Extracellular vesicle drug delivery system and preparation method and application thereof

Abstract

The present invention relates to the field of biomedical technology, and specifically to an extracellular vesicle drug delivery system, a preparation method thereof, and an application thereof. The extracellular vesicle drug delivery system comprises extracellular vesicles, and CircRNA and anti-tuberculosis drugs loaded in the extracellular vesicles. The present invention uses extracellular vesicles as co-carriers of CircRNA and anti-tuberculosis drugs, efficiently loads CircRNA and anti-tuberculosis drugs with anti-tuberculosis immune regulation functions, and then further enhances the targeting of the extracellular vesicle drug delivery system by performing surface targeted modification on the extracellular vesicles. The extracellular vesicle drug delivery system more effectively kills and eliminates Mycobacterium tuberculosis in host cells by combining the anti-tuberculosis immune regulation function of CircRNA and the Mycobacterium tuberculosis inhibitory effect of anti-tuberculosis drugs, thereby providing a new strategy for the development of safe and efficient anti-tuberculosis drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an extracellular vesicle drug-carrying system and a preparation method and application thereof. Background Art

[0002] Current drug delivery systems, such as nano-drug delivery systems, usually use various stabilizers to prepare stable nano-systems. After loading drugs, they can actively or passively aggregate to target organs, cells or organelles to enhance the targeting of drugs. Using nanoparticles to load drugs can also improve the stability and solubility of drugs, promote the transmembrane transport of drugs, and prolong the circulation time of drugs to improve drug efficacy, and reduce the toxicity of free drugs to non-target organs / cells.

[0003] Nanomedicine can promote the clinical transformation of drugs and improve the side effects of patient treatment, but currently only a few polymeric nanodrugs have been approved by the FDA and are used in clinical practice. Currently approved nanodrugs are rarely recommended as first-line treatments, and some drugs only show better efficacy in a small number of patients. Although existing engineered nanotechnology can break through the limitations of traditional drug delivery technology and show the potential to enhance drug efficacy. However, there are also some shortcomings, such as liposome-loaded drugs are prone to leakage and poor stability; inorganic nanoparticles often use heavy metal preparations, which have low solubility and high risk of toxic side effects. Therefore, the development of highly safe and targeted nanosystems as delivery carriers is still a major scientific challenge in the biomedical field.

[0004] Extracellular vesicles (EVs) are nano-sized vesicles of cell origin, mainly including exosomes and microvesicles. Under different types of endogenous or exogenous stimulation, the plasma membrane can be shed to produce or secreted by endosomes to produce vesicles of different sizes, 100-1000nm is called microvesicles, and 30-150nm is called exosomes. Extracellular vesicles are distributed in biological fluids, can carry biological molecules such as nucleic acids, proteins and lipids to circulate in body fluids, and can pass through the blood-brain barrier, playing an important role in intercellular communication; at the same time, extracellular vesicles have a typical lipid bilayer structure, which can protect nucleic acids from degradation, and are ideal carriers of nucleic acids (such as siRNA and miRNAs) in gene therapy, so they have the potential to be used as biological molecule delivery carriers. In addition, after being released into the extracellular environment, extracellular vesicles can be taken up by different types of cells, mainly through two ways of cell signal transduction and communication: one is binding to specific cell membrane molecules; the other is entering target cells through endocytosis (such as clathrin / caveolin-mediated endocytosis, macropinocytosis, phagocytosis, lipid raft-mediated uptake, etc.). After entering the cell, the extracellular vesicle can release its cargo into the target cell and perform its biological function. Because the extracellular vesicle carries the phospholipid bilayer structure of the source cell during cell shedding, it can fuse with the receptor cell more effectively; in addition, more efficient targeting can be achieved by modifying the surface of the extracellular vesicle, enabling them to deliver drugs, proteins and other biological molecules to the target tissue.

[0005] In summary, extracellular vesicles are ideal carriers for drug and biomolecule delivery. Compared with synthetic carriers such as liposomes or other nanoparticles, extracellular vesicles, as cell products, have better biocompatibility, low immunogenicity, and the ability to cross biological barriers such as the blood-brain barrier. Extracellular vesicles have great prospects as nanocarriers for the treatment of various diseases, which is conducive to improving clinical efficacy and safety.

