Injectable exosomes and their use in repair of injury

Abstract

The application discloses an injectable exosome and application thereof in injury repair, and the gel is prepared by compounding a temperature-sensitive chitosan hydrogel and umbilical cord mesenchymal stem cell exosomes; a temperature-sensitive carrier is obtained by mixing chitosan acid solution with a specific molecular weight and sodium beta-glycerophosphate in an ice bath, the low-temperature liquid can be injected, and the exosome is formed in situ and released at 37 DEG C. The preparation is free of heavy metals, is simple to prepare, can long-acting protect the activity of the exosome, and can efficiently repair endometrial injury and improve the conception and littering rate.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical preparations, specifically providing an injectable exosome and its application in damage repair. Background Technology

[0002] Exosomes are double-membrane extracellular vesicles secreted by living cells. As a core component of intercellular communication, they play a regulatory role in the transport of bioactive macromolecules such as cytoplasmic proteins, nucleic acids, and lipids. They are widely present in bodily fluids such as saliva, plasma, pleural effusion, tears, lymph, urine, and ascites. They can regulate immunity, promote cell regeneration and repair, and play biological roles and functions in the development of various diseases. Exosomes are characterized by low immunogenicity and high tolerability, making them suitable as drug carriers for delivering therapeutic substances.

[0003] However, the route of administration of exosomes directly determines their in vivo retention efficiency and lesion enrichment effect: free exogenous exosomes disappear rapidly in the body through fluid circulation. Intravenously injected exosomes disappear rapidly from the bloodstream, intraperitoneally injected exosomes preferentially accumulate in the pancreas and gastrointestinal tract, while subcutaneous injection results in low enrichment in all organs. This is one of the key issues restricting the application of exosome therapeutic carriers and the development of targeted technologies.

[0004] Chitosan is obtained by partial deacetylation of chitin. It has strong bioadhesion and permeability, is non-toxic, biodegradable, and biocompatible. It also possesses antibacterial, hemostatic, tissue regeneration-accelerating, and fiber synthesis-promoting properties, making it applicable in pharmaceuticals, medicine, and agricultural processing, such as surgical sutures and wound healing materials. Chitosan thermosensitive gel, prepared from chitosan and sodium glycerophosphate, remains liquid at low temperatures, facilitating the incorporation of bioactive substances or cells. It transforms into a gel at normal human body temperature (37°C), making it a biodegradable material suitable for use as an injectable scaffold material, facilitating manipulation.

[0005] Existing technologies include research protocols for repairing endometrial damage using gel carriers loaded with stem cell-derived exosomes. The mainstream carriers fall into two categories: one is polyethylene glycol-based silver ion hydrogels, which rely on the gel carrier to immobilize exosomes and achieve intrauterine damage repair. However, the gel components contain silver heavy metal ions, which are difficult to metabolize normally and accumulate in the body after degradation, failing to meet the clinical needs for high-dose, repeated administration. The other is type I collagen-based chitosan gels, which, while eliminating the heavy metal risk, involve complex raw material pretreatment and gel forming processes, a lengthy freeze-drying preparation cycle, and high industrial production costs. Furthermore, the freeze-drying process can easily damage the exosome membrane structure, leading to a decrease in exosome bioactivity. In addition, existing sodium alginate and hyaluronic acid-based exosome hydrogels are mostly used for bone defect repair, and conventional chitosan thermosensitive gels are only applied to skin wound healing. Currently, there is no mature technology for preparing injectable formulations specifically for endometrial damage repair using chitosan-sodium glycerophosphate thermosensitive gels combined with exosomes. Summary of the Invention

[0006] When free exosomes are administered in liquid form, there are problems such as short residence time, easy loss or drying, low enrichment concentration, and low activity. Among the existing exosome-loaded repair gels, polyethylene glycol silver-based gel contains heavy metals, which limits its in vivo use. Collagen-chitosan composite gel is complicated to prepare, has high production costs, and is prone to damaging exosome activity. Moreover, there are no existing chitosan thermosensitive gels for endometrial injury repair, which cannot meet the clinical need for long-term repair of endometrial injury.

