Targeted carrier with property of crossing blood-brain barrier, pharmaceutical composition and use thereof

Abstract

To provide a targeted carrier with the property of crossing the blood-brain barrier, a pharmaceutical composition and uses thereof.SOLUTION: Provided is a targeted carrier with the property of crossing the blood-brain barrier, comprising an extracellular vesicle with a human leukocyte antigen-G antibody bound on its surface. The targeted carrier can serve as a pharmaceutical composition for promoting apoptosis of brain tumor cells, inhibiting growth of brain tumor cells, or reducing expression of O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells, thereby achieving the effect of treating glioblastoma multiforme (GBM).SELECTED DRAWING: Figure 2B

Description

[Technical field]

[0001] The present invention relates to a drug carrier, particularly a drug carrier having blood-brain barrier penetrating properties, and uses thereof. [Background technology]

[0002] Glioblastoma multiforme (GBM) is the most common cerebral malignant tumor. This type of tumor cell is difficult to treat, and conventionally, surgery and postoperative radiation therapy can only remove a portion of the cancer cells. Furthermore, GBM contributes to the development of drug resistance to Temodal, the only oral chemotherapy drug, due to the expression of O6-methylguanine-DNA methyltransferase (MGMT), so the average life expectancy of patients after treatment is only 10 months to 1 year, and the survival rate is less than 5%. Therefore, an effective treatment for GBM is urgently needed in current medical practice. Summary of the Invention [Problem to be solved by the invention]

[0003] Provided are a drug carrier having blood-brain barrier penetrating properties, a pharmaceutical composition, and uses thereof. [Means for solving the problem]

[0004] Provided is a drug carrier having blood-brain barrier passing properties, which comprises extracellular vesicles having human leukocyte antigen-G antibodies on the surface thereof.

[0005] The extracellular vesicles bind to the human leukocyte antigen-G antibody via a transmembrane protein.

[0006] The membrane-transducing protein comprises a single domain antibody fragment that binds to the human leukocyte antigen-G antibody.

[0007] The single domain antibody fragment is a variable domain of a heavy chain of a heavy-chain antibody (VHH) or a single-chain variable fragment (single-chain variable fragment, scFv).

[0008] The transmembrane protein is selected from the group consisting of CD68, CD81 and CD9.

[0009] The drug carrier contains a down-regulated gene or drug.

[0010] Furthermore, the present invention also provides a use of the drug carrier for promoting apoptosis of brain tumor cells, inhibiting the growth of brain tumor cells, or reducing the expression of O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells. Effect of the Invention

[0011] The drug carrier provided by the present invention can effectively cross the blood-brain barrier, specifically reach the location of tumor cells, release the drug, and achieve excellent therapeutic effect in promoting apoptosis of tumor cells. [Brief description of the drawings]

