Central nervous system targeted drug composition

Abstract

The present invention provides a targeted drug composition that can specifically bind to the central nervous system and brain regions. [Solution] The present invention provides a central nervous system-targeted pharmaceutical composition that can be used to treat various forms of brain injury, wherein an integrin antibody is conjugated to a transmembrane protein of an exosome through genetic engineering, and the exosome is used as a carrier to encapsulate the therapeutic substance.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition, particularly a central nervous system target pharmaceutical composition produced by genetic engineering and capable of being used for damaged brain nerves.

Background Art

[0002] Brain injury and brain nerve aging are common as central nervous system (CNS) degenerative diseases in the brain. The symptoms of this disease usually progress gradually over time, affect the motor nervous system, and are incurable. Conventionally, the deterioration of the disease has been delayed by the treatment method of drug administration. However, with the degeneration and loss of nerves and the blockade of the blood-brain barrier, the drug treatment method also gradually becomes ineffective. In the current technology, there is a technology that encapsulates drugs, proteins, nucleotides, etc. by using exosomes, and has transport and biocompatibility, and shows delivery and drug release effects.

Summary of the Invention

Problems to be Solved by the Invention

[0003] However, the technology of binding transmembrane proteins to antibodies has not been fully established yet. Using this technology to achieve specific binding to the central nervous system and further enter the brain region through the blood-brain barrier has not been observed either. Therefore, the development of exosomes in the medical industry is restricted. In view of this, the development of a target pharmaceutical composition that can specifically bind to the central nervous system and the brain region is an urgent issue in the related fields.

Means for Solving the Problems

[0004] In order to develop a target pharmaceutical composition that can specifically bind to the central nervous system and the brain region, the present invention provides exosomes in which an integrin antibody is bound to a transmembrane protein.

[0005] The transmembrane protein is selected from the group consisting of any one of CD68, CD81, and CD9.

[0006] The aforementioned exosomes contain miRNA-BDNF. [Effects of the Invention]

[0007] The exosomes provided by this invention possess the ability to cross the blood-brain barrier and can achieve the treatment of neurological injury diseases or the mitigation of their progression, and can also be used for the treatment of spinal cord injury diseases or the mitigation of their progression. Furthermore, they achieve the effect of treating motor neurons and promoting the activation of neurofilament protein expression. [Brief explanation of the drawing]

[0008] [Figure 1A] This is a microscopic image showing the size and shape of the ITExo support. [Figure 1B] This is a diagram showing the results after the ITExo support underwent a Western blot experiment. [Figure 2A] This is a schematic diagram of a blood-brain barrier cell culture model. [Figure 2B] This is a diagram analyzing the target effect of ITexo carriers in a blood-brain barrier cell culture model. [Figure 2C] This is a diagram analyzing the target effect of ITexo carriers in a blood-brain barrier cell culture model. [Figure 3A] This is a diagram analyzing the miRNA encapsulation effect of the ITExo carrier. [Figure 3B] This is a diagram analyzing the miRNA encapsulation effect of the ITExo carrier. [Figure 4A] This is an analysis of inflammatory factor expression in a central nervous system injury cell model. [Figure 4B] This is an analysis of inflammatory factor expression in a central nervous system injury cell model. [Figure 5] This is an analysis diagram of the mNSS behavioral test in an animal model of stroke. [Figure 6] This is a diagram analyzing the targeted effect of ITexo carriers in an animal model of stroke. [Figure 7A] This is a TUNEL detection analysis diagram of an animal model of stroke. [Figure 7B]It is an analysis diagram of BDNF expression in a stroke animal model. [Figure 8A] It is an analysis diagram of the mNSS behavioral test in a dementia animal model. [Figure 8B] It is an analysis diagram of the mNSS behavioral test in a dementia animal model. [Figure 8C] It is an analysis diagram of the mNSS behavioral test in a dementia animal model. [Figure 9] It is an analysis diagram of the target effect of the ITExo carrier in a spinal cord injury animal model. [Figure 10A] It is an analysis diagram of the BBB behavioral expression in a spinal cord injury animal model. [Figure 10B] It is an analysis diagram of the gait analysis system in a spinal cord injury animal model. [Figure 10C] It is an analysis diagram of the gait analysis system in a spinal cord injury animal model. [Figure 10D] It is an analysis diagram of the gait analysis system in a spinal cord injury animal model. [Figure 10E] It is an analysis diagram of the gait analysis system in a spinal cord injury animal model. [Figure 10F] It is an analysis diagram of the gait analysis system in a spinal cord injury animal model. [Figure 11A] It is an analysis diagram of protein expression at the spinal cord injury site in a spinal cord injury animal model. [Figure 11B] It is an analysis diagram of protein expression at the spinal cord injury site in a spinal cord injury animal model. [Figure 11C] It is an analysis diagram of protein expression at the spinal cord injury site in a spinal cord injury animal model. [Figure 11D] It is an analysis diagram of protein expression at the spinal cord injury site in a spinal cord injury animal model.

