A nebulized inhalation composition based on stem cell extracellular vesicles, its preparation method and application

Abstract

The present invention belongs to the technical field of stem cell therapeutic drugs, and specifically relates to an aerosol inhalation composition based on extracellular vesicles of stem cells, as well as its preparation method and application. The composition is delivered to the lungs by aerosol inhalation. The synergistic effect between the components can reduce the levels of inflammatory factors IL-1β and TNF-α, and repair respiratory lung damage. It has the advantages of being non-invasive, highly safe, and having significant effects. It is a new treatment method in the field of regenerative medicine and provides an innovative treatment solution for respiratory diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cell therapeutic drugs, and specifically relates to an aerosol inhalation composition based on stem cell extracellular vesicles, and a preparation method and application thereof. Background Art

[0002] Respiratory diseases are common and frequently occurring diseases. The main lesions occur in the trachea, bronchi, lungs and chest cavity. Patients with mild lesions often experience coughing, chest pain and affected breathing. Severe cases can cause difficulty breathing, hypoxia, and even respiratory failure, leading to death.

[0003] The use of traditional drugs, such as glucocorticoids and antibiotics, can alleviate symptoms, but they have defects such as systemic toxicity, drug resistance, and inability to promote tissue regeneration. Surgical treatments, such as lung transplantation or mechanical ventilation, are only suitable for critically ill patients and are invasive, expensive, and have low survival rates. The direct transplantation of stem cells has problems with immune rejection, tumorigenicity, and low survival rates, and it is difficult to precisely regulate the paracrine effects of stem cells. Extracellular vesicles (EVs) are key mediators of the paracrine effects of stem cells, and can regulate inflammation and promote repair by delivering active ingredients such as mRNA, miRNA, and proteins. However, existing EVs preparations have the following problems:

[0004] (1) A single EV component is difficult to fully regulate the complex lung injury microenvironment;

[0005] (2) Conventional intravenous or local injection results in poor lung targeting and low bioavailability;

[0006] (3) Single EVs are not stable enough and are easily cleared by the respiratory mucus barrier. Summary of the Invention

[0007] To solve the problems raised by the background technology, the present invention provides an aerosol inhalation composition based on stem cell extracellular vesicles, and a preparation method and application thereof.

[0008] The technical solutions of the present invention are as follows:

[0009] The present invention provides an aerosol inhalation composition based on stem cell extracellular vesicles, the composition comprising:

[0010] Stem cell extracellular vesicles, KL4 peptide, N-acetylcysteine, recombinant human DNase;

[0011] The mass ratios of stem cell extracellular vesicles to KL4 peptide, N-acetylcysteine, and recombinant human DNA enzyme were 1:0.01-0.05, 1:0.01-0.05, and 1:0.01-0.02, respectively.

[0012] Furthermore, the mass ratios of stem cell extracellular vesicles to KL4 peptide, N-acetylcysteine, and recombinant human DNA enzyme were 1:0.025, 1:0.025, and 1:0.015, respectively.

[0013] The particle size of stem cell extracellular vesicles is 30-200 nm, the average particle size is 127.5 nm, the peak particle size is 120 nm, and the particle concentration is 10 billion particles / mL.

[0014] The stem cell extracellular vesicles are derived from mesenchymal stem cells, specifically:

[0015] (1) Mesenchymal stem cells were isolated from human umbilical cord, expanded under hypoxic conditions, cultured in serum-free medium until the fifth generation, and the cell supernatant was collected and stored at 4°C for later use;

[0016] (2) The supernatant was centrifuged at 3000 × g for 20 min and then filtered through a 0.22 μm filter membrane;

[0017] The filtrate was centrifuged at 100,000 × g at 4°C for 2 h, the supernatant was discarded, and the precipitate was resuspended in phosphate-buffered saline to obtain the crude product;

[0018] (3) The crude product was further purified by sucrose gradient centrifugation;

[0019] High-purity stem cell extracellular vesicles are separated using size exclusion chromatography, and the target peak is collected to obtain purified stem cell extracellular vesicles.

[0020] The composition further comprises a lyoprotectant.

[0021] The composition is used in the form of an aerosol, and the concentration of the composition in the aerosol is 500-2000 ug / mL.

[0022] The present invention also provides a method for preparing the stem cell extracellular vesicle-based aerosol inhalation composition, comprising:

[0023] The purified stem cell extracellular vesicles were incubated with N-acetylcysteine, and then KL4 peptide was coupled to the stem cell extracellular vesicles, and recombinant human DNA enzyme was added and mixed.

