CN117064877A

CN117064877A - Exosome composition containing baicalein or derivative thereof and application of exosome composition in treatment of rhinitis

Info

Publication number: CN117064877A
Application number: CN202311227597.9A
Authority: CN
Inventors: 张建民; 龙立; 陈雪
Original assignee: Guodian Beijing Medicine Technology Co ltd
Current assignee: Guodian Beijing Medicine Technology Co ltd
Priority date: 2023-09-22
Filing date: 2023-09-22
Publication date: 2023-11-17
Anticipated expiration: 2043-09-22
Also published as: CN117064877B

Abstract

The application relates to the use of an exosome composition comprising baicalein or a derivative thereof for the treatment of nasal-sinusitis, both of which can achieve a synergistic therapeutic effect, wherein the baicalein or a derivative thereof in the exosome composition is partially or fully loaded in the exosome, and the nasal-sinusitis, in particular eosinophilic chronic sinusitis. The exosomes are used for loading baicalein or derivatives thereof, so that the solubility of the baicalein or derivatives thereof can be improved, the in vivo circulation time of the baicalein or derivatives thereof can be prolonged, the therapeutic activity of medicaments can be reserved, and the permeability of the baicalein or derivatives thereof in nasal mucosa can be enhanced.

Description

Exosome composition containing baicalein or derivative thereof and application of exosome composition in treatment of rhinitis

Technical Field

The application relates to the technical field of medicines, in particular to an exosome composition containing baicalein or a derivative thereof and application of the exosome composition, in particular to application in treating rhinitis.

Background

The anatomy of the nasal cavity and the sinuses are interrelated, so clinically, although rhinitis and sinusitis are distinguished, they are often interrelated, often with concomitant morbidity. After the European allergy and clinical immunology of 2007 were issued according to the evidence-based diagnosis and treatment guidelines for sinusitis and nasal polyps, rhinitis and sinusitis were commonly referred to as rhinosinusitis (including nasal polyps), which refers to inflammation of the nasal cavity and sinuses, and which is characterized clinically by two or more of nasal and sinus inflammation, wherein at least one of nasal obstruction/congestion and/or nasal leakage (front and rear nasal leakage) should be present, with or without facial pressure pain/swelling sensation, with or without hyposmia/loss; nasal endoscopy finds nasal polyps and/or mucopurulent secretions from the middle nasal passages and/or swelling or blockage of the mucous membranes of the middle nasal passages; nasal CT examination revealed mucosal alterations in the osteomeatal complex or sinus. The symptoms are classified into acute and chronic according to the time that they continue to occur. Acute rhinosinusitis refers to symptoms that are less than 12 weeks in duration and can be completely alleviated, while chronic rhinosinusitis refers to symptoms that are more than 12 weeks in duration and that are not completely alleviated or may be exacerbated. The 2020 edition European sinusitis and nasal polyp opinion book divides chronic sinusitis into type 1 (Th 1 type), type 2 (Th 2 type) and type 3 (Th 17 type) according to different immune inflammation types, wherein type 2 is also commonly called eosinophilic chronic sinusitis in clinic, type 1 and type 3 are also called non-type 2, and non-eosinophilic chronic sinusitis.

Although the current conventional clinical treatment for chronic sinusitis is still to use glucocorticoid locally as the first choice of treatment, and no more accurate symptomatic treatment is performed for different subtypes, because the chronic sinusitis subtypes have different immune responses and inflammatory characteristics, such as type 2 inflammation and non-type 2 inflammation, the therapeutic effects of these treatments are greatly different, and many patients with insensitive responses receive treatments with poor actual therapeutic effects.

Baicalein is one of flavonoid compounds with highest content in radix Scutellariae, and baicalin is glycoside compound formed by combining baicalein and one molecule of glucuronic acid, and both are simultaneously present in radix Scutellariae. Baicalein and baicalin have similar pharmacological activities including anti-inflammatory, antioxidant, free radical scavenging and the like, and have wide biotransformation in vivo.

The induced pluripotent stem cells are similar to the mesenchymal stem cells and have strong differentiation and regeneration capacity, but the induced pluripotent stem cells have the characteristic of unlimited proliferation and can differentiate into various cell types required by various organs and tissues of human bodies. The induced pluripotent stem cells are not directly derived from human organs and tissues, but are stem cells obtained by gene editing, so that social ethical problems do not exist in terms of sources, and the induced pluripotent stem cells are easier to obtain than mesenchymal stem cells. Therefore, the induced pluripotent stem cell exosome is easier to produce and prepare in a large scale and the source is easier than the mesenchymal stem cell exosome.

Exosomes are extracellular vesicles secreted by cells, having unique properties of low immunogenicity, innate stability, high delivery efficiency and ability to cross the blood brain barrier, as well as a number of unique advantages of tissue repair, anti-inflammatory, etc. Exosomes have also gained extensive attention and research as nanocarriers for drugs.

Disclosure of Invention

The present application relates to an exosome composition comprising baicalein or a derivative thereof and its use in the treatment of nasal-sinusitis, in particular eosinophilic chronic sinusitis. The exosome is used to encapsulate baicalein or its derivative, which can improve the solubility of baicalein or its derivative, prolong the circulation time in vivo, retain the therapeutic activity of the medicine, and enhance the permeability of baicalein or its derivative in nasal mucosa. The exosome composition comprising baicalein or its derivative of the present application has better therapeutic effect on treating nasal-sinusitis than that of exosome alone, baicalein or its derivative alone, and independent exosome and independent baicalein or its derivative are administered sequentially or simultaneously.

