Preparation process and application of cationized exosome nucleic acid delivery system

Abstract

The invention discloses a preparation process and application of a cationized exosome nucleic acid delivery system, the delivery system is prepared from an exosome and a cationic material through charge non-covalent interaction, and a nucleic acid therapeutic agent is loaded in the delivery system; the surface of the exosome is modified and transformed, so that the cellular uptake is enhanced, the delivery efficiency of the nucleic acid carrying drug of the exosome can be remarkably improved, and particularly, the targeted delivery effect of the exosome on the respiratory tract part is improved; by optimizing the modification ratio of the exosome to cations, the stability and expression level of the exosome carrying nucleic acid medicine are improved, and the exosome carrying nucleic acid medicine has efficient lung delivery capacity, shows remarkable treatment potential in tumor treatment, has a certain clinical application prospect, and has a wide application prospect. The invention further provides a preparation method of the efficient nucleic acid cationization exosome delivery system and application of the delivery system to treatment and / or prevention of related diseases or symptoms.

Description

Technical Field

[0001] The present invention relates to the field of drug delivery, and in particular to a cationized exosome nucleic acid delivery system, and also to applications of the system. Background Art

[0002] As a natural intercellular communication carrier, exosomes have broad application prospects in the fields of drug delivery and gene therapy due to their low immunogenicity, good biocompatibility, and ability to carry a variety of bioactive substances (such as nucleic acids, proteins, etc.). However, due to the negative charge on the surface of the exosome membrane, conventional exosome delivery systems face problems such as unstable carrier load and low transfection efficiency when delivering nucleic acids. Therefore, it is necessary to develop a new strategy to improve the delivery efficiency of exosomes as nucleic acid carriers.

[0003] In the prior art, surface modification of exosomes usually involves chemical covalent modification, which may affect the biocompatibility and functional integrity of exosomes. Cationic modification is a common means to improve the efficiency of delivery systems. By introducing cationic modification on the surface of the exosome membrane, it can be made easier to bind to negatively charged nucleic acids, thereby improving the loading efficiency and delivery effect of nucleic acids. At the same time, cationic modified exosomes can also improve their binding ability and entry efficiency with cell membranes, further enhancing their application value in gene therapy. Therefore, it is extremely necessary to develop a new method that can maintain the natural state of exosomes and significantly improve their nucleic acid delivery ability, and provide more efficient delivery vectors for gene therapy, vaccine development and other fields. Summary of the invention

[0004] In view of this, one of the objects of the present invention is to provide a cationized exosome nucleic acid delivery system; a second object of the present invention is to provide a method for preparing the cationized exosome nucleic acid delivery system; a third object of the present invention is to provide the use of the cationized exosome nucleic acid delivery system in the preparation of drugs for gene therapy, vaccine delivery, tumor therapy or cell therapy.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. A cationized exosome nucleic acid delivery system, wherein the exosome nucleic acid delivery system is made of nucleic acid, exosomes and cationic materials through non-covalent interactions.

[0007] In the present invention, the exosomes are extracted from bovine milk or biological cells, wherein the mass ratio of the exosomes to the nucleic acid in the composition is 0.5:1-60:1.

[0008] Preferably, the biological cells of the present invention are mammalian cells or other biological cells.

[0009] Preferably, the nucleic acid encodes therapeutic agents such as therapeutic proteins, vaccine antigens, antibodies, regulators, gene repair factors, etc., and the nucleic acid is selected from at least one of the following: messenger RNA, self-amplifying RNA, circular RNA, small interfering RNA, short hairpin RNA and micro RNA, primary-miRNA, antisense oligonucleotides, transfer RNA, plasmid DNA, single-stranded DNA, double-stranded DNA, deoxyribozymes, ribozymes, nucleic acid aptamers, clustered regularly interspaced short palindromic repeats-related nucleic acids, single guide RNA, CRISPR-RNA, trans-activated crRNA, guide RNA, single-stranded RNA and double-stranded RNA.

[0010] Preferably, the cationic material is chitosan, cationic lipid, poly-β-amino ester, polyethyleneimine, polypeptide / protein rich in basic amino acids, polyarginine, polylysine and dendrimer;