[0024] Specifically:

[0025] (1) The purified stem cell extracellular vesicles were electroporated with N-acetylcysteine at 37°C for 1 h, with a voltage of 200 V and a pulse time of 10 ms.

[0026] (2) After incubation, the extracellular vesicles of the stem cells were mixed with the KL4 peptide in a phosphate buffered saline solution, and then 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h and centrifuged to obtain the coupled product.

[0027] (3) The coupled product is mixed with recombinant human DNA enzyme.

[0028] The present invention also provides a use of the stem cell extracellular vesicle-based aerosol inhalation composition in the preparation of medicines or preparations for respiratory diseases.

[0029] Furthermore, the aerosol inhalation composition based on stem cell extracellular vesicles is used in the preparation of respiratory disease drugs or preparations for reducing the levels of inflammatory factors IL-1β and TNF-α.

[0030] Beneficial effects: The aerosol inhalation composition based on stem cell extracellular vesicles provided by the present invention is delivered to the lungs by aerosol inhalation. The synergistic effect between the components can reduce the levels of inflammatory factors IL-1β and TNF-α, and repair respiratory lung damage. It has the advantages of being non-invasive, highly safe, and having significant effects. It is a new treatment method in the field of regenerative medicine and provides an innovative treatment plan for respiratory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 (a) is a transmission electron microscopy photograph of the purified stem cell extracellular vesicles, (b) is the particle size distribution, and (c) is the marker protein detection result.

[0032] Figure 2 (a) shows the expression of IL-1β in lung tissue of different groups in animal experiments, and (b) shows the expression of TNF-α.

[0033] Figure 3 Photos of lung tissue from different groups of animal experiments were taken.

[0034] Figure 4 HE pathological images of lung tissues in different groups of animal experiments. DETAILED DESCRIPTION

[0035] The following examples are intended to illustrate the present invention rather than to further limit the present invention.

[0036] The present invention provides an aerosol inhalation composition based on stem cell extracellular vesicles, the composition comprising:

[0037] Stem cell extracellular vesicles, KL4 peptide, N-acetylcysteine, and recombinant human DNase.

[0038] Among them, the mass ratios of stem cell extracellular vesicles to KL4 peptide, N-acetylcysteine, and recombinant human DNA enzyme are 1:0.01-0.05, 1:0.01-0.05, and 1:0.01-0.02, respectively.

[0039] Preferably, the mass ratios of stem cell extracellular vesicles to KL4 peptide, N-acetylcysteine, and recombinant human DNA enzyme are 1:0.025, 1:0.025, and 1:0.015, respectively.

[0040] The particle size of stem cell extracellular vesicles is 30-200 nm, the average particle size is 127.5 nm, the peak particle size is 120 nm, and the particle concentration is 10 billion particles / mL.

[0041] The composition further comprises a lyoprotectant.

[0042] The composition is used in the form of an aerosol, and the concentration of the composition in the aerosol is 500-2000 ug / ml.

[0043] The present invention also provides a method for preparing the stem cell extracellular vesicle-based aerosol inhalation composition, comprising:

[0044] The purified stem cell extracellular vesicles were incubated with N-acetylcysteine, and then KL4 peptide was coupled to the stem cell extracellular vesicles, and recombinant human DNA enzyme was added and mixed.

[0045] The purification process of stem cell extracellular vesicles (EVs) is as follows:

[0046] (1) Stem cell culture

[0047] Isolate human umbilical cord mesenchymal stem cells (hUC-MSCs) and expand them under hypoxic (5% O2) conditions in serum-free medium (e.g., DMEM / F12) until passage 5. Collect the cell supernatant and store at 4°C until use.

[0048] (2) Extracellular vesicle enrichment

[0049] The supernatant was centrifuged at 3000 × g for 20 min to remove cell debris and then filtered through a 0.22 μm filter membrane to remove large particles.

[0050] EVs were pelleted using an ultracentrifuge (100,000 × g, 4°C, 2 h), the supernatant was discarded, and the pellet was resuspended in PBS.

[0051] (3) Extracellular vesicle purification

[0052] The crude EVs were further purified by sucrose gradient centrifugation (density 1.15-1.18 g / mL) to remove impurities.