In a first aspect of the present application, there is provided an exosome composition comprising baicalein or a derivative thereof, comprising as an active ingredient a combination of baicalein or a derivative thereof and an exosome. In one embodiment of the present application, only a portion of the baicalein or a derivative thereof in the exosome composition is loaded in the exosome. In one embodiment of the present application, the baicalein or a derivative thereof in the exosome composition is entirely loaded in the exosome. In the present application, "loaded in the exosome" includes at least that baicalein or a derivative thereof permeates into the exosome and that baicalein or a derivative thereof associates with the exosome in two existing forms.

In some embodiments of the present application, the loading concentration of baicalein or a derivative thereof is 2 to 500 μg/mL in terms of baicalein, for example, 2.1. Mu.g/mL, 2.5. Mu.g/mL, 3. Mu.g/mL, 4. Mu.g/mL, 5. Mu.g/mL, 6. Mu.g/mL, 7. Mu.g/mL, 8. Mu.g/mL, 9. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 30. Mu.g/mL, 40. Mu.g/mL, 50. Mu.g/mL, 60. Mu.g/mL, 70. Mu.g/mL, 80. Mu.g/mL, 90. Mu.g/mL, 100. Mu.g/mL, 110. Mu.g/mL, 120. Mu.g/mL, 130. Mu.g/mL, 140. Mu.g/mL, 150. Mu.g/mL, 160. Mu.g/mL, 170. Mu.g/mL, 180. Mu.g/mL, 190. Mu.g/mL, 200. Mu.g/mL, 210. Mu.g/mL, and 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 495 μg/mL, and the like. In the present application, "loading concentration" refers to the concentration of baicalein or a derivative thereof loaded in an exosome composition.

In some embodiments of the application, the amount of baicalein or derivative thereof loaded in the exosomes in the exosome composition is 5% -100%, e.g., 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, etc., based on the total amount of baicalein or derivative thereof in the exosome composition.

A second aspect of the present application provides the use of the exosome composition comprising baicalein or a derivative thereof in the manufacture of a medicament for treating nasal-sinusitis;

providing: the use of exosomes and baicalein or derivatives thereof in the manufacture of a medicament for the treatment of nasal-sinusitis.

In a third aspect, the present application provides a method for preparing the exosome composition comprising baicalein or a derivative thereof.

In some embodiments of the application, the exosome composition comprising baicalein or a derivative thereof is prepared by a method of incubation, sonication, freeze thawing, extrusion, or saponin treatment.

In one embodiment of the application, the method of incubating comprises: baicalein or its derivative is prepared into solution with proper concentration, and incubated with exosome. In one embodiment of the application, appropriate concentrations of baicalein are co-incubated with exosomes; preferably, the incubation system comprises, in an incubation system, the final concentration of baicalein is 5-500 μg/mL (e.g., 6 μg/mL, 7 μg/mL, 8 μg/mL, 9 μg/mL, 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 45 μg/mL, 50 μg/mL, 55 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, 100 μg/mL, 110 μg/mL, 120 μg/mL, 130 μg/mL, 140 μg/mL, 150 μg/mL, 160 μg/mL, 170 μg/mL, 180 μg/mL, 190 μg/mL, 200 μg/mL, 210 μg/mL) 220. Mu.g/mL, 230. Mu.g/mL, 240. Mu.g/mL, 250. Mu.g/mL, 260. Mu.g/mL, 270. Mu.g/mL, 280. Mu.g/mL, 290. Mu.g/mL, 300. Mu.g/mL, 310. Mu.g/mL, 320. Mu.g/mL, 330. Mu.g/mL, 340. Mu.g/mL, 350. Mu.g/mL, 360. Mu.g/mL, 370. Mu.g/mL, 380. Mu.g/mL, 390. Mu.g/mL, 400. Mu.g/mL, 410. Mu.g/mL, 420. Mu.g/mL, 430. Mu.g/mL, 440. Mu.g/mL, 450. Mu.g/mL, 460. Mu.g/mL, 470. Mu.g/mL, 480. Mu.g/mL, 490. Mu.g/mL, 495. Mu.g/mL, etc.), for example 50-300 mug/mL, the final concentration of the exosomes is 2-3000 μg/mL (e.g., 3 μg/mL, 4 μg/mL, 5 μg/mL, 6 μg/mL, 7 μg/mL, 8 μg/mL, 9 μg/mL, 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 45 μg/mL, 50 μg/mL, 55 μg/mL, 60 μg/mL, 70 μg/mL, 80 μg/mL, 90 μg/mL, 100 μg/mL, 110 μg/mL, 120 μg/mL, 130 μg/mL, 140 μg/mL, 150 μg/mL, 160 μg/mL, 170 μg/mL, 180 μg/mL, 190 μg/mL, 200 μg/mL, 210 μg/mL, 220 μg/mL, 230. Mu.g/mL, 240. Mu.g/mL, 250. Mu.g/mL, 260. Mu.g/mL, 270. Mu.g/mL, 280. Mu.g/mL, 290. Mu.g/mL, 300. Mu.g/mL, 350. Mu.g/mL, 400. Mu.g/mL, 450. Mu.g/mL, 500. Mu.g/mL, 550. Mu.g/mL, 600. Mu.g/mL, 650. Mu.g/mL, 700. Mu.g/mL, 750. Mu.g/mL, 800. Mu.g/mL, 850. Mu.g/mL, 900. Mu.g/mL, 950. Mu.g/mL, 1000. Mu.g/mL, 1500. Mu.g/mL, 2000. Mu.g/mL, 2500. Mu.g/mL, 2600. Mu.g/mL, 2700. Mu.g/mL, 2800. Mu.g/mL, 2900. Mu.g/mL, 2950. Mu.g/mL, 2960. Mu.g/mL, 2970 μg/mL, 2980 μg/mL, 2990 μg/mL, 2995 μg/mL, etc.), for example, 50-200 μg/mL; more preferably, the incubation conditions are from 0℃to 5℃for 1 to 25 hours, or from 20℃to 35℃for 1 to 4 hours.