[0011] The cationic lipid comprises at least one selected from the group consisting of DOTMA, DOSPA, DOTAP, ePC, DODAP, DODMA, DDAB, DSDMA, DODAC, DOAP, DMRIE, DOGS, DMOBA, HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4005, DLin-MC3-DMA, DLin-KC2-DMA, Acuitas ALC-0315, AcuitasA9, Acuitas Lipid 2,2, Moderna Lipid H, Moderna Lipid 5. A2-Iso5-2DC18, BAME-O16B, 9A1P9, C12-200, cKK-E12, OF-Deg-Lin, 306Oi10, TT3, FTT5, Lipid319, 5A2-S C8, GenevantCL1, DLinDMA, DLenDMA, ClinDMA, CpLinDMA, imidazole cholesteryl ester, RE-1, RE-2, RE-3, GL-67, 5A2-SC8, Acuitas A9.Arcturus Lipid 2,2(8,8)4C CH3, OF-02, A18-Iso5-2DC18, BAME-O16B, A6, 98N12-5, L319, L343, 304O13, 306O138, 306O12B, 30 6-O12B, LP01, G0-C14, 7C1, Cephalin, Dlin-EG-DMA, DLinAP, DLin-MPZ, DLin-C-DAP, DLin-2-DMAP , Dlin-S-DMA, DLinDAP, DLin-MA, DLin-DAC, DLin-K-DMA, DLin-K-MPZ, DLin-K-DMA, DLin-K6-C4-DMA, DLin-K-C4-DMA, DLin-K-C3-DMA, CpLinDMA, DOcarbDAP, DLincarbDAP, C12-(2-3-2), Genevant Lipid CL1, XTC, ALNY-100, NC98-5 and their derivatives;

[0012] The molar ratio of nitrogen (amine) groups in the cationic material to phosphate groups in the nucleic acid in the composition is 3.0-15.0;

[0013] The polypeptide / protein rich in basic amino acids is preferably protamine; and the dendritic polymer is preferably polyurethane dendritic polymer (PAMAM).

[0014] 2. The preparation method of the cationic exosome nucleic acid delivery system comprises the following steps:

[0015] (1) loading nucleic acid into the isolated exosomes by electroporation;

[0016] (2) modifying and compounding the cationic material with the exosomes, so that the cationic material is combined with the exosomes through electrostatic interaction or copolymerization reaction to obtain a cationic exosome nucleic acid delivery system; or

[0017] (1) incubating the cationic material with the nucleic acid to form a complex through electrostatic interaction or copolymerization reaction;

[0018] (2) combining the complex with exosomes by electroporation or incubation to obtain a cationized exosome nucleic acid delivery system; or

[0019] (1) combining the separated exosomes with the cationic material through an incubation method to form a complex;

[0020] (2) The nucleic acid is loaded into the cationic material-exosome complex by electroporation or incubation to obtain a cationic exosome nucleic acid delivery system.

[0021] Preferably, the electroporation conditions of the present invention are: the electroporation voltage is 150-400V, the pulse length is 5-10ms, the pulse interval is 0.5-2s, and the number of pulses is 1-5 times; the preferred conditions are: the electroporation voltage is 200V, the pulse length is 10ms, the pulse interval is 1s, and the number of pulses is 3 times.

[0022] Preferably, the method for modifying and compounding the cationic material with exosomes is as follows: a cationic material solution is mixed with exosomes loaded with nucleic acid, incubated for 30-60 minutes, and then centrifuged to collect the precipitate to obtain cationized exosomes.

[0023] Preferably, the cationic material is chitosan, cationic lipid, poly-β-amino ester, polyethyleneimine, polypeptide / protein rich in basic amino acids, polyarginine, polylysine and polyurethane dendrimer; the chitosan solution is prepared with 1M acetic acid solution, the poly-β-amino ester solution is dissolved with DMSO and then prepared with 25mM NaAc solution, the polyethyleneimine solution, polyarginine, polylysine, protamine and polyurethane dendrimer are all dissolved with nuclease-free water, and the cationic lipid is dissolved with ethanol.

[0024] 4. Application of the cationic exosome nucleic acid delivery system in the preparation of drugs for gene therapy, vaccine delivery, tumor therapy or cell therapy.

[0025] The beneficial effects of the present invention are as follows: the present invention provides a cationized exosome nucleic acid delivery system, which is made of exosomes loaded with mRNA and cationic materials through charge non-covalent interaction; due to the modification and transformation on the surface of the exosomes, the cell uptake is enhanced, and the delivery efficiency of the exosome-loaded nucleic acid drug can be significantly improved, especially the targeted delivery effect in the lung is improved; the present invention optimizes the ratio of exosomes to cationic modifications, and the present invention achieves the improvement of the stability and expression level of the exosome-loaded nucleic acid drug, has efficient lung delivery ability, shows significant therapeutic potential in tumor treatment, and has broad clinical application prospects. The present invention also provides a preparation method of the efficient nucleic acid cationized exosome delivery system, and the preparation method is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0027] Figure 1 This is a schematic diagram of the modification process of cationic exosomes in Example 1;

[0028] Figure 2 This is a diagram showing the expression effects of different cationic exosomes on F-Luc in cells;

[0029] Figure 3 The lung delivery ability of different cationic exosomes is shown in the figure. The effect of F-Luc reporter gene transfection in mouse lungs;

[0030] Figure 4 The results of TEM (A), particle size (B), potential (C) and marker protein (D) of different cationic exosomes (Exo / CS-Exo / PBAE-Exo) in Example 4 are shown;

[0031] Figure 5 This is a graph showing the potential stability analysis of different cationized exosomes;

[0032] Figure 6 Figure 2 shows the toxicity evaluation of different cationic exosomes in different lung-related cells.