[0012] [Figure 1A] FIG. 1 is an analysis of GBM cell lines according to a preferred embodiment of the present invention. [Figure 1B] FIG. 2 is a fluorescence profile of a GBM cell line according to a preferred embodiment of the present invention. [Figure 2A] FIG. 2 is a surface plasmon resonance test diagram of a drug carrier according to a preferred embodiment of the present invention. [Figure 2B] FIG. 2 is a microscopic image of a drug carrier according to a preferred embodiment of the present invention. [Figure 2C] FIG. 2 is a tracking analysis diagram of nanoparticles of a drug carrier according to a preferred embodiment of the present invention. [Figure 2D]FIG. 2 is a quantitative analysis diagram of membrane-permeable proteins of a drug carrier according to a preferred embodiment of the present invention. [Figure 2E] FIG. 2 is an expression analysis diagram of a membrane-permeable protein of a drug carrier according to a preferred embodiment of the present invention. [Figure 2F] FIG. 1 is a quantitative analysis diagram of HLA-G antibodies of a drug carrier according to a preferred embodiment of the present invention. [Diagram 3] FIG. 2 is a fluorescent staining diagram of a GBM cell line according to a preferred embodiment of the present invention. [Figure 4A] FIG. 2 is an analysis diagram of the loading capacity of the drug carrier of a preferred embodiment of the present invention. [Figure 4B] FIG. 1 shows an analysis of endocytosis in GBM cell lines according to a preferred embodiment of the present invention. [Diagram 5] FIG. 1 is a protein expression analysis diagram of a GBM cell line according to a preferred embodiment of the present invention. [Figure 6] FIG. 1 is an illustration of a blood-brain barrier cell culture model according to a preferred embodiment of the present invention. [Figure 7] FIG. 1 is a trend diagram of mortality rates of GBM cell lines according to a preferred embodiment of the present invention. [Figure 8A] FIG. 2 is a fluorescent staining diagram of BAX protein expression in a GBM cell line according to a preferred embodiment of the present invention. [Figure 8B] FIG. 1 is an analysis of apoptotic factors in GBM cell lines according to a preferred embodiment of the present invention. [Figure 9] FIG. 1 is an expression analysis diagram of MGMT protein according to a preferred embodiment of the present invention. [Figure 10A] FIG. 2 is an image of drug carrier distribution in an animal model according to a preferred embodiment of the present invention. [Figure 10B] FIG. 2 is a trend graph of body weight of mice in an animal model according to a preferred embodiment of the present invention. [Figure 10C] FIG. 2 is a trend diagram of survival rate in an animal model of a preferred embodiment of the present invention. [Figure 10D] FIG. 1 shows the growth trend of GBM cell lines in an animal model according to a preferred embodiment of the present invention. [Figure 10E] FIG. 2 is a stained image of a tissue section of an animal model according to a preferred embodiment of the present invention. [Figure 11A] FIG. 2 is a fluorescence diagram of a first sample in a preferred embodiment of the present invention. [Figure 11B] FIG. 2 is an expression analysis diagram of a drug carrier of the first sample according to a preferred embodiment of the present invention. [Figure 11C] FIG. 1 is an expression analysis diagram of MGMT protein of the first sample in a preferred embodiment of the present invention. [Figure 11D] FIG. 2 is a trend diagram of cell mortality rate of the first specimen in a preferred embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] As used herein, "extracellular vesicles (EVs)" refer to bilayer lipid vesicles secreted by cells into the extracellular space. Extracellular vesicles include exosomes and microvesicles, and as a new method of intercellular communication, they have become a new research field in physiology, pathology, nanoscience, etc. in recent years. Extracellular vesicles are highly heterogeneous endoplasmic reticulum secreted by cells, and their contents include proteins, mRNA / miRNA, DNA, lipids, etc., and they are classified based on their biological origin, release route, size, content and function, and purification methods are distinguished according to particle size.

[0014] The present invention will be described below in combination with the specification, drawings and specific examples, but the examples do not impose any formal limitations on the present invention. Unless otherwise specified, the reagents, methods and devices used in the present invention are common reagents, methods and devices in the technical field. The drawings should be expressed in color, but are converted to black and white due to the limitations of the expression means of electronic application software.

[0015] Referring to Figure 1A and Figure 1B, the present invention firstly proves the high expression of O6-methylguanine-DNA methyltransferase (MGMT) protein in three different glioblastoma multiforme (GBM) cell lines (1.U87, 2.8901 and 3.DBTRG) by Western blotting and fluorescent staining techniques, respectively. In addition, human leukocyte antigen G (HLA-G) is an important immune regulatory molecule in the body, and HLA-G is abnormally highly expressed on the surface of various tumor cells, and many studies have found that the expression of HLA-G can suppress the immune response of tumor cells and prevent tumor cells from being attacked by the immune system.

[0016] As can be seen from the Western blotting results in Figure 1A, U87, 8901 and DBTRG cells all show expression of MGMT and HLA-G. In the fluorescent staining in Figure 1B, F-actin is marked with red fluorescence to define the cell range, nuclei are marked with blue fluorescence to define the position of the cell nucleus, and HLA-G protein is marked with green to confirm the expression of HLA-G. It can be seen that all HLA-G proteins are expressed in the cells.

[0017] The present invention provides a drug carrier, which is prepared by binding human leukocyte antigen-G antibody (HLA-G antibody) to the surface of extracellular vesicles, and the drug carrier achieves the effect of specifically binding to the HLA-G protein of GBM tumor cells via the HLA-G antibody, and can promote the endocytosis of GBM tumor cells while avoiding the binding reaction of normal cells to the drug carrier (αHLA-GEV).

[0018] Here, the extracellular vesicles are bilayer lipid vesicles secreted into the extracellular space by parent cells. After culturing the cells, the medium is collected and the extracellular vesicles can be extracted from the medium by conventional separation techniques such as centrifugation or filtration.