Modes for Carrying Out the Invention

[0009] As a pathological characteristic of known brain injury or neurodegeneration (such as stroke, dementia, amyotrophic lateral sclerosis, and spinal cord injury), it is known that the expression level of integrin increases for the purpose of repair. Referring to Figure 1, in order to obtain a specific therapeutic effect, the present invention provides a method to obtain an integrin-targeted exosome (hereinafter referred to as an ITexo carrier) on the surface secreted by the HEK-293 cell line, by gene transgenicizing the HEK-293 cell line so that at least a portion of the gene sequence of the transgenic HEK-293 cell line contains an integrin antibody (anti-Integrin) sequence.

[0010] The ITexo carrier produced in this invention can achieve specific binding to brain-damaged or degenerated nerve cells via anti-integrin, effectively promoting the phagocytic behavior of nerve cells and avoiding the binding reaction to exosomes of normal cells.

[0011] Here, the protein sequence of the integrin antibody (anti-Integrin) includes an anti-integrin single-chain variable region fragment (scFv) and contains one of SEQ ID No:1 to SEQ ID No:5.

[0012] Here, the integrin-targeted exosomes bind to anti-integrin via transmembrane proteins such as CD63, CD81, and CD9, and the CD63 binding site of the exosome carrier will be explained below as an example.

[0013] The ITexo carrier provided by the present invention can optionally carry a drug inside it to produce a biocompatible pharmaceutical composition. Here, the drug includes, but is not limited to, gene fragments (DNA, RNA), protein sequences, chemical agents, or chemical compounds.

[0014] In this embodiment, the step of preparing the ITexo carrier includes the following: Step S1, Construction of integrin antibody plasmid: A carrier gene expressing an integrin antibody is prepared, and the anti-integrin single-chain variable region fragment (scFv) of the integrin antibody is inserted into the target gene of the transmembrane protein via genetic engineering techniques, and the fragment gene and the target gene are joined to form the carrier gene. Here, the anti-integrin single-chain variable region fragment (scFv) is inserted into the gene sequence corresponding to the second large extracellular loop of CD63.

[0015] Next, the carrier gene is incorporated into a plasmid via gene cloning. In this embodiment, the plasmid is a pEXO plasmid (Addgene, Watertown, MA, USA).

[0016] Step S2: Transfect the parent cell line with the carrier gene and culture it: The plasmid containing the carrier gene is introduced into the parent cell line via cell transfection technology to form an ITExo cell line, and the integrin antibody is highly expressed in the ITExo cell line. The cell transfection technology includes electroporation, cell compression, ultrasound, viral transfection, or chemical transfection. In this example, lipofection (Lipofectamine 3000, L3000015, Invitrogen, Waltham, MA, USA) was selected.

