Nasal administration preparation containing extracellular vesicles and acellular matrix hydrogel and preparation method thereof

Abstract

The invention relates to a nasal administration preparation containing extracellular vesicles and acellular matrix hydrogel and a preparation method of the nasal administration preparation, and belongs to the technical field of extracellular vesicles. According to the nasal administration preparation, the nasal administration preparation contains extracellular vesicles, acellular matrix hydrogel and auxiliary materials, and the auxiliary materials contain at least one of a lipophilic solvent, a humectant, a thickening agent and an osmotic pressure regulator. The EVs nasal administration dosage form is prepared, the EVs has the capacity of passing through a blood brain barrier, can be delivered to a brain central nervous system and has high brain administration capacity, the human placenta source ECM forms nanofibers through structural regulation and control, the nanofibers are used as a carrier material of the EVs, long-term storage of the biological activity of the EVs can be achieved, the local treatment time of the EVs is prolonged, and the EVs nasal administration dosage form can be applied to the brain. EVs can be efficiently absorbed in the nasal cavity, and compared with similar products, the effective component acting time is longer.

Description

Technical Field

[0001] The present invention relates to the technical field of extracellular vesicles, and particularly relates to a nasal administration preparation containing extracellular vesicles and acellular matrix hydrogel and a preparation method thereof. Background Art

[0002] Extracellular vesicles (EVs) are nanoscale vesicles secreted by cells with a lipid bilayer structure, with a diameter of 40-160 nm (average 100 nm), containing abundant substances such as proteins, lipids, and nucleic acids. They can serve as intercellular messengers to reflect the physiological and pathological states of the source cells, and are of great significance in the early prevention, diagnosis, and prognosis judgment of diseases. EVs derived from mesenchymal stem cells have many advantages compared to cell therapy: they belong to cell-free therapy, have no tumorigenicity, no immunogenicity, and can expand the biodistribution and permeability of the blood-brain barrier. The potential therapeutic mechanisms of EVs include antioxidant, pro-angiogenic, immunomodulatory, and neuroplasticity regulatory processes, and EVs have drug-loading ability and cell type specificity. In addition, EVs are involved in many biological processes and may be used as neuroprotection and transport to treat neurological disorders including ischemic stroke. Importantly, they may be used in combination with currently available tissue-type plasminogen activator (tPA) and / or endovascular therapy. Previous studies have found that injecting EVs derived from mesenchymal stem cells into rodent models can improve limb ischemia, but there is no significant reduction in infarct volume, and it has improved disease progression and promoted neuronal recovery in the treatment of mechanically occluded stroke.

[0003] During the treatment of central nervous system diseases, intravenous injection of drugs is extremely easy to degrade in the blood, lose biological activity, and is extremely easy to spread throughout the body, increasing the dose requirement and reducing the local accumulation of lesions, making the treatment effect unable to reach the maximum. Local injection can lead to an increased risk of brain trauma and infection. When administered orally, due to the degradation effect of the gastrointestinal tract and the first-pass effect of the liver, the curative effect is often poor, and at the same time, there is an easy accumulation of therapeutic drugs in the liver. Nasal administration is an effective and safe way to deliver drugs to the central nervous system. Based on its own anatomical structure, there are many microvilli on the surface of the nasal mucosa, which can increase the absorption surface area, and there is a rich vascular network in the nasal cavity, which can accelerate the entry of drugs into the blood circulation. EVs have the ability to cross the blood-brain barrier and can be transported to the central nervous system through the olfactory mucosa via the nasal-brain pathway. However, the current nasal administration preparations have a low action time at the target site and a short biological activity time, making it difficult to ensure a good treatment effect. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a nasal administration preparation containing extracellular vesicles and acellular matrix hydrogel with an extended biological activity time and a preparation method thereof.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a nasal administration preparation, which contains extracellular vesicles, acellular matrix hydrogel and excipients, and the excipients contain at least one of a lipophilic solvent, a humectant, a thickening agent and an osmotic pressure regulator.

[0007] The present invention has prepared a nasal administration preparation containing extracellular vesicles (EVs), and confirmed that EVs can reach the central nervous system, laying a scalable program foundation for the application of mesenchymal stem cell-derived EVs in the clinical treatment of nervous system diseases. For example, the nanodrug delivery ability of EVs can be exerted to meet the needs of neuroprotection and regeneration treatment.

