using an open field test. Next, an injection of oxydopamine (6-hydroxydopamine, 6-OHDA) was performed into the left dorsal striatum of the rats to destroy dopamine neurons in the dorsal striatum, thus creating an animal model of Parkinson's disease that simulates the symptoms of the disease. The unit volume of each injection of the hydroxydopamine (6-hydroxydopamine, 6-OHDA) agent is 3.2 μL, and each unit volume of the hydroxydopamine (6-hydroxydopamine, 6-OHDA) agent contains 11 μg of hydroxydopamine (6-hydroxydopamine, 6-OHDA) powder.

[0056] While the specificity of DATEV delivery in an animal model of Parkinson's disease has not been confirmed, this invention uses the first embodiment as an example. In this Parkinson's disease animal model, Cur@DATEV and Cur@EV were administered intravenously. Prior to intravenous injection, Cur@DATEV and Cur@EV were reacted with a lipophilic fluorescent dye (XenoLight DiR, PerkinElmer) to impart fluorescence properties. Twenty-four hours after injection, the absorption of Cur@DATEV and Cur@EV in various organs of the Parkinson's disease animal model was detected using an In Vivo Imaging System (IVIS Imaging System). Figure 8 shows the fluorescence values ​​measured in various organs of the Parkinson's disease animal model after standardization based on the group administered Cur@EV. It can be seen that in the animal model administered Cur@DATEV, a clearly measurable fluorescence response was observed in the brain. This not only demonstrates the specificity of DATEV but also proves that DATEV has the ability to cross the blood-brain barrier. In addition, it was discovered that DATEV can remain in the brain for up to 7 days (results not shown).

[0057] Experiment 3 Having confirmed that Cur@DATEV can cross the blood-brain barrier in this Parkinson's disease animal model, this experiment further confirms, using the first embodiment, whether Cur@DATEV can release curcumin within the Parkinson's disease animal model and alter the Parkinson's disease symptoms, thereby slowing disease progression or even producing a therapeutic effect. The Parkinson's disease animal model was treated with intravenous injection every 7 days from the day of surgery (week 0) to week 5, and was divided into the following groups: The healthy control group consisted of individuals who underwent sham surgery in the dorsal striatum region of the brain and received no drug treatment. This animal model of Parkinson's disease, which had not undergone any drug treatment, served as the first control group (PD). The Parkinson's disease animal model treated with EV vector served as a second control group (EV). The Parkinson's disease animal model treated with the DATEV vector served as the third control group (DATEV). This Parkinson's disease animal model treated with curcumin served as the fourth control group (Cur). The Parkinson's disease animal model treated with an EV vector loaded with curcumin served as the fifth control group (Cur@EV). In both the fourth and fifth control groups (Cur and Cur@EV), 1 mg of curcumin per 100 g of mouse weight was administered. The Parkinson's disease animal model treated with the DATEV vector loaded with curcumin was used as the experimental group (Cur@DATEV) to confirm whether curcumin was released in the brain of the Parkinson's disease animal model after Cur@DATEV treatment and the effectiveness of the treatment.

[0058] Please refer to Figure 9. Each group began the rotarod test at week 0 (W0), and the test was conducted at weeks 2 (W2), 4 (W4), 6 (W6), and 8 (W8) to observe the performance of motor values ​​in each group. These motor values ​​included the duration of time spent on the rotarod. The results showed that, compared to the healthy control group, all motor values ​​in the Parkinson's disease animal models of the first to fifth control groups and the experimental group were significantly lower.

[0059] Furthermore, compared with the second (EV), third (DATEV), fourth (Cur), and fifth control group (Cur@EV), only in the experimental group (Cur@DATEV) did the motor function of the Parkinson's disease animal model significantly increase with the number of Cur@DATEV treatments and weeks. Notably, after the final intravenous injection treatment in week 5, the motor function of the Parkinson's disease animal model in the experimental group (Cur@DATEV) not only did not decline in weeks 6 to 8, but even showed a trend of further improvement.