[0017] Here, there are no restrictions on the selection of the parent cell line, and it can be selected based on the cellular characteristics of the parent cell line itself. For example, the Human Embryonic Kidney Cells 293 (HEK-293) cell line (hereinafter referred to as the HEK-293 cell line) has high efficiency during cell transfection, a fast growth rate, and a simple culture method, allowing for the acquisition of a large number of cells and the extracellular vesicles they secrete in a short time. Alternatively, since mesenchymal stem cells (MSCs) inherently possess abundant growth factors and anti-inflammatory factors, the extracellular vesicles they secrete can simultaneously carry abundant growth factors and anti-inflammatory factors, achieving an effect that can be directly used for therapeutic purposes.

[0018] 2 x 10 8 The ITExo cells were distributed in a culture dish (CelCradle® benchtop bioreactor, ESCO Aster, Singapore) and cultured in 500 mL of Dulbecco's modified Eagle's medium (DMEM) culture medium. The DMEM culture medium contained exosome-depleted fetal bovine serum (exosome-depleted FBS, Gibco, Grand Island, NY, USA) and 1% antibiotic (penicillin / streptomycin / amphotericin B solution).

[0019] Step S3, Collecting DATEV Carriers: After culturing ITExo cells for 3-4 days, collect the culture medium from the ITExo cell line and filter it through a 0.22 μm filter membrane. Next, concentrate and purify the mixture using a tangential flow filtration system (MAP.03-plus TFF System; Lefo Science) with a 300 kDa molecular weight cutoff value. Then, elute the supernatant obtained by chromatography and concentrate it through a 30 kDa molecular weight cutoff membrane. Finally, collect the ITExo carriers released into the culture medium and resuspend them in phosphate buffer (PBS).

[0020] The ITexo support obtained through the above steps was further confirmed by surface plasmon resonance (SPR), and its affinity for integrins was found to be KD: 3.6 × 10⁻⁶ -9 It is possible to reach M.

[0021] In Figure 1A, it was demonstrated that the ITExo carrier exhibited expression of the integrin antibody (anti-Integrin), and that the ITExo carrier bound to the integrin antibody (anti-Integrin) did not show any change in its overall size or shape compared to a typical exosome (exo carrier). Western blot analysis verified that the ITExo carrier possessed the labeling proteins (CD63, CD81, CD9, and Alix) found in typical exosomes, and clearly confirmed the enriched expression of CD63 and the integrin antibody (anti-Integrin) in the ITExo carrier.

[0022] Refer to Figures 2A to 2C. To demonstrate that the ITexo carrier can be effectively applied in vivo, a blood-brain barrier cell culture model 10 was provided. The purpose of this model is to simulate the blood-brain barrier environment in vivo and to ensure that the ITexo carrier can pass through the blood-brain barrier and specifically bind to nerve cells 20.

[0023] Here, the nerve cells 20 are formed by the differentiation of human-induced pluripotent stem cells (iPSCs) after induction. The step includes inducing the inducible pluripotent stem cells into neural stem cells (NSCs) through culture using PSC neural induction medium (Gibco, A1647801), and then differentiating the NSCs cells into mature nerve cells (NSCs) through culture in differentiation medium. The differentiation medium includes neurobasal medium (Gibco, 17504044), nerve cell supplement (B-27™ supplement, Gibco, 35050061), and L-alanyl-L-glutamine dipeptide supplement (GlutaMAX™ supplement, Gibco, 35050061).

[0024] Here, the blood-brain barrier cell culture model 10 includes a first culture region 11 and a second culture region 12, and the first culture region 11 and the second culture region 12 are connected by a plurality of perforations 13. The first culture region 11 and the second culture region 12 are filled with culture medium A, and the nerve cells 20 are cultured at the bottom of the first culture region 11, while primary microglia 30 are placed at the bottom of the second culture region 12 adjacent to the plurality of perforations 13, thereby effectively establishing the impedance environment of the blood-brain barrier. In subsequent experiments, an Exo carrier and an ITExo carrier are sequentially administered to the second culture region 12 of the blood-brain barrier cell culture model 10 as needed, and experimental analysis is performed on the primary microglia 30 in the second culture region 12 and the nerve cells 20 in the first culture region 11, respectively, to confirm the effectiveness of the ITExo carrier passing from the second culture region 12 to the first culture region 11.