[0008] Further, the lipophilic solvent includes glycerol; the humectant includes polyethylene glycol; the thickening agent includes carboxymethyl cellulose; the osmotic pressure regulator includes sodium chloride.

[0009] Nasal administration can avoid the degradation effect of the gastrointestinal tract and the first-pass effect of the liver during oral administration. It is a natural transporter and will not cause the accumulation of therapeutic substances in the liver. When preparing a nasal administration preparation, the solute carrier, i.e., the solvent, needs to be considered, which is related to the nature of the solute. EVs are phospholipid bilayer vesicles containing soluble proteins, lipids, RNAs, etc., and are lipophilic. Therefore, the lipophilic solvent glycerol is selected. Carboxymethyl cellulose is used as a thickening agent to adjust its viscosity and increase its contact opportunity in the nasal cavity. Polyethylene glycol, a humectant, needs to be added to the nasal preparation to avoid nasal irritation and thus does not affect drug absorption.

[0010] Further, by volume, the nasal administration preparation contains 1-2 parts of extracellular vesicles, 1-2 parts of acellular matrix hydrogel, 85-90 parts of glycerol, 4 parts of polyethylene glycol, 3-4 parts of carboxymethyl cellulose, and 0-6 parts of sodium chloride.

[0011] Preferably, by volume, the nasal administration preparation contains 1.5 parts of extracellular vesicles, 1.5 parts of acellular matrix hydrogel, 87 parts of glycerol, 4 parts of polyethylene glycol, 3.5 parts of carboxymethyl cellulose, and 2.5 parts of sodium chloride.

[0012] Further, the volume ratio of the extracellular vesicles to the acellular matrix hydrogel is 1:1-2, preferably 1:1.

[0013] Further, the concentration of the extracellular vesicles is 5-5.2×10 11 cells / mL, preferably 5.2×10 11 cells / mL.

[0014] Further, the extracellular vesicles are mesenchymal stem cell extracellular vesicles.

[0015] Furthermore, the acellular matrix hydrogel is a placenta-derived acellular matrix hydrogel.

[0016] In a second aspect, the present invention provides a method for preparing the nasal administration preparation as described above, comprising the following steps:

[0017] S1: Mix the extracellular vesicles with the acellular matrix hydrogel and self-crosslink to obtain an acellular matrix hydrogel encapsulating the extracellular vesicles;

[0018] S2: Mix the lipophilic solvent, humectant, thickening agent with the acellular matrix hydrogel encapsulating the extracellular vesicles obtained in step S1, and adjust the pH to 6.5 - 7.5 with an osmotic pressure regulator to obtain the nasal administration preparation.

[0019] The pH value of the nasal administration preparation has a great influence on drug absorption. An appropriate pH value can avoid irritating the nasal mucosa and prevent the growth of pathogenic bacteria in the nasal cavity. For example, lysozyme is a nasal secretion that can kill bacteria in an acidic pH environment but has no effect in an alkaline environment, which makes the nasal tissue vulnerable to bacterial infection. Sodium chloride is selected as the osmotic pressure regulator and also as the pH regulator to adjust the pH value between 6.5 and 7.5.

[0020] Furthermore, in step S1, the self-crosslinking temperature is 36.5 - 37.5 °C and the time is 3 - 5 min. Preferably, the self-crosslinking temperature is 37 °C and the time is 3 min.

[0021] Furthermore, the method for preparing extracellular vesicles in step S1 is as follows: Centrifuge the cell culture supernatant at 1500 - 2000 g for 20 - 30 min, take the supernatant, and ultracentrifuge the supernatant at 100000 - 120000 g for 1 - 1.5 h, centrifuge 1 - 2 times, and take the precipitate as the extracellular vesicles.

[0022] Preferably, in step S1, centrifuge the cell culture supernatant at 2000 g for 20 min, take the supernatant, ultracentrifuge the supernatant at 100000 g for 1.5 h, centrifuge 2 times, and take the precipitate as the extracellular vesicles.

