Exosome atomized preparation as well as preparation method and application thereof

Abstract

The invention discloses an exosome atomized preparation as well as a preparation method and application thereof, and belongs to the technical field of exosome technology and respiratory disease treatment. The problems that antibiotics and anti-inflammatory drugs adopted in the prior art have the defects of drug resistance, large side effects and the like in lung inflammation are solved. The exosome is derived from stem cells which are subjected to gp96 pretreatment and overexpress gp96 antigen information, the gp96 activates antigen presenting cells, the delivery efficiency of immunomodulatory factors (such as IL-10 and TGF-beta) carried by the exosome is enhanced, proinflammatory factors (such as TNF-alpha and IL-6) are inhibited, and in combination with the administration mode of an atomized preparation, through aerosol inhalation, the exosome can be used for preparing the immunomodulatory factor immunomodulatory agent. The exosome can directly reach a lung lesion area, so that the local drug concentration is improved, and the systemic side effects are reduced.

Description

Technical Field

[0001] The present invention belongs to the technical fields of exosome technology and respiratory disease treatment, and specifically relates to an exosome aerosol preparation, a preparation method thereof, and an application thereof. Background Art

[0002] Exosomes are nanoscale vesicles secreted by cells, which can carry bioactive molecules such as proteins and nucleic acids, and have important potential in intercellular communication and disease treatment. In the prior art, exosomes have been used for the treatment of osteoarthritis (such as patent CN112190592A). However, if exosomes are to be used for the treatment of lung inflammation, they still face problems such as low delivery efficiency and insufficient targeting.

[0003] Therefore, the current traditional treatment methods for lung inflammation (such as pneumonia, chronic obstructive pulmonary disease, etc.) rely on antibiotics and anti-inflammatory drugs, but have defects such as drug resistance and large side effects. Summary of the Invention

[0004] Aiming at the problems of drug resistance and large side effects in the treatment of lung inflammation with antibiotics and anti-inflammatory drugs in the prior art, the present invention provides an exosome aerosol preparation, a preparation method thereof, and an application thereof.

[0005] The technical solution adopted by the present invention is as follows:

[0006] An exosome aerosol preparation is obtained by subjecting a prepared aerosol solution to aerosolization treatment, and the aerosol solution includes exosomes and a solvent, and the exosomes are derived from stem cells that have overexpressed gp96 antigen information after being pretreated with gp96.

[0007] Preferably, the concentration of the aerosol solution is 1×10 8 -1×10 9 particles / mL.

[0008] Preferably, the aerosol solution is converted into aerosol particles of 1-5 μm through aerosolization treatment to obtain the exosome aerosol preparation.

[0009] After adopting this technical solution, the reason why the exosome aerosol solution is converted into 1-5 μm aerosol particles is mainly that particles in this particle size range can be effectively inhaled and deposited in the lungs, thereby improving the treatment efficiency and effect.

[0010] Specifically:

[0011] Lung deposition efficiency: The particle size of aerosol particles has a significant impact on their deposition efficiency in the lungs. Particles with too large a size are difficult to penetrate deep into the bronchioles of the lungs, while particles with too small a size, although able to reach finer bronchi, have strong Brownian motion and are easily exhaled, resulting in a low lung deposition rate. Therefore, a particle size range of 1-5 μm ensures that the exosome nebulization solution can reach most areas of the lungs while maintaining a relatively high lung deposition rate. Specifically: Maximizing drug efficacy: By converting the exosome nebulization solution into aerosol particles with a size of 1-5 μm, the drug efficacy can be maximized. Particles in this size range can directly reach the alveoli, and the bioactive molecules in the exosomes can directly act on target cells, regulate physiological functions, and transmit signals, thereby exerting a therapeutic effect. Safety and tolerance: Using aerosol particles with a size of 1-5 μm can reduce adverse irritation to the respiratory tract and improve the safety of treatment and the patient's tolerance. Aerosols in this size range have been widely used in the treatment research of respiratory diseases, demonstrating their safety and efficacy. In summary, converting the exosome nebulization solution into aerosol particles with a size of 1-5 μm is based on their particle size effect to ensure that the drug can efficiently reach the lungs and exert its therapeutic effect while ensuring the safety and good tolerance of the treatment.