[0025] Refer to Figures 2B and 2C. The Exo carrier and ITExo carrier were labeled with green fluorescence using immunofluorescence technology. The expression results of the ITExo carrier in primary microglia 30 in the second culture region 12 and in the nerve cells 20 in the first culture region 11 were observed using flow cytometry technology. The results showed that the rate of ITExo carrier entry into microglia was 86.0%, which is significantly higher than the rate of Exo carrier entry (27.1%). Similar results were observed in the nerve cells 20, further demonstrating that the ITExo carrier possesses excellent targeting ability for brain nerves and the ability to cross the blood-brain barrier (BBB).

[0026] Brain-derived neurotrophic factor (BDNF) is an active protein that can regulate the growth, survival, and synapse formation of nerve cells in the brain. It is considered a very important nutrient protein in the brain, and studies have shown that BDNF has potential in treating brain injury and delaying aging.

[0027] To demonstrate the efficacy of BDNF in neuronal cell therapy, the present invention further encapsulated miRNA-BDNF, a microribonucleic acid (mMicro ribonucleic acid, abbreviated as miRNA) used for BDNF expression, using an ITexo carrier and a general exosome (Exo carrier), and then conducted subsequent cell and animal experiments to confirm the specificity and functionality of the ITexo carrier and the efficacy of BDNF in neuronal cell therapy for brain injury or neuronal degeneration.

[0028] The central nervous system was constructed as a central nervous system injury model, and the aforementioned blood-brain barrier cell culture model 10 was cultured for 48 hours at 37°C in a hypoxic environment consisting of a mixed gas of 92% N2, 3% O2, and 5% CO2. By confirming the cellular inflammation index of primary microglia (Figure 3) and the nerve cells 20 (Figure 4), the physiological expression of the central nervous system during stroke was simulated.

[0029] To demonstrate the efficacy of BDNF@ITExo for neuronal cell therapy, the present invention subsequently further treats stroke cell models in groups, including the following: Control group 1 (Ctl): Group that did not undergo hypoxic environment culture; Control group 2 (Hyp): Group that underwent culture in a hypoxic environment; Control group 3 (Exo): A group that underwent co-culture treatment by administering the Exo carrier in a hypoxic environment; Control group 4 (ITExo): A group that underwent co-culture treatment by administering an ITexo carrier in a hypoxic environment; Control group 5 (BDNF@Exo): This group underwent co-culture treatment in a hypoxic environment by administering an Exo carrier carrying miRNA-BDNF; Experimental group 6 (BDNF@ITExo): This group administered an ITExo carrier loaded with miRNA-BDNF in a hypoxic environment and performed co-culture treatment.

[0030] When comparing experimental group 6 with the other groups, it was observed that when primary microglia and the nerve cells 20 were cultured in the hypoxic environment and simultaneously treated with BDNF@ITExo, the expression levels of the inflammatory factors TNF-α and IL-1β were significantly reduced in both primary microglia and the nerve cells 20.

[0031] In addition, the present invention further confirmed the relative expression relationships of regenerative factors related to regeneration among the groups. In experimental group 6, the group treated with BDNF@ITExo not only had lower expression levels of the inflammatory factors compared to the stroke cell models of the other groups, but the expression of the regenerative factors (BDNF, GDNF, MCP-1, and IL-10, etc.) was also significantly higher than that of the stroke cell models of the other groups (not shown).