[0006] Cellular RNA plays an important regulatory role in physiological and pathological environments. Therefore, exploring the specific RNA expression profiles in various disease states and exploring their pathogenic mechanisms is expected to use specific RNA as candidate biomarkers and potential therapeutic targets for diseases. In addition to mRNA, many non-coding RNAs (ncRNAs) or circular RNAs (circRNAs) synergistically control gene expression, constituting a variety of potential targets for drug development. Currently, RNA molecules or analogs used as therapeutic drugs mainly use chemically synthesized single-stranded oligonucleotides to complement the target mRNA base pairs, block its action and inhibit gene expression; or use vectors to deliver miRNAs to enhance the expression of disease-related defects. However, RNA drugs are easily degraded in the blood and need to pass through the cell membrane barrier to reach the intracellular target; in addition, RNA drugs usually require vector delivery and use extensive chemical modifications to improve RNA stability, but they also face the risk of inducing immunogenic reactions, which limits their clinical application. Studies have shown that the development of natural RNA drugs prepared in living cells is expected to minimize the risk of inducing immune responses. Therefore, using extracellular vesicles as co-carriers of RNA and drugs can achieve drug stability and tissue-specific targeting, avoid or minimize off-target effects and immunogenicity, and help improve clinical efficacy and safety.

[0007] Studies have shown that specific RNAs, such as miRNAs and CircRNAs, can be selectively enriched in extracellular vesicles. As an endogenous, tissue-specific RNA expressed by the eukaryotic transcriptome, CircRNAs do not have a 5' end cap and a 3' end poly (A) tail. They are formed by connecting the 3' and 5' ends through exon or intron cyclization to form a covalent ring structure that is resistant to RNase R digestion. Through high-throughput RNA sequencing and microarray analysis, different CircRNAs contained in extracellular vesicles from different sources can be identified, and studies have shown that CircRNAs in extracellular vesicles can play extracellular functions and key roles in recipient cells. Based on its advantage of being more stable than other types of RNA, CircRNAs are therefore more likely to be used in the biomedical field.

[0008] Based on this, the present invention proposes to use extracellular vesicles as co-carriers of circRNA and anti-tuberculosis drugs, thereby forming an extracellular vesicle drug delivery system, providing a new strategy for the preparation of anti-tuberculosis drugs or anti-tuberculosis treatment. Summary of the invention

[0009] The purpose of the present invention is to provide an extracellular vesicle drug delivery system and a preparation method and application thereof in order to solve the above-mentioned deficiencies of the prior art.

[0010] The objective of the present invention is achieved through the following technical scheme: The present invention provides an extracellular vesicle drug delivery system, comprising an extracellular vesicle and CircRNA and an anti-tuberculosis drug loaded in the extracellular vesicle.

[0011] Furthermore, the anti-tuberculosis drug is rifampicin.

[0012] Furthermore, the extracellular vesicles are exosomes or microvesicles.

[0013] Furthermore, the extracellular vesicles are surface-targeted modified exosomes. In the present invention, the natural exchange characteristics of phospholipids in the exosome membrane structure are utilized to perform surface-targeted modification on exosomes with DSPE-PEG-MAN, thereby enhancing the targeting of the extracellular vesicle drug delivery system.

[0014] Furthermore, the CircRNA is circTRAPPC6B.

[0015] In the present invention, by utilizing extracellular vesicles that highly express specific CircRNA and loading them with anti-tuberculosis drugs, the extracellular vesicles are used as co-carriers of CircRNA and anti-tuberculosis drugs, thereby forming the extracellular vesicle drug delivery system; further, in order to enhance the system targeting of the extracellular vesicles, the surface of the extracellular vesicles is further modified for targeting. The extracellular vesicle drug delivery system of the present invention is expected to provide new strategies and methods for the development of new anti-tuberculosis drugs or the treatment of anti-tuberculosis by combining the advantages of host-oriented therapy and drug therapy.