[0012] Preferably, the solvent is physiological saline.

[0013] Preferably, the stem cells are human placenta mesenchymal stem cells, gp96 is encoded by the HSP90B1 gene, and the protein sequence number is: P14625.1.

[0014] Preferably, the exosomes are in a cup-like structure with a particle size of 30-200 nm.

[0015] A method for preparing an exosome nebulization preparation, comprising the following steps:

[0016] S1: Prepare exosomes;

[0017] S2: Mix the exosomes and the solvent to obtain a nebulization solution;

[0018] S3: Subject the nebulization solution to nebulization treatment to obtain an exosome nebulization preparation.

[0019] Preferably, the specific steps of S1 are as follows:

[0020] S101: Isolate the tissue mass containing stem cells, and culture the tissue mass containing stem cells together with the gp96 antigen complex;

[0021] S102: Change the culture medium at a set time, passage when the cell density reaches the set value, and recover the culture supernatant of the 3rd to 5th generations;

[0022] S103: Isolate exosomes from the culture supernatant to obtain the exosomes.

[0023] Furthermore, the gp96 antigen complex is a protein complex derived from human placental tissue, and the antigen information contained in the gp96 antigen complex includes ARHGAP33, ACTR5, MAML1, MUC3B, MAMDC4, LRP2, KCNT1, ITPR2, FBXO34, FLRT3, DHCR24, C1orf115.

[0024] Furthermore, the specific steps of S101 are as follows:

[0025] S1011: Obtain a fresh and intact placenta, wash the placenta, and then peel off the amniotic tissue on the placenta;

[0026] S1012: Wash the amniotic tissue and cut it into tissue blocks of 0.5 - 1.0 cm 2 in size;

[0027] S1013: Spread the tissue blocks flat in a T75 culture flask and let them adhere to the wall in a cell culture incubator for 1 - 3 h;

[0028] S1014: Add 10 - 15 mL of serum-free stem cell medium supplemented with the gp96 antigen complex and culture it together with the tissue blocks.

[0029] Furthermore, the medium in S1014 includes the following components: 450 - 475 mL of stem cell serum-free medium, 25 - 50 mL of platelet lysate with a volume fraction of 5 - 10%, 2.5 - 10 mL of penicillin-streptomycin with a concentration of 0.5 - 2%, and the final concentration of the gp96 antigen complex used in the medium is 3 μg / mL - 15 μg / mL.

[0030] Furthermore, in S102, the medium is changed every 3 - 5 days, and subculture is performed when the density of adherent cells around the tissue blocks reaches 80 - 90%. When subculturing, trypsin is used to recover the cells, and the subcultured cells are cultured at a density of 1 - 2.5×10⁴ / cm 2 in a 25 cm 2 culture flask, the medium is changed every 3 - 5 days, and when the cell growth density is 80% - 90%, subculture is performed at a ratio of 1:2 or 1:3.

[0031] Furthermore, the specific steps of S103 are as follows:

[0032] S1031: Centrifuge the supernatant obtained in S102 at 2 - 8°C and 300 - 500 g for 5 - 10 minutes, and then take the supernatant;

[0033] S1032: Centrifuge the supernatant obtained in S1031 at 2 - 8°C and 2000 - 4000 g for 10 - 20 minutes, then take the supernatant;

[0034] S1033: Centrifuge the supernatant obtained in S1032 at 2 - 8°C and 10000 - 13000 g for 30 - 60 minutes, then take the supernatant;

[0035] S1034: Perform ultrafiltration and concentration on the supernatant obtained in S1033 to obtain an ultrafiltrate;

[0036] S1035: Centrifuge the ultrafiltrate at 2 - 8°C and 5000 - 10000 g for 20 - 30 minutes to obtain a supernatant;

[0037] S1036: Filter and sterilize the supernatant obtained in S1035 with a 0.22 μm filter to obtain a concentrated solution containing the exosomes.