[0023] Furthermore, in step S1, the ultracentrifugation temperature is 0 - 4 °C, preferably 4 °C.

[0024] Furthermore, after the first ultracentrifugation, resuspend with PBS buffer and then perform the next ultracentrifugation.

[0025] The method for preparing extracellular vesicles (EVs) of the present invention improves the separation efficiency of EVs. Transmission electron microscopy examination of EVs shows their spherical double-membrane-bound morphology, and the diameter is 40 - 100 nm, and all have EV surface markers CD9, CD63, and TSG101.

[0026] Furthermore, the method for preparing the acellular matrix hydrogel in step S1 includes the following steps:

[0027] S11: Digest the cells with trypsin and EDTA solution;

[0028] S12: Mix Triton X-100 with the cells digested in step S11, stir and react, then mix with sodium deoxycholate and stir and react to obtain an acellular matrix scaffold;

[0029] S13: Digest the acellular matrix scaffold with pepsin and / or hydrochloric acid, and neutralize the pH to obtain the acellular matrix hydrogel.

[0030] Furthermore, in step S11, the concentration of trypsin is 0.02 - 0.05% w / v, preferably 0.04% w / v.

[0031] Furthermore, in step S11, the concentration of EDTA is 0.1 - 0.15% w / v, preferably 0.1% w / v.

[0032] Furthermore, in step S11, digest the cells with trypsin and EDTA solution by stirring at 35 - 42°C for 3 - 6 h, preferably stir and digest the cells at 37°C for 4 h.

[0033] Furthermore, in step S12, the concentration of Triton X-100 is 2 - 5% v / v, preferably 3% v / v. Triton X-100 can dissolve lipids and improve the permeability of cell membranes, thereby removing cell components.

[0034] Furthermore, in step S12, the mixing time of Triton X-100 with the cells digested in step S21 by stirring is 3 - 4.5 h, preferably 4 h.

[0035] Furthermore, in step S12, the concentration of sodium deoxycholate is 2 - 6% w / v, preferably 4% w / v. Sodium deoxycholate can promote cell lysis.

[0036] Furthermore, in step S12, the mixing time of sodium deoxycholate by stirring is 2 - 3 h, preferably 2.5 h.

[0037] Furthermore, in step S12, after the stirring reaction of Triton X-100, wash it and then carry out the stirring reaction with sodium deoxycholate.

[0038] Furthermore, after washing and soaking the acellular matrix scaffold prepared in step S12 with deionized water, remove the chemical reagents, then carry out freeze-drying and grind it into powder, and then digest it with pepsin and / or hydrochloric acid.

[0039] Further, the acellular matrix scaffold prepared in step S12 is washed with deionized water 2 - 3 times, 5 - 15 minutes each time, preferably 10 minutes, and soaked in deionized water for 6 - 8 hours, preferably 6 hours.

[0040] Further, in step S13, NaOH is used to neutralize the pH, and the concentration of the NaOH can be 0.1 - 0.2 mol / L, preferably 0.1 mol / L.

[0041] Further, in step S13, after neutralizing the pH, it is placed in an incubator at 37°C with 4% CO2 for 5 - 10 minutes to obtain the acellular matrix hydrogel, preferably 5 minutes.

[0042] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0043] 1. In the preparation method of the EVs intranasal administration preparation of the present application, EVs are a cell-free therapy with low immunogenicity and are natural transporters. Compared with liposomes, their toxic effects and immune responses are smaller, and they are suitable for repeated administration. The method for separating and purifying EVs of the present invention can achieve efficient separation and extraction of EVs. The EVs carried by human placenta-derived ECM nanofibers in the preparation are easy to store and can be stored at -80°C for a long time without losing their biochemical activity. At the same time, EVs can be enriched in large quantities in the culture medium to achieve mass production, effectively improving the curative effect. EVs therapy is a treatment method with high safety and good stability. The EVs prepared by this method can exist in the intranasal administration preparation at a higher concentration, improving the absorption and utilization rate of the main active ingredient.