In one embodiment of the application, a method of ultrasound comprises: mixing the exosome suspension with baicalein or its derivative solution, and ultrasonically treating the mixed solution with ultrasonic instrument to obtain exosome composition containing baicalein or its derivative.

In one embodiment of the application, a method of freeze thawing comprises: mixing exosome suspension and baicalein or its derivative solution, and freezing in refrigerator. And thawing, and freezing again, and repeating the steps for several times to obtain the exosome composition containing baicalein or the derivative thereof.

In a fourth aspect, the application provides a pharmaceutical composition comprising an exosome composition according to the first aspect of the application, or further comprising a pharmaceutically acceptable excipient. In one embodiment of the present application, only a portion of the baicalein or a derivative thereof in the pharmaceutical composition is loaded in the exosomes. In one embodiment of the present application, the baicalein or a derivative thereof in the pharmaceutical composition is entirely loaded in the exosomes. According to the present application, the pharmaceutical composition may take various formulation forms known in the art, such as: liquid formulations (including but not limited to suspensions), solid formulations (including but not limited to tablets, powder injection, etc.), semi-solid formulations (including but not limited to ointments).

In one embodiment of the present application, the pharmaceutical composition is a suspension, and may further contain a buffer in addition to the exosome composition comprising baicalein or its derivative, which may be a buffer commonly used in the art, including but not limited to PBS buffer, DPBS buffer, tris buffer, hepes buffer, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, and the like.

In one embodiment of the present application, the pharmaceutical composition is a lyophilized formulation, and may further contain a cryoprotectant, which may be a cryoprotectant commonly used in the art, including, but not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, etc., in addition to the exosome composition comprising baicalein or a derivative thereof. For lyophilized formulations, water for injection or buffer may be added to reconstitute the suspension prior to use.

According to the present application, the content of exosomes and the content of baicalein or its derivatives (calculated as baicalein) in the pharmaceutical composition may independently be 0.1 to 500 μg respectively, for example 0.2. Mu.g, 0.3. Mu.g, 0.4. Mu.g, 0.5. Mu.g, 0.6. Mu.g, 0.7. Mu.g, 0.8. Mu.g, 0.9. Mu.g, 1. Mu.g, 2. Mu.g, 3. Mu.g, 4. Mu.g, 5. Mu.g, 6. Mu.g, 7. Mu.g, 8. Mu.g, 9. Mu.g, 10. Mu.g, 20. Mu.g, 30. Mu.g, 40. Mu.g, 50. Mu.g, 60. Mu.g, 70. Mu.g, 80. Mu.g, 90. Mu.g, 100. Mu.g, 110. Mu.g, 120. Mu.g, 130. Mu.g, 140. Mu.g, 150. Mu.g, 160. Mu.g, 170. Mu.g 180 μg, 190 μg, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 360 μg, 370 μg, 380 μg, 390 μg, 400 μg, 410 μg, 420 μg, 430 μg, 440 μg, 450 μg, 460 μg, 470 μg, 480 μg, 490 μg, 495 μg, etc. When the pharmaceutical composition is a suspension, the concentration of the exosomes and the concentration of baicalein or its derivatives calculated as baicalein may independently be 2-500 μg/mL respectively, for example, 2.1. Mu.g/mL, 2.5. Mu.g/mL, 3. Mu.g/mL, 4. Mu.g/mL, 5. Mu.g/mL, 6. Mu.g/mL, 7. Mu.g/mL, 8. Mu.g/mL, 9. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 30. Mu.g/mL, 40. Mu.g/mL, 50. Mu.g/mL, 60. Mu.g/mL, 70. Mu.g/mL, 80. Mu.g/mL, 90. Mu.g/mL, 100. Mu.g/mL, 110. Mu.g/mL, 120. Mu.g/mL, 130. Mu.g/mL, 140. Mu.g/mL, 150. Mu.g/mL, 160. Mu.g/mL, 170. Mu.g/mL, 180. Mu.g/mL, 190. Mu.g/mL, 200. Mu.g/mL, 60. Mu.g/mL 210. Mu.g/mL, 220. Mu.g/mL, 230. Mu.g/mL, 240. Mu.g/mL, 250. Mu.g/mL, 260. Mu.g/mL, 270. Mu.g/mL, 280. Mu.g/mL, 290. Mu.g/mL, 300. Mu.g/mL, 310. Mu.g/mL, 320. Mu.g/mL, 330. Mu.g/mL, 340. Mu.g/mL, 350. Mu.g/mL, 360. Mu.g/mL, 370. Mu.g/mL, 380. Mu.g/mL, 390. Mu.g/mL, 400. Mu.g/mL, 410. Mu.g/mL, 420. Mu.g/mL, 430. Mu.g/mL, 440. Mu.g/mL, 450. Mu.g/mL, 460. Mu.g/mL, 470. Mu.g/mL, 480. Mu.g/mL, 490. Mu.g/mL, 495. Mu.g/mL, etc.