[0007] To address the shortcomings of the existing technologies, this invention proposes an injectable exosome that solves the problems of existing exosome-loaded repair gels, such as polyethylene glycol silver-based gel containing heavy metals and limited in vivo drug use, and collagen-chitosan composite gel which is complex to prepare, has high production costs, and is prone to damaging exosome activity. This invention also achieves clinical efficacy in treating endometrial damage.

[0008] One aspect of the present invention provides an exosome in situ gel for damage repair, which is prepared by mixing a thermosensitive chitosan hydrogel with exosomes; The chitosan hydrogel was prepared by mixing an acidic chitosan solution with an aqueous solution of sodium β-glycerophosphate in an ice bath; the acidic chitosan solution had a mass fraction of 1.5-3 wt%, the sodium β-glycerophosphate solution had a mass fraction of 40-60 wt%, and the volume ratio of the two was 3-9:1. The exosomes are exosomes derived from umbilical cord mesenchymal stem cells.

[0009] Furthermore, the chitosan acidic solution is prepared by dispersing chitosan in an acidic solution.

[0010] Furthermore, the number-average molecular weight of the chitosan is 40,000-60,000, preferably 45,000-55,000.

[0011] Furthermore, the acidic solution is an acetic acid solution or a hydrochloric acid solution.

[0012] Furthermore, the concentration of the acidic solution is 0.06–0.12 M.

[0013] Furthermore, the chitosan acid solution contains 1.5 to 3 wt% chitosan by mass.

[0014] Furthermore, the thermosensitive chitosan hydrogel is mixed with exosomes in a volume ratio of 1:10-10:1, preferably 1:5-5:1, for example 1:1, 1:2, 1:3, 1:4, 1:5, 5:1, 4:1, 3:1, 2:1.

[0015] Furthermore, the particle concentration of the exosomes is 10. 8 ~10¹ 0 per mL.

[0016] A second aspect of this invention provides a method for preparing the above-mentioned exosome in situ gel for damage repair, comprising the following steps: S1. Dissolve chitosan in an acidic solution, stir and filter, store in an ice bath, and prepare an acidic chitosan solution. S2. Prepare an aqueous solution of sodium β-glycerophosphate; S3. Under ice bath conditions, add sodium β-glycerophosphate solution to chitosan solution at a volume ratio of 3-9:1 and mix well to obtain blank thermosensitive chitosan gel. S4. Take exosomes and mix them with an equal volume of blank thermosensitive gel, incubate to form a gel, and obtain the target gel.

[0017] Furthermore, in step S1, the number-average molecular weight of the chitosan is preferably 45,000-55,000.

[0018] Furthermore, in step S2, the prepared sodium β-glycerophosphate aqueous solution is sterilized by filtration through a 0.2 μm filter membrane.

[0019] Furthermore, in step S4, the preferred volume ratio of the thermosensitive chitosan hydrogel to the exosomes is 1:5-5:1, for example, 1:1, 1:2, 1:3, 1:4, 1:5, 5:1, 4:1, 3:1, or 2:1.

[0020] Furthermore, the concentration of exosome particles used in step S4 is 10. 8 ~10¹ 0 per mL.

[0021] Furthermore, in step S4, the mixed system is incubated at 37°C for 10–30 min, resulting in a thermosensitive phase transition and the formation of a solid in-situ gel.

[0022] Furthermore, the exosomes derived from umbilical cord mesenchymal stem cells are obtained by serum-free culture of 3rd to 5th generation umbilical cord mesenchymal stem cells, and the cell supernatant is collected and purified by differential ultracentrifugation or ultrafiltration.

[0023] The third aspect of the present invention provides the application of the above-mentioned exosome in situ gel for damage repair in the preparation of drugs for repairing endometrial damage, improving fertility due to endometrial damage, and improving the embryo implantation ability of the endometrium. Furthermore, the exosomes are derived from human / animal-derived mesenchymal stem cells.

[0024] A fourth aspect of the present invention provides a pharmaceutical composition comprising the above-described exosome in situ gel for damage repair; Furthermore, the pharmaceutical composition is formulated as an injection.