[0038] An application of an exosome nebulization preparation for treating pulmonary inflammation.

[0039] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0040] In the present invention, the exosomes are derived from stem cells that overexpress gp96 antigen information after gp96 pretreatment. Gp96 activates antigen - presenting cells, enhances the delivery efficiency of immune regulatory factors (such as IL - 10, TGF - β) carried by exosomes, and inhibits pro - inflammatory factors (such as TNF - α, IL - 6). Therefore, the exosomes play a role in treating pneumonia mainly through the following mechanisms: Immunomodulatory effect: Bioactive molecules such as cytokines and growth factors in exosomes can interact with immune cells to regulate the immune response and reduce tissue damage caused by excessive immune responses. Improvement of lung function: Exosomes can directly act on the lungs, improve alveolar and interstitial lung lesions, reduce pulmonary fibrosis, and improve lung function. At the same time, combined with the administration method of the nebulization preparation, through nebulization inhalation, exosomes can directly reach the lung lesion area, increase the local drug concentration, and reduce systemic side effects. Description of the Drawings

[0041] Figure 1 It is a morphological diagram of exosomes after nebulization.

[0042] Figure 2 It is a diagram characterizing the particle size and particle concentration of exosomes after nebulization.

[0043] Figure 3 It is a diagram characterizing the particle size and particle concentration of exosomes before nebulization.

[0044] Figure 4 It is a morphological diagram of exosomes before nebulization.

[0045] Figure 5 It is a graph of the detection result of biomarker expression before atomization.

[0046] Figure 6 It is a graph of the detection result of biomarker expression after atomization.

[0047] Figure 7 It is an optical microscope image of the normal control group.

[0048] Figure 8 It is an optical microscope image of the LPS model control group.

[0049] Figure 9 It is an optical microscope image of the common exosome control group.

[0050] Figure 10 It is an optical microscope image of the exosome atomization preparation group. Specific implementation manners

[0051] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative efforts shall fall within the scope of protection of the present application.

[0052] Preparation examples

[0053] Preparation of exosomes:

[0054] I. The preparation method of the gp96 antigen complex is as follows. It should be noted that the centrifuge model used for centrifugation treatment in the present invention is the LG-21M large-capacity refrigerated centrifuge (Sichuan Shuke Instrument Co., Ltd.):

[0055] 1. Pretreatment of human-derived ex vivo placental tissue

[0056] (1) If the placenta has been cryopreserved, it needs to be thawed first, and then the enveloping substances and connective tissues are removed.

[0057] (2) Preparation of lysis solution: It is composed of pre-cooled NaHCO3 and phenylmethylsulfonyl fluoride (PMSF), with a pH value of 7.0 and a molar ratio of 30:1 between the two.

[0058] (3) Remove the enveloping substances and connective tissues from the ex vivo placental tissue, cut it into pieces, add the lysis solution, and grind it into a homogenate.

[0059] 2. Centrifugation of the homogenate:

[0060] (1) Centrifuge the prepared homogenate at 4°C at a speed of 10,000 rpm for 1 hour, and then centrifuge again under the same conditions for 0.5 hour. After that, take the supernatant for standby.

[0061] 3. GP96 monoclonal antibody-Sepharose 4B affinity chromatography:

[0062] (1) Load the supernatant onto a GP96 monoclonal antibody-Sepharose 4B affinity chromatography column at a flow rate of 0.8 ml / min.

[0063] (2) Elute the chromatography column with 0.1 M Tris-HCl buffer solution containing 0.5 M NaCl, and then elute the chromatography column with glycine-HCl as the eluent.

[0064] (3) Add NaHCO3 to the collected eluate to neutralize it to pH = 7.0.