[0044] 2. In the preparation method of the EVs intranasal administration preparation of the present application, the main components of the EVs intranasal administration preparation are glycerol, carboxymethyl cellulose, EVs, polyethylene glycol, sodium chloride, and human placenta-derived ECM nanofibers. Sodium chloride and human placenta-derived ECM nanofibers are used as an osmotic pressure regulator and an EVs carrier material, respectively. The preparation obtained by this formulation can achieve efficient absorption of EVs in the nasal cavity and has a longer action time of the active ingredient compared with similar products.

[0045] 3. In the method for delivering mesenchymal stem cell-derived EVs to the brain by intranasal administration of the present application, EVs have the ability to cross the blood-brain barrier and can be delivered to the central nervous system of the brain, with strong brain drug delivery ability. Human placenta-derived ECM forms nanofibers through structural regulation and is used as a carrier material for EVs, which can achieve long-term preservation of the biological activity of EVs and extend the local treatment time of EVs, providing a new strategy for the application of mesenchymal stem cell-derived EVs in the clinical treatment of stroke. Description of the Drawings

[0046] Figure 1Structures and characterizations of isolated and purified EVs. Among them, A is a diagram of purified EVs under a transmission electron microscope, with a scale bar of 100 nm; B is the particle size distribution range of purified EVs; C is a comparison of the expression of surface markers of EVs extracted in the present invention with that of standard EVs.

[0047] Figure 2 Schematic diagram of the preparation process and administration mode of the intranasal administration preparation of EVs.

[0048] Figure 3 Physical diagram of the intranasal administration preparation of EVs.

[0049] Figure 4 Diagram of fluorescent labeling of EVs with DiR.

[0050] Figure 5 Fluorescent in vivo imaging tracing diagram of EVs in rats. Among them, A is the fluorescent in vivo imaging tracing diagram of the brain; B is the bar chart of the fluorescent distribution in the brain; C is the fluorescent in vivo imaging tracing diagram of different organs; D is the bar chart of the fluorescent distribution in different organs.

[0051] Figure 6 Bar chart of the fluorescent distribution of EVs carried by Matrigel and human placenta-derived ECM nanofibers acting on the central nervous system respectively. Specific implementation manners

[0052] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Other materials, reagents, etc. used in the embodiments can be obtained from commercial channels without special instructions.

[0053] Example 1 Extraction method of mesenchymal stem cell-derived EVs

[0054] 1. Cell culture: Human placenta mesenchymal stem cells are cultured in a serum-free basal medium for human mesenchymal stem cells (manufacturer: TBDscience, product number: SC2013-G-A). The cell viability is detected by trypan blue staining > 99%. After culturing for 2 - 3 days in a 37°C, 5% CO2 incubator, the cell culture supernatant is collected and stored at -80°C.

[0055] 2. Isolation and purification of EVs:

[0056] (1) Centrifuge the cell culture supernatant in step 1 at 2000g for 20 min, take the supernatant, and remove debris;

[0057] (2) Centrifuge the supernatant from step (1) at 4°C and 100,000 g for 1.5 h in an ultracentrifuge (manufacturer: Hitachi, model: CP100NX). After removing the supernatant with a pipette, rinse the bottom of the centrifuge tube with 1 mL of PBS buffer (phosphate buffer, pH 7.4, manufacturer: servicebio) to obtain a PBS resuspension.

[0058] (3) Centrifuge the PBS resuspension from step (2) for the second time under the same centrifugation conditions as in step (2). Remove the supernatant to obtain a precipitate (EVs). Dissolve the EVs with 100 μL of PBS to obtain an EVs solution, and store it at -80°C for later use.

[0059] Through the steps of separating and purifying EVs in the present invention, efficient separation of EVs is achieved, ensuring that the concentration of EVs in the components after subsequent preparation compatibility dilution reaches 5.2×10 11 per mL.

[0060] Example 2 Identification and Physicochemical Characterization of EVs

[0061] I. Experimental Methods

[0062] Use the identification criteria of the International Society for Extracellular Vesicles, including protein content determination and evaluation of the morphological structure and distribution density of EVs. The specific steps are as follows:

[0063] 1. Protein content determination: Use a MicroBCA protein detection kit (manufacturer: Thermo Fisher Scientific, USA, catalog number: 23235) to measure the absorbance of the EVs separated in Example 1 at 562 nm to determine the protein content of the EVs.

