Drug carrier with blood brain barrier crossing characteristic, pharmaceutical composition and application of drug carrier and pharmaceutical composition

Abstract

The invention discloses a drug carrier with a blood-brain barrier crossing characteristic, which comprises an extracellular vesicle, and the surface of the extracellular vesicle is provided with a human leukocyte antigen-G antibody. Wherein the drug carrier can be further used as a pharmaceutical composition to achieve the effects of promoting the apoptosis of brain tumor cells, inhibiting the growth of the brain tumor cells, or reducing the expression of O6-methylguanine-DNA (Deoxyribose Nucleic Acid) methylated metastasis (O6-MGMT) in the brain tumor cells, so as to achieve the effect of treating glioblastoma multiforme (GBM).

Description

Technical Field

[0001] The present invention relates to a drug carrier, and in particular to a drug carrier capable of crossing a blood-brain barrier and a use thereof. Background Art

[0002] Glioblastoma multiforme (GBM) is the most common malignant tumor in the brain. This tumor cell is difficult to treat. Traditionally, surgery plus postoperative radiotherapy can only eliminate some cancer cells. In addition, GBM is resistant to the only oral chemotherapy drug Temodal due to the expression of O6-methylguanine-DNAmethyltransferase (MGMT). As a result, the average life expectancy of patients after treatment is only 10 months to one year, and the survival rate is <5%. Therefore, the development of effective treatment for glioblastoma multiforme (GBM) is an urgent direction for current medical development. Summary of the invention

[0003] A drug carrier with the property of crossing the blood-brain barrier comprises an extracellular vesicle with a human leukocyte antigen-G antibody on its surface.

[0004] The extracellular vesicle is combined with the human leukocyte antigen-G antibody via a transmembrane protein.

[0005] The transmembrane protein expresses a single domain antibody fragment that binds to the human leukocyte antigen-G antibody.

[0006] The single-domain antibody fragment is a variable domain of heavy chain of heavy-chain antibody (VHH) or a single-chain variable fragment (scFv).

[0007] Wherein, the transmembrane protein is selected from the group consisting of any one of CD68, CD81 and CD9.

[0008] The drug carrier contains a down-regulated gene or a drug.

[0009] Furthermore, the present invention also provides uses of the above-mentioned drug carrier, including promoting apoptosis of a brain tumor cell, inhibiting the growth of a brain tumor cell, or reducing the expression of O6-methylguanine-DNAmethyltransferase (MGMT) in a brain tumor cell.

[0010] The drug carrier provided by the present invention can effectively cross the blood-brain barrier, specifically reach the location of tumor cells, and release drugs to achieve the excellent therapeutic effect of promoting tumor cells to apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A This is a diagram of GBM cell line analysis in a preferred embodiment of the present invention;

[0012] Figure 1B This is a fluorescence image of a GBM cell line in a preferred embodiment of the present invention;

[0013] Figure 2A This is a test diagram of surface plasmon resonance of a drug carrier according to a preferred embodiment of the present invention;

[0014] Figure 2B This is a microscope image of a drug carrier according to a preferred embodiment of the present invention;

[0015] Figure 2C This is a tracking analysis diagram of drug carrier nanoparticles in a preferred embodiment of the present invention;

[0016] Figure 2D This is a quantitative analysis diagram of the transmembrane protein of the drug carrier in a preferred embodiment of the present invention;

[0017] Figure 2E This is a performance analysis diagram of the drug carrier transmembrane protein of a preferred embodiment of the present invention;

[0018] Figure 2F This is a quantitative analysis diagram of HLA-G antibody in a drug carrier of a preferred embodiment of the present invention;

[0019] Figure 3 This is a fluorescence staining image of a GBM cell line in a preferred embodiment of the present invention;

[0020] Figure 4A This is a diagram analyzing the loading capacity of the drug carrier in a preferred embodiment of the present invention;

[0021] Figure 4B This is a diagram of endocytosis analysis of GBM cell lines in a preferred embodiment of the present invention;

[0022] Figure 5 This is a protein expression analysis diagram of a GBM cell line in a preferred embodiment of the present invention;

[0023] Figure 6 This is a schematic diagram of a blood-brain barrier cell culture model according to a preferred embodiment of the present invention;