[0019] Preferably, the parent cell may be Human Embryonic Kidney Cells 293 (HEK293) or a derived cell line thereof, such as HEK293T, HEK293F, HEK293A, or HEK293E.

[0020] Preferably, the parent cell may be a mesenchymal stem cell (MSC).

[0021] Here, the extracellular vesicles can bind to HLA-G antibodies via a membrane-permeable protein, which may be CD63, CD81, or CD9.

[0022] Here, the sequence of the HLA-G antibody can be inserted into the extracellular vesicles to produce the extracellular vesicles expressing the HLA-G antibody.

[0023] Preferably, the membrane-permeable protein of the extracellular vesicle expresses a single domain antibody fragment, and the single domain antibody fragment serves as a site of specific binding between the membrane-permeable protein and the HLA-G antibody.

[0024] Here, the single domain antibody fragment is a variable domain of a heavy chain of a heavy-chain antibody (VHH) or a single-chain variable fragment (scFv). Furthermore, the expression of the single domain antibody fragment of the membrane-transducing protein can be achieved by techniques such as gene transfer and gene processing.

[0025] In addition, in this embodiment, a synthetic sequence of tandem HLA-G antibody / single domain antibody fragment / membrane-transmitting protein is synthesized by molecular cloning technology, and the synthetic sequence is inserted into pcDNA3.4 plasmid (ThermoScientific) to create an HLA-G antibody expression plasmid. Next, the HLA-G antibody expression plasmid is introduced into the parent cells using Lipofectamine3000 reagent (ThermoScientific) and cultured, and the extracellular vesicles expressing the HLA-G antibody secreted from the parent cells are collected.

[0026] The present invention will next be described as examples in which the membrane-permeable protein CD63 and the HLA-G antibody expressing the single domain antibody fragment are used, and as a comparative example in which conventional extracellular vesicles (EVs) that do not bind to the HLA-G protein are used.

[0027] Referring to FIG. 2A, the surface plasmon resonance (SPR) technique was used to detect the response units of the drug carriers at different concentrations, and the affinity parameter (Affinity, K D In this embodiment, the concentration range of the drug carrier used for surface plasmon resonance technology detection is 25 to 0.78125 nM, and after detection and numerical conversion, the affinity parameter of the drug carrier is K D Value 7.18x10 -9 (molar concentration, M), and K D A value of 10 -9 A lower affinity than this indicates that the antibody has high affinity.

[0028] FIG. 2B shows that, using a transmission electron microscope (TEM), there is not much difference in particle size between the conventional extracellular vesicles (EVs) and the drug carrier (αHLA-GEVs), and it can be determined that the two have similar properties.

[0029] 2C, nanoparticle tracking analysis (NTA) can be used to confirm whether the particle sizes of the common extracellular vesicles (EVs) and the drug carriers (αHLA-GEVs) are comparable, and further detect whether the concentrations of the common extracellular vesicles (EVs) and the drug carriers (αHLA-GEVs) after extraction are similar, demonstrating that both the common extracellular vesicles (EVs) and the drug carriers (αHLA-GEVs) belong to the extracellular vesicles.

[0030] Figures 2D and 2E further analyze the overall expression ratio of the transmembrane protein CD63 after extraction of the normal extracellular vesicles (EVs) and the drug carrier (αHLA-GEVs). It can be seen that the expression ratio of the transmembrane protein CD63 in the entire drug carrier (αHLA-GEVs) carrying the HLA-G antibody reaches 90%. Similarly, the results of Western blotting show that the expression levels of the transmembrane proteins CD63 and CD81 in the drug carrier (αHLA-GEVs) are significantly higher than those in the normal extracellular vesicles (EVs). Figure 2F further demonstrates that the drug carrier (αHLA-GEVs) retains the HLA-G antibody.

[0031] Figure 3 shows the endocytosis effect of the drug carrier (αHLA-GEV) on the GBM cell lines 8901 and DBTRG, respectively, and marks F-actin with red fluorescence to define the cell range, and marks extracellular vesicles with green to confirm whether the extracellular vesicles have entered the cell range. The results of fluorescent staining show that the expression level of the drug carrier in the GBM cell lines is significantly increased, proving that the drug carrier has enhanced affinity with the GBM cell lines by carrying HLA-G antibodies.