[0065] 4. HiTrap Q Sepharose ion exchange chromatography:

[0066] (1) Load the eluate neutralized to pH = 7.0 onto a HiTrap Q Sepharose ion exchange chromatography column at a flow rate of 1.5 ml / min.

[0067] (2) Wash the ion column with 0.02 M Tris-HCl buffer solution containing 0.2 M NaCl, and then gradually increase the NaCl concentration to 0.8 M for elution.

[0068] 5. Ultrafiltration concentration:

[0069] Ultrafilter and concentrate the eluate through a 30 kD membrane package, and the resulting solution is the solution containing the gp96 antigen complex.

[0070] II. Preparation of exosomes

[0071] S101: Isolate the tissue mass containing stem cells, and culture the tissue mass containing stem cells together with the prepared gp96 antigen complex as follows:

[0072] S1011: Obtain a fresh and intact pregnant woman's placenta. After obtaining the placenta, store it aseptically, and it needs to be used within 3 hours after obtaining the placenta; when using, first rinse the placenta tissue 3 times with normal saline containing 1-3% penicillin-streptomycin double antibody (in this example, normal saline containing 2% penicillin-streptomycin double antibody is used) to remove residual blood and blood clots as much as possible. After the cleaning is completed, bluntly dissect the amniotic membrane tissue on the placenta;

[0073] S1012: Wash the stripped amniotic membrane tissue 3 times with PBS buffer to remove the blood clots adhering to the surface of the amniotic membrane. Cut the rinsed amniotic membrane tissue into pieces with a sterilized ophthalmic scissors, into 0.5 cm 2 tissue pieces, and add 15 mL of serum-free stem cell medium containing gp96 antigen complex for culture;

[0074] S1013: Spread the cut tissue pieces evenly in a T75 culture flask until the bottom of the T75 culture flask is covered. Place the T75 culture flask in a 5% CO2, 37 °C cell culture incubator and let it stand for 2 h to allow the tissue pieces to adhere to the wall;

[0075] S1014: Then add 15 mL of serum-free stem cell medium supplemented with gp96 antigen complex and culture it together with the tissue pieces;

[0076] S102: During the growth of adherent cells at the edge of the tissue pieces, change the medium once every 3 days. When the density of adherent cells around the tissue pieces reaches 80%, passage the cells. Use 0.25% trypsin to recover the cells, and culture the passage cells at a density of 1X105 / cm 2 in a 25 cm 2 culture flask, change the medium every 3 days, and passage at a ratio of 1:2 when the cell growth density reaches 80%. The third to fifth generations of placental mesenchymal stem cells are used for experiments, and the culture supernatant is collected;

[0077] S103: Isolate exosomes from the culture supernatant to obtain engineered exosomes containing gp96 antigen information, specifically as follows:

[0078] S1031: Centrifuge the supernatant obtained in S102 at 4 °C and 300 g for 5 minutes, and then take the supernatant;

[0079] S1032: Centrifuge the supernatant obtained in S1031 at 4 °C and 2000 g for 10 minutes, and then take the supernatant;

[0080] S1033: Centrifuge the supernatant obtained in S1032 at 4 °C and 10000 g for 30 minutes, and then take the supernatant;

[0081] S1034: Ultrafilter and concentrate the supernatant obtained in S1033 using a 100 kd ultrafiltration tube to obtain an ultrafiltrate;

[0082] S1035: Centrifuge the ultrafiltrate to obtain a supernatant;

[0083] S1036: Filter and sterilize the supernatant obtained in S305 with a 0.22 μm filter to obtain a concentrated solution of engineered exosomes containing gp96 antigen information, and use it immediately or store it at -80 °C.