[0024] Figure 7 This is a trend chart of the mortality rate of the GBM cell line in a preferred embodiment of the present invention;

[0025] Fig. 8A This is a fluorescence staining image of BAX protein expression in GBM cell line in a preferred embodiment of the present invention;

[0026] Figure 8B This is a graph showing apoptosis factor analysis of GBM cell lines in a preferred embodiment of the present invention;

[0027] Fig. 9 This is a diagram showing the expression of MGMT protein in cells according to a preferred embodiment of the present invention;

[0028] Fig. 10A This is an image diagram of drug carrier distribution in an animal model of a preferred embodiment of the present invention;

[0029] Fig. 10B This is a weight trend chart of the animal model mice in the preferred embodiment of the present invention;

[0030] Fig. 10C This is a survival rate trend chart of the animal model in the preferred embodiment of the present invention;

[0031] Fig. 10D This is a growth trend diagram of the GBM cell line in the animal model of the preferred embodiment of the present invention;

[0032] Fig.10E This is a staining diagram of the tissue section of the animal model of the preferred embodiment of the present invention;

[0033] Fig.11A This is a fluorescence image of the first specimen in a preferred embodiment of the present invention;

[0034] Fig. 11B The first specimen of the preferred embodiment of the present invention shows a fluorescence image of the drug carrier;

[0035] Fig. 11C This is a graph showing the expression of MGMT protein in the first sample of the preferred embodiment of the present invention;

[0036] Fig.11D This is a trend diagram of the cell death rate of the first specimen in a preferred embodiment of the present invention.

[0037] Explanation of symbols:

[0038] 10 Blood-brain barrier cell culture model

[0039] 11The first cultivation area

[0040] 12 Second Training Area

[0041] 13. Piercing

[0042] A Hydrogel

[0043] 20 Endothelial cells

[0044] 30 weeks cells

[0045] 40Stellate cells DETAILED DESCRIPTION

[0046] It is understood that the "extracellular vesicles (EV)" used herein refers to bilayer lipid vesicles secreted by cells into the extracellular space. Extracellular vesicles include exosomes and microvesicles. As a new form of intercellular communication, they are a new field of research in physiology, pathology, nanoscience, etc. in recent years. Extracellular vesicles are highly heterogeneous vesicles secreted by cells, and their contents contain proteins, mRNA / miRNA, DNA, lipids, etc. They are classified according to their biogenesis, release pathway, size, content and function, and the purification method is mainly distinguished by particle size.

[0047] It is to be understood that the present invention is described below in conjunction with the description, drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0048] Please refer to Figure 1A And 1B, the present invention firstly uses Western blotting and fluorescent staining techniques to respectively prove the high expression of O6-methylguanine-DNAmethyltransferase (MGMT) protein in three different glioblastoma multiforme (GBM) cell lines (1.U87, 2.8901 and 3.DBTRG). In addition, human leukocyte antigen G (HLA-G) is an important immune regulatory molecule in the body. Many studies have found that HLA-G is abnormally highly expressed on the surface of various tumor cells. The expression of HLA-G can inhibit the immune response of tumor cells and prevent tumor cells from being attacked by immune system.

[0049] At Figure 1A The Western blotting results show that MGMT and HLA-G can be seen in U87, 8901 and DBTRG cells. Figure 1B In the fluorescent staining, red fluorescence was used to identify F-actin to define the cell range; blue fluorescence was used to identify the nucleus to define the nucleus position; and green fluorescence was used to identify HLA-G protein to confirm HLA-G expression. It can be found that HLA-G protein is expressed in cells.

[0050] The present invention provides a drug carrier, which combines a human leukocyte antigen-G antibody (HLA-G antibody) on the surface of an extracellular vesicle to prepare the drug carrier (αHLA-G EV). The drug carrier can achieve a specific binding effect with the HLA-G protein in GBM tumor cells through the HLA-G antibody, and promote the GBM tumor cells to perform endocytosis, while avoiding the binding reaction of normal cells to the drug carrier (αHLA-G EV).

[0051] The extracellular vesicles are bilayer lipid vesicles secreted into the extracellular space by a source mother cell. After the cells are cultured, the culture medium is collected and the extracellular vesicles can be extracted from the culture medium by conventional separation techniques such as centrifugation and filtration.