[0033] Figure 7 Comparative diagram of the in vivo distribution of different cationic exosomes and their quantitative analysis (A: in vivo imaging; B: fluorescence intensity at different times).

[0034] Figure 8 Specific serum IgG of mice after immunization with different cationic exosomes in the lung (A: mouse immunization and sampling process; B: OVA-specific serum IgG absorbance (OD450) at each time point after immunization).

[0035] Fig. 9The different cationic exosomes in Example 9 were used to treat B16-F10 melanoma (A: tumor model construction and treatment process; B: weight changes of mice in each group at each time point; C: quantification of bioluminescent signals in mice in each group at each time point). DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0037] Example 1. Preparation method of exosomes from different sources modified with different cations

[0038] Extraction and separation of exosomes from different sources

[0039] (1) Extraction of exosomes from bovine milk

[0040] Commercially available milk, skim milk and acetic acid were mixed (100:1, v / v) at room temperature for 5 min, then centrifuged at 12,000 × g for 20 min at 4 ° C, and the supernatant was filtered with a 0.22 μm membrane to obtain whey. The whey was centrifuged at 210,000 × g for 70 min at 4 ° C. The EXO pellet was resuspended in PBS and ultracentrifuged again. After washing, the pellet was resuspended in PBS and centrifuged at 10,000 × g for 5 min at 4 ° C to remove the residual precipitate, and the EXO was stored at -80 ° C until use.

[0041] (2) Extraction of cell-derived exosomes

[0042] Human embryonic kidney HEK293T cells and human non-small cell lung cancer A549 cell lines were purchased commercially and cultured in minimal essential medium containing 10% FBS, 1% L-glutamine, 0.5% gentamicin and 0.18% 2-mercaptoethanol, and the medium was refreshed every other day. As described previously, before generating serum-free secretomes, HEK293 and A549 cells were allowed to reach 70-80% confluence and then switched to serum-free medium for three days.

[0043] The supernatant of each cell was collected and centrifuged at low speed of 300×g / min for 10min to separate the cells; the supernatant was centrifuged at 2000×g / min for 10min to remove dead cells. The remaining supernatant was centrifuged at 10000×g / min for 30min to remove cell debris; then the supernatant was taken again and centrifuged at 100,000×g / min for 60min to obtain the exosome precipitate, and the exosome precipitate was resuspended with sterile PBS and centrifuged again at 100,000×g / min for 70min to obtain the exosomes.

[0044] Preparation of different cationized exosome preparations

[0045] (1) Exosome mRNA loading

[0046] The extracted exosomes from various sources were mixed with mRNA and loaded using an electroporation system. 9 The volume of exosome particles was diluted in Gene Pulser electroporation buffer at a ratio of 1:9 with buffer, and then 10 μg of mRNA was added. The mixture was transferred to a 4 mm MicroPulser electroporation cuvette and pre-cooled on ice. The electroporation conditions were set as follows: electroporation voltage was 200 V, pulse length was 10 ms, pulse interval was 1 s, and pulse number was 5 times. After the electroporation, the electroporation solution was allowed to stand at 37 ° C for more than 10 min, and the electroporation buffer and concentrated exosomes were removed to the expected volume using ultrafiltration and a centrifugal filter device (cutoff value was 100 kDa).

[0047] (2) Cationization of exosomes after loading with nucleic acid

[0048] Cationic materials: commercially available chitosan (CS), poly-β-amino ester (PBAE), polyethyleneimine (PEI), polylysine (PLL), polyarginine (Parg), protamine (Protamine), polyurethane dendrimer (PAMAM), and cationic lipid (DOTAP).

[0049] Chitosan-modified exosomes (CS-Exo) were prepared, and the modification process was as follows Figure 1 shown.

[0050] Preparation of chitosan (CS) solution: Prepare 1 M acetic acid solution, add 0.35 g chitosan to the acetic acid solution, stir until fully dissolved, and leave at room temperature overnight until it is completely dissolved.

[0051] Exo and CS were mixed in a mass ratio of 5:3 and incubated in a shaker at 25-37°C for 30-60 min. After the incubation, the mixture was centrifuged at 100,000 g for 70 min, the supernatant was removed, and the retained precipitate was the chitosan-modified exosomes (CS-Exo).

[0052] (3) Preparation of poly-β-amino ester modified exosomes (PBAE-Exo)

[0053] Preparation of poly (β-amino ester) (PBAE) solution: PβAE dissolved in DMSO was mixed with NaAc solution (25 mM, pH=5) to form a polymer suspension (“milky white” appearance). After sonication, the sample was incubated at room temperature on an orbital shaker for 1 hour.

[0054] Exo and PBAE were mixed at a mass ratio of 1:6, incubated at 25-37°C in a shaker for 30-60 min, and after incubation, centrifuged at 100,000 g for 70 min, the supernatant was removed, and the retained precipitate was poly-β-amino ester-modified exosomes (PBAE-Exo).