[0016] The present invention provides a method for preparing an extracellular vesicle drug delivery system, comprising the following steps:

[0017] (1) Extraction of highly expressed circRNA exosomes:

[0018] (2) loading the anti-tuberculosis drug into the highly expressed circRNA exosomes prepared in step (1) by a simple incubation method or an electroporation method;

[0019] (3) Using an ultrafiltration tube to wash away free anti-tuberculosis drugs, adding PBS for washing, centrifuging multiple times until the free anti-tuberculosis drugs are completely removed, and then resuspending the ultrafiltration tube with PBS to collect the circRNA and anti-tuberculosis drug co-loaded exosomes;

[0020] (4) performing surface targeted modification on the exosomes co-loaded with circRNA and anti-tuberculosis drugs obtained in step (3), thereby obtaining the extracellular vesicle drug delivery system.

[0021] Furthermore, in step (2), the anti-tuberculosis drug is rifampicin, and the specific steps of the electroporation method are: take 100 μL of the highly expressed CircRNA exosomes with a concentration of 1 mg / mL prepared in step (1) into an EP tube, add 100 μL of rifampicin with a concentration of 2 mg / mL, invert and mix well, and place on ice, set the operating conditions of the electroporation instrument to 1000 V, 10 ms and 2 pulses, electroporate 200 μL of the mixture of highly expressed CircRNA exosomes and rifampicin, collect the exosomes after electroporation, and incubate at 37° C. for 30 min to restore the exosome membrane structure.

[0022] Furthermore, in step (4), the specific operation of surface targeted modification is as follows: take 1 mL of 500 μg / mL circRNA and anti-tuberculosis drug co-loaded exosomes prepared in step (3), add 1 mL of 20 μg / mL DSPE-PEG-MAN, invert to mix, place in a shaker at 4°C in the dark and incubate overnight; then use an ultrafiltration tube to wash away the free DSPE-PEG-MAN, add PBS to wash, and repeat the centrifugation several times.

[0023] The present invention also provides application of the extracellular vesicle drug-carrying system in anti-tuberculosis drugs.

[0024] The beneficial effects of the present invention are as follows: the present invention proposes an extracellular vesicle drug delivery system and a preparation method thereof, wherein the extracellular vesicle is used as a co-carrier of CircRNA and anti-tuberculosis drugs, the extracellular vesicle has good biocompatibility, and can efficiently load the anti-tuberculosis immune regulatory function CircRNA and anti-tuberculosis drugs, and the targeting of the extracellular vesicle drug delivery system can be further enhanced by surface targeted modification of the extracellular vesicle. The extracellular vesicle drug delivery system can more effectively kill and eliminate Mycobacterium tuberculosis in host cells by combining the anti-tuberculosis immune regulatory function of CircRNA and the Mycobacterium tuberculosis inhibitory effect of anti-tuberculosis drugs, thereby providing a new strategy for the development of safe and efficient anti-tuberculosis drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The fluorescence expression of normal untransfected cells and cells transfected with highly expressed CircRNA lentivirus was observed under a fluorescence microscope;

[0026] Figure 2 RT-qPCR was used to detect the expression level of circTRAPPC6B in normal untransfected cells and cells transfected with lentivirus with high expression of circTRAPPC6B;

[0027] Figure 3Western blot was used to detect the expression of positive proteins (ALIX, CD63, TSG101) and negative proteins (GRP94) in exosomes secreted by normal untransfected cells and cells transfected with high-expression CircRNA lentivirus;

[0028] Figure 4 The particle size analyzer detects the particle size of exosomes secreted by normal untransfected cells and cells transfected with high-expression CircRNA lentivirus;

[0029] Figure 5 UV spectrophotometry (480 nm) was used to detect the drug concentration in the circRNA and anti-tuberculosis drug co-loaded exosomes prepared by simple incubation method and electroporation method (n=5);

[0030] Figure 6 RT-qPCR was used to detect the expression level of circTRAPPC6B in exosomes secreted by normal untransfected cells and cells transfected with lentivirus with high expression of circTRAPPC6B, as well as in exosomes co-loaded with surface targeted modified circRNA and anti-tuberculosis drugs;

[0031] Figure 7 Western blot was used to detect the expression of positive proteins (ALIX, CD63, TSG101) and negative proteins (GRP94) in exosomes loaded with circRNA alone and exosomes co-loaded with surface-targeted modified circRNA and anti-tuberculosis drugs;

[0032] Figure 8 The particle size analyzer detects the particle size of exosomes loaded with circRNA alone and exosomes loaded with surface-targeted modified circRNA and anti-tuberculosis drugs.