[0064] 2. Evaluation of EVs morphological structure and distribution density: Fix the EVs separated in Example 1 with 2.5% (v / v) glutaraldehyde for 2 h. After washing, ultracentrifuge the EVs and suspend them in 100 μL of electron microscopy blocking solution HAS. Negative stain with 3% (w / v) phosphotungstic acid on a carbon-coated grid for 1 min, and observe the morphological structure of the EVs with a transmission electron microscope (manufacturer: Hitachi, model: H-7650). Perform a Western blot assay to determine the surface markers CD9, CD63, and TSG101 of the EVs. Use a NanoSight NS300 system for nanoparticle tracking analysis (NTA) to detect the size distribution and concentration of the EVs.

[0065] II. Experimental Methods

[0066] Transmission electron microscopy examination of the purified EVs showed their spherical double-membrane-bound morphology ( Figure 1 A), and the diameter was 40 - 100 nm ( Figure 1B), both have the EV surface markers CD9, CD63, and TSG101( Figure 1 C). Characterization of EVs showed that the EVs derived from mesenchymal stem cells extracted in Example 1 met the standards of the International Society for Extracellular Vesicles and were able to maintain uniformity.

[0067] Preparation method of human placenta-derived ECM nanofibers in Example 3

[0068] 1. Collect fresh placental tissues without any clinical diseases from full-term pregnant women under 28 years old. Cut the collected placental tissues into slices (2 - 3 cm 3 ), and place them into a mixed solution of 150 mL of 0.04% (w / v) trypsin and 150 mL of 0.1% (w / v) EDTA, and stir at 37 °C for 4 h.

[0069] 2. Rinse the placental tissues treated in step 1 three times with deionized water, and transfer them to 300 mL of 3% (v / v) Triton X-100 solution, and stir at room temperature for 4 h.

[0070] 3. After washing the placental tissues treated in step 2 with deionized water for 15 min, transfer the placenta to 300 mL of sodium deoxycholate (4% w / v) solution, and stir evenly at room temperature for 2.5 h to obtain a decellularized dECM (decellularized extracellular matrix) scaffold.

[0071] 4. Wash the dECM scaffold in step 3 three times with deionized water, 10 min each time, and soak it in deionized water for 6 h to remove traces of chemical reagents.

[0072] 5. Freeze-dry the dECM scaffold treated in step 4 for 48 h, chop it, and put it into a sterile grinding steel tank to grind it into powder (grinding frequency 60 Hz, grinding time 2 min) to obtain human placenta-derived dECM powder.

[0073] 6. Digest the human placenta-derived dECM powder in step 5 with 100 mL of pepsin-hydrochloric acid solution for 2 days to prepare a pre-gel, and store it at 4 °C under sterile conditions.

[0074] 7. Neutralize the pre-gel in step 6 with NaOH (0.1 moL / L) solution and 10×PBS to maintain its pH at 7 - 8, and culture it in a 37 °C 5% CO2 incubator for 30 min to prepare a human placenta-derived ECM nanofiber hydrogel.

[0075] Preparation method of EVs intranasal administration preparation in Example 4

[0076] The preparation process flow diagram and administration mode description of the EVs intranasal administration preparation of the present invention are as Figure 2 shown.

[0077] 1. Dissolve 3 g of carboxymethyl cellulose and 4 g of polyethylene glycol in 90 g of glycerol, and obtain a homogeneous solution 1 after mixing evenly.

[0078] 2. Encapsulate and wrap EVs with the human placenta-derived ECM nanofiber hydrogel prepared in Example 3. Mix the human placenta-derived ECM nanofiber hydrogel prepared in Example 3 and the EVs solution prepared in Example 1 at a volume ratio of 1:1, and perform thermal cross-linking in a 37 °C and 5% CO₂ incubator for 3 min to obtain a human placenta-derived ECM nanofiber hydrogel encapsulating EVs; mix the human placenta-derived ECM nanofiber hydrogel encapsulating EVs with the homogeneous solution 1 in step 1 to obtain a homogeneous solution 2, ensuring that the concentration of EVs in the preparation reaches 5.2×10 11 per mL. EVs are phospholipid bilayer vesicle structures, which are lipophilic and can be dissolved in glycerol.