[0055] (4) Preparation of polyethyleneimine modified exosomes (PEI-Exo)

[0056] Prepare polyethyleneimine (PEI) solution: take a corresponding amount of PEI, add an appropriate amount of warm water (30-40°C), stir with a magnetic stirrer until the solution is completely uniform, and finally filter through a 0.22 μm membrane to remove undissolved particles and store at 4°C.

[0057] Exo and PEI were mixed at a mass ratio of 1:1-1:10, incubated at 25-37°C in a shaker for 30-60 min, and after incubation, centrifuged at 100,000 g for 70 min, the supernatant was removed, and the retained precipitate was polyethyleneimine-modified exosomes (PEI-Exo).

[0058] (5) Preparation of polylysine-modified exosomes (PLL-Exo)

[0059] Prepare polylysine (PLL) solution: Take a corresponding amount of PLL, gradually add deionized water, stir while adding water to dissolve the polylysine, use a magnetic stirrer to continue stirring until the solution is completely uniform, and finally filter through a 0.22 μm membrane to remove undissolved particles and store at 4°C.

[0060] Exo and PLL were mixed at a mass ratio of 1:3-1:7, incubated at 25-37°C in a shaker for 30-60 min, and after incubation, centrifuged at 100,000 g for 70 min, the supernatant was removed, and the retained precipitate was the polylysine-modified exosomes (PLL-Exo).

[0061] (6) Preparation of polyarginine-modified exosomes (Parg-Exo)

[0062] Prepare polyarginine (Parg) solution: Take a corresponding amount of Parg, gradually add deionized water, stir while adding water to dissolve the polyarginine, use a magnetic stirrer to continue stirring until the solution is completely uniform, and finally filter through a 0.22μm membrane to remove undissolved particles and store at 4°C.

[0063] Exo and Parg were mixed in a mass ratio of 1:2-1:8, incubated in a shaker at 25-37°C for 30-60 min, and after incubation, centrifuged at 100,000 g for 70 min, the supernatant was removed, and the retained precipitate was the polyarginine-modified exosomes (Parg-Exo).

[0064] (7) Preparation of protamine-modified exosomes (Protamine-Exo)

[0065] Prepare protamine solution: Take a corresponding amount of protamine, gradually add deionized water, stir while adding water to dissolve the protamine, use a magnetic stirrer to continue stirring until the solution is completely uniform, finally filter through a 0.22μm membrane to remove undissolved particles and store at 4°C.

[0066] Exo and Protamine were mixed in a mass ratio of 1:0.01-1:1, incubated in a shaker at 25-37°C for 30-60 min, and after incubation, centrifuged at 100,000 g for 70 min, the supernatant was removed, and the retained precipitate was polyarginine-modified exosomes (Protamine-Exo).

[0067] (8) Preparation of polyurethane dendrimer-modified exosomes (PAMAM-Exo)

[0068] Prepare polyurethane dendrimer (PAMAM) solution: take a corresponding amount of PAMAM, gradually add deionized water, stir while adding water to dissolve PAMAM, use a magnetic stirrer to continue stirring until the solution is completely uniform, finally filter through a 0.22 μm membrane to remove undissolved particles and store at 4°C.

[0069] Exo and PAMAM were mixed at a mass ratio of 1:2-1:10, incubated at 25-37°C in a shaker for 30-60 min, and after incubation, centrifuged at 100,000 g for 70 min, the supernatant was removed, and the retained precipitate was polyurethane dendrimer-modified exosomes (PAMAM-Exo).

[0070] (9) Preparation of cationic lipid-modified exosomes (DOTAP-Exo)

[0071] Preparation of cationic lipid (DOTAP) solution: Take a corresponding amount of DOTAP in ethanol, sonicate moderately to dissolve it, and incubate at 37° C. until the solution is completely uniform.

[0072] According to the molar ratio of nitrogen (amine) groups to phosphate groups of nucleic acids (N / P ratio) of 3.0-15.0, DOTAP and mRNA were mixed by microfluidics at a volume ratio of 1:3 to obtain a DOTAP / mRNA complex.

[0073] Exo and DOTAP / mRNA complex were mixed at a mass ratio of 1:0.01-1:1, incubated at 25-37°C in a shaker for 30-60 min, and after incubation, centrifuged at 100,000 g for 70 min, the supernatant was removed, and the retained precipitate was the cationic lipid-modified exosomes (DOTAP-Exo).

[0074] Subsequent administration concentration of cationic exosomes: The cationic exosomes carrying mRNA were collected by centrifugation at 100,000 g for 70 min, resuspended in PBS, and the particle concentration was adjusted to 1×10 11 / mL, which can be used for subsequent dosing.

[0075] Example 2: Evaluation of the cellular expression of different cationized exosomes

[0076] This example mainly illustrates that Fluc (Firefly Luciferase) mRNA delivered by different cationic exosomes can express a large amount of Firefly Luciferase protein in cells.