[0033] Fig. 9 The morphology of exosomes loaded with circRNA alone and exosomes loaded with surface-targeted circRNA and anti-tuberculosis drugs was observed by transmission electron microscopy (magnification 40000), **P<0.01, ***P<0.001;

[0034] Fig.10 It is the change of fluorescence intensity in cells detected by flow cytometry after macrophages were co-incubated with two kinds of stained exosomes for a certain period of time;

[0035] Fig.11 is the analysis of the changes in fluorescence values ​​in macrophages, ***P<0.001;

[0036] Fig.12 The particle size analysis was performed to determine the particle sizes of two types of microvesicles produced by normal untransfected cells and cells transfected with lentivirus that highly expressed circTRAPPC6B;

[0037] Fig.13 is RT-qPCR to detect the expression level of circTRAPPC6B in the two microvesicles;

[0038] Fig.14 The drug concentration in the microvesicles was detected by UV spectrophotometry (480 nm) (n=5), *P<0.05. DETAILED DESCRIPTION

[0039] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments, and the contents mentioned in the implementation modes are not intended to limit the present invention.

[0040] Example 1

[0041] The specific operation of extracting highly expressed circRNA exosomes is as follows: (1) Design and construct a highly expressed lentivirus of specific circRNA (taking circRNA-circTRAPPC6B with anti-tuberculosis immune regulation function as an example), and use the lentivirus to transfect 293T cells. Count 1×10 cells per well of a 24-well plate. 5 After 24 h of culture, 500 μL of 2×10 6 TU lentivirus (MOI = 10) serum-containing medium (10 μL lentivirus (1×10 8 TU / mL) was added to 490 μL of serum-containing medium) and cultured for 48 hours. Obvious green fluorescence expression was visible under a fluorescence microscope 48 hours after transfection. The cells were cultured for 3-4 days, digested with trypsin, collected, centrifuged at 1000rpm for 5 minutes, the supernatant was discarded, the precipitate was resuspended with 1mL of PBS, the centrifugation was repeated twice, and the cell precipitate was resuspended with 500 μL of PBS and collected in a flow tube. All cells containing green fluorescence were sorted out by a flow sorting instrument, and the sorting efficiency was 89%, thereby screening out all cell lines with stable and high expression of CircRNA containing green fluorescence.

[0042] (2) The cell lines with stable and high expression of circRNA were cultured at 37°C and 5% CO2 for a period of time, and the cell culture supernatant within 48 hours was collected. The supernatant was collected by centrifugation at 300×g at 4℃ for 10min; the supernatant was collected by centrifugation at 2000×g at 4℃ for 10min; the supernatant was collected by centrifugation at 10000×g at 4℃ for 10min, and filtered through a 0.22μm filter. The cell supernatant after filtering was added to a 100KD ultrafiltration tube for ultrafiltration, and the concentrated solution after ultrafiltration was collected. The reagent Exoquick-TC of the exosome precipitation kit was added (the volume ratio of the concentrated solution to Exoquick-TC was 5:1), and the solution was shaken and placed at 4℃ for precipitation for 24h. The solution was centrifuged at 3000×g for 10min and the supernatant was discarded; the solution was centrifuged at 3000×g for 5min and the residual supernatant was discarded. The tube wall was washed 3 times with PBS, the supernatant was discarded, and 500μL of PBS was added to resuspend the precipitate to obtain highly expressed CircRNA exosomes, which were stored at -80℃ for the following experiments.

[0043] In this example, normal untransfected cells were used as the normal control group, and fluorescence microscopy was used to observe the fluorescence expression of the sorted cells. The results showed that compared with normal untransfected cells (NC-cells), the successfully transfected cells (OE-cells) stably expressed green fluorescent protein with high fluorescence intensity ( Figure 1 ). The expression levels of CircRNA in normal non-transfected cells (NC-cells) and CircRNA stably transfected cells (OE-cells) were detected by RT-qPCR. The results showed that the expression level of CircRNA in CircRNA stably transfected cells was significantly higher than that in the normal control group ( Figure 2 The above results show that this example has successfully constructed a cell line with stable and high expression of CircRNA.