A fifth aspect of the application provides the use of the pharmaceutical composition for the manufacture of a medicament for the treatment of nasal-sinusitis.

The administration of the exosome composition comprising baicalein or a derivative thereof according to the present application may be performed using various administration modes known in the art, such as oral administration, nasal administration, subcutaneous injection, external administration, intravenous injection, etc. In a preferred embodiment, nasal administration is employed. The dosage of exosome and the dosage of baicalein or its derivative calculated as baicalein are respectively 0.1-500 μg independently, for example, 0.2. Mu.g, 0.3. Mu.g, 0.4. Mu.g, 0.5. Mu.g, 0.6. Mu.g, 0.7. Mu.g, 0.8. Mu.g, 0.9. Mu.g, 1. Mu.g, 2. Mu.g, 3. Mu.g, 4. Mu.g, 5. Mu.g, 6. Mu.g, 7. Mu.g, 8. Mu.g, 9. Mu.g, 10. Mu.g, 20. Mu.g, 30. Mu.g, 40. Mu.g, 50. Mu.g, 60. Mu.g, 70. Mu.g, 80. Mu.g, 90. Mu.g, 100. Mu.g, 110. Mu.g, 120. Mu.g, 130. Mu.g, 140. Mu.g, 150. Mu.g, 160. Mu.g, 170. Mu.g, 180. Mu.g 190 μg, 200 μg, 210 μg, 220 μg, 230 μg, 240 μg, 250 μg, 260 μg, 270 μg, 280 μg, 290 μg, 300 μg, 310 μg, 320 μg, 330 μg, 340 μg, 350 μg, 360 μg, 370 μg, 380 μg, 390 μg, 400 μg, 410 μg, 420 μg, 430 μg, 440 μg, 450 μg, 460 μg, 470 μg, 480 μg, 490 μg, 495 μg, etc.

In a sixth aspect, the present application provides a method of treating a disease, comprising administering to a patient in need thereof an effective amount of an exosome composition comprising baicalein or a derivative thereof according to the present application, or a pharmaceutical composition according to the present application.

According to the present application, the baicalein or a derivative thereof is selected from one or more of the group consisting of: baicalin, pharmaceutically acceptable salt of baicalin, baicalin and pharmaceutically acceptable salt of baicalin.

According to the application, the pharmaceutically acceptable salt of baicalin can be selected from the salts of organic acid and inorganic acid respectively. The inorganic acid may be hydrochloric acid, sulfuric acid, phosphoric acid, biphosphoric acid, hydrobromic acid or nitric acid. The organic acid may be acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid or oxalic acid.

In some embodiments of the application, the nasal-sinusitis is chronic nasal-sinusitis, preferably eosinophilic chronic sinusitis. The eosinophilic chronic sinusitis is accompanied or not accompanied by nasal polyps.

According to the application, the treatment comprises reducing or alleviating or inhibiting inflammation of the mucous membranes of the nasal cavities and/or sinuses, repairing the mucous membranes of the nasal cavities and/or sinuses, inhibiting the thickness of the mucous membranes of the nasal cavities and/or sinuses.

According to the application, the exosomes may be exosomes of human or mammalian cells.

Such mammals include, but are not limited to, ungulates such as cows, sheep, horses, pigs, rodents such as rabbits, rats, mice, guinea pigs, primates such as monkeys, gorillas, cats, dogs, and the like. In one embodiment of the application, the exosomes are exosomes of human cells.

The cells may be normal somatic cells or/and stem cells including, but not limited to, vascular endothelial cells, dendritic cells, T cells (e.g., foxp3 ⁺ Treg cells), neurons, oligodendrocytes, astrocytes, microglia, embryonic stem cells, adult stem cells (e.g., hematopoietic stem cells, neural stem cells, mesenchymal stem cells derived from bone marrow, umbilical cord, fat, umbilical cord blood, amniotic membrane, (placenta) chorion, dental pulp, thymus, synovium, etc.), and the like.

Terminally differentiated somatic cells can also be reprogrammed to pluripotent stem cells by introducing specific genes (e.g., oct4, sox2, klf4, c-Myc, etc.) using the induced pluripotent stem cell (Induced pluripotent stem cells, iPSCs) technique to obtain the exosomes derived from iPSCs, which are abbreviated as "iPS-EV" in the present application. The iPSS technology can be used for preparing the proprietary induced pluripotent stem cells by using the somatic cells of the patient, and the exosomes are obtained by using the proprietary induced pluripotent stem cells of the patient, so that the possibility of immune rejection can be greatly reduced when the iPSS technology is used for the patient.

In some embodiments of the application, the exosomes are multipotent stem cell-derived exosomes. In some embodiments of the application, the pluripotent stem cell exosomes are induced pluripotent stem cell-derived exosomes, mesenchymal stem cell-derived exosomes, or embryonic pluripotent stem cell-derived exosomes. In some embodiments of the application, the pluripotent stem cell exosomes are human pluripotent stem cell exosomes, e.g., human induced pluripotent stem cell-derived exosomes, human mesenchymal stem cell-derived exosomes, or human embryonic pluripotent stem cell-derived exosomes.

The exosomes can be prepared by culturing corresponding cells in vitro and separating the cells from a cell culture solution in a cell autocrine manner. The culturing of each cell type may be performed using methods and media known in the art. For example: the human skin fibroblast medium may be dmem+15% fbs+neaa, the umbilical cord mesenchymal stem cell medium may be dmem+10% fbs, and the neuron cell medium may be b27+neurobasal+glutamax.