[0084] (1) Detection of cell phenotypes of placental amniotic mesenchymal stem cells:

[0085] Take the placental mesenchymal stem cells obtained from 0.5 mL of S102, add HLA-DR\CD34\CD19\CD14\CD45\CD90\CD105\CD73 flow cytometry fluorescent antibodies for labeling, incubate in the dark at 4°C for 30 min, wash twice with PBS and then resuspend in 400 μL of PBS, and detect and analyze on a flow cytometer. The detection results are shown in Table 1. As can be seen from Table 1, the proportion of positive expression markers of stem cells such as CD90\CD105\CD73 is more than 99%, and the proportion of negative expression markers such as HLA-DR\CD34\CD19\CD14\CD45 is less than 0.5%, meeting the property requirements of stem cells.

[0086] Table 1

[0087]

[0088] (2) Identification of exosomes containing gp96 antigen information:

[0089] The morphology of exosomes was detected by electron microscopy, and the detection results are as Figure 1 shown. As can be seen from Figure 1 it, the exosomes are cup-shaped structures with a clear membrane structure;

[0090] The particle size and number of exosomes were detected by NTA, and the detection results are as Figure 2 shown. As can be seen from Figure 2 it, the particle size of exosomes is 30 - 200 nm;

[0091] After the exosomes were ultrasonically disrupted, the protein concentration was detected by BCA. The detection results are shown in Table 2. As can be seen from Table 2, the protein concentration in this batch of exosomes is 1.30 mg / ml;

[0092] Table 2

[0093] Number OD562 OD562 OD562 Average concentration mg / ml Standard deviation of concentration mg / ml 240903 0.232 0.232 0.234 1.304773 0.013553

[0094] The content of stem cell factor was detected by ELISA method, and the detection results are as Figure 3 shown; detected by electrophoresis method, and the detection results are as Figure 4 shown. As can be seen from Figure 4 it, there is a 96 kDa protein band in the electrophoresis;

[0095] The gene sequence of the submitted protein was monitored, and the results are shown in Table 3. As can be seen from Table 3, this protein gene is HSP90B1, with a molecular weight of 92.468 kDa, 803 amino acid residues, and a purity of 73.53% in the total protein.

[0096] Table 3

[0097]

[0098]

[0099] The exosomes prepared above are used to prepare an exosome nebulization preparation, and the specific steps are as follows:

[0100] S1: Obtain exosomes through the above preparation examples;

[0101] S2: Mix the exosomes and normal saline to obtain an aerosol solution with a concentration of 1×10 9 particles / mL;

[0102] S3: Use an ultrasonic nebulizer or a vibrating orifice nebulizer (a vibrating orifice nebulizer in this embodiment) to convert the aerosol solution into 1-5 μm aerosol particles, ensuring the activity of exosomes and achieving deep lung deposition.

[0103] The reason for selecting a sol particle size of 1-5 μm for exosome nebulization is mainly closely related to its delivery efficiency and targeted deposition ability in the respiratory system. The setting of this particle size range is based on the following scientific basis and actual application requirements:

[0104] 1. Respiratory tract anatomy and aerosol deposition mechanism

[0105] The structure of the respiratory tract has selectivity for the deposition positions of particles with different particle sizes:

[0106] Particles > 5 μm:

[0107] Due to inertial impaction, they are mainly deposited in the upper respiratory tract (such as the nasal cavity, pharynx, and trachea) and are difficult to reach the deep lungs.

[0108] They are easily cleared by ciliary movement and mucus, resulting in a decrease in delivery efficiency.

[0109] Particles of 1-5 μm:

[0110] Can penetrate deep into the lower respiratory tract (bronchi, bronchioles, and alveoli) with the airflow and be efficiently deposited in the alveolar region through gravitational sedimentation or Brownian motion.

[0111] The alveoli have a large surface area (about 100 m 2 ) and no cilia, which is conducive to the long-term retention of exosomes and their uptake by target cells.

[0112] Particles < 1 μm:

[0113] Are easily suspended in the air due to the diffusion effect and are exhaled out of the body with exhaled air, resulting in a significant decrease in the deposition rate.