[0060] Please refer to Figures 10A to 10C. Each group began the experiment at week 0, and open field analysis was conducted at weeks 2, 4, 6, and 8 to observe the performance of motor values ​​in each group. The motor values ​​described in this experiment include speed, total distance traveled, and total rest time. Results corresponding to the rotarod test were obtained. In the experimental group treated with curcumin-DATEV, the motor values ​​(speed, total distance traveled, and total rest time) of the Parkinson's disease animal model significantly improved with increasing weeks and the number of Cur@DATEV treatments. This indicates that treatment with curcumin-DATEV not only alleviated the symptoms of Parkinson's disease (decreased motor ability) but also potentially restored the behavioral performance of the Parkinson's disease animal model.

[0061] Experiment 4 Since Experiment 3 confirmed that treatment with the curcumin-coated DATEV vector could restore the behavioral performance of the Parkinson's disease animal model, this experiment further conducted biochemical analyses on brain slices and blood samples from each of the aforementioned groups of Parkinson's disease animal models.

[0062] In this study, striatum brain slices were obtained from each group of animal models of Parkinson's disease, and antibody antigen reactions were used to detect 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.

[0063] As can be seen from the results in Figure 11A, in the experimental group of the Parkinson's disease animal model treated with Cur@DATEV, the dorsal striatum brain region not only had significantly less accumulation of α-synuclein (α-syn) than the first to fifth control groups, but also showed significantly higher levels of TH and DAT proteins. Apart from this, there was no significant difference in the expression of α-syn, TH, and DAT proteins between the experimental group (Cur@DATEV) and the healthy control group (Health).

[0064] After obtaining blood from each of the Parkinson's disease animal models in each group, biochemical analysis was performed on a protein microarray using antigen labeling technology to observe the expression of proteins related to nerve regeneration and inflammatory response in the blood of each Parkinson's disease animal model after treatment.

[0065] The proteins related to nerve regeneration include BDNF, NGF, VEGF, IL-10, and CNTF; the proteins related to inflammatory responses include INF-γ, IL-1β, IL-6, and TNF-α. The values ​​of the aforementioned items obtained by detection are standardized and analyzed and plotted based on the values ​​obtained from the healthy control group (Health).

[0066] Figure 11B shows the protein analysis results related to nerve regeneration in this Parkinson's disease animal model in each group. The results show that, compared to the first to fifth control groups, the experimental group exhibited an increasing trend in the expression of proteins related to nerve regeneration, especially BDNF, IL-10, and CNTF. Notably, the expression of the nerve regeneration-related protein BDNF in the experimental group was even significantly higher than that in the healthy control group.

[0067] Figure 11C shows the results of protein analysis related to inflammation in the Parkinson's disease animal model in each group. The results show that, compared to the first to fifth control groups, the experimental groups exhibited a decreasing trend in the expression of proteins related to inflammation, particularly INF-γ, IL-1β, IL-6, and TNF-α. Notably, the expression of TNF-α in the experimental groups was even significantly lower than that in the healthy control group.

[0068] Figure 11D compares the expression of neural stem cell-related proteins in brain slices from the dorsal striatum of this Parkinson's disease animal model between the first control group (PD) and the experimental group (Cur@DATEV) using immunofluorescence staining. The neural stem cell-related proteins include Nestin and Ki67 protein. The results show that the expression of neural stem cell-related proteins in the experimental group (Cur@DATEV) of the Parkinson's disease animal model is significantly higher than that in the first control group (PD), indicating that the curcumin-encapsulated DATEV carrier can maintain the activity of neural stem cells in the dorsal striatum brain region to achieve the effect of dopamine-induced neuronal regeneration and differentiation.

[0069] Based on the above results, it can be comprehensively evaluated that after treatment with curcumin-encapsulated DATEV carrier, curcumin can not only cross the blood-brain barrier via DATEV to be delivered to the dorsal striatum brain region, thereby reducing the secretion and accumulation of synuclein proteins and alleviating the production of inflammatory responses, but also promote the decline of the expression of inflammatory response-related proteins and the increase of the expression of neural regeneration-related proteins. At the same time, it can maintain and sustain the activity of neural stem cells, achieve the effect of dopamine-induced neural cell regeneration and differentiation, and effectively improve the motor ability of the rat model of Parkinson's disease.