[0032] The present invention then constructed an animal model of stroke and tested the behavioral changes of the stroke animal model after treatment with BDNF@ITExo. After collecting blood from the tail of a rat, the blood was injected into the rat's brain, and the motor, sensory, reflex, and balance functions of the stroke animal model were analyzed using a modified neurological severity score (mNSS).

[0033] The stroke animal models were then divided into groups, and each group was administered to the stroke animal models via intravenous injection for five consecutive days, using physiological saline (placebo) (Saline), Exo carrier, ITexo carrier, BDNF@Exo carrier, and BDNF@ITExo carrier, respectively, in 100 microliters of 5 × 10⁶ solutions each time. 9 Participants were injected with saline solution containing the particles, and the mNSS behavioral test was performed daily. As shown in Figure 5, the mNSS scores in the BDNF@ITExo treatment group were significantly lower than those in the other groups (this was particularly pronounced on days 2 and 3).

[0034] Furthermore, exosomes were labeled in each organ of the stroke animal model using the near-infrared lipophilic dye DiR, and each organ was imaged using the IVIS bio-optical imaging system. The optical signals obtained from the images were then quantified to compare the absorption effects of BDNF@ITExo and BDNF@Exo in each organ after administration of BDNF@Exo and BDNF@ITExo, respectively.

[0035] Figure 6 shows the results after quantifying the optical signals, and it was observed that the labeling reaction of the exosome in the brain could be clearly measured in the animal model group administered BDNF@ITExo. Not only was the specificity of ITExo demonstrated, but it was also proven that ITExo possesses the ability to cross the blood-brain barrier.

[0036] In Figure 7A, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed on rat brain sections in the stroke animal model groups administered with physiological saline and BDNF@ITExo, respectively, and the proportion of cells exhibiting apoptotic characteristics in the sections was evaluated. It was observed that the proportion of apoptotic cells in the stroke animal model group treated with BDNF@ITExo was significantly lower than that in the group treated with physiological saline. In Figure 7B, the expression of the regenerative factor BDNF protein was detected in rat brain sections of the stroke animal model groups administered with physiological saline and BDNF@ITExo, respectively, using IHC staining technology. From the results, it was found that BDNF expression could be clearly measured in the brain sections of the stroke animal model group treated with BDNF@ITExo. Based on the results described above, we were able to comprehensively evaluate that BDNF crosses the blood-brain barrier via ITexo and is transmitted to the brain region affected by brain injury, thereby reducing the secretion of the inflammatory factor, mitigating the onset of the inflammatory response, and increasing the expression of the regenerative factor and BDNF, thereby improving the motor function of the stroke animal model rat.

[0037] Furthermore, to demonstrate that the ITexo carrier can similarly exert excellent effects on other brain injury diseases, we constructed further animal models of dementia and confirmed the behavioral changes of these dementia animal models after BDNF@ITExo treatment. We performed hindbrain surgery on the rats to induce vascular dementia, and analyzed the motor, sensory, reflex, and balance functions of the stroke animal models using the Modified Neurological Severity Score (mNSS). We also confirmed the cognitive function of each dementia animal model using a novel object recognition (NOR) test.

[0038] The aforementioned dementia animal models were divided into groups, and the stroke animal models in each group were administered saline (Saline) and BDNF@ITExo, respectively, by intravenous injection for four consecutive days. Healthy rats were used as the control group, and the mNSS behavioral test and NOR test were performed daily. As shown in Figure 8A, the mNSS scores, which began on the third day, were significantly lower in the groups treated with BDNF@ITExo than in the other groups. Figures 8B and 8C further analyze the Rotarod test and the Two-object recognition task in the mNSS behavioral test. The Rotarod test monitored the time each group of stroke animal models spent performing Rotarod movements on the Rotarod. The two-object recognition task evaluated the time each group of stroke animal models spent searching for a familiar object and an unknown object. The time difference ΔT, obtained by subtracting the time T1 for searching for the familiar object from the time T2 for searching for the unknown object, was used as the ability of the stroke animal models to distinguish between new and old objects. In both cases, the group treated with BDNF@ITExo showed superior performance compared to the dementia animal models administered with saline. Notably, in the Rotarod test, the time the group treated with BDNF@ITExo maintained Rotarod movements gradually increased, and this overall trend was similar to that of the control group, confirming that the group treated with BDNF@ITExo still possessed a certain level of motor and learning ability.