[0052] Preferably, the source mother cell can be a human embryonic kidney cell 293 cell line (HEK293) or a derived cell line thereof, such as HEK293T, HEK293F, HEK293A, or HEK293E.

[0053] Preferably, the source mother cell can be a mesenchymal stem cell (MSC).

[0054] The extracellular vesicle can be bound to the HLA-G antibody via a transmembrane protein, which can be CD63, CD81 or CD9.

[0055] The sequence of the HLA-G antibody can be taken and inserted into the extracellular vesicle to produce an extracellular vesicle expressing the HLA-G antibody.

[0056] Preferably, the transmembrane protein of the extracellular vesicle expresses a single-domain antibody fragment, so that the single-domain antibody fragment can serve as a site for the transmembrane protein to specifically bind to the HLA-G antibody.

[0057] Wherein, the single-domain antibody fragment is a heavy chain variable region fragment (variable domain of heavy chain of heavy-chain antibody, VHH) or a single-chain variable region antibody fragment (single-chain variable fragment, scFv). Furthermore, the transmembrane protein expressing a single-domain antibody fragment can be achieved by gene transfer, genetic engineering and other technologies.

[0058] Furthermore, in this embodiment, a synthetic sequence of tandem HLA-G antibody / single domain antibody fragment / transmembrane protein was synthesized by molecular cloning technology, and the synthetic sequence was inserted into a pcDNA3.4 plasmid (Thermo Scientific) to create an HLA-G antibody expression plasmid. Then, the HLA-G antibody expression plasmid was transfected into the source mother cells using Lipofectamine 3000 reagent (Thermo Scientific) and cultured, and the extracellular vesicles secreted by the source mother cells expressing the HLA-G antibody were collected.

[0059] The present invention will be described later using the transmembrane protein CD63 expressing the single domain antibody fragment and the HLA-G antibody as an example, and using a common extracellular vesicle (EV) as a comparative example not binding the HLA-G protein antibody.

[0060] refer to Figure 2A In the present study, the surface plasmon resonance (SPR) technique was used to detect the response unit of the drug carrier at different concentrations to obtain an affinity parameter (Affinity, K) of the drug carrier (αHLA-G EV). D In this embodiment, the concentration range of the drug carrier used for surface plasmon resonance technology is between 25nM and 0.78125nM. After detection and numerical conversion, it can be found that the affinity parameter of the drug carrier is K D Value 7.18x 10 -9 (Molar concentration,

[0061] M), K D Values ​​below 10 -9 The following indicates that the antibody has high affinity.

[0062] Figure 2B Transmission electron microscope (TEM) observations revealed little difference in particle size between the common extracellular vesicle (EV) and the drug carrier (αHLA-G EV), indicating that the two have similar characteristics.

[0063] Figure 2CNanoparticle tracking analysis (NTA) can not only confirm whether the particle sizes of the common extracellular vesicles (EV) and the drug carrier (αHLA-G EV) are similar, but also further detect whether the concentrations of the common extracellular vesicles (EV) and the drug carrier (αHLA-G EV) after extraction are similar. This confirms that the common extracellular vesicles (EV) and the drug carrier (αHLA-G EV) are both extracellular vesicles.

[0064] Figure 2D And 2E, further analysis of the common extracellular vesicles (EV) and the drug carrier (αHLA-G EV) after extraction, the overall expression of the transmembrane protein CD63 ratio. It can be found that the drug carrier (αHLA-G EV) with the HLA-G antibody expresses the transmembrane protein CD63 at a rate of up to 90%. Similarly, it can be seen in the Western blotting results that the expression of the transmembrane proteins CD63 and CD81 in the drug carrier (αHLA-G EV) is significantly higher than that in the common extracellular vesicles (EV). Figure 2F This further confirmed that the drug carrier (αHLA-G EV) carried the HLA-G antibody.

[0065] Figure 3 The endocytosis effect of the drug carrier (αHLA-G EV) on the glioblastoma multiforme (GBM) cell line 8901 and DBTRG was tested respectively. F-actin was marked with red fluorescence to define the cell range; extracellular vesicles were marked with green to confirm whether the extracellular vesicles entered the cell range. The results of fluorescent staining showed that the degree of expression of the drug carrier by the GBM cell line increased significantly, proving that the drug carrier increased its affinity with the GBM cell line by carrying HLA-G antibodies.