[0070] Experiment 5 This experiment used the DATEV vector from the second embodiment as an example to confirm whether BDNF@DATEV could release mRNA-BDNF-CY3 within the Parkinson's disease model cell line 50 and effectively downregulate BDNF expression. The Parkinson's disease animal model was treated with intravenous injection every 7 days from the day of surgery (week 0) to week 5, and was divided into the following groups: The healthy control group consisted of individuals who underwent sham surgery in the dorsal striatum region of the brain and received no drug treatment. This animal model of Parkinson's disease, which had not undergone any drug treatment, served as the first control group (PD). An animal model of Parkinson's disease treated with an EV vector loaded with mRNA-BDNF-CY3 served as a second control group (BDNF@EV); and The experimental group (BDNF@DATEV) was treated with DATEV vector loaded with mRNA-BDNF-CY3 to confirm whether mRNA-BDNF-CY3 was released after the Parkinson's disease model cell line engulfed BDNF@DATEV and the effectiveness of the treatment.

[0071] Please refer to Figures 12A and 12B. The above-mentioned groups of Parkinson's disease animal models began at week 0 (W0) and underwent the rotarod test and open field trajectory analysis test at weeks 2 (W2), 4 (W4), 6 (W6), 8 (W8), and 12 (W12) to observe the performance of motor values ​​in each group. The motor values ​​included the duration of time spent on the rotarod and the total distance traveled.

[0072] Results from both the roller running test and the open field trajectory analysis experiment revealed that the motor values ​​of the Parkinson's disease animal models in the first control group, the second control group, and the experimental group were significantly lower than those in the healthy control group. However, it is noteworthy that the motor values ​​of the experimental group gradually increased over time, especially at week 12. The duration of the roller running test in the experimental group increased by more than 60% compared to week 0, and the total distance traveled in the open field trajectory analysis experiment increased by more than 32% compared to week 0. This indicates that the BDNF@DATEV treatment in the experimental group not only alleviated the symptoms of Parkinson's disease (decreased motor ability) but also had a restorative effect on the behavioral performance of the Parkinson's disease animal models.

[0073] Figure 12C compares the expression of neural stem cell-related proteins in brain slices from the dorsal striatum of the Parkinson's disease animal models in the above groups using immunofluorescence staining. F-actin filaments are identified by red fluorescence to confirm the cytoskeleton; the cell nucleus is identified by blue fluorescence; and green fluorescence identifies alpha-synuclein (α-syn) accumulation. The figure clearly shows that the experimental group treated with BDNF@DATEV showed a significant improvement in α-syn accumulation compared to the second control group (BDNF@EV). This result corresponds to the relevant experimental results obtained in the first embodiment.

[0074] Experiment Six Furthermore, to demonstrate that the DATEV vector can produce the same excellent effects on human cells, Figures 13A to 13 show experiments conducted after differentiating induced pluripotent stem cells (iPSCs) from Parkinson's disease patients into neural cells (PDs). In Figure 13A, Western blotting was used to compare the PDs with normal cells (Health). The high expression of α-synuclein (α-syn) in the differentiated neural cells (PDs) confirmed that the neural cells differentiated from induced pluripotent stem cells (iPSCs) from Parkinson's disease patients have protein physiological indicators consistent with those of Parkinson's disease patients.

[0075] Next, the differentiated neurons were treated with Cur@DATEV, and the performance of synuclein (α-syn), Parkin, and DJ-1 proteins was observed on day 10. Figure 13B shows that, consistent with the previous experimental results, the Cur@DATEV-treated group exhibited decreased synuclein performance, while the performance of Parkin and DJ-1 proteins tended to increase.

[0076] Please refer to Figures 14A and 14B to demonstrate that DATEV can exhibit different functions depending on the cellular characteristics of the parent cell line. Similarly, Parkinson's disease model cell line 50 was cultured in blood-brain barrier cell culture model 10 to demonstrate the therapeutic efficacy of the third embodiment.