[0077] Materials and methods: human bronchial epithelial cells (16HBE); Luciferase mRNA; CS-Exo / PBAE-Exo / PEI-Exo / PLL-Exo / Parg-Exo / Protamine-Exo / PAMA-Exo / DOTAP-Exo (preparation method is the same as Example 1), 16HBE cell culture medium is DMEM culture medium containing 10% fetal bovine serum.

[0078] Promega dual luciferase reporter kit was used to detect the protein expression of Fluc-mRNA delivered by CS-Exo / PBAE-Exo / PEI-Exo / PLL-Exo / Parg-Exo / Protamine-Exo / PAMA-Exo / DOTAP-Exo in 16HBE cells. 16HBE cells were cultured in DMEM (GIBCO) medium containing 10% fetal bovine serum in a 37°C, 5% CO2 incubator. The cells were observed to be in good growth state. After culturing to the logarithmic growth phase, the cells were seeded into 96-well plates and 1×10 4 / well, cultured in a 37°C, 5% CO2 incubator, and transfection can be performed when the cell confluence reaches 80%. The experiment was divided into a cell control group, a CS-Exo group, a PBAE-Exo group, a PEI-Exo group, a PLL-Exo group, a Parg-Exo group, a Protamine-Exo group, a PAMA-Exo group, a DOTAP-Exo group, and a Lipofectamine3000 / Fluc-mRNA group. The 96-well plate was taken out, and different liposomes containing 400ngFluc-mRNA were added, and cultured in a 37°C, 5% CO2 incubator. After 6 hours, the culture medium was discarded, and the cells were lysed according to the instructions of the dual luciferase kit, and the lysate was transferred to a 96-well white plate, and detected using a microplate chemiluminometer, and 50μL of lysate and 50μL of luciferase substrate were added to each well.

[0079] result:

[0080] The CS-Exo / PBAE-Exo / PEI-Exo / PLL-Exo / Parg-Exo / Protamine-Exo / PAMA-Exo / DOTAP-Exo groups all showed high expression of Firefly luciferase protein at 24 h and were much higher than those in the Exo group (e.g. Figure 2 As shown). In summary, Fluc-mRNA delivered by CS-Exo / PBAE-Exo / PEI-Exo / PLL-Exo / Parg-Exo / Protamine-Exo / PAMA-Exo / DOTAP-Exo can express a large amount of Firefly luciferase in cells, and can be efficiently stabilized and stably expressed in cells.

[0081] Example 3: Evaluation of the lung transfection effects of different cationic exosomes

[0082] This example mainly illustrates that mRNA delivered by different cationic exosomes can express proteins in BALB / c mice.

[0083] Materials and methods: female BALB / c mice, 20 g; Luciferase mRNA; CS-Exo / PBAE-Exo / PEI-Exo / PLL-Exo / Parg-Exo / Protamine-Exo / PAMA-Exo / DOTAP-Exo (preparation method is the same as Example 1).

[0084] The cationic exosomes carrying Luciferase mRNA were used as the experimental group, and Exo was used as the control group. The drug concentration was 1×1011 / mL, 50μL / mouse. Balb / c female mice aged 6 to 8 weeks were anesthetized with isoflurane, with three mice in each group. After 6 hours of drug administration, 1.1% sodium pentobarbital solution was injected intraperitoneally at 120μL / mouse. After complete anesthesia, the mice were killed by dislocating the neck, the abdominal and thoracic cavities were opened, the liver, spleen and whole lungs were removed, rinsed with 1×PBS, and placed in the living imaging system to detect chemiluminescent signals. The light flux of the circled area was calculated using Living Image software.

[0085] After pulmonary administration of cationic exosomes containing the Fluc reporter gene, there were stronger chemiluminescent signals than the Exo control group, and the chemiluminescent signal intensity in the lung tissue of the CS-Exo group was the highest. The above results show that the nanoparticles of each group of CS-Exo / PBAE-Exo / PEI-Exo / PLL-Exo / Parg-Exo / Protamine-Exo / PAMA-Exo / DOTAP-Exo can efficiently express the target mRNA in the lung tissue of mice after respiratory administration, and the effects of the two groups of nanoparticles of CS-Exo and PBAE-Exo are the most significant. Therefore, in the subsequent examples, the two groups of preparations of CS-Exo and PBAE-Exo are selected to continue the subsequent experiments (such as Figure 3 shown).

[0086] Example 4: Characterization of the physicochemical properties of different cationized exosomes

[0087] The morphology, particle size distribution, potential size and surface specific proteins of the blank exosomes (Exo) and cationic exosomes (PBAE-Exo and CS-Exo) prepared in Example 1 were characterized.

[0088] The exosomes extracted in Example 1 showed a typical vesicular and disc-shaped double-layer membrane structure in the transmission electron microscope (TEM) image. The cationic exosomes (PBAE-Exo and CS-Exo) prepared in Example 1 were evenly distributed under the transmission electron microscope ( Figure 4 , A).