[0114] 2. Particularities of exosome delivery:

[0115] Exosomes themselves have a particle size of 30 - 200 nm and need to be encapsulated in atomized droplets to form 1 - 5 μm sol particles to prevent individual exosomes from being exhaled due to their small size.

[0116] Droplets in this particle size range can carry a sufficient number of exosomes while maintaining their structural integrity and biological activity; specifically, in terms of particle size, the average particle size of exosomes before and after atomization is 135.3 nm ( Figure 3 ) and 138.7 nm ( Figure 2 ), respectively, with no significant difference (p > 0.05); in terms of morphological structure, NTA shows that exosomes still maintain a complete vesicle structure after atomization ( Figure 1 after atomization, Figure 4 before atomization); in terms of biomarker expression, the Western blot results show that both before atomization ( Figure 5 ) and after atomization ( Figure 6 ) highly express CD63, CD9, and TSG101 proteins, indicating that atomization does not damage the membrane integrity of exosomes. It can be seen from this that forming 1 - 5 μm sol particles can maintain the structural integrity and biological activity of exosomes.

[0117] 3. Compatibility of atomization technology

[0118] There are significant differences in the particle size distributions generated by different atomization devices:

[0119] Vibrating mesh nebulizer:

[0120] Generates uniform 1 - 5 μm droplets through high-frequency vibration, with low shear force, suitable for sensitive biomolecules such as exosomes.

[0121] Ultrasonic nebulizer:

[0122] May cause local high temperature or pressure changes due to cavitation effects, and parameters need to be optimized to avoid exosome damage.

[0123] Spray drying method:

[0124] It is necessary to control the drying conditions to prevent the particle size from exceeding the ideal range (such as forming aggregated particles > 5 μm).

[0125] 4. Balance between function and activity

[0126] Avoid aggregation and degradation:

[0127] Droplets with too small a particle size (such as < 1 μm) may cause exosome aggregation due to surface tension or accelerate oxidative damage.

[0128] Droplets with too large a particle size (> 5 μm) may settle on the inner wall of the nebulizer or the upper respiratory tract due to gravity, resulting in waste of exosomes.

[0129] Delivery dose control:

[0130] Droplets of 1 - 5 μm can carry multiple exosomes (10 2 ~10 3 exosomes per single droplet), balancing the delivery efficiency and the therapeutic dose requirement.

[0131] The above - mentioned exosome nebulization preparation is used for treating pulmonary inflammation, and the usage method and treatment effect are as follows:

[0132] Usage method:

[0133] Nebulize and inhale 1 - 2 times a day, with each dose being 0.5×10 9 -1×10 9 particles, and continuously treat for 7 - 14 days to inhibit the pulmonary inflammatory response and promote tissue repair.

[0134] Experimental method:

[0135] 1. Select experimental mice: Usually select healthy, non - pregnant female C57BL / 4 mice, weighing 18 g - 20 g. Before the experiment, the mice go through a certain adaptation period to ensure their physiology.

[0136] 2. Test period: 5 days. Administer the drug every day and conduct detection on the 5th day.

[0137] 3. Experimental groups

[0138] Construct a lipopolysaccharide (LPS) - induced acute pneumonia model in mice: Infect by dripping 10 mg of LPS into the nasal cavity to establish an acute LPS - infected mouse model.

[0139] LPS model control group: LPS model group, infect by dripping 10 mg of LPS into the nasal cavity to establish an acute LPS - infected mouse model (no treatment is done after the model is established).

[0140] Ordinary exosome control group: Exosomes extracted from normal stem cells group (non - engineered exosomes, that is, exosomes derived from stem cells that have not been pretreated with gp96 and over - expressed gp96 antigen information), administer the drug by the way of reinfusion, with 1×109 particles administered each time, that is, use exosomes extracted from normal stem cells to treat the constructed lipopolysaccharide (LPS) - induced acute pneumonia model in mice.

[0141] Exosome nebulization preparation group: That is, use the exosome nebulization preparation prepared by the present invention to treat the constructed lipopolysaccharide (LPS) - induced acute pneumonia model in mice. This group inhales exosomes containing 1×109 particles every day.