[0066] In order to prove that the drug carrier (αHLA-G EV) can exhibit the ability of drug release after entering the cell via endocytosis, the present invention further loads the down-regulating gene miRNA-181 (miR-181) that inhibits the expression of MGMT into the drug carrier to ensure that the drug carrier can exhibit the effect of inhibiting MGMT after entering the GBM cell line. Among them, Figure 4AAfter the drug carrier is coated with miR-181, the expression level of miR-181 in the drug carrier is confirmed by reverse transcription-PCR (RT-PCR). In this experiment, the expression level of miR-181 of 50ng confirmed by reverse transcription-polymerase chain reaction (RT-PCR) is set as the standard value to 1, and the drug carrier without miR-181 (αHLA-G EV) and the drug carrier loaded with 25ng miR-181 (miR-181@αHLA-G

[0067] EV (25g)), and the drug carrier loaded with 50ng of miR-181 (miR-181@αHLA-GEV (50g)). It can be demonstrated in the group of the drug carrier loaded with 50ng of miR-181 (miR-181@αHLA-GEV (50g)) that the performance of the drug carrier is equivalent to that of the same amount of miR-181 (50ng) when not coated, proving the effectiveness of the drug carrier in loading small molecules (such as miR-181, proteins or drugs, etc.) after binding to the HLA-G antibody.

[0068] At Figure 4B In the experiment, RT-PCR was used to confirm the expression of miR-181 in each GBM cell line after the GBM cell line 8901 and DBTRG engulfed the drug carrier through endocytosis. Among them, the group that did not react was used as a first control group (Clt), and it was set as a standard value of 1; the group that reacted with GBM cell lines after loading miR-181 with Lipo3000, a drug carrier commonly used in current technology, was used as a second control group (Lipo3000); the group that reacted with the drug carrier and GBM cell lines was used as a third control group (αHLA-G EV). The results showed that the GBM cell line was treated with the drug carrier loaded with miR-181 (miR-181@αHLA-G EV). The expression of miR-181 in the GBM cell line was high, even higher than that of Lipo3000, a drug carrier commonly used in current technology. In addition to proving the loading effectiveness of the drug carrier, it also proves that it has the ability to release.

[0069] Figure 5In the experiment, the group without reaction was used as the first control group (Clt); the drug carrier reacted with GBM as the second control group (αHLA-G EV); the miRNA was directly reacted as the third control group (miR-181); the drug carrier loaded with miRNA was used as the experimental group (miR-181@αHLA-G EV), and the above groups were cultured with each GBM cell line for 24 hours, and the protein expression in each GBM cell line in each group was confirmed by Western blot. The results showed that in each GBM cell line in the experimental group, the expression of MGMT, an indicator of resistance to the chemotherapy drug Temodal (TMZ), was significantly decreased, which can prove the excellent down-regulation effect of miR-181. It is worth noting that the expression of Bcl-2 protein, an indicator of inhibiting cell apoptosis, was also significantly decreased, while the expression of BAX protein, an indicator of promoting cell apoptosis, was significantly increased, indicating that miR-181 reaction also helps GBM cell lines develop towards apoptosis.

[0070] To prove that the drug carrier can be effectively applied to organisms, Figure 6 A blood-brain barrier cell culture model 10 is provided, the purpose of which is to simulate the blood-brain barrier environment in an organism to ensure that the drug carrier can cross the blood-brain barrier into the brain area and specifically bind to the GBM cells. The blood-brain barrier cell culture model 10 includes a first culture area 11 and a second culture area 12, and the first culture area 11 and the second culture area 12 are connected by a plurality of perforations 13. The first culture area 11 and the second culture area 12 are filled with a hydrogel A, wherein the GBM cell line U87 is cultured in the hydrogel A of the first culture area 11, and the second culture area 12 is sequentially provided with an endothelial cell (Endothelium) 20, a pericyte (Pericyte) 30 and an astrocyte (Astrocytes) 40, wherein a resistance value of the blood-brain barrier cell culture model 10 is set to 2144Ω.cm 2 In the subsequent experiments, the drug carrier is administered to the second culture area 12 of the blood-brain barrier cell culture model 10, and the GBM cell line U87 in the first culture area 11 is used for experimental analysis to confirm the effectiveness and therapeutic effect of the drug carrier passing through the second culture area 12 to the first culture area 11.