[0039] In addition, the present invention further constructed an animal model of spinal cord injury and confirmed the behavioral changes in the spinal cord injury model after BDNF@ITExo treatment. A laminectomy was performed on the T9 thoracic vertebral body of the rats, followed by induction of spinal cord contusion using an impact device (parameter settings: 250 kilodyne force, 2.5 m / s velocity, and 1.7 mm displacement). This damaged the spinal cord and destroyed its motor function, which was then evaluated through the Basso, Beattie, and Bresnahan (BBB) ​​scores.

[0040] Furthermore, exosomes were labeled in each organ of the spinal cord injury animal model using the near-infrared lipophilic dye DiR, and the absorption effects of BDNF@ITExo and BDNF@293Exo in each organ after administration of BDNF@Exo and BDNF@ITExo, respectively, were compared. In Figure 9, after normalizing each organ by the fluorescence value of BDNF@Exo, it was measured that the labeling reaction of BDNF@ITExo in the spinal cord was approximately five times that of the BDNF@Exo group. This confirmed the accumulation of integrin expression at the site of nerve injury lesions and demonstrated the specificity of ITExo and its ability to cross the blood-brain barrier.

[0041] The spinal cord injury animal models were then divided into groups, and the spinal cord injury animal models within each group were administered by intravenous injection for four consecutive days, using saline, exocarcinoma, iTexocarcinoma, BDNF@exocarcinoma, and BDNF@itexocarcinoma respectively, with each injection being 100 microliters of 5 × 10⁶. 9 Saline solution containing individual particles was injected, and BBB behavioral tests were performed on days 7, 14, 21, and 28. As shown in Figure 10A, the group treated with BDNF@ITExo showed significantly higher BBB scores starting from day 7 compared to the other groups.

[0042] Figure 10B shows the results of a grid walk test, where gait accuracy and bilateral coordination were evaluated for the gait parameters of the spinal cord injury animal models in each group described above. The success rate was defined as the ratio of whether the claw placement was accurate or not. Simultaneously, referring to Figures 10C to 10F, quantitative analysis was performed on the gait parameters of the spinal cord injury animal models in each group using a fully automated rat gait analysis system (Catwalk) to evaluate gait coordination and strength. This includes the angle of incline of the hind limb sole contact with the ground for each group, analyzed using healthy rats as the baseline in Figure 10C; the regular index of each group, analyzed after normalizing the saline administration group to a value of 0, as shown in Figure 10D; the swing speed of each group, detected in Figure 10E; and the stride length of the hind limb, as shown in Figure 10F. In all of the results described above, the group treated with BDNF@ITExo showed the most significant improvements in gait accuracy, hindlimb coordination, and trunk stability, and its success rate was observed to be higher than that of the control group and the mBDNF@EV group (p < 0.001). This demonstrates the targeting ability of the ITexo carrier and the significant improvement effect of mBDNF on the aforementioned spinal cord injury animal model.