[0032] In order to prove the ability of the drug carrier (αHLA-GEV) to release the drug after entering the cell through endocytosis, the present invention further carries the down-regulating gene miRNA-181 (miR-181) that suppresses the expression of MGMT in the drug carrier, ensuring that the drug carrier can exert the effect of suppressing MGMT when entering the GBM cell line. Here, Figure 4A shows the expression level of miR-181 in the drug carrier after the drug carrier is coated with miR-181 by reverse transcription-polymerase chain reaction (RT-PCR). In this experiment, reverse transcription polymerase chain reaction (RT-PCR) was used to compare the expression level of 50ng of miR-181 with the standard value of 1, and the drug carriers without miR-181 (αHLA-GEV), the drug carriers with 25ng of miR-181 (miR-181@αHLA-GEV(25g)), and the drug carriers with 50ng of miR-181 (miR-181@αHLA-GEV(50g)) were compared. It was proved that the expression level of the drug carriers with 50ng of miR-181 (miR-181@αHLA-GEV(50g)) was equivalent to that of the same amount of miR-181 (50ng) not coated in the drug carriers, proving the effectiveness of the drug carriers to carry small molecules (such as miR-181, proteins or drugs) after binding to HLA-G antibodies.

[0033] In FIG. 4B, the expression level of miR-181 in GBM cell lines 8901 and DBTRG was confirmed by RT-PCR after the drug carrier was taken up through endocytosis. Here, the group without reaction was the first control group (Clt), which was set as the reference value with a value of 1. The group in which miR-181 was loaded onto the drug carrier Lipo3000, which is commonly used in the current technology, and reacted with GBM cell lines was the second control group (Lipo3000). The group in which the drug carrier was reacted with GBM cell lines was the third control group (αHLA-GEV). The results show that in the experimental group (miR-181@αHLA-GEV) after the GBM cell lines were treated with the drug carrier loaded with miR-181, the expression level of miR-181 in the GBM cell lines was high, which was higher than that of Lipo3000, which is a drug carrier commonly used in the current technology. In addition to the proven efficacy of loading the drug carriers, they also have proven release capabilities.

[0034] In Figure 5, the group without reaction was the first control group (Clt). The drug carrier was reacted with GBM to form the second control group (αHLA-GEV). Direct reaction with miRNA was performed to form the third control group (miR-181). The drug carrier was loaded with miRNA as the experimental group (miR-181@αHLA-GEV), and the above groups were cultured with each GBM cell line for 24 hours, and the protein expression of each GBM cell line in each group was confirmed by Western blotting. The results showed that the expression of MGMT, an indicator protein of drug resistance of the chemotherapy drug Temodal (TMZ), was significantly reduced in each GBM cell line in the experimental group, proving the excellent downregulation effect of miR-181. In addition, the expression of Bcl-2 protein, an indicator of apoptosis inhibition, was also significantly reduced, and the expression of BAX protein, an indicator of apoptosis promotion, was significantly increased, which explains that the GBM cell line is also useful for promoting apoptosis after the reaction of miR-181.

[0035] In order to prove that the drug carrier can be effectively applied in the living body, FIG. 6 shows a blood-brain barrier cell culture model 10, which simulates the blood-brain barrier environment in the living body and ensures that the drug carrier can reliably pass through the blood-brain barrier to enter the brain region and specifically bind to the GBM cells. 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 communicated with each other by a plurality of through-holes 13. The first culture region 11 and the second culture region 12 are filled with hydrogel A, where the GBM cell line U87 is cultured in the hydrogel A of the first culture region 11, and the second culture region 12 is sequentially provided with endothelium cells 20, pericytes 30, and astrocytes 40, and the resistance value of the blood-brain barrier culture cell model 10 is 2144 Ω·cm 2 In the subsequent experiment, the drug carrier is administered to the second culture region 12 of the blood-brain barrier cell culture model 10, and an experimental analysis is performed on the GBM cell line U87 in the first culture region 11 to confirm the efficacy and therapeutic effect of the drug carrier passing through the second culture region 12 to the first culture region.