In order to increase the production of exosomes in the cell culture, the content of chemicals in its medium can be adjusted according to the characteristics of the cells being cultured, for example: the chemical substances necessary for the cell growth in the culture medium are reserved, and the unnecessary chemical substances or the content thereof for the cell growth in the culture are removed or reduced, so that the cell growth is in a starvation state, and the effect of improving the yield of exosomes in the culture is achieved. For example, in the case of culturing stem cells to obtain stem cell exosomes, L-ascorbic acid or a salt thereof, selenium or a salt thereof, insulin or the like may be added to a basic medium of stem cells, such as DMEM, F12 or DMEM/F12, to induce stem cells to secrete exosomes in a large amount. An exosome secretion inducer as described, for example, in chinese patent application CN112920991a, which is incorporated herein in its entirety.

In the present application, the exosomes may be isolated from the cell culture broth using various exosome isolation methods in the art.

The exosomes can precipitate different impurity components in the sample under different centrifugal forces through differential ultracentrifugation to obtain purer exosomes, and are the most commonly used exosome separation and concentration methods at present. The method can improve product purity and reduce exosome aggregation by ultrafiltration with 0.22 μm or 0.45 μm pore size filter membrane.

Exosomes are used as a subgroup of extracellular vesicles, the diameters of the exosomes are concentrated to 30-150 nm, and the exosomes can be separated based on particle size, and the methods comprise ultrafiltration, size exclusion chromatography, hydrostatic filtration dialysis, asymmetric field flow separation and the like.

Commercial exosome extraction kits such as Exo-spin can also be used ^TM 、ExoQuick ^TM 、Invitrogen ^TM And the like to separate exosomes.

The extracted exosomes may be suspended in a buffer, e.g. phosphate buffer, and stored frozen at-80-4 ℃. It is also possible to preserve in lyophilized form by lyophilization with the addition of cryoprotectants such as trehalose and the like.

Definition of terms:

and/or is to be taken as a specific disclosure of each of two specified features or components with or without the other. Thus, the term "and/or" as used in phrases such as "a and/or B" is intended to include "a and B", "a or B", "a" (alone) and "B" (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following aspects: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

"comprising" and "including" have the same meaning and are intended to be open and allow for the inclusion of additional elements or steps but not required. When the terms "comprising" or "including" are used herein, the terms "consisting of" and/or "consisting essentially of … …" are therefore also included and disclosed.

By "pharmaceutically acceptable excipient" is meant any ingredient other than exosomes described herein, and having substantially non-toxic and non-inflammatory properties in the patient, including, but not limited to, any and all solvents, dispersion media or other liquid carriers, dispersion or suspension aids, diluents, isotonic agents, preservatives, colorants, sweeteners or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmolality adjusting agents, pH adjusting agents, buffers, chelating agents, cryoprotectants and/or fillers, as appropriate for the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the compositions are known in the art. Exemplary antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzethonium chloride, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, and the like, and combinations thereof. Exemplary preservatives include, but are not limited to, vitamin a, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, and the like, and combinations thereof. Exemplary buffers to control pH may include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, and the like, and/or combinations thereof. Exemplary cryoprotectants include, but are not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, and the like, and combinations thereof.

Drawings

Fig. 1: standard graph of baicalein concentration versus absorbance.

Fig. 2: h & E staining section of mouse sinus tissue.

Fig. 3: statistical plot of nasal mucosa thickness of olfactory epithelium of nasal sinus tissue of mice.

Detailed Description

The application is further described below with reference to examples. It should be noted that the examples should not be construed as limiting the scope of the present application, and those skilled in the art will understand that any modifications and variations based on the present application are within the scope of the present application.

Conventional reagents used in the following examples are all commercially available. The biological experiments are all routine in the art, and can be performed according to the instruction of the corresponding experimental manual or the instruction of the kit.

Example 1 preparation of exosomes derived from human-induced pluripotent stem cells

(1) Cell culture: iPSC cells (commodity of national (Beijing) medical science and technology Co., ltd.) were subjected to expansion culture (conventional expansion culture using Essential 8TM Medium kit (available from Thermo Fisher, cat# A1517001)) to a logarithmic growth phase and were well conditioned, and small cell masses (3-10 cells) were digested according to the technical manual of the Versene Solution kit (available from Thermo Fisher Co., cat# 15040066), and then spread on a 10cm cell culture dish coated with vitronectin (available from Peprotech Co., recombinant Human Vitronectin, cat# 140-09), and the coverage rate after adherence reached 60% -70%, 37 ℃,95% air and 5% CO ₂ The culture was carried out overnight under an atmosphere. When the iPSC has good adherence, the liquid is changed into GDEV culture medium (commodity of national dictionary (Beijing) medical science and technology Co., ltd.), the cell culture supernatant is collected after 24 hours, the previous operation is repeated, and a batch of cells can be collected for 3 times.

(2) Isolation of exosomes: and (3) centrifuging the cell culture supernatant obtained by the induced culture cells in the step (1) at a room temperature of 3000g for 15min, and removing dead cells and cell fragments. Collecting supernatant, and filtering with 0.22 μm filter; the filtrate was transferred to a Centricon Plus-70 (100 kDa) ultrafiltration tube and concentrated, and centrifuged at 3000g for 30min at room temperature. The concentrate was prepared with DPBS (dolby phosphate buffer) according to a ratio of about 1:100 was diluted and then concentrated again using the same apparatus to give an exosome solution.