[0053] High-purity EVs were separated using size exclusion chromatography (SEC, Sepharose CL-4B column), and the target peak was collected.

[0054] The morphology of the purified stem cell extracellular vesicles was observed using transmission electron microscopy. Figure 1 As shown in (a).

[0055] The ZetaView Nanoparticle Tracking Analyzer (NTA) was used to detect the particle size distribution of purified stem cell extracellular vesicles. Figure 1 As shown in (b), it is mainly 30-200 nm.

[0056] Western Blot was used to detect the purified stem cell extracellular vesicle marker proteins (CD63, TSG101, HSP70). Figure 1 As shown in (c).

[0057] During the preparation process, KL4 peptide is coupled to stem cell extracellular vesicles using EDC / NHS chemical cross-linking. EDC is 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, and NHS is N-hydroxysuccinimide.

[0058] Specifically:

[0059] (1) The purified stem cell extracellular vesicles were electroporated with N-acetylcysteine at 37°C for 1 h, with a voltage of 200 V and a pulse time of 10 ms.

[0060] (2) After incubation, the extracellular vesicles of the stem cells were mixed with the KL4 peptide in a phosphate buffered saline solution, and then 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h and centrifuged to obtain the coupled product.

[0061] (3) The coupled product is mixed with recombinant human DNA enzyme.

[0062] The preparation process also includes adding a freeze-drying protective agent to the mixture, pre-freezing and then vacuum drying.

[0063] The present invention also provides an aerosol inhalation composition based on stem cell extracellular vesicles and the use of the aerosol inhalation composition prepared by the preparation method in the preparation of medicines or preparations for respiratory diseases.

[0064] Specifically, the respiratory diseases include acute pneumonia, lung injury, pulmonary fibrosis or chronic obstructive pulmonary disease.

[0065] Preferably, the stem cell extracellular vesicle-based aerosol inhalation composition is used in the preparation of respiratory disease drugs or preparations for reducing the levels of inflammatory factors IL-1β and TNF-α.

[0066] The aerosol inhalation composition based on extracellular vesicles of stem cells provided by the present invention is delivered to the lungs by aerosol inhalation. The synergistic effect between the components can significantly repair lung damage in the respiratory system. It has the advantages of being non-invasive, highly safe, and having significant effects. It is a new treatment method in the field of regenerative medicine and provides an innovative treatment solution for respiratory diseases.

[0067] Example 1

[0068] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into extracellular vesicles of stem cells.

[0069] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0070] The stem cell extracellular vesicles and KL4 peptide were mixed in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0071] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.015;

[0072] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0073] Example 2

[0074] Compared with Example 1, the difference is that the content of N-acetylcysteine is changed, as follows:

[0075] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.01 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into the extracellular vesicles of stem cells.

[0076] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0077] The stem cell extracellular vesicles and KL4 peptide were mixed in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0078] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.015;

[0079] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0080] Example 3

[0081] Compared with Example 1, the difference is that the content of N-acetylcysteine is changed, as follows:

[0082] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.05 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into the extracellular vesicles of stem cells.

[0083] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0084] The stem cell extracellular vesicles and KL4 peptide were mixed in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0085] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.015;

[0086] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0087] Example 4

[0088] Compared with Example 1, the difference is that the content of KL4 peptide is changed, as follows:

[0089] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into extracellular vesicles of stem cells.

[0090] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0091] The stem cell extracellular vesicles were mixed with KL4 peptide in PBS at a mass ratio of 1:0.01, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0092] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.015;

[0093] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0094] Example 5

[0095] Compared with Example 1, the difference is that the content of KL4 peptide is changed, as follows:

[0096] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into extracellular vesicles of stem cells.

[0097] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0098] The stem cell extracellular vesicles were mixed with KL4 peptide in PBS at a mass ratio of 1:0.05, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0099] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.015;

[0100] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0101] Example 6

[0102] Compared with Example 1, the difference is that the content of recombinant human DNA enzyme is changed, as follows:

[0103] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into extracellular vesicles of stem cells.

[0104] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0105] The stem cell extracellular vesicles and KL4 peptide were mixed in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0106] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.01;

[0107] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0108] Example 7

[0109] Compared with Example 1, the difference is that the content of recombinant human DNA enzyme is changed, as follows:

[0110] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into extracellular vesicles of stem cells.