[0036] In this experiment, the group that was not reacted was the first control group (Clt). The drug carrier with HLA-G antibody bound to the surface was the second control group (αHLA-GEV). The third control group (miR-181) was reacted directly with miRNA. The fourth control group (TMZ) was reacted directly with the chemotherapy drug Temodal (TMZ). The drug carrier was loaded with miRNA to form the first experimental group (miR-181@αHLA-GEV). The drug carrier was loaded with a chemotherapy drug to form the second experimental group (TMZ@αHLA-GEV). The drug carrier loaded with miRNA and the drug carrier loaded with the chemotherapy drug were simultaneously reacted in a 1:1 ratio to form the third experimental group (miR-181@αHLA-GEV+TMZ@αHLA-GEV). The above groups were tested in the blood-brain barrier cell culture model 10 to confirm the cell death percentage after treatment with each group of the GBM cells on days 1, 2, and 3, respectively. From the results in FIG. 7, it was found that the GBM cells in the group treated with the chemotherapy drug Temodal (TMZ) had significant cell death (see the fourth control group (TMZ), the second experimental group (TMZ@αHLA-GEV), and the third experimental group (miR-181@αHLA-GEV+TMZ@αHLA-GEV)), while the group given only miR-181 had limited cell death (see the third control group (miR-181), the first experimental group (miR-181@αHLA-GEV)).

[0037] In addition, the fourth control group and the second experimental group were treated with the chemotherapy drug Temodal (TMZ), which successfully induced cell death in both groups, but the cell death tended to be mild by the third day, and drug resistance tended to develop. However, in the third experimental group (miR-181@αHLA-GEV+TMZ@αHLA-GEV), in which Temodal (TMZ) and the chemotherapy drug carrying miR-181 were administered simultaneously, not only was a clear toxic effect observed on the first day, but the cell death rate (cell death%) of the GBM cells was also significantly increased on the third day.

[0038] Furthermore, the first control group (Clt), the second control group (αHLA-GEV), the fourth control group (TMZ), the second experimental group (TMZ@αHLA-GEV) and the third experimental group (miR-181@αHLA-GEV+TMZ@αHLA-GEV) were used to analyze the expression of proteins in the GBM cell lines through Western blotting and fluorescent staining techniques. In the fluorescent staining in Figure 8A, BAX protein is marked with red fluorescence, and GBM cells are marked with green fluorescence to define the cell range. The results showed that only the GBM cells treated with the chemotherapy drug Temodal (TMZ) showed significant fluorescent expression of HLA-G protein, and the expression rate of BAX protein in the third experimental group of the GBM cell lines treated with both miRNA-181 and TMZ was significantly higher than that of the fourth control group and the second experimental group. FIG. 8B not only shows the results corresponding to those mentioned above, but also shows that the expression of Bcl-2 protein, which is an indicator of apoptosis inhibition in the third experimental group, was significantly reduced, demonstrating that the GBM cell lines in the third experimental group promoted apoptosis.

[0039] Figure 9 shows that the drug carriers carrying miR181 at concentrations of 100ng / ml, 200ng / ml and 400ng / ml were reacted with cells overexpressing MGMT protein (MGMT overexpression), and the drug carriers effectively overcome the blood-brain barrier cell culture model 10 and promote miR181 to down-regulate the expression of MGMT gene and protein. This result reflects the previous experiment, and it is confirmed that as the concentration of the drug carrier carrying miR181 increases, the MGMT protein concentration in cells overexpressing MGMT protein (MGMT overexpression) decreases.

[0040] Taking the above experimental results into consideration, it is proven that the drug carrier can effectively overcome the blood-brain barrier environment simulated by the blood-brain barrier cell culture model 10, promote the release of miR181 and the anti-cancer drug, increase the expression of apoptosis factors in GBM cell lines, reduce the drug resistance response of GBM cell lines, and improve the poisoning effect of anti-cancer drugs.

[0041] To prove the actual effect of the drug carrier in the organism, 10 6 The GBM cell line (U87) was transplanted into each mouse at a cell count of 100 cells to establish an animal model of glioblastoma multiforme. Seven days after the transplantation, the chemotherapy drug and / or the drug carrier were intravenously injected into each mouse, and the growth of the chemotherapy drug and / or the drug carrier and the GBM cell line in the mouse brain was confirmed by imaging.

[0042] In Figure 10A, the control group (TMZ@EV) was loaded with an anti-cancer drug in the conventional extracellular vesicles, and the experimental group (TMZ@αHLA-GEV) was loaded with the anti-cancer drug in the drug carrier. 60 minutes (min) after intravenous injection, the conventional extracellular vesicles in the control group were widely distributed in the upper body region of the experimental mouse (especially the lung region), whereas the drug carriers in the experimental group (TMZ@αHLA-GEV) were clearly concentrated in the brain region of the experimental mouse, demonstrating that the drug carrier (αHLA-GEV) has targeting (specificity).