Example 2 preparation of exosomes derived from human umbilical mesenchymal Stem cells

(1) Sample processing: fresh 10cm umbilical cord tissue was harvested using normal saline containing penicillin-streptomycin solution (diabody) (100×) (available from Gibco)Cleaning, cutting with an ophthalmic scissors to 1mm ² Transferring to a cell culture flask, adding DMEM/F12 medium (all available from Gibco) containing 10% by volume of serum replacement, penicillin-streptomycin, at 5% CO ₂ And (3) standing and culturing in an incubator at 37 ℃.

(2) Cell culture: in the primary culture process of the step (1), liquid is changed for the first time after 1 week, and then liquid is changed for 1 time after 3-4 days. When the cells grow to 80% -90% fusion, the cells are digested and passaged by trypsin/EDTA digestive fluid (purchased from Gibco), and transferred into a new culture dish for culture.

(3) Isolation of exosomes: when the cell concentration grows to 80-90%, the human mesenchymal stem cells are replaced by serum-free medium (MSCXF Medium, available from Biological Industries), continuing culturing, changing the liquid after 24 hours to collect the cell culture supernatant, repeating the previous step, and collecting 3 times of a batch of cells; the 3-fold harvested cell culture supernatant was centrifuged for 20min (3000 g/min), cells and cell debris were removed, and the exosomes were obtained by centrifugation using a Centricon Plus-70 (100 kDa) ultrafiltration tube (purchased from Sigma) at 3000g/min for 30min at room temperature.

Example 3 preparation of exosome composition comprising baicalein

1.1 baicalein powder 5mg was weighed, and 1.15mL of DMSO was added to obtain a DMSO stock solution at a concentration of 16 mM. Subpackaging into 10 tubes, and storing in a refrigerator at-80deg.C in dark place.

1.2 take out Density 10 in refrigerator at-80 deg.C ¹¹ personal/mL (density 10) ¹¹ The concentration of individual/mL of the exosome was about 1 mg/mL), 100. Mu.L of iPSC exosome was thawed at room temperature, 290. Mu.L of PBS solution was added, then 10. Mu.L of 16mM baicalein DMSO solution in step 1.1 was added, at which time the concentration of baicalein in the system was 0.4mM (about 108. Mu.g/mL), the system contained 2.5% DMSO, and the mixture was shaken and mixed, left at room temperature in the absence of light for 2 hours, or left at 4℃overnight in a refrigerator.

1.3 transfer the solution to a 100K ultrafiltration tube, set to 14000 Xg for 5min.

1.4 into an ultrafiltration tube, 400. Mu.L of PBS containing 2.5% DMSO was added, and the centrifugation was carried out for 5min at 14000 Xg. This washing step was repeated four times and the last wash was collected.

1.5 400. Mu.L of PBS solution without DMSO was added, and the mixture was centrifuged at 14000 Xg for 5min.

1.6 carefully sucking the drug-loaded exosomes in the ultrafiltration tube by using a 20-mu L pipette, and transferring the exosomes into a 0.2mL centrifuge tube to obtain the drug-loaded exosomes. Can be stored in-80deg.C refrigerator.

Example 4 detection of the drug load of baicalein in an exosome composition comprising baicalein

1.1. Preparing baicalein standard solution.

1) 10. Mu.L of 16mM baicalein in DMSO was added to the solution to obtain 8mM baicalein in DMSO.

2) 10. Mu.L of the 8mM baicalein solution was added to 390. Mu.L of PBS solution to obtain a standard solution having a concentration of 200. Mu.M.

3) 200. Mu.L of the 200. Mu.M standard solution was added with 200. Mu.L of a PBS solution containing 2.5% DMSO to obtain a 100. Mu.M standard solution.

4) According to the dilution method, standard solutions with concentrations of 50. Mu.M, 25. Mu.M, 10. Mu.M, 5. Mu.M, 1. Mu.M, and 0. Mu.M were obtained by dilution in this order.

1.2 method for measuring ultraviolet visible absorbance photometry by enzyme-labeled instrument

1) Preparing a standard curve solution.

Taking 4.5 mu L of each standard solution prepared in 1.1, placing into a 0.2mL centrifuge tube, and respectively adding into a particle number of 10 ¹¹ 3 μl of iPSC exosomes per mL. The gradient concentration of baicalein in the system was 120. Mu.M, 60. Mu.M, 30. Mu.M, 15. Mu.M, 6. Mu.M, 3. Mu.M, 0.6. Mu.M, and 0. Mu.M (blank). All contained 1.5% DMSO.

2) Diluting the sample solution

The exosome composition containing baicalein prepared in example 3 was taken as a sample, 3. Mu.L was taken, and 4.5. Mu.L of PBS solution containing 2.5% DMSO was added. The DMSO content at this time was 1.5%.

3) The standard curve solution and the diluted sample solution (3 parallel wells) were each applied in 6. Mu.L on a microporous ELISA plate. Then, in an M5 enzyme-labeled instrument, 338nm is set as absorption wavelength, and OD value is measured. Absorbance values for 3 parallel wells of the sample solution are shown in table 1.

4) In the origin8.0 software, the concentration of the standard curve solution is taken as the abscissa, the OD value of the blank absorption is subtracted as the ordinate, and linear fitting is carried out, and a linear equation and R are obtained ² Values, as shown in figure 1.

5) And calculating the concentration of baicalein in the diluted sample according to a standard curve equation. The concentration multiplied by 2.5 is the loading concentration of baicalein in the baicalein-containing exosome composition prepared in example 3.