[0071] In this experiment, the group without reaction was used as a first control group (Clt); the drug carrier with HLA-G antibody bound to the surface was used as a second control group (αHLA-G EV); the miRNA was directly reacted as a third control group (miR-181); a chemotherapy drug Temodal (TMZ) was directly reacted as a fourth control group (TMZ); the drug carrier loaded with miRNA was used as a first experimental group (miR-181@αHLA-G EV); the drug carrier loaded with the chemotherapy drug was used as a second experimental group (TMZ@αHLA-G EV); and the drug carrier loaded with miRNA and the drug carrier loaded with the chemotherapy drug in a 1:1 ratio was used as a third experimental group (miR-181@αHLA-G EV+

[0072] TMZ@αHLA-G EV). The above groups were tested in the blood-brain barrier cell culture model 10, and the cell death rate (cell death %) of the GBM cells after being treated with each group was confirmed on the 1st, 2nd and 3rd day respectively. Figure 7 The results showed that the GBM cells had significant cell death rates in the groups treated with the chemotherapy drug Temodal (TMZ) (such as the fourth control group (TMZ), the second experimental group (TMZ@αHLA-G EV) and the third experimental group (miR-181@αHLA-GEV+

[0073] TMZ@αHLA-G EV), while in the group given only miR-181, cell death was limited (as shown in the third control group (miR-181) and the first experimental group (miR-181@αHLA-G EV)).

[0074] It is worth noting that although the fourth control group and the second experimental group were successfully treated with the chemotherapy drug Temodal (TMZ), the cell death tended to be milder by the third day, and there was a tendency for drug resistance. However, in the third experimental group (miR-181@αHLA-G EV+TMZ@αHLA-G EV) that was simultaneously administered with the chemotherapy drug Temodal (TMZ) and miR-181, not only was there a significant toxic effect on the first day, but even on the third day, the cell death rate (cell death%) of the GBM cells was significantly increased.

[0075] The first control group (Clt), the second control group (αHLA-G EV), the fourth control group (TMZ), the second experimental group (TMZ@αHLA-G EV) and the third experimental group (miR-181@αHLA-G EV+TMZ@αHLA-G EV) were further analyzed by Western blotting and fluorescent staining techniques to analyze the expression of proteins in the GBM cell line. Fig. 8A In the fluorescence staining, BAX protein was marked with red fluorescence, and GBM cells were marked with green to define the cell range. The results also showed that HLA-G protein was obviously expressed in GBM cells treated with the chemotherapy drug Temodal (TMZ), and the expression ratio of BAX protein in the GBM cell line treated with miRNA-181 and TMZ in the third experimental group was significantly higher than that in the fourth control group and the second experimental group. Figure 8B In addition to the above results, the expression of Bcl-2 protein, an indicator of inhibiting cell apoptosis, in the third experimental group also decreased significantly, confirming that the GBM cell line in the third experimental group was heading towards apoptosis.

[0076] Fig. 9 The drug carrier loaded with 100ng / ml, 200ng / ml and 400ng / ml miR181 was reacted with cells overexpressing MGMT protein. It has been confirmed that the drug carrier can effectively overcome the blood-brain barrier cell culture model 10 and promote miR181 to reduce the expression of MGMT gene and protein. The results can echo the above-mentioned experimental confirmation that as the concentration of miR181 attached to the drug carrier increases, the MGMT protein level of cells overexpressing MGMT protein decreases accordingly.

[0077] The above experimental results can prove that the drug carrier can effectively overcome the blood-brain barrier environment in the organism simulated by the blood-brain barrier cell culture model 10, and promote the release of miR181 and the anticancer drug, so as to enhance the expression of apoptotic factors in GBM cell lines, reduce the drug resistance response of GBM cell lines, and enhance the toxic effect of anticancer drugs.

[0078] To verify the actual effect of the drug carrier in vivo, another experiment was conducted in which mice were given 10 6The GBM cell line (U87) was implanted with 100 cells per mouse to establish an animal model of glioblastoma multiforme. Seven days after the implantation, the chemotherapeutic drug and / or the drug vector was intravenously injected into each mouse, and the growth of the chemotherapeutic drug and / or the drug vector and the GBM cell line in the mouse brain was confirmed by imaging.