[0043] 10B to 10D, in the animal model, a group administered with saline is designated as the first control group (Clt), a group administered with the drug carrier having HLA-G antibody bound to its surface is designated as the second control group (αHLA-GEV), a group administered with the chemotherapy drug Temodal (TMZ) is designated as the third control group (TMZ), a group administered with the drug carrier carrying miRNA is designated as the first experimental group (miR-181@αHLA-GEV), a group administered with the drug carrier carrying the chemotherapy drug is designated as the second experimental group (TMZ@αHLA-GEV), and a group administered with a 1:1 mixture of drug carriers carrying miRNA and the chemotherapy drug is designated as the third experimental group (miR-181@αHLA-GEV+TMZ@αHLA-GEV). The weight, survival rate, and growth status of the GBM cell line of the experimental mice in each group were confirmed by images every 7 days.

[0044] There is no significant difference in the bodies of the experimental mice in each group in Figure 10B, and there is no significant change in physiological status due to the administration of different injections. However, as can be seen from Figure 10C, the mice in the first control group (Clt), the second control group (αHLA-GEV), and the first experimental group (miR-181@αHLA-GEV) all died within about 30 days after administration, and the survival rate of the experimental mice tended to increase in the groups treated with the chemotherapy drug Temodal (TMZ) (e.g., the third control group and the second experimental group). However, in the third experimental group, Temodal (TMZ) and a chemotherapy drug carrying miR-181 were administered simultaneously, and the survival rate of the experimental mice was significantly improved in the third experimental group (miR-181@αHLA-GEV+TMZ@αHLA-GEV), and was significantly improved even after 60 days or more.

[0045] FIG. 10D and FIG. 10E show the fluorescence expression (Intensity (10 6)) and hematoxylin and eosin staining (H&E Stain) techniques were used to analyze the staining of tissue sections showing tissue morphology, and to further analyze the growth status of the GBM cells. Compared with the first control group (Clt) and the second control group (αHLA-GEV), the growth status of the GBM cell lines in the third control group, the first experimental group (miR-181@αHLA-GEV) and the second experimental group (TMZ@αHLA-GEV) gradually increased, but still remained suppressed. However, in the third experimental group (miR-181@αHLA-GEV+TMZ@αHLA-GEV), the fluorescence response of the GBM cells tended to be generally flat and consistent, and even tended to decline on the 21st day, and the staining of tissue sections showed that the distribution area of ​​the GBM cells in the third experimental group was significantly smaller than that of the other groups. It was demonstrated that simultaneous administration of the drug carriers carrying miR-181 and TMZ to the experimental mice produced a significant additive effect in inhibiting the growth of the GBM cell line.

[0046] In addition, to prove that the drug carrier can produce similar excellent effects on human cells, Figures 11A to 11D provide further confirmation on tumor cells of a brain tumor patient, in which the brain tumor patient has developed drug resistance to the anti-cancer drug TMZ after anti-cancer drug treatment, and in the fluorescent staining of Figure 11A, F-actin is marked with red fluorescence to define the cell range, the cell nuclei are marked with blue fluorescence to define the position of the cell nuclei, and HLA-G protein is marked with green, and the expression of a large number of HLA-G proteins in the tumor cells can be observed.

[0047] Next, the tumor cells were cultured and then treated with the drug carrier carrying miR181. In the fluorescent staining of Figure 11B, F-actin was marked with purple fluorescence to define the cell range, the cell nuclei was marked with blue fluorescence to define the cell nucleus position, the drug carrier was marked with green, and miR181 was marked with red fluorescence, proving that the tumor cells had taken in and expressed the drug carrier and miR181 in the cells. In addition, Figure 11C demonstrated that after the tumor cells were treated with the drug carrier carrying miR181 (miR-181@αHLA-GEV), the expression of MGMT was significantly reduced compared with the drug carrier alone (αHLA-GEV) or the group treated with miR181 alone, the expression of the apoptosis inhibitor BCL2 was also reduced, and the expression of the apoptosis promoter BAA showed a clear tendency to increase.