[0089] Next, the particle size of PBAE-Exo and CS-Exo was analyzed using nanoparticle tracking analysis (NTA). The results showed that the particle size distribution of PBAE-Exo and CS-Exo was basically consistent with that of blank exosomes, with a slight increase in the average particle size ( Figure 4 , B). At the same time, the surface of the exosomes is cationized, the proteins are shielded, and the potential increases ( Figure 4 , C).

[0090] Western Blot analysis was used to analyze the expression of Exo-specific markers. The results of immunoblotting analysis showed that the expression of surface-specific proteins CD63 and CD81 in PBAE-Exo and CS-Exo was basically consistent with that in blank exosomes ( Figure 4 , D). The above results confirm that the modification method of the present invention has no effect on the main physical and chemical properties of exosomes.

[0091] Example 5: Evaluation of the stability of exosomes with different cationization properties

[0092] In Examples 1 and 2, different cationized exosomes have been successfully prepared and characterized. However, considering the complex environment in the body, cationic modifiers (cationized exosomes) with changed surface charge may be adversely affected. Therefore, after modification using the method of the present invention, the stability of the binding of the cationic modifier is crucial.

[0093] Materials and Methods

[0094] Exo / CS-Exo / PBAE-Exo (preparation method is the same as Example 1)

[0095] In this example, the cationized exosome (PBAE-Exo and CS-Exo) suspension and blank exosome prepared in Example 1 were added to 2% FBS and stored at room temperature. Subsequently, the PBAE-Exo and CS-Exo in FBS were re-purified by ultrafiltration (molecular weight cutoff 100KDa) at specific time points (1, 2, 4, 8, 12 and 24h) and their Zeta potential was measured.

[0096] The results showed that the surface Zeta potential fluctuations of PBAE-Exo and CS-Exo were relatively small and remained relatively stable, confirming the stability of the surface potential of the cationized exosomes prepared by the method of the present invention (e.g. Figure 5 shown).

[0097] Example 6: Evaluation of the cytotoxicity of different cationized exosomes

[0098] This example mainly evaluates the cytotoxicity of cationic exosomes (CS-Exo / PBAE-Exo). Considering that the cationic exosomes prepared in Example 1 will be used for pulmonary administration later, lung-related cells are preferred for the experiment. Considering that the yield of cell-derived exosomes currently studied is extremely low and the preparation cost is high, which greatly limits the large-scale application of exosomes as nucleic acid delivery vectors, subsequent experiments use high-yield milk exosomes (Milk-Exo) as a vector for example display.

[0099] Materials and methods: 16HBE cells and A549 cells, Exo / CS-Exo / PBAE-Exo (preparation method is the same as in Example 1), 16HBE cells and A549 cells were cultured in DMEM containing 10% fetal bovine serum.

[0100] 1×10 4 Cells were inoculated at a density of 10 cells / well in a 96-well plate. After the cells adhered to the wall, complete medium containing different cationic exosomes was added and cultured for 24h and 60h. Then the drug-containing medium was removed, and complete medium containing 5mg / ml tetrazolium salt (MTT) was added and cultured for 4h. The medium containing MTT was removed, and 200ulDMSO was added to each well to dissolve the formazan crystals in the well, and the absorbance was detected by the microplate reader at a wavelength of 490nm. The wells without drug administration were used as the control group, and the wells without cells were used as the blank group. The cell survival rate was calculated using the following formula: Cell viability (%) = ((A drug-A background) / (A no drug-A background)) × 100%.

[0101] Results: The carrier material (EXO) and the modified material (CS, PBAE) had no obvious toxicity to lung-related cells, but CS-Exo did show a slight inhibitory effect on tumor cells (A549), which may be due to the anti-tumor activity of chitosan (CS) manifesting itself by interfering with the metabolic growth of tumor cells (e.g. Figure 6 shown).

[0102] Example 7: Evaluation of the in vivo distribution of different cationized exosomes

[0103] This example mainly illustrates the tissue distribution characteristics of CS-Exo / PBAE-Exo in vivo

[0104] Materials and methods: Fluorescent dye 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR), Exo / CS-Exo / PBAE-Exo (preparation method is the same as Example 1), female BALB / c mice, 20 g.

[0105] Exo / CS-Exo / PBAE-Exo were labeled with the fluorescent dye DiR (dye concentration, 0.05 mM). After incubation at 37°C for 2 h, unbound dye was separated using an exosome spin column (Invitrogen) and removed by centrifugation at 10,000 g and 4°C for 15 min. 9 The particle number dose was injected into the lungs through the trachea. The fluorescence signal was observed using the IVIS spectral imaging system, and fluorescence images were taken at 6h, 24h, 48h, and 72h for each group.

[0106] In vivo imaging showed that the fluorescence intensity of the Exo and CS-Exo / PBAE-Exo groups in the lungs was strong after 6 hours. However, after 24 hours, the distribution and retention of the CS-Exo / PBAE-Exo group in the lungs were significantly higher than those of the Exo group. At the same time, CS-Exo / PBAE-Exo showed obvious retention in the lungs after 24 hours, while the Exo group decreased rapidly after 24 hours (e.g. Figure 7 shown).