[0111] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0112] The stem cell extracellular vesicles and KL4 peptide were mixed in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0113] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.02;

[0114] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0115] Comparative Example 1

[0116] Compared with Example 1, the difference is that KL4 peptide is not added, specifically as follows:

[0117] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into extracellular vesicles of stem cells.

[0118] (2) The incubated product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.015;

[0119] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0120] Comparative Example 2

[0121] Compared with Example 1, the difference is that N-acetylcysteine is not added, specifically as follows:

[0122] (1) KL4 peptide was coupled to purified stem cell extracellular vesicles using EDC / NHS chemical cross-linking method, specifically:

[0123] The purified stem cell extracellular vesicles were mixed with KL4 peptide in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0124] (2) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.015;

[0125] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0126] Comparative Example 3

[0127] Compared with Example 1, the difference is that no recombinant human DNA enzyme is added, specifically as follows:

[0128] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into extracellular vesicles of stem cells.

[0129] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0130] The stem cell extracellular vesicles and KL4 peptide were mixed in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0131] (3) Add lyophilization protective agent (5% trehalose + 1% PVP) to the coupled product, pre-freeze and vacuum dry to prepare lyophilized powder.

[0132] Comparative Example 4

[0133] Compared with Example 1, the difference is that KL4 peptide and N-acetylcysteine are not added, specifically as follows:

[0134] (1) Mix the purified stem cell extracellular vesicles with recombinant human DNA enzyme at a mass ratio of 1:0.015;

[0135] (2) Add lyophilization protective agent (5% trehalose + 1% PVP), pre-freeze and vacuum dry to make lyophilized powder.

[0136] Comparative Example 5

[0137] Compared with Example 1, the difference is that KL4 peptide and recombinant human DNA enzyme are not added, specifically as follows:

[0138] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into extracellular vesicles of stem cells.

[0139] (2) Add lyophilization protective agent (5% trehalose + 1% PVP), pre-freeze and vacuum dry to make lyophilized powder.

[0140] Comparative Example 6

[0141] Compared with Example 1, the difference is that N-acetylcysteine and recombinant human DNA enzyme are not added, specifically as follows:

[0142] (1) The purified stem cell extracellular vesicles were mixed with KL4 peptide in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0143] (2) Add lyophilization protective agent (5% trehalose + 1% PVP), pre-freeze and vacuum dry to make lyophilized powder.

[0144] Comparative Example 7

[0145] Compared with Example 1, the difference is that KL4 peptide, N-acetylcysteine and recombinant human DNA enzyme are not added, specifically as follows:

[0146] Lyophilization protectant (5% trehalose + 1% PVP) was added to the purified extracellular vesicles of stem cells, and the cells were pre-frozen and vacuum-dried to prepare lyophilized powder.

[0147] Comparative Example 8

[0148] Compared with Example 1, the difference is that the content of N-acetylcysteine is changed (below the limit value), as follows:

[0149] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.005 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into the extracellular vesicles of stem cells.

[0150] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0151] The stem cell extracellular vesicles and KL4 peptide were mixed in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0152] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.015;

[0153] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0154] Comparative Example 9

[0155] Compared with Example 1, the difference is that the content of N-acetylcysteine is changed (higher than the limit value), as follows:

[0156] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.1 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into the extracellular vesicles of stem cells.

[0157] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0158] The stem cell extracellular vesicles and KL4 peptide were mixed in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0159] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.015;

[0160] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0161] Comparative Example 10

[0162] Compared with Example 1, the difference is that the KL4 peptide content is changed (below the limit value), as follows:

[0163] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into extracellular vesicles of stem cells.

[0164] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0165] The stem cell extracellular vesicles were mixed with KL4 peptide in PBS at a mass ratio of 1:0.005, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0166] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.015;

[0167] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0168] Comparative Example 11

[0169] Compared with Example 1, the difference is that the KL4 peptide content is changed (higher than the limit value), as follows:

[0170] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into extracellular vesicles of stem cells.

[0171] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0172] The stem cell extracellular vesicles and KL4 peptide were mixed in PBS at a mass ratio of 1:0.1, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0173] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.015;

[0174] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0175] Comparative Example 12

[0176] Compared with Example 1, the difference is that the recombinant human DNA enzyme (below the limit value) is as follows:

[0177] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into extracellular vesicles of stem cells.