[0044] Furthermore, Western blot was used to detect the expression of positive proteins ALIX, CD63, TSG101 and negative protein GRP94 in highly expressed CircRNA exosomes ( Figure 3 ), the results showed that the exosomes secreted by normal non-transfected cells (Exo) and the exosomes secreted by CircRNA stably transfected cells (CircRNA@Exo) can highly express positive proteins, while negative proteins are not expressed, thus confirming the purity of the above two types of exosomes. The particle size of the above two types of exosomes was detected by a particle size analyzer ( Figure 4 ), the results showed that the particle sizes of the above two exosomes were distributed in the range of 60-120 nm. The above methods jointly confirmed that the above preparation steps in this example can successfully extract stable and highly expressed CircRNA exosomes.

[0045] Example 2

[0046] This embodiment provides a method for preparing an extracellular vesicle drug-carrying system (surface-targeted modified CircRNA and anti-tuberculosis drugs co-loaded exosomes) comprising the following steps:

[0047] (1) Extracting highly expressed circRNA exosomes according to the procedures of Example 1;

[0048] (2) Rifampicin was loaded into the highly expressed CircRNA exosomes obtained in step (1) by simple incubation and electroporation, respectively. The specific operation of the single incubation was as follows: 100 μL of the highly expressed CircRNA exosomes obtained in Example 1 (1 mg / mL) was placed in an EP tube, 100 μL of rifampicin (2 mg / mL) was added, the tube was inverted to mix, and incubated at 37°C for 30 min.

[0049] The specific operation of the electroporation method is as follows: take 100 μL of the highly expressed CircRNA exosomes (1 mg / mL) obtained in Example 1 into an EP tube, add 100 μL of rifampicin (2 mg / mL), invert and mix well and place on ice, use an electroporation instrument (1000 V, 10 ms, 2 pulses) to electroporate 200 μL of the highly expressed CircRNA exosomes and rifampicin mixture, collect the exosomes after electroporation, and incubate at 37 ° C for 30 min to restore the exosome membrane structure.

[0050] (3) Use a 100KD ultrafiltration tube to wash away the free rifampicin in the exosomes collected by the simple incubation method and the electroporation method in step (2), centrifuge at 3000×g for 15 min each time, add PBS for washing, repeat centrifugation 6 times, and use ultraviolet spectrophotometry to detect whether the flow-through of the last wash contains rifampicin (absorbance is 480nm) until it is confirmed that the rifampicin is completely washed. Take 500μL of PBS to resuspend the exosomes collected in the ultrafiltration tube, and obtain circRNA and anti-tuberculosis drug co-loaded exosomes.

[0051] (4) Utilizing the natural exchange characteristics of phospholipids in the exosome membrane structure, DSPE-PEG-MAN (1 mL, 20 μg / mL) was used to perform surface targeted modification on the circRNA and anti-tuberculosis drug co-loaded exosomes (1 mL, 500 μg / mL) obtained in step (3). The specific operation was as follows: 1 mL of the circRNA and anti-tuberculosis drug co-loaded exosomes (500 μg / mL) obtained in step (3) was taken, 1 mL of DSPE-PEG-MAN (20 μg / mL) was added, the mixture was mixed by inversion, and the mixture was incubated overnight in a shaker at 4°C in the dark. The free DSPE-PEG-MAN was washed away using a 100KD ultrafiltration tube, centrifuged at 3000×g for 15 min each time, washed with PBS, and centrifuged three times to obtain surface targeted modified circRNA and anti-tuberculosis drug co-loaded exosomes (Man-CircRNA@Rif@Exo), which were stored at -80°C in the dark.