[0043] Furthermore, sections were prepared from the spinal cord injury sites of each of the spinal cord injury animal models in the saline (Saline) administration group, the BDNF@Exo carrier group, and the BDNF@ITExo carrier group, respectively. Immunofluorescence staining was performed, and astrocytes (green) were labeled with glial fibrillary acidic protein (GFAP), and neurons (red) were labeled with neurofilament protein to evaluate the state of nerve fiber growth and repair at the spinal cord injury site in each of the spinal cord injury animal models. As shown in Figure 11A, the spinal cord injury site (white dashed line) in the group treated with BDNF@ITExo showed not only a smaller wound space due to the injury than the other groups, but also weaker astrocyte fluorescence intensity than in the saline (Saline) administration group and the BDNF@Exo carrier group. This confirmed that administration of the BDNF@ITExo carrier can reduce the accumulation of astrocytes at the spinal cord injury site. Of particular note is the abundant expression of neurofilament proteins at the spinal cord injury site in the group administered the BDNF@ITExo carrier. Figure 11B quantifies the percentage of the area of ​​the spinal cord injury site (the area to the right of the white dashed line or the area between the two white dashed lines) occupied by the red fluorescence area of ​​neurofilament proteins in Figure 11A, and evaluates the expression status of neurofilament proteins. The results showed that the growth and repair status of nerve fibers at the spinal cord injury site was superior in the group described above. Figures 11C and 11D similarly measure the expression of inflammatory factors (IFN-γ, IL-6, IL-8, IL-1β, and TNF-α) and regenerative factors (BDNF, NGF, GDNF, IL-10, and VEGF-A, etc.) at the spinal cord injury site in each of the above groups, and the results are consistent with the above experiment.

[0044] The ITexo carrier provided by the present invention can carry drugs and pass through the blood-brain barrier, achieve specific binding to central nervous system cells after injury, effectively reduce the expression of inflammatory factors (INF-γ, IL-1β, IL-6, TNF-α), improve the expression of regenerative factors (BDNF, NGF, VEGF, IL-10), improve motor function in animal models of brain injury or central nervous system injury, and achieve the effect of repairing nerve cells. [Explanation of symbols]

[0045] 10 Cell culture models 11 1st culture area 12 Second culture area 13 perforation 20 nerve cells 30 First Generation Microglia

Claims

1. A central nervous system-targeted pharmaceutical composition in which an integrin antibody is conjugated on an exosome transmembrane protein, The exosomes are obtained by secretion from a parent cell line after gene transgenicization, and at least a portion of the protein sequence of the integrin antibody contains one of SEQ ID No: 1 to SEQ ID No: 5, comprising a central nervous system targeted pharmaceutical composition.

2. The central nervous system targeted pharmaceutical composition according to claim 1, wherein the transmembrane protein is selected from one of CD68, CD81, and CD9.

3. The central nervous system targeted pharmaceutical composition according to claim 2, wherein the exosome encapsulates miRNA-BDNF.

4. A central nervous system-targeted pharmaceutical composition for treating or alleviating central nervous system injury diseases, comprising an exosome on which an integrin antibody is conjugated to a transmembrane protein, The exosomes are obtained by secretion from the parental cell line after gene transgenicization, and at least a portion of the protein sequence of the integrin antibody contains one of SEQ ID No: 1 to SEQ ID No:

5. The aforementioned exosomes contain miRNA-BDNF and are a central nervous system-targeted pharmaceutical composition for treating or alleviating central nervous system injury diseases.

5. The central nervous system targeted pharmaceutical composition according to claim 4, wherein the transmembrane protein is selected from one of CD68, CD81, and CD9.

6. The central nervous system-targeted pharmaceutical composition according to claim 5, wherein the central nervous system injury disease includes stroke, dementia, or spinal cord injury disease.

7. A central nervous system targeted pharmaceutical composition for activating neuronal filament protein expression, comprising an exosome on which an integrin antibody is bound to a transmembrane protein, The exosomes are obtained by secretion from the parental cell line after gene transgenicization, and at least a portion of the protein sequence of the integrin antibody contains one of SEQ ID No: 1 to SEQ ID No:

5. The exosome is a central nervous system-targeted pharmaceutical composition that encapsulates miRNA-BDNF and activates the expression of neuronal filament proteins.

8. The central nervous system targeted pharmaceutical composition according to claim 7, wherein the transmembrane protein is selected from one of CD68, CD81, and CD9.

Images