[0178] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0179] The stem cell extracellular vesicles and KL4 peptide were mixed in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0180] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.005;

[0181] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0182] Comparative Example 13

[0183] Compared with Example 1, the difference is that the recombinant human DNA enzyme (higher than the limit value) is as follows:

[0184] (1) The purified extracellular vesicles of stem cells were incubated with N-acetylcysteine at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of N-acetylcysteine into extracellular vesicles of stem cells.

[0185] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0186] The stem cell extracellular vesicles and KL4 peptide were mixed in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0187] (3) The coupled product was mixed with recombinant human DNA enzyme at a mass ratio of 1:0.03;

[0188] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0189] Comparative Example 14

[0190] Compared with Example 1, the difference is that the recombinant human DNA enzyme is replaced by RNA enzyme, and N-acetylcysteine is replaced by vitamin C, as follows:

[0191] (1) The purified extracellular vesicles of stem cells were incubated with vitamin C at a mass ratio of 1:0.025 at 37°C for 1 h, and electroporation (voltage 200 V, pulse time 10 ms) was used to promote the loading of vitamin C into extracellular vesicles of stem cells.

[0192] (2) After incubation, the KL4 peptide was coupled to the stem cell extracellular vesicles using the EDC / NHS chemical cross-linking method, specifically:

[0193] The stem cell extracellular vesicles and KL4 peptide were mixed in PBS at a mass ratio of 1:0.025, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h, and the unreacted KL4 peptide was removed by centrifugation.

[0194] (3) The coupled product was mixed with RNA enzyme at a mass ratio of 1:0.015;

[0195] A freeze-dried protective agent (5% trehalose + 1% PVP) was added, and the mixture was pre-frozen and then vacuum-dried to prepare a freeze-dried powder.

[0196] Experimental analysis

[0197] 1. In vitro cell experiments

[0198] Cell line: RAW264.7 mouse monocyte-macrophage cell line (routinely cultured in DMEM medium containing 10% FBS).

[0199] Culture medium: DMEM high glucose medium + 10% fetal bovine serum (FBS) + 1% penicillin / streptomycin (double antibody).

[0200] LPS solution: Dissolve LPS (from Escherichia coli O111:B4, Sigma) in sterile PBS or culture medium to a stock solution (e.g., 1 mg / mL). Aliquot and store at -20°C. The working concentration is 100 ng / mL.

[0201] Cell plating: RAW264.7 mouse mononuclear macrophages were counted and plated at 1×10 6 Plate cells / well (6-well plate, 2 mL medium per well) and allow cells to adhere for at least 6 hours (or overnight) before stimulation to ensure a stable state.

[0202] LPS stimulation: Aspirate the old culture medium and add fresh culture medium containing LPS (100 ng / mL LPS). Stimulation lasts for 24 hours (37°C, 5% CO2). Collect cell culture fluid samples for ELISA testing of inflammatory cytokines IL-1β and TNF-α.

[0203] The lyophilized powders of Examples 1-7 and Comparative Examples 1-14 were reconstituted with physiological saline to a concentration of 100 μg / ml of extracellular vesicles of stem cells and applied to an in vitro cell inflammation model. The groups were as follows:

[0204] Model group: LPS (lipopolysaccharide)-induced macrophages;

[0205] Example 1: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Example 1 to dissolve the liquid;

[0206] Example 2: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Example 2 to dissolve in liquid;

[0207] Example 3: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Example 3 to dissolve the liquid;

[0208] Example 4: LPS-induced macrophages co-incubated with the reconstituted lyophilized powder of Example 4 dissolved in liquid;

[0209] Example 5: LPS-induced macrophages co-incubated with the reconstituted lyophilized powder of Example 5 dissolved in liquid;

[0210] Example 6: LPS-induced macrophages co-incubated with the reconstituted lyophilized powder of Example 6 dissolved in liquid;

[0211] Example 7: LPS-induced macrophages co-incubated with the reconstituted lyophilized powder of Example 7 dissolved in liquid;

[0212] Comparative Example 1: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Comparative Example 1;

[0213] Comparative Example 2: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Comparative Example 2;

[0214] Comparative Example 3: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Comparative Example 3;

[0215] Comparative Example 4: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Comparative Example 4;

[0216] Comparative Example 5: LPS-induced macrophages co-incubated with the reconstituted lyophilized powder of Comparative Example 5;

[0217] Comparative Example 6: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Comparative Example 6;

[0218] Comparative Example 7: LPS-induced macrophages co-incubated with the reconstituted lyophilized powder of Comparative Example 7;