[0052] In this example, normal untransfected cells were used as the normal control group, and the drug concentrations of circRNA and anti-tuberculosis drugs co-loaded exosomes prepared by simple incubation and electroporation were detected by ultraviolet spectrophotometry (480 nm). Figure 5 ), the results showed that both the simple incubation method and the electroporation method can successfully load the anti-tuberculosis drug (rifampicin) into exosomes, but compared with the simple incubation method, it can be confirmed that the electroporation method is more efficient in loading the exosomes with anti-tuberculosis drugs. The expression levels of circRNA in exosomes secreted by normal untransfected cells (Exo) and exosomes secreted by circRNA stably transfected cells (CircRNA@Exo, Man-CircRNA@Rif@Exo) were detected by RT-qPCR. The results showed that the expression level of circRNA in exosomes of circRNA stably transfected cells was significantly higher than that in the normal control group ( Figure 6 ), which confirmed that exosomes can successfully load CircRNA.

[0053] Western blot was used to detect the expression of positive and negative proteins in exosomes loaded with CircRNA alone (CircRNA@Exo) and exosomes co-loaded with surface-targeted modified CircRNA and anti-tuberculosis drugs (Man-CircRNA@Rif@Exo). Figure 7 ), the results showed that the positive proteins of the two exosomes were expressed, but the negative proteins were not expressed, and the purity of the two exosomes was confirmed. The particle size of the two exosomes was detected by a particle size analyzer ( Figure 8), the results showed that compared with the particle size of exosomes loaded with circRNA alone (99.0±4.1nm), the particle size of exosomes loaded with surface targeted circRNA and anti-tuberculosis drugs was larger (114.4±8.3nm). The morphology of the above two exosomes was observed by transmission electron microscopy ( Fig. 9 ), the results showed that both exosomes had membrane vesicle structures. Therefore, the above three methods combined to confirm that the surface-targeted modified circRNA and anti-tuberculosis drugs co-loaded exosomes were successfully prepared.

[0054] Example 3

[0055] This example verifies the enhanced cell targeting of the extracellular vesicle drug-carrying system (surface-targeted modified CircRNA and anti-tuberculosis drugs co-loaded exosomes) prepared in Example 2, and the specific operation is as follows:

[0056] The lipophilic dye DIO was used to stain and trace the surface-targeted CircRNA and anti-tuberculosis drug co-loaded exosomes successfully prepared in Example 2 (with unmodified CircRNA and drug co-loaded exosomes as a control). 1 mL of the surface-targeted CircRNA and anti-tuberculosis drug co-loaded exosomes (1 mg / mL) prepared in Example 2 was taken, 10 μL of DIO (1 mM) was added, inverted and mixed, and incubated at 37 ° C in the dark for 30 min. The free dye was washed away using a 100KD ultrafiltration tube, centrifuged at 14000 × g for 10 min each time, and PBS was added for washing, and the centrifugation was repeated 6 times. The flow-through liquid of the last wash was detected by ultraviolet spectrophotometry to determine whether it contained DIO dye (absorbance of 480 nm) until the dye was completely washed. 500 μL of PBS was taken to resuspend the stained exosomes collected in the ultrafiltration tube, and the collected stained exosomes were evenly divided into two tubes, that is, the volume of each tube was 250 μL. Add 250 μL of DSPE-PEG-MAN (20 μg / mL) to one tube and 250 μL of DSPE-mPEG (20 μg / mL) to the other tube as the control group, invert and mix, and incubate overnight at 4°C in the dark. Use a 100KD ultrafiltration tube to wash away the free DSPE-PEG-MAN or DSPE-mPEG, centrifuge at 14000×g for 10 min each time, add PBS to wash, and repeat centrifugation 3 times. Finally, take 200 μL of PBS to resuspend the stained exosomes modified with DSPE-PEG-MAN and DSPE-mPEG collected in the ultrafiltration tube, and store them at 4°C in the dark.

[0057] (3) Count 4×10 5THP-1 macrophages were plated and cultured at 37°C in an environment containing 5% CO2 for 24 h. 50 μL of the stained exosomes modified with DSPE-PEG-MAN and stained exosomes modified with DSPE-mPEG (200 μg / mL) obtained in step (2) were taken respectively and incubated with THP-1 macrophages in a 37°C incubator for 1 h, 3 h and 6 h respectively. The cells were washed twice with PBS, digested with trypsin, collected, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 1 mL of PBS was added to resuspend and wash the precipitate, centrifuged at 1000 rpm for 5 min, repeated twice, and finally the precipitate was collected into a flow tube, 500 μL of PBS was added to resuspend the cells, and the fluorescence value MFI in THP-1 macrophages was detected by flow cytometry.