[0079] 3. Since the pH of the nasal cavity is slightly acidic, the osmotic pressure of the liquid is adjusted with sodium chloride and the pH is adjusted with sodium hydroxide to control the pH of the homogeneous solution 2 between 6.5 and 7.5, and the preparation of the present application is obtained ( Figure 3 ).

[0080] Table 1 Components and specific functions of the EVs intranasal administration preparation

[0081]

[0082]

[0083] Therapeutic effect of the EVs intranasal administration preparation in Example 5

[0084] I. Experimental method

[0085] 1. Fluorescently label the EVs isolated in Example 1 with the cell membrane staining reagent DiR (manufacturer: ThermoFisher Scientific, Invitrogen, catalog number: D12731) to obtain dark red fluorescence ( Figure 4 ).

[0086] 2. Prepare a 1 - 5 mM DMSO or EtOH stock solution.

[0087] 3. Preparation of working solution: Dilute the stock solution in step 1 with a suitable buffer (such as: serum-free medium, HBSS or PBS) to prepare a working solution with a concentration of 1 - 5 μM.

[0088] 4. Resuspend the EVs fluorescently labeled in step 1 with an appropriate volume of the working solution, and incubate human placenta mesenchymal stem cells at 37 °C for 2 - 20 min. The optimal culture time for different cells is different. Take 20 min as the starting incubation time, and then optimize the system to obtain a uniform labeling result.

[0089] 5. Ultracentrifuge the EVs after incubation in Step 4 at 1,000,000 g for 70 min at 4 °C to remove excess dye.

[0090] 6. Dilute the EVs treated in Step 4 with PBS to a concentration of 5.2×10 11 per mL to prepare an EVs solution.

[0091] 7. Prepare the EVs solution treated in Step 5 into a nasal administration preparation according to the method of Example 4.

[0092] Select male SD rats aged 7 - 8 weeks for animal experiments. The animals are divided into 2 groups: Group 1 is the EVs nasal administration preparation group, and Group 2 is the control preparation 1. The preparation method of the control preparation 1 is as follows: Do not encapsulate and wrap the EVs with human placenta-derived ECM nanofiber hydrogel, directly add the EVs solution into the mixture 1 to obtain the mixture 3, and other preparation methods are the same as those of the EVs nasal administration preparation.

[0093] The nasal administration dose of rats is 100 μL, that is, 100 μL of the EVs nasal administration preparation and 100 μL of the control preparation 1 are respectively dropped into the bilateral nasal cavities of each group of rats.

[0094] 8. Perform in vivo fluorescence live imaging (IVIS) at 0 h, 24 h, 48 h, and 72 h after administration respectively to observe the fluorescence distribution and intensity of EVs in vivo.

[0095] II. Experimental Results

[0096] Fluorescence live imaging confirmed that EVs can reach the central nervous system. As shown in Figure 5 A and Figure 5 B, in the EVs nasal administration preparation group, EVs are absorbed in the nasal capillaries, enter the central nervous system of the brain through the blood-brain barrier, and the fluorescence gradually weakens over time. EVs are degraded. The fluorescence intensity of the pure EVs group is lower than that of the EVs nasal administration preparation group at the same time period, indicating that the EVs nasal administration preparation can prevent more EVs from being degraded in vitro, that is, in the nasal cavity, and maintain longer biological activity.

[0097] The fluorescence in the brains of rats in Group 2 is significantly weaker than that in Group 1, and the fluorescence in the brains of Group 2 is not obvious after 48 h, while the fluorescence in the brains of Group 1 is still significant, indicating that the EVs nasal administration preparation slows down the degradation time of EVs in the brain and extends the biological activity time.

[0098] In addition, as shown in Figure 5 C and Figure 5 D, fluorescence live imaging shows that except for the brain region, the fluorescence level in other body regions of rats is relatively weak, indicating that the EVs nasal administration preparation has strong brain delivery ability.

[0099] In the intranasal administration preparation of EVs, human placenta-derived ECM nanofibers as the carrier material of EVs are of great significance for achieving long-term retention of the biological activity of EVs in vivo and ensuring the therapeutic effect of the preparation.