[0219] Comparative Example 8: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Comparative Example 8;

[0220] Comparative Example 9: LPS-induced macrophages co-incubated with the reconstituted lyophilized powder of Comparative Example 9;

[0221] Comparative Example 10: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Comparative Example 10;

[0222] Comparative Example 11: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Comparative Example 11;

[0223] Comparative Example 12: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Comparative Example 12;

[0224] Comparative Example 13: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Comparative Example 13;

[0225] Comparative Example 14: LPS-induced macrophages were co-incubated with the reconstituted lyophilized powder of Comparative Example 14;

[0226] The LPS concentration for macrophage induction was 500 ng / ml, and the stimulation duration was 24 hours. The incubation time for all groups was 24 hours. The concentration of the composition in each group was 100 μg / ml of stem cell extracellular vesicles. After the experiment, ELISA kits were used to measure IL-1β and TNF-α secretion. The test results are shown in Tables 1 and 2.

[0227] Table 1 IL-1β expression levels in different groups

[0228]

[0229] Table 2 TNF-α expression levels in different groups

[0230]

[0231] As shown in Tables 1 and 2, compared with the model group and the comparative example 1-14 groups, the compositions of Examples 1-7 of the present application can significantly reduce the levels of cellular inflammatory factors IL-1β and TNF-α (P<0.01).

[0232] 2. Animal Experiments

[0233] The lyophilized powder of Example 1 was reconstituted with physiological saline and prepared into 5 ml of aerosol solution for standby use, thereby obtaining sprays of EVs compositions of three concentrations (500 ug / ml, 1000 ug / ml, and 2000 ug / ml) for subsequent experiments.

[0234] Twenty-five healthy 6-week-old male C57BL / 6 mice weighing 20-25 g were selected and divided into five groups.

[0235] After one week of adaptive feeding, the mouse acute pneumonia model was established. The specific procedures are as follows:

[0236] Prepare 2 ml of 1 g / L LPS solution on a clean bench;

[0237] Mice were anesthetized with an intraperitoneal injection of the anesthetic sodium pentobarbital (1%, 50 mg / kg, IP);

[0238] The mouse was placed supine on a flat surface at a 45° angle. The tongue was pulled with forceps, and 70 μl of LPS solution was pipetted onto the posterior pharyngeal wall. The nose was immediately pinched. After 20 seconds, the tongue and nose were released, and the mouse was placed in a cage to recover naturally. All models were successfully established using the above procedures.

[0239] Experimental groups:

[0240] NS: normal mice instilled with normal saline;

[0241] LPS+NS: mice with acute pneumonia model induced by LPS (lipopolysaccharide) instillation were treated with normal saline nebulization;

[0242] LPS+EVs500ug / ml: LPS-induced acute pneumonia model mice were treated with a nebulized EVs composition at a concentration of 500ug / ml;

[0243] LPS+EVs1000ug / ml: LPS-induced acute pneumonia model mice were treated with aerosolization of EVs composition at a concentration of 1000ug / ml;

[0244] LPS+EVs2000ug / ml: Mice with acute pneumonia model induced by LPS were treated with aerosolization of EVs composition at a concentration of 2000ug / ml.

[0245] Experiment according to group:

[0246] LPS enters the lungs through the respiratory tract and causes inflammation. Atomized inhalation of sprays of EVs compositions of different concentrations was used to treat acute pneumonia in mice to observe the therapeutic effect. Specifically, two hours after the mice inhaled LPS, they were treated with atomized inhalation of the EVs composition, and the inhalation amount was controlled according to each concentration. Each mouse in each group was atomized 1 ml each time, and the atomization treatment was performed once a day (atomization process parameters: using a vibrating mesh nebulizer (MMAD 2.5μm), controlling the atomization pressure to 0.5-1.0 MPa), for ten minutes each time, for 7 consecutive days, and the condition of the mice was observed after the atomization treatment. After atomization inhalation treatment, the activity state of the mice in the LPS+EVs composition group was significantly improved, the brightness of the fur was significantly restored, and the overall condition was significantly restored.

[0247] After 7 consecutive days of aerosol treatment, mice were anesthetized with sodium pentobarbital (1%, 50 mg / kg, IP), and their eyeballs were removed for blood collection. After standing at 4°C for 2 h, the blood was centrifuged at 4°C and 5000 rpm for 10 min to extract serum, which was then frozen at -80°C. ELISA kits were used to detect the levels of inflammatory markers IL-1β and TNF-α (see Figure 2 ), and the mouse lung tissue was removed and photographed (see Figure 3 ), lung tissue was fixed with paraformaldehyde and HE pathological examination was performed (see Figure 4 ).