TABLE 1 absorbance values for 3 parallel wells of sample solution after dilution

Parallel holes	Parallel holes 1	Parallel holes 2	Parallel holes 3
				Absorbance value (a.u.)	0.0320	0.0306	0.0317

The absorbance values of 3 parallel wells of the diluted sample solution were brought into the standard curve y=0.0016x, giving a concentration of baicalein in the diluted sample of 20 μm, 19.13 μm, 19.81 μm, and an average concentration of 19.65 μm. The loading concentration of baicalein in the baicalein-containing exosome composition prepared in example 3 was 19.65 μm×2.5=49.12 μm.

The loading ratio of baicalein is%49.12 μΜ/400 μΜ×100% = 12.28%.

By adopting the method, the applicant changes the incubation conditions, different final exosome concentrations and different baicalein concentrations in an incubation system, and the loading concentration result of baicalein in the exosome composition containing baicalein is measured as follows:

(1) PBS system containing 2.5X10 ⁸ Individual exosomes/mL, 200 μm baicalein

(2) PBS system containing 3.0X10 ¹¹ Individual exosomes/mL, 1mM baicalein

Temperature (temperature)	Incubation time	Concentration of baicalein loaded in exosomes	Drug loading rate
				RT	3h	420μM	42.0％

Example 5 treatment of eosinophilic sinusitis with an exosome composition comprising baicalein

Modeling protocol for murine eosinophilic sinusitis: an appropriate amount of papain is weighed and dissolved in PBS solution to prepare papain solution with the final concentration of 1 mg/ml. The mice were alternately dropped with 5. Mu.L of noses in the morning and evening, respectively, per day, and were subjected to molding, 20. Mu.L per day.

Grouping scheme: mice were nasally induced with papain (days 0-2 and 7-10) to model eosinophilic rhinitis and then divided into PBS group, PC (i.e., positive control) group, EXO-1 (i.e., exosome) group, baicalein (hereinafter abbreviated as BC) group, and BC+EXO-1 (i.e., exosome composition containing Baicalein) group, respectively.

Additional normal mice were induced with normal saline (days 0-2 and 7-10) as a Sham normal control group.

On days 7-13 after modeling, the Sram group, the PBS group, the PC group, the EXO-1 group, the BC group and the BC+EXO-1 group were respectively used for nasal drip of PBS, inner Shu Na, iPSC exosomes, baicalein and an exosome composition containing baicalein, intervention was performed in the morning and evening, nasal sinus tissues of mice were taken on day 14, paraffin embedding was performed, and the nasal mucosa conditions were observed by hematoxylin & eosin staining.

Grouping arrangement and drug formulation instructions

1) sham group: normal control group, using normal saline to make model, PBS administration;

2) PBS group: placebo group, PBS administration using papain-induced rhinitis model;

3) PC group: a positive control group, which was given with papain-induced rhinitis model (internal Shu Na: mometasone furoate nasal spray) at 10. Mu.L/time, once a day in the morning and at night;

4) EXO-1 group: in the exosome treatment group, using a papain-induced rhinitis model, iPSC exosomes are administered at a final concentration of 100 μg/ml, and 10 μl/time (exosome content of 1 μg) is dropped in the morning and evening;

5) BC group: baicalein treatment group, using papain-induced rhinitis model, contains baicalein (2% DMSO as solvent) at concentration of 100 μg/mL, and is used for nasal drip 1 μg/time, about 10 μl/time, each time in the morning and evening;

6) Bc+exo-1 group: the treatment group of the exosome composition containing baicalein was incubated with 100. Mu.g/mL of iPSC exosomes (20 h at 4 ℃ C., or 3h at room temperature) at a final concentration of 100. Mu.g/mL (2% DMSO as vehicle) using a papain-induced rhinitis model, and 10. Mu.L/time (exosomes content of 1. Mu.g, baicalein content of 1. Mu.g) was dropped on the nose, each time in the morning and evening.

Experimental procedure

1) 30 male C57BL/6J mice, 18-22g, animals numbered after one week of adaptation, were randomly divided into 6 groups of 5 animals according to Table 1.

2) Referring to the following table, on days 0-2, 7-10, groups 2-6 (PBS group and multiple treatment groups), nasal papain was dropped alternately left and right, 20 μl/unit, nasal equivalent volumes of physiological saline were dropped in group 1 (sham group), respectively;

3) On days 7-13, groups 3-6 alternately drop nasal PC, EXO-1, BC and BC+EXO-1, respectively, groups 1 (sham group) and 2 (PBS group) drop nasal equal volumes of PBS solution;

4) On day 14, nasal sinus tissues of the mice are taken, fixed, paraffin embedded, HE staining is carried out subsequently to observe the morphology, the nasal mucosa thickness is counted at 3 positions selected for each slice, and the data statistics is carried out after the nasal mucosa thickness at 3 positions of each slice is averaged. Animal experiment group:

experimental results

Epithelial cell damage was assessed by H & E staining sections. As can be seen in fig. 2, the nasal mucosa olfactory epithelium of the Sham group was intact and cilia were intact. The PBS group nasal mucosa is seriously damaged, the nasal mucosa is obviously atrophic by olfactory epithelium mucosa, cells are loose and damaged, partial cell nuclei are deeply contracted and dyed, vacuole is changed, and cilia fall off. Nasal mucosa olfactory epithelium mucosa of PC group is atrophic, cell is loose and broken, cell nucleus is contracted and deeply stained, vacuole is changed, but part of cilia is lodged. The nasal mucosa of the BC group had intact olfactory epithelial mucosa, but cilia were shed. The nasal mucosa of EXO-1 group had cilia shed in the olfactory epithelial mucosa. The nasal mucosa and olfactory epithelium mucosa of the BC+EXO-1 group are intact, and cilia are intact. As can be seen from the H & E stained sections, the intact cilia of the nasal mucosa were not shed in the BC+EXO-1 group, and the extent of nasal mucosa damage was reduced compared to the PBS group, the PC group, and the BC or EXO-1 group alone.