[0077] Experiment 7 Provided separately in the blood-brain barrier cell culture model 10: The group that received no additional treatment was used as the primary control group (PD). A second control group (EV) was added, consisting of cells treated with the standard EV vector collected from these 293 cells. A group treated with a standard EV vector collected from these mesenchymal stem cells was added as a third control group (MSCEV); The group treated with DATEV vector collected from DAT-293 cells served as the fourth control group (293-DATEV); DATEV vector collected via DAT-MSC cells was added as the experimental group (MSC-DATEV). The endocytosis efficiency of the Parkinson's disease model cell line was confirmed after reacting with each of the above groups for 24 hours, and the therapeutic effect was confirmed after 48 hours of reaction when the Parkinson's disease model cell line phagocytosed DATEV collected via DAT-MSC cells.

[0078] Immunofluorescence staining and extracellular vesicle protein labeling technology (Protein EV Labeling Kit (Red), ExoGlow™, System Biosciences, Palo Alto, CA, USA) enabled the fluorescence identification of these exosomal proteins of MSCEV and DATEV in the Parkinson's disease model cell line. Flow cytometry analysis was then used to determine the efficiency of MSCEV or DATEV uptake in the Parkinson's disease model cell line. As shown in Figure 16A, the results for the third control group (MSCEV) and the experimental group (MSC-DATEV) indicate that the Parkinson's disease model cell line 50 can efficiently take up extracellular vesicles secreted by MSC cells. In particular, the uptake rate in the experimental group (MSC-DATEV) can reach over 95%.

[0079] After reacting the first control group (PD), the third control group (MSCEV), and the experimental group (MSC-DATEV) for 48 hours, the protein expression of α-synuclein (α-syn), Parkinson's protein, and TH protein in the Parkinson's disease model cell line 50 was detected in each group (Figure 14B). Notably, the experimental group (MSC-DATEV) effectively downregulated the expression of α-syn and increased the expression of Parkin and TH proteins without loading any drugs, which is conducive to the development of cell repair.

[0080] Experiment 8 Further animal experiments confirmed that the DATEV vector obtained from DAT-MSC cells could directly exert physiological effects on this Parkinson's disease animal model. The Parkinson's disease animal model was treated with intravenous injection every 7 days from the day of surgery (week 0) to week 5, and was divided into the following groups: The healthy control group consisted of individuals who underwent sham surgery in the dorsal striatum region of the brain and received no drug treatment. This animal model of Parkinson's disease, which had not undergone any drug treatment, served as the first control group (PD). The group treated with conventional EV vectors collected from these mesenchymal stem cells served as the second control group (MSCEV). DATEV vectors collected via DAT-MSC cells were administered as the experimental group (MSC-DATEV) to confirm the physiological mechanisms of the Parkinson's disease animal model after MSCEV treatment, as well as the protein expression of Parkinson's disease-related factors α-syn, neurogenesis-related protein IL-10, and inflammatory response-related protein NF-γ in the dorsal striatum brain region.

[0081] Please refer to Figures 15A and 15B. The above-mentioned groups of Parkinson's disease animal models were subjected to open field trajectory analysis experiments starting at week 0 (W0), and at weeks 2 (W2), 4 (W4), 6 (W6), 8 (W8), and 12 (W12) to observe the performance of the motor values ​​of each group. The motor values ​​include the duration of time spent on the roller and the total distance traveled.

[0082] Results from both the roller running test and the open field trajectory analysis experiment revealed that the motor values ​​of the Parkinson's disease animal models in the first control group, the second control group, and the experimental group were significantly lower than those in the healthy control group. However, it is noteworthy that the motor values ​​of the Parkinson's disease animal models in the experimental group gradually increased over time, especially at week 12. The duration of the roller running test in the experimental group increased by more than 48% compared to week 0, while the total distance traveled in the open field trajectory analysis experiment increased 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 produced a restorative effect on the behavioral performance of the Parkinson's disease animal models.

[0083] As shown in Figure 15B, with the extension of time, the expression of α-syn, a Parkinson's disease-related factor, decreased in the dorsal striatum brain region of the experimental group of Parkinson's disease animal model, while the expression of IL-10, a protein related to nerve regeneration, and NF-γ, a protein related to inflammation, showed an increasing trend. This confirms that extracellular vesicles secreted by DAT-MSC cells have the effect of directly regulating physiological mechanisms.