[0079] Fig. 10A In the experiment, the ordinary extracellular vesicles were loaded with the anticancer drug as the control group (TMZ@EV) and the drug carrier was loaded with the anticancer drug as the experimental group (TMZ@αHLA-G EV). At 60 minutes (min) after intravenous injection, it was found that the ordinary extracellular vesicles in the control group were widely distributed in the upper body area of ​​the experimental mice (especially the lungs), while the drug carriers in the experimental group (TMZ@αHLA-G EV) were obviously concentrated in the brain area of ​​the experimental mice, confirming that the drug carrier (αHLA-G EV) has targeting (specificity).

[0080] Figures 10B to 10D In the animal model, the group injected with normal saline was used as a first control group (Clt); the group injected with the drug carrier with HLA-G antibody bound on the surface was used as a second control group (αHLA-G EV); the group injected with the chemotherapy drug Temodal (TMZ) was used as a third control group (TMZ); the group injected with the drug carrier loaded with miRNA was used as a first experimental group (miR-181@αHLA-G EV); the group injected with the drug carrier loaded with the chemotherapy drug was used as a second experimental group (TMZ@αHLA-G EV); and the group injected with the drug carrier loaded with miRNA and the chemotherapy drug in a 1:1 mixture was used as a third experimental group (miR-181@αHLA-G EV+TMZ@αHLA-G EV), and the weight of the experimental mice, the survival rate and the growth status of the GBM cell line in each group were confirmed by imaging every 7 days.

[0081] Fig. 10B There were no significant differences in the body mass of the experimental mice in each group, and the different injections did not cause serious changes in their physiological conditions. Fig. 10CIt can be found that the mice in the first control group (Clt), the second control group (αHLA-G EV) and the first experimental group (miR-181@αHLA-G EV) all died within about 30 days after injection. In the groups treated with the chemotherapy drug Temodal (TMZ) (such as the third control group and the second experimental group), the survival rate of the experimental mice showed an upward trend. However, it is worth noting that in the third experimental group (miR-181@αHLA-G EV+TMZ@αHLA-G EV) that was given the chemotherapy drug carrying Temodal (TMZ) and miR-181 at the same time, the survival rate of the experimental mice can be greatly improved, even up to more than 60 days.

[0082] Fig. 10D as well as Fig.10E The next step is to analyze the fluorescence performance of the GBM cell line in the brain area of ​​the experimental mice in each group (Intensity (10 6 )) analysis and tissue section staining of tissue morphology presented by hematoxylin-eosin staining (H&E Stain) technology were used to analyze the growth of the GBM cells. Compared with the first control group (Clt) and the second control group (αHLA-G EV), the growth of the GBM cell line in the third control group, the first experimental group (miR-181@αHLA-G EV) and the second experimental group (TMZ@αHLA-G EV) gradually improved, but was still inhibited. However, in the third experimental group (miR-181@αHLA-G EV+TMZ@αHLA-G EV), it can be found that the fluorescence response of the GBM cells tends to be flat and consistent as a whole, and even has a downward trend on the 21st day. In the tissue section staining diagram, it can be seen that the distribution area of ​​the GBM cells in the third experimental group is significantly smaller than that of other groups. It was confirmed that the simultaneous administration of the drug carrier loaded with miR-181 and TMZ to the experimental mice could significantly produce a synergistic effect in inhibiting the growth of the GBM cell line.

[0083] Furthermore, to prove that the drug carrier can produce the same excellent effect on human cells, FIG. 11A to FIG. 11D In the present invention, a tumor cell of a brain tumor patient is further confirmed. The brain tumor patient develops resistance to the anticancer drug TMZ after anticancer drug treatment, such as Fig.11A In the fluorescent staining, F-actin is marked with red fluorescence to define the cell range; the nucleus is marked with blue fluorescence to define the position of the nucleus; and HLA-G protein is marked with green fluorescence, so that the large amount of HLA-G protein in the tumor cells can be observed.