[0248] Depend on Figure 2 It can be seen that compared with the LPS+NS group, the levels of inflammatory factors IL-1β and TNF-α in mice were reduced after using the EVs composition of the present application.

[0249] Depend on Figure 3 It can be seen that compared with the LPS+NS group, after using the EVs composition of the present application, the EVs composition of the present invention can significantly inhibit acute pneumonia and improve lung damage caused by acute pneumonia.

[0250] Depend on Figure 4 The results of HE pathological analysis of lung tissue show that the use of the EVs composition of the present application for aerosol inhalation can effectively relieve acute pneumonia, and has a significant effect on regenerating and repairing respiratory tract damage and inflammation, with good therapeutic effects.

Claims

1. A composition for aerosol inhalation based on extracellular vesicles of stem cells, characterized in that: The composition comprises: Stem cell extracellular vesicles, KL4 peptide, N-acetylcysteine, recombinant human DNase; The mass ratios of stem cell extracellular vesicles to KL4 peptide, N-acetylcysteine, and recombinant human DNA enzyme were 1:0.01-0.05, 1:0.01-0.05, and 1:0.01-0.02, respectively.

2. The aerosol inhalation composition based on stem cell extracellular vesicles according to claim 1, characterized in that The mass ratios of stem cell extracellular vesicles to KL4 peptide, N-acetylcysteine, and recombinant human DNA enzyme were 1:0.025, 1:0.025, and 1:0.015, respectively.

3. The aerosol inhalation composition based on stem cell extracellular vesicles according to claim 1, characterized in that The particle size of stem cell extracellular vesicles is 30-200 nm, the average particle size is 127.5 nm, the peak particle size is 120 nm, and the particle concentration is 10 billion particles / mL.

4. The aerosol inhalation composition based on stem cell extracellular vesicles according to claim 1, characterized in that The stem cell extracellular vesicles are derived from mesenchymal stem cells, specifically: (1) Mesenchymal stem cells were isolated from human umbilical cord, expanded under hypoxic conditions, cultured in serum-free medium until the fifth generation, and the cell supernatant was collected and stored at 4°C for later use; (2) The supernatant was centrifuged at 3000 × g for 20 min and then filtered through a 0.22 μm filter membrane; The filtrate was centrifuged at 100,000 × g at 4°C for 2 h, the supernatant was discarded, and the precipitate was resuspended in phosphate-buffered saline to obtain the crude product; (3) The crude product was further purified by sucrose gradient centrifugation; High-purity stem cell extracellular vesicles are separated using size exclusion chromatography, and the target peak is collected to obtain purified stem cell extracellular vesicles.

5. The aerosol inhalation composition based on stem cell extracellular vesicles according to claim 1, characterized in that The composition further comprises a lyoprotectant.

6. The aerosol inhalation composition based on stem cell extracellular vesicles according to claim 1, characterized in that The composition is used in the form of an aerosol, and the concentration of the composition in the aerosol is 500-2000 ug / mL.

7. A method for preparing the aerosol inhalation composition based on stem cell extracellular vesicles according to claim 1, characterized in that: include: The purified stem cell extracellular vesicles were incubated with N-acetylcysteine, and then KL4 peptide was coupled to the stem cell extracellular vesicles, and recombinant human DNA enzyme was added and mixed.

8. The method for preparing the aerosol inhalation composition based on stem cell extracellular vesicles according to claim 7, characterized in that: Specifically: (1) The purified stem cell extracellular vesicles were electroporated with N-acetylcysteine at 37°C for 1 h, with a voltage of 200 V and a pulse time of 10 ms. (2) After incubation, the extracellular vesicles of the stem cells were mixed with the KL4 peptide in a phosphate buffered saline solution, and then 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide were added. The mixture was reacted at room temperature for 2 h and centrifuged to obtain the coupled product. (3) The coupled product is mixed with recombinant human DNA enzyme.

9. Use of the stem cell extracellular vesicle-based aerosol inhalation composition according to claim 1 or the aerosol inhalation composition prepared by the preparation method according to claim 7 in the preparation of a drug or preparation for acute pneumonia.

Images