[0084] The DATEV carrier provided by this invention can carry drugs across the blood-brain barrier and can specifically bind to dopamine nerve cells, thereby achieving excellent therapeutic effects in regulating the secretion of Parkinson's disease marker proteins and delaying the progression of Parkinson's disease.

[0085] Further loading curcumin into the DATEV vector can effectively reduce the accumulation of synuclein and the expression of inflammation-related proteins (INF-γ, IL-1β, IL-6, TNF-α), enhance the expression of Parkin protein, DJ-1 protein, TH protein, and neuroregeneration-related proteins (BDNF, NGF, VEGF, IL-10, CNTF), and maintain the activity of neural stem cells (nesin and Ki67 protein) in the dorsal striatum. The DATEV vector can also load BDNF mRNA, thereby delaying the symptoms of Parkinson's disease by downregulating BDNF expression and reducing the accumulation of synuclein. It can even achieve the effects of dopamine neuron regeneration and differentiation, showing great potential for the treatment of Parkinson's disease.

[0086] 10: Blood-brain barrier cell culture model 11: First Cultivation Area 12: Second Cultivation Area 13: Perforation A: Culture medium 20: Endothelial cells 30: Pericytes 40: stellate cells 50: Parkinson's disease model cell line S1-S3: Steps

[0087] TW202604577A_114125732_SEQL.xml

Claims

1. A targeting vector comprising an extracellular vesicle, wherein an anti-dopamine transporter antibody is bound to a transmembrane protein of the extracellular vesicle, the extracellular vesicle being secreted by a cell transfected with a vector gene, and at least a portion of the vector gene comprising SEQ ID No:

1.

2. The targeting vector as described in claim 1, wherein the transmembrane protein is CD63, and the anti-dopamine transporter antibody forms an extracellular loop between the third and fourth transmembrane domains of the transmembrane protein.

3. The targeting vector as described in claim 1, wherein the cell is HEK-293 cell or mesenchymal stem cell.

4. The targeting vector as described in claim 1, wherein the transmembrane protein is CD63, and at least a portion thereof contains SEQ ID No:

5.

5. The targeting vector as described in claim 1, wherein the transmembrane protein is CD63, at least a portion of which contains SEQ ID No:

6.

6. A targeting vector comprising an extracellular vesicle wherein an anti-dopamine transporter antibody is bound to a transmembrane protein, the extracellular vesicle being secreted by a cell genetically transfected with a vector gene, and at least a portion of the vector gene comprising SEQ ID No:

6.

7. A pharmaceutical preparation comprising the targeting vector provided by any one of claims 1 to 6, wherein, The targeting vector contains a drug, which may include gene fragments (DNA, RNA), protein sequences, or chemical agents.

8. The pharmaceutical preparation as described in claim 7, wherein the extracellular vesicle is coated with curcumin.

9. Use of a pharmaceutical preparation comprising the method provided in claim 7 or 8 for preparing a protein synuclein accumulation in the striatum brain region that reduces dopamine-induced neuronal degeneration or lesions.

10. Use of a pharmaceutical preparation comprising the ingredients provided in claim 7 or 8 for preparing a preparation to enhance the expression of Parkin protein, DJ-1 protein, and TH protein in the striatum brain region of a brain region with dopamine-producing neuronal degeneration or lesions.

11. Use of a pharmaceutical preparation comprising the method provided in claim 7 or 8 for preparing a protein expression related to neural regeneration in the blood of an organism with degenerative or diseased dopamine neurons in the striatum brain region, wherein, Proteins involved in nerve regeneration include BDNF, NGF, VEGF, IL-10, and CNTF.

12. Use of a pharmaceutical preparation comprising the method provided in claim 7 or 8 for preparing a protein expression associated with inflammation in the blood of an organism with degenerative or diseased dopamine neurons in the striatum brain region, wherein, Inflammation-related proteins include INF-γ, IL-1β, IL-6, and TNF-α.

13. Use of a pharmaceutical preparation comprising the method provided in claim 7 or 8 for preparing a preparation to enhance or maintain the activity of neural stem cells in the striatum brain region where dopamine neurons are degenerated or diseased.

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