[0084] Next, the tumor cells were cultured and treated with the drug carrier loaded with miR181. Fig. 11B In the fluorescent staining, purple fluorescence was used to identify F-actin to define the cell range; blue fluorescence was used to identify the nucleus to define the nucleus position; green fluorescence was used to identify the drug carrier; and red fluorescence was used to identify miR181, which can prove that the tumor cells express the drug carrier and miR181 in the cell through endocytosis. Fig. 11C It can also be confirmed that after the tumor cells were treated with the drug carrier loaded with miR181 (miR-181@αHLA-G EV), the expression of MGMT was significantly decreased compared with the group treated with only the drug carrier (αHLA-G EV) or only miR181, and the apoptosis inhibitor BCL2 also showed a decreased expression, while the apoptosis promoter BAA showed a significant upward trend.

[0085] Fig.11D The tumor cell mortality was compared after treatment under various conditions. In this experiment, the group that did not react was used as the first control group (Clt); the drug carrier with HLA-G antibody bound to the surface was used as the second control group (αHLA-G EV); the miRNA was directly reacted as the third control group (miR-181); the chemotherapy drug Temodal (TMZ) was directly reacted as the fourth control group (TMZ); the drug carrier loaded with miRNA was used as the first experimental group (miR-181@αHLA-G EV); the drug carrier loaded with the chemotherapy drug was used as the second experimental group (TMZ@αHLA-G EV); and the drug carrier loaded with miRNA and the drug carrier loaded with the chemotherapy drug in a 1:1 ratio was used as the third experimental group (miR-181@αHLA-G EV+

[0086] TMZ@αHLA-G EV). Similar to the above paragraph, the third experimental group showed an excellent effect of promoting apoptosis of tumor cells.

[0087] The drug carrier provided by the present invention can effectively cross the blood-brain barrier, specifically reach the location of tumor cells, and release drugs to achieve the excellent therapeutic effect of promoting tumor cells to apoptosis.

Claims

1. A drug carrier having the property of crossing the blood-brain barrier, characterized in that: It comprises an extracellular vesicle with a human leukocyte antigen-G antibody bound to its surface.

2. The drug carrier according to claim 1, characterized in that The extracellular vesicle is bound to the human leukocyte antigen-G antibody via a transmembrane protein.

3. The drug carrier according to claim 2, characterized in that The transmembrane protein comprises a single domain antibody fragment combined with the human leukocyte antigen-G antibody.

4. The drug carrier according to claim 2, characterized in that The transmembrane protein is selected from the group consisting of CD68, CD81 and CD9.

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

6. The drug carrier according to any one of claims 1 to 5, characterized in that The drug carrier contains a down-regulated gene or a drug.

7. The drug carrier according to claim 6, characterized in that The down-regulated gene comprises miRNA-181; and the drug comprises Temodal.

8. A use of the drug carrier having the property of crossing the blood-brain barrier as claimed in claim 1 to reduce the expression of O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells, characterized in that: The drug carrier contains a down-regulated gene, which is miRNA-181.

9. The use according to claim 8, characterized in that The brain tumor cells are glioblastoma multiforme (GBM).

10. A use of the drug carrier having the property of crossing the blood-brain barrier as claimed in claim 1 to promote apoptosis of brain tumor cells, characterized in that: The drug carrier contains a down-regulated gene or a drug.

11. The use according to claim 10, characterized in that The downregulated gene includes miRNA-181, and the drug includes Temodal.

12. The use according to claim 10 or 11, characterized in that The expression of a Bcl-2 protein in these brain tumor cells was decreased, while the expression of a BAX protein was increased.

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

14. A use of the drug carrier having the property of crossing the blood-brain barrier as claimed in claim 1 to inhibit the growth of brain tumor cells, characterized in that: The drug carrier contains a down-regulated gene or a drug, the down-regulated gene contains miRNA-181, and the drug contains Temodal.

15. The use according to claim 14, characterized in that The brain tumor cells are glioblastoma multiforme (GBM).

16. A pharmaceutical composition, characterized in that The drug carrier comprising the drug carrier having the property of crossing the blood-brain barrier as claimed in claim 1.

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

18. Use of a drug carrier to cross the blood-brain barrier, characterized in that: The drug carrier comprises an extracellular vesicle, the surface of which is bound with a human leukocyte antigen-G antibody.

19. The use according to claim 18, characterized in that A transmembrane protein of the extracellular vesicle comprises a single domain antibody fragment, and the single domain antibody fragment binds to the human leukocyte antigen-G antibody.

20. The use according to claim 18 or 19, characterized in that 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 fragment (scFv).

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