[0142] Normal control group: Normal mice, and this group of mice is given a nebulization preparation prepared with an equal amount of normal saline every day.

[0143] For each of the above groups, there were 5 mice, and the test result on the fifth day was the average of the test results of the 5 mice.

[0144] Therapeutic effect:

[0145] Under an optical microscope, the changes in the inflammatory lesions of the lung tissues of mice in the normal control group, LPS model control group, ordinary exosome control group, and exosome aerosol preparation group could be clearly observed. The results are as Figures 7 - 10 shown. As Figure 7 shown, the lung tissue structure of the mice in the normal control group was clear and complete, the distance between alveoli was not expanded and the structure was easy to identify, there was no obvious inflammatory fluid secretion in the upper respiratory tract and alveolar cavity, and no inflammatory cell infiltration was seen. As Figure 8 shown, the lung tissue structure of the mice in the LPS model control group was damaged to varying degrees, with relatively severe diffuse pulmonary edema, the alveolar wall and the distance between alveoli increased and thickened, the capillaries dilated and the alveolar cavity narrowed, and a large number of inflammatory cells infiltrated in the lung tissue and bleeding of different sizes occurred. As Figure 9 shown, compared with the LPS model control group, the lung tissue damage of the mice in the ordinary exosome control group was significantly improved after administration, the symptoms of diffuse pulmonary edema were reduced, the alveolar wall and the distance between alveoli were reduced, and the inflammatory cell infiltration and bleeding were greatly reduced. As Figure 10 shown, the degree of pathological improvement of the lung of the infected mice in the exosome aerosol preparation group was better than that in the ordinary exosome control group. Specifically Figure 9 compared with Figure 10 , Figure 10 the alveolar structure was clearer and more obvious, and the symptoms of diffuse pulmonary edema in the alveoli were reduced, the alveolar wall and the distance between alveoli were significantly reduced, and the inflammatory cell infiltration and bleeding were reduced a lot.

[0146] Determination of serum cytokine content

[0147] The contents of IL-6, IFN-γ and TNF-α in the serum of mice were measured separately by ELISA method. At the same time, the OD value was measured strictly according to the requirements in the ELISA method instruction manual, and then the contents of IL-6, IFN-γ and TNF-α in the serum of each group of mice were calculated.

[0148] From the comparison of the data in Table 4, on the 5th day after modeling, there were also differences in varying degrees in the serum cytokines IL-6, IFN-γ, and TNF-α of each group of mice. After successful infection of the mice, it was measured on the 5th day after administration that the data of TNF-α in the LPS model control group of mouse serum was much higher than that of the normal control group (*P<0.01), indicating that LPS could induce a large release of TNF-α in the mouse body. At the same time, the level of TNF-α in the exosome aerosol preparation group was significantly lower than that in the LPS model control group (*P<0.01), indicating that the engineered exosome aerosol had a certain inhibitory effect on the large release of TNF-α induced by LPS in the mouse body. Moreover, the level of TNF-α in the exosome aerosol preparation group was significantly lower than that in the ordinary exosome control group, indicating that the exosome aerosol preparation group had a better inhibitory effect. On the 5th day after successful infection of the mice and after administration, it was observed in the measurement results that the data of serum II-6 in the model group of mice was also much higher than that of the control group (*P<0.01), indicating that LPS could induce a large release of inflammatory factors in the mouse body; at the same time, the data in the exosome aerosol preparation group was significantly smaller than that of the mice in the LPS model control group (*P<0.01), and was lower than that in the ordinary exosome control group, indicating that the aerosolized engineered exosomes could better inhibit the inflammatory response caused by LPS.