[0048] Figure 11D compares the mortality rate of the tumor cells after treatment under various conditions. In this experiment, the group without reaction was the first control group (Clt), the drug carrier with HLA-G antibody bound to the surface was the second control group (αHLA-GEV), the one directly reacted with miRNA was the third control group (miR-181), the one directly reacted with the chemotherapy drug Temodal (TMZ) was the fourth control group (TMZ), the one with miRNA loaded on the drug carrier was the first experimental group (miR-181@αHLA-GEV), the one with chemotherapy drug loaded on the drug carrier was the second experimental group (TMZ@αHLA-GEV), and the one with miRNA loaded on the drug carrier and the one with chemotherapy drug loaded on the drug carrier were simultaneously administered in a 1:1 ratio and reacted with each other was the third experimental group (miR-181@αHLA-GEV+TMZ@αHLA-GEV). Similar to the previous paragraph, the third experimental group showed a good effect on promoting apoptosis of tumor cells.

[0049] The drug carrier provided by the present invention can effectively cross the blood-brain barrier, specifically reach the location of tumor cells, release the drug, and achieve excellent therapeutic effect in promoting apoptosis of tumor cells. [Explanation of symbols]

[0050] 10. Blood-brain barrier cell culture model 11 1st culture area 12 Second culture area 13 Through hole A Hydrogel 20 endothelial cells 30 pericytes 40 Astrocytes

Claims

1. A drug carrier having blood-brain barrier passing properties, comprising extracellular vesicles having human leukocyte antigen-G antibodies bound to their surfaces.

2. The drug carrier described in claim 1, wherein the extracellular vesicle binds to the human leukocyte antigen-G antibody via a membrane-permeable protein.

3. The drug carrier of claim 2, wherein the membrane-permeable protein comprises a single domain antibody fragment that binds to the human leukocyte antigen-G antibody.

4. The drug carrier according to claim 2, wherein the membrane-permeable protein is selected from the group consisting of CD68, CD81 and CD9.

5. The drug carrier according to claim 3, wherein the single domain antibody fragment is a variable domain of heavy chain of heavy-chain antibody (VHH) or a single-chain variable fragment (scFv).

6. A drug carrier according to any one of claims 1 to 5, comprising a down-regulated gene or drug within the drug carrier.

7. The drug carrier of claim 6 , wherein the downregulated gene includes miRNA-181 and the drug includes Temodal.

8. The use of the drug carrier having blood-brain barrier passing properties according to claim 1, comprising the down-regulated gene being miRNA-181, for decreasing the expression of O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells.

9. The use according to claim 8, wherein the brain tumor cells are glioblastoma multiforme (GBM).

10. Use of a drug carrier having blood-brain barrier passing properties as described in claim 1, which contains a down-regulated gene or drug, for promoting apoptosis of brain tumor cells.

11. The use according to claim 10, wherein the downregulated gene comprises miRNA-181 and the drug comprises Temodal.

12. The use according to claim 10 or 11, wherein the expression of Bcl-2 protein is decreased and the expression of BAX protein is increased in the brain tumor cells.

13. The use according to claim 10 or 11, wherein the brain tumor cells are glioblastoma multiforme (GBM).

14. The use of a drug carrier having blood-brain barrier passing properties as described in claim 1, comprising a down-regulated gene or drug, the down-regulated gene comprising miRNA-181, and the drug comprising Temodal, for inhibiting the growth of brain tumor cells.

15. The use according to claim 14, wherein the brain tumor cells are glioblastoma multiforme (GBM).

16. A pharmaceutical composition comprising a drug carrier having the blood-brain barrier passing property described in claim 1.

17. 17. Use of the pharmaceutical composition according to claim 16 for crossing the blood-brain barrier.

18. Use of a drug carrier comprising extracellular vesicles having surface-bound human leukocyte antigen G antibodies to cross the blood-brain barrier.

19. The use of claim 18, wherein the permeabilization protein of the extracellular vesicle comprises a single domain antibody fragment, which binds to the human leukocyte antigen-G antibody.

20. The use according to claim 18 or 19, wherein the transmembrane protein is selected from the group consisting of CD68, CD81 and CD9, and the single domain antibody fragment is a variable domain of heavy chain of heavy-chain antibody (VHH) or a single-chain variable domain antibody fragment (single-chain variable fragment (scFv)).

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