[0058] In this example, normal non-transfected cells (NC-Exo) were used as the normal control group, and the fluorescence value MFI in THP-1 macrophages was detected by flow cytometry. The results showed that the surface-targeted CircRNA and anti-tuberculosis drug co-loaded exosomes (Man-Exo) prepared in this example can be taken up more by macrophages, which is beneficial to the surface-targeted CircRNA and anti-tuberculosis drug co-loaded exosomes to play a regulatory role in host cells ( Figure 10-11 ).

[0059] Example 4

[0060] This embodiment provides a method for preparing an extracellular vesicle drug-carrying system (circRNA and anti-tuberculosis drugs co-loaded microvesicles) comprising the following steps:

[0061] (1) By designing and constructing a high-expression lentivirus of a specific circRNA (taking circRNA-circTRAPPC6B with anti-tuberculosis immune regulation function as an example), 293T cells were transfected with the lentivirus. 1×10 cells were counted in each well of a 24-well plate. 5 After 24 h of culture, 500 μL of 2×10 6 TU lentivirus (MOI = 10) serum-containing medium (10 μL lentivirus (1×10 8TU / mL) was added to 490 μL of serum-containing culture medium) and cultured for 48 hours. Obvious green fluorescence expression was visible under a fluorescence microscope 48 hours after transfection. Culture was continued for 3-4 days, digested with trypsin, collected cells, centrifuged at 1000rpm for 5 minutes, discarded the supernatant, resuspended the precipitate with 1mL of PBS, repeated centrifugation twice, and finally resuspended the cell precipitate with 500 μL of PBS and collected in a flow tube. All cells containing green fluorescence were sorted out by flow sorting instrument, with a sorting efficiency of 89%, thereby screening out all cell lines with stable and high expression of CircRNA containing green fluorescence;

[0062] (2) Two cell lines, normal untransfected (normal control group) and circRNA stably highly expressed, were cultured at 37°C in an environment containing 5% CO2. 4×10 cells were counted in each 10 cm culture dish. 6 The cells were plated and cultured for 24 hours. Then, 5 mL of serum-free medium containing high concentration of rifampicin (final concentration of 1 mg / mL) was added in the form of liquid replacement (50 μL of rifampicin with a concentration of 100 mg / mL was added to 4950 μL of serum-free medium). The cells were co-incubated with rifampicin for 24 hours to induce cell apoptosis, and the cell culture supernatant was collected. The supernatant was collected by centrifugation at 4°C 600×g for 10 minutes; 4°C 2700×g for 10 minutes, the supernatant was collected; 4°C 14000×g for 2 minutes, the supernatant was collected; 4°C 14000×g for 60 minutes, the supernatant was discarded, and the precipitate was washed with PBS 3 times, each time at 4°C 14000×g for 60 minutes, and the precipitate was resuspended with 500 μL of PBS for the last time to obtain circRNA and anti-tuberculosis drug co-loaded microvesicles, which were stored at -80°C.

[0063] In this example, the particle size of the cell microvesicles (MPs) produced by normal untransfected cells and the cell microvesicles (CircRNA@MPs) produced by CircRNA stably transfected cells was confirmed by particle size analysis ( Fig.12 ), and the results confirmed that the above preparation steps of this example can successfully extract two types of microvesicles (microvesicles produced by normal non-transfected cells and microvesicles produced by CircRNA stably transfected cells). The expression level of CircRNA in the two types of microvesicles was detected by RT-qPCR. The results showed that the expression level of CircRNA in the microvesicles of CircRNA stably transfected cells was significantly higher than that in the normal control group ( Fig.13 ), which confirmed that the cell microvesicles could be successfully loaded with CircRNA. The drug concentration of the cell microvesicles was then detected by ultraviolet spectrophotometry (480nm). Fig.14 ), which confirmed that the cell microvesicles can be successfully loaded with anti-tuberculosis drugs (rifampicin).