Nasal mucosa thickening was assessed by H & E staining sections. Thickening of the nasal mucosa generally indicates chronic inflammation of the nasal cavity, recurrent episodes of rhinitis or chronic local irritation over time. As can be seen from fig. 3, in the normal saline modeling Sham group, the nasal mucosa thickness is about 30 μm, the nasal mucosa is thickened to about 40 μm due to continuous stimulation of the edema by the PBS group, and the inner Shu Na PC group and the BC group using baicalein alone do not show obvious conditions of inhibiting the thickening of the nasal mucosa compared with the PBS group, the overall average thickness of the nasal mucosa of the exosome EXO-1 group is reduced compared with the PBS group, but the therapeutic effect of the exosome composition bc+exo-1 group containing baicalein is the best, and after the treatment, the nasal mucosa thickness of the bc+exo-1 group mice reaches the level of the Sham group, which indicates that the exosome containing baicalein can effectively inhibit the inflammatory reaction of the nasal mucosa, and has a better therapeutic effect on chronic sinusitis.

The embodiments of the present application have been described above. However, the present application is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. An exosome composition comprising baicalein or a derivative thereof, characterized by comprising, as an active ingredient, a combination of baicalein or a derivative thereof and an exosome;

preferably, only a portion of the baicalein or derivative thereof in the exosome composition is loaded in the exosome;

preferably, the baicalein or a derivative thereof in the exosome composition is fully loaded in exosomes;

preferably, the loading concentration of the baicalein or the derivative thereof is 2-500 mug/mL calculated by baicalein;

preferably, the amount of baicalein or a derivative thereof loaded in the exosome composition is 5% -100% of the total amount of the baicalein or the derivative thereof in the exosome composition, based on the baicalein.

2. The exosome composition of claim 1, wherein the exosome composition is obtained by mixing the exosome with the baicalein or derivative thereof and incubating the mixture.

3. The exosome composition of claim 2, wherein the incubating comprises: co-incubating baicalein with proper concentration with exosomes; in the incubation system, the final concentration of baicalein is 5-500 μg/mL, such as 50-300 μg/mL, and the final concentration of exosome is 2-3000 μg/mL, such as 50-200 μg/mL; the incubation conditions are 0 ℃ to 5 ℃ for 1 to 25 hours, or 20 ℃ to 35 ℃ for 1 to 4 hours.

4. Use of an exosome composition comprising baicalein or a derivative thereof according to any one of claims 1-3 in the manufacture of a medicament for treating nasal-sinusitis.

5. A pharmaceutical composition comprising the exosome composition of any one of claims 1-3;

preferably, the pharmaceutical composition is a suspension containing a buffer in addition to an exosome composition comprising baicalein or a derivative thereof, preferably, the exosome concentration and the concentration of baicalein or a derivative thereof calculated as baicalein are independently 2 to 500 μg/mL, respectively;

preferably, the pharmaceutical composition is a lyophilized preparation containing a cryoprotectant in addition to an exosome composition containing baicalein or a derivative thereof, preferably, the content of exosome and the content of baicalein Ji Huangcen or a derivative thereof are independently 0.1-500 μg, respectively.

6. An exosome composition according to any one of claims 1-3 or the use of claim 4 or the pharmaceutical composition of claim 5, wherein the baicalein or derivative thereof is selected from one or more of the group consisting of: baicalein, pharmaceutically acceptable salts of baicalein, baicalin and pharmaceutically acceptable salts of baicalin;

preferably, the pharmaceutically acceptable salt of baicalin is the salt of organic acid and the salt of inorganic acid respectively;

preferably, the inorganic acid is hydrochloric acid, sulfuric acid, phosphoric acid, diphosphoric acid, hydrobromic acid or nitric acid;

preferably, the organic acid is acetic acid, maleic acid, fumaric acid, tartaric acid, succinic acid, lactic acid, p-toluenesulfonic acid, salicylic acid or oxalic acid.

7. The exosome composition according to any one of claims 1-3 or the use according to claim 4 or the pharmaceutical composition according to claim 5, wherein the exosome is of a human or mammalian cell;

preferably, the exosomes are exosomes of human cells; preferably, the cell is a normal somatic cell or a stem cell;

preferably, the exosomes are multipotent stem cell-derived exosomes; preferably, the multipotent stem cell exosomes are induced multipotent stem cell-derived exosomes, mesenchymal stem cell-derived exosomes, or embryonic multipotent stem cell-derived exosomes; preferably, the pluripotent stem cell exosomes are human pluripotent stem cell exosomes, for example, human induced pluripotent stem cell-derived exosomes, human mesenchymal stem cell-derived exosomes, or human embryonic pluripotent stem cell-derived exosomes.

8. The use according to claim 4, wherein the nasal-sinusitis is chronic nasal-sinusitis;

preferably, the nasal-sinusitis is eosinophilic chronic sinusitis;

preferably, the eosinophilic chronic sinusitis is with or without nasal polyps; and/or the number of the groups of groups,

preferably, the treatment is to reduce or alleviate or inhibit inflammation of, repair, and/or inhibit the thickness of the mucosa of the nasal cavity and/or the sinus.