[0149] Table 4

[0150]

[0151] The data in Table 4 showed that after the 5th day of modeling, the level of IFN-γ in the LPS model control group was significantly lower than that in the normal control group (*P<0.01), indicating that LPS might affect the immune regulation of infected mice, inhibit their immune response, and cause functional damage to the mouse body. Compared with the LPS model control group, the data of the exosome aerosol preparation group and the ordinary exosome control group showed that the exosome aerosol preparation group could slightly increase the expression level of IFN-γ, suggesting that aerosol inhalation of engineered exosomes might counteract the symptoms caused by LPS by improving the defense of the immune barrier in the mouse body and increasing the expression of immune factors.

[0152] The above-described embodiments only represent the specific implementation manners of the present application, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation to the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several deformations and improvements can still be made, and these all belong to the protection scope of the present application.

Claims

1. An exosome aerosol preparation, characterized in that: The prepared atomized liquid is obtained by atomizing the atomized liquid, and the atomized liquid includes exosomes and a solvent, and the exosomes are derived from stem cells that have been pre-treated with gp96 and over-expressed gp96 antigen information.

2. The exosome aerosol preparation according to claim 1, characterized in that: The concentration of the atomized liquid is 1×10 8 -1×10 9 Particles / ml.

3. The exosome aerosol preparation according to claim 1, characterized in that: The atomized liquid is converted into aerosol particles of 1-5 μm through atomization treatment to obtain the exosome atomized preparation.

4. An exosome aerosol preparation according to any one of claims 1 to 3, characterized in that: The solvent is physiological saline.

5. An exosome aerosol preparation according to any one of claims 1 to 3, characterized in that: The stem cells are human placental mesenchymal stem cells, gp96 is encoded by the HSP90B1 gene, and the protein sequence number is: P14625.

1.

6. An exosome aerosol preparation according to any one of claims 1 to 3, characterized in that: The exosomes are cup-like structures with a particle size of 30-200 nm.

7. A method for preparing the exosome aerosol preparation according to any one of claims 1 to 6, characterized in that: The following steps are involved: S1: Exosomes are prepared; S2: mixing the exosomes and the solvent to obtain a nebulized liquid; S3: The nebulized liquid is subjected to nebulization treatment to obtain an exosome nebulized preparation.

8. The method for preparing an exosome aerosol preparation according to claim 7, characterized in that: The specific steps of S1 are as follows: S101: separating a tissue block containing stem cells, and culturing the tissue block containing the stem cells with a complex containing a gp96 antigen; S102: changing the medium at the set time, performing subculture when the cell density reaches the set value, and collecting the culture supernatant of the 3rd to 5th generations; S103: separating exosomes from the culture supernatant to obtain the exosomes.

9. The method for preparing an exosome aerosol preparation according to claim 8, characterized in that: The gp96 antigen complex is a protein complex derived from human placental tissue. The antigen information contained in the gp96 antigen complex includes ARHGAP33, ACTR5, MAML1, MUC3B, MAMDC4, LRP2, KCNT1, ITPR2, FBXO34, FLRT3, DHCR24, and C1orf115; The specific steps of S103 are as follows: S1031: centrifuge the supernatant obtained in S102 at 2-8°C and 300-500g for 5-10 minutes, and take the supernatant; S1032: centrifuge the supernatant obtained in S1031 at 2000-4000 g for 10-20 minutes at 2-8°C, and collect the supernatant; S1033: centrifuging the supernatant obtained in S1032 at 2 to 8°C and 10,000 to 13,000 g for 30 to 60 minutes, and taking the supernatant; S1034: performing ultrafiltration and concentration treatment on the supernatant obtained in S1033 to obtain an ultrafiltrate; S1035: centrifuging the ultrafiltrate at 2 to 8°C and 5,000 to 10,000 g for 20 to 30 minutes to obtain a supernatant; S1036: The supernatant obtained in S1035 is subjected to 0.22-0.40 μm filtration sterilization treatment to obtain a concentrated solution containing the exosomes.

10. A use of the exosome aerosol preparation according to any one of claims 1 to 6, characterized in that: Used to treat lung inflammation.

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