[0100] Therapeutic efficiency of different nanofibers transporting EVs in Comparative Example 1

[0101] Most of the existing nanofibers used as drug carriers are synthetic fibers, which are composed of inorganic materials such as metals and semiconductors or organic materials such as phospholipids and synthetic polymers, and have properties such as biocompatibility, biodegradability, and low toxicity. However, during the process of transporting EVs with such nanofiber materials, it is difficult to provide a suitable environment for transporting EVs, and it is difficult to preserve and stably maintain the activity of EVs for a long time, resulting in a reduction in therapeutic effect. Therefore, scientists prefer to use nanofibers of biological materials for drug transportation, such as Matrigel matrix gel derived from mouse tumors. Matrigel matrix gel can provide rich nutrients and support the growth and development of various cell types, but there are large differences between batches and it lacks bioactive factors, so it cannot ensure stable therapeutic effects. The human placenta-derived ECM nanofiber hydrogel, as a biomimetic tissue bio-nanofiber, is prepared from human placenta. The extracellular matrix skeleton is completely retained through decellularization technology, providing the original microenvironment for maintaining the biological activity of EVs, and realizing stable targeted drug delivery without affecting the biological activity of EVs.

[0102] I. Experimental method

[0103] Replace the human placenta-derived ECM nanofiber hydrogel in Example 4 with Matrigel (manufacturer: Corning, product number: 356234) to prepare Comparative Preparation 2, and perform intranasal administration according to the method of Example 5.

[0104] II. Experimental results

[0105] As shown by Figure 6 , the radiation efficiency of EVs in the human placenta-derived ECM nanofiber hydrogel is significantly better than that of EVs in the Matrigel matrix gel, and the preservation time of EVs is significantly longer, that is, the human placenta-derived ECM nanofiber hydrogel is more suitable for transporting EVs.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A preparation for nasal administration, characterized in that: The nasal administration preparation contains extracellular vesicles, acellular matrix hydrogel and excipients, wherein the excipients contain at least one of a lipophilic solvent, a moisturizer, a thickener and an osmotic pressure regulator.

2. The nasal administration preparation according to claim 1, characterized in that: The lipophilic solvent includes glycerol; the moisturizer includes polyethylene glycol; the thickener includes carboxymethyl cellulose; and the osmotic pressure regulator includes sodium chloride.

3. The nasal administration preparation according to claim 2, characterized in that: The nasal administration preparation contains, by volume, 1-2 parts of extracellular vesicles, 1-2 parts of decellularized matrix hydrogel, 85-90 parts of glycerol, 4 parts of polyethylene glycol, 3-4 parts of carboxymethyl cellulose and 0-6 parts of sodium chloride.

4. The nasal administration preparation according to claim 3, characterized in that: The nasal administration preparation contains, by volume, 1.5 parts of extracellular vesicles, 1.5 parts of decellularized matrix hydrogel, 87 parts of glycerol, 4 parts of polyethylene glycol, 3.5 parts of carboxymethyl cellulose and 2.5 parts of sodium chloride.

5. The nasal administration preparation according to claim 1, characterized in that: The volume ratio of the extracellular vesicles to the acellular matrix hydrogel is 1:1-2.

6. The nasal administration preparation according to claim 1, characterized in that: The concentration of the extracellular vesicles is 5 to 5.2×10 11 Pieces / mL.

7. The nasal administration preparation according to claim 1, characterized in that: The exovesicles are mesenchymal stem cell exovesicles.

8. The nasal administration preparation according to claim 1, characterized in that: The decellularized matrix hydrogel is placenta-derived decellularized matrix hydrogel.

9. The method for preparing the nasal preparation according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1: mixing extracellular vesicles with acellular matrix hydrogel, and self-crosslinking to obtain acellular matrix hydrogel encapsulating extracellular vesicles; S2: Mixing the lipophilic solvent, moisturizer, thickener and the decellularized matrix hydrogel encapsulating the extracellular vesicles described in step S1, and adjusting the pH to 6.5-7.5 with an osmotic pressure regulator to obtain the nasal administration preparation.

10. The preparation method according to claim 9, characterized in that: In step S1, the self-crosslinking temperature is 36.5-37.5° C. and the time is 3-5 min.

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