[0064] In summary, the present invention uses extracellular vesicles as co-carriers of CircRNA and anti-tuberculosis drugs, efficiently loads anti-tuberculosis immune regulatory function CircRNA and anti-tuberculosis drugs, and then further enhances the targeting of the extracellular vesicle drug delivery system by surface targeted modification of the extracellular vesicles, thereby obtaining the extracellular vesicle drug delivery system. The extracellular vesicle drug delivery system combines the anti-tuberculosis immune regulatory function of CircRNA and the inhibitory effect of anti-tuberculosis drugs on Mycobacterium tuberculosis, and more effectively kills and eliminates Mycobacterium tuberculosis in host cells, thereby providing a new strategy for the development of safe and efficient anti-tuberculosis drugs.

[0065] The above specific embodiments are further explanations of the technical solutions and beneficial effects of the present invention, and are not limitations of the implementation methods. For those skilled in the art, any obvious replacements that do not depart from the concept of the present invention are within the protection scope of the present invention.

Claims

1. An extracellular vesicle drug delivery system, characterized in that: It includes extracellular vesicles, CircRNA loaded in the extracellular vesicles, and an anti-tuberculosis drug; the anti-tuberculosis drug is rifampicin; the CircRNA is circTRAPPC6B; The preparation method of the extracellular vesicle drug delivery system comprises the following steps: (1) Extraction of highly expressed circRNA exosomes: (2) using a simple incubation method or an electroporation method to load the anti-tuberculosis drug into the highly expressed circRNA exosomes prepared in step (1); (3) Wash away the free anti-tuberculosis drugs, add PBS for washing, centrifuge several times until the free anti-tuberculosis drugs are completely removed, and then resuspend the ultrafiltration tube with PBS to collect the circRNA and anti-tuberculosis drug co-loaded exosomes; (4) The circRNA and anti-tuberculosis drug co-loaded exosomes obtained in step (3) are subjected to surface targeted modification to obtain the extracellular vesicle drug delivery system.

2. A method for preparing an extracellular vesicle drug delivery system, characterized in that: The steps include: (1) Extracting highly expressed circRNA exosomes: the circRNA is circTRAPPC6B; (2) using a simple incubation method or an electroporation method to load the anti-tuberculosis drug into the highly expressed circRNA exosomes prepared in step (1); the anti-tuberculosis drug is rifampicin; (3) Wash away the free anti-tuberculosis drugs, add PBS for washing, centrifuge several times until the free anti-tuberculosis drugs are completely removed, and then resuspend the ultrafiltration tube with PBS to collect the circRNA and anti-tuberculosis drug co-loaded exosomes; (4) The circRNA and anti-tuberculosis drug co-loaded exosomes obtained in step (3) are subjected to surface targeted modification to obtain the extracellular vesicle drug delivery system.

3. The method for preparing an extracellular vesicle drug delivery system according to claim 2, characterized in that: In step (2), the specific steps of the electroporation method are as follows: take 100 μL of the highly expressed CircRNA exosomes with a concentration of 1 mg / mL prepared in step (1) into an EP tube, add 100 μL of rifampicin with a concentration of 2 mg / mL, invert and mix well, and place on ice. Set the operating conditions of the electroporation instrument to 1000 V, 10 ms and 2 pulses, electroporate 200 μL of the mixture of highly expressed CircRNA exosomes and rifampicin, collect the exosomes after electroporation, and incubate at 37°C for 30 min to restore the exosome membrane structure.

4. The method for preparing an extracellular vesicle drug delivery system according to claim 2, characterized in that: In step (4), the specific operation of surface targeted modification is as follows: take 1 mL of 500 μg / mL circRNA and anti-tuberculosis drug co-loaded exosomes prepared in step (3), add 1 mL of 20 μg / mL DSPE-PEG-MAN, invert to mix, place in a shaker at 4°C in the dark and incubate overnight; then use an ultrafiltration tube to wash away the free DSPE-PEG-MAN, add PBS to wash, and repeat the centrifugation several times.

5. Use of the extracellular vesicle drug delivery system as claimed in claim 1 in the preparation of anti-tuberculosis drugs.

6. Use of the preparation method of the extracellular vesicle drug delivery system as described in any one of claims 2 to 4 in the preparation of anti-tuberculosis drugs.

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