[0107] Example 8: Investigation of the levels of specific antibodies induced by different cationic exosomes in mice

[0108] Materials and methods: Female C57BL / 6N mice, B16-F10-Luc cells, Exo / CS-Exo / PBAE-Exo (preparation method is the same as Example 1).

[0109] Twenty-four Balb / c female mice aged 6 to 8 weeks were randomly selected and divided into three groups. CS-Exo / PBAE-Exo@OVA-mRNA and 1×PBS solution groups were administered into the lungs at a concentration of 1×10 11 / mL, 50μl / mouse, and set as experimental group and negative control group. The immunization and sampling time points are as follows Figure 8 shown.

[0110] Serum collection: 40 μl of blood was collected from the tail vein of the experimental mice. The blood sample was kept at 37°C for 1 h, centrifuged at 8000 rpm for 20 min, and the upper serum was aspirated and stored at -80°C for later use.

[0111] Add 100 μL / well of coating buffer containing 5 mg / LOVA protein to a 96-well plate and coat overnight at 4°C. Discard the liquid in the wells, add 250 μL 1×PBST to each well to wash the plate, let it stand for 2 minutes, then blot dry, repeat three times. Add 250 μL 1×PBST containing 1% BSA to the wells, block overnight at 4°C, and wash the plate. Use 1×PBST containing 0.5% BSA to dilute serum and bronchoalveolar lavage fluid samples as required, add 100 μL to each well, and incubate at 37°C for 1 hour. Wash the plate, add the diluted goat anti-mouse IgG to the serum sample well, 100 ul / well, and incubate at 37°C for 40 minutes. Wash the plate, add 100 μL / well of TMB colorimetric solution in the dark, add 100 ul stop solution to each well after color development for 5 minutes, and use an enzyme reader to detect the absorbance at a wavelength of 450 nm.

[0112] Mouse Press Figure 8After administration and sampling according to the immunization scheme shown in the figure, the specific IgG level of the CS-Exo / PBAE-Exo@OVA-mRNA group was significantly higher than that of the PBS group in the serum samples at 7, 21, 28, 35, and 42 days, and the serum IgG level entered a plateau phase from day 35. The above results indicate that vaccination with CS-Exo / PBAE-Exo@OVA-mRNA nanoparticles can effectively induce mice to produce a certain level of antigen-specific serum IgG.

[0113] Example 9: Study on the treatment of melanoma in mice with different cationized exosomes

[0114] Female C57BL / 6N mice, mouse skin melanoma cells (B16-F10-Luc), Exo / CS-Exo / PBAE-Exo (preparation method is the same as Example 1).

[0115] C57BL / 6N mice aged 6-8 weeks were selected and inoculated with 1×10 6 The B16 melanoma model was constructed by intraperitoneal injection of 3 mg / mouse luciferase substrate on the 3rd day. After 10 minutes, the bioluminescent signals in the lungs of mice were observed using a small animal in vivo imaging system to confirm the presence of tumors. The tumor-bearing mice were then randomly divided into: PBS control group, Exo group (EGFR), CS-Exo group (EGFR) and PBAE-Exo group (EGFR). The dosing and treatment time points of mice are shown in the figure below. The dosing concentration was 1×10 11 The weight of mice was monitored and their survival was recorded every day. A 20% decrease in weight was considered as death. The growth of tumors at specific time points was monitored using IVIS, and the bioluminescent signal in the lungs was quantitatively analyzed using Living Image software.

[0116] For metastatic lung tumors, a B16-F10-Luc cell-induced lung metastatic melanoma model and Exo / CS-Exo / PBAE-Exo(EGFR) administration were established. Compared with the PBS and Exo groups, CS-Exo / PBAE-Exo(EGFR) significantly inhibited tumor development and showed moderate weight gain, while the lung weight of CS-Exo / PBAE-Exo(EGFR)-treated mice decreased accordingly as the tumor burden decreased. Compared with the PBS and Exo groups, the size of mouse melanoma was significantly reduced after CS-Exo / PBAE-Exo(EGFR) treatment (according to quantitative analysis), indicating that CS-Exo / PBAE-Exo(EGFR) can effectively inhibit the growth of mouse melanoma (such as Fig. 9 shown).

[0117] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A cationic exosome nucleic acid delivery system, characterized in that: The exosome nucleic acid delivery system is made of nucleic acids, exosomes and cationic materials through non-covalent interactions.

2. The cationized exosome nucleic acid delivery system according to claim 1, characterized in that: The exosomes are extracted from cow milk or biological cells; the mass ratio of exosomes to nucleic acids in the composition is 0.5:1-60:

1.

3. The cationized exosome nucleic acid delivery system according to claim 2, characterized in that: The biological cells are mammalian cells or other biological cells.

4. The cationized exosome nucleic acid delivery system according to claim 1, characterized in that: The nucleic acid encodes therapeutic agents such as therapeutic proteins, vaccine antigens, antibodies, regulators, gene repair factors, etc. The nucleic acid is selected from at least one of the following: messenger RNA, self-amplifying RNA, circular RNA, small interfering RNA, short hairpin RNA and micro RNA, primary-miRNA, antisense oligonucleotide, transfer RNA, plasmid DNA, single-stranded DNA, double-stranded DNA, deoxyribozymes, ribozymes, nucleic acid aptamers, clustered regularly interspaced short palindromic repeats-related nucleic acids, single guide RNA, CRISPR-RNA, trans-activated crRNA, guide RNA, single-stranded RNA and double-stranded RNA.

5. The cationized exosome nucleic acid delivery system according to claim 1, characterized in that: The cationic material comprises at least one selected from the group consisting of chitosan, cationic lipids, poly-β-amino esters, polyethyleneimine, polypeptides / proteins rich in basic amino acids, polyarginine, polylysine, and dendrimers; The cationic lipid comprises at least one selected from the group consisting of DOTMA, DOSPA, DOTAP, ePC, DODAP, DODMA, DDAB, DSDMA, DODAC, DOAP, DMRIE, DOGS, DMOBA, HGT5000, HGT5001, HGT5002, HGT4001, HGT4002, HGT4003, HGT4005, DLin-MC3-DMA, DLin-KC2-DMA, Acuitas ALC-0315, AcuitasA9, Acuitas Lipid 2,2, Moderna Lipid H, Moderna Lipid 5. A2-Iso5-2DC18, BAME-O16B, 9A1P9, C12-200, cKK-E12, OF-Deg-Lin, 306Oi10, TT3, FTT5, Lipid319, 5A2-S C8, GenevantCL1, DLinDMA, DLenDMA, ClinDMA, CpLinDMA, imidazole cholesteryl ester, RE-1, RE-2, RE-3, GL-67, 5A2-SC8, Acuitas A9.Arcturus Lipid 2,2(8,8)4C CH3, OF-02, A18-Iso5-2DC18, BAME-O16B, A6, 98N12-5, L319, L343, 304O13, 306O138, 306O12B, 30 6-O12B, LP01, G0-C14, 7C1, Cephalin, Dlin-EG-DMA, DLinAP, DLin-MPZ, DLin-C-DAP, DLin-2-DMAP , Dlin-S-DMA, DLinDAP, DLin-MA, DLin-DAC, DLin-K-DMA, DLin-K-MPZ, DLin-K-DMA, DLin-K6-C4-DMA, DLin-K-C4-DMA, DLin-K-C3-DMA, CpLinDMA, DOcarbDAP, DLincarbDAP, C12-(2-3-2), Genevant Lipid CL1, XTC, ALNY-100, NC98-5 and their derivatives; The molar ratio of nitrogen groups in the cationic material to phosphate groups in the nucleic acid in the composition is 3.0-15.

0.

6. The method for preparing the cationized exosome nucleic acid delivery system according to any one of claims 1 to 5, characterized in that: The steps include: (1) loading nucleic acid into the isolated exosomes by electroporation; (2) modifying and compounding the cationic material with the exosomes, so that the cationic material is combined with the exosomes through electrostatic interaction or copolymerization reaction to obtain a cationic exosome nucleic acid delivery system; or (1) incubating the cationic material with the nucleic acid to form a complex through electrostatic interaction or copolymerization reaction; (2) combining the complex with exosomes by electroporation or incubation to obtain a cationic exosome nucleic acid delivery system; or (1) combining the separated exosomes with the cationic material through an incubation method to form a complex; (2) The nucleic acid is loaded into the cationic material-exosome complex by electroporation or incubation to obtain a cationic exosome nucleic acid delivery system.

7. The preparation method according to claim 6, characterized in that: The conditions of the electroporation method are as follows: the electroporation voltage is 150-400 V, the pulse length is 5-10 ms, the pulse interval is 0.5-2 s, and the pulse number is 1-5 times.

8. The preparation method according to claim 6, characterized in that: The method for modifying and compounding the cationic material with exosomes is as follows: a cationic material solution is mixed with exosomes loaded with nucleic acid, incubated for 30-60 minutes, and then centrifuged to collect the precipitate to obtain cationic exosomes.

9. The preparation method according to claim 6, characterized in that: The cationic materials are chitosan, cationic lipids, poly-β-amino esters, polyethyleneimine, polypeptides / proteins rich in basic amino acids, polyarginine, polylysine and polyurethane dendrimers; the chitosan solution is prepared with a 1M acetic acid solution, the poly-β-amino ester solution is dissolved with DMSO and then prepared with a 25mM NaAc solution, the polyethyleneimine solution, polyarginine, polylysine, protamine and polyurethane dendrimers are all dissolved with nuclease-free water, and the cationic lipids are dissolved with ethanol.

10. Use of the cationized exosome nucleic acid delivery system according to any one of claims 1 to 5 in the preparation of drugs for gene therapy, vaccine delivery, tumor therapy or cell therapy.

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