[0025] Beneficial effects 1. The carrier raw materials of this invention are only chitosan and sodium β-glycerophosphate, without heavy metal components such as silver, with high biosafety, no metabolic burden of heavy metal accumulation in the body, and support for large-dose multiple administration. 2. The overall preparation steps are simple, without the need for a long freeze-drying process, the production cycle is short, the raw materials are readily available, and large-scale mass production can be achieved, resulting in lower preparation costs. 3. The formulation is liquid at low temperatures, allowing for both intrauterine injection and non-invasive topical application, providing flexibility in administration. At a physiological temperature of 37°C, it quickly gels and anchors at the site of injury, effectively preventing the loss of exosomes. 4. The gel matrix can protect the structure and bioactivity of exosomes, and achieve long-term sustained release of exosomes by relying on the carrier for gradual degradation, which greatly increases the concentration and duration of exosomes at the lesion site and enhances the tissue repair effect. 5. Animal experiments have confirmed that the formulation of this invention can significantly repair endometrial damage in rats and greatly increase the litter size of rats after injury. 6. It can be used with exosomes from various sources, and the amount of exosomes added can be adjusted as needed. The finished product can be stored at low temperature and used immediately. It has no immune side effects related to cell preparations and is suitable for multi-tissue damage repair scenarios. Attached Figure Description

[0026] Figure 1 shows the characterization of exosomes. The left side is an image of exosome nanoparticle tracking, and the right side is a transmission electron microscope (TEM) image with a scale bar of 100 nm. It can be seen that the vesicles are typically round and the particle size is consistent with the 30–150 nm characteristics of exosomes, confirming the successful preparation of exosomes derived from the target umbilical cord mesenchymal stem cells.

[0027] Figure 2 shows a series of characterization images of the thermosensitive chitosan gel: the SEM image on the left shows that the blank gel has a three-dimensional porous interconnected structure, which is conducive to exosome loading and sustained release of nutrients and active substances in vivo; the NTA particle size distribution curve on the right verifies the exosome particle size range; the test tube photo below visually shows the thermosensitive phase change characteristics of the system, which is a flowable liquid at low temperature and rapidly solidifies into a semi-solid gel at 37°C, meeting the requirements for in-situ molding after injection.

[0028] Figure 3 shows a comparison of the morphology of the isolated uterus after rats were caged together. The left side is the damaged uterus of the blank gel control group, with severe endometrial atrophy and tissue fibrosis. The right side is the uterus of the exosome-loaded gel intervention group of this invention, where embryo formation can be seen, the thickness of the endometrium in the uterine cavity is significantly restored, and the tissue is full and plump. This directly proves that the composite gel can effectively repair ethanol-induced organic damage to the endometrium. Detailed Implementation

[0029] The following detailed description of the present invention through specific embodiments illustrates the preferred aspects of the invention. It should be noted that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. The following detailed description of the preferred features and performance of the present invention, in conjunction with the embodiments, provides further details.

[0030] In this invention, the exosomes are exosomes derived from human umbilical cord mesenchymal stem cells, prepared according to conventional methods in the art: 3rd to 5th generation human umbilical cord mesenchymal stem cells are cultured in serum-free medium for 48 hours, and the cell supernatant is collected. The cells are then separated and purified by differential ultracentrifugation or ultrafiltration, and used after BCA protein quantification.

[0031] Example 1: Preparation of blank thermosensitive chitosan gel 1. Preparation of blank thermosensitive chitosan gel in acetic acid system Chitosan with a number average molecular weight of 50,000 was added to a 0.09 mol / L aqueous acetic acid solution and stirred until fully dissolved to prepare a 2.0% (w / w) chitosan solution. After filtering to remove insoluble impurities, the solution was stored in an ice bath. A 50% (w / w) sodium β-glycerophosphate aqueous solution was prepared using triple-distilled water and filtered through a 0.2 μm filter membrane for sterilization. Under ice bath conditions, sodium β-glycerophosphate solution was slowly added to the chitosan solution at a volume ratio of 5:1 (chitosan solution:sodium β-glycerophosphate solution) and stirred until homogeneous to obtain a low-temperature liquid blank chitosan thermosensitive gel.

[0032] 2. Preparation of blank thermosensitive chitosan gel in hydrochloric acid system Chitosan with a number average molecular weight of 50,000 was dissolved in 0.07 mol / L hydrochloric acid solution, stirred and filtered to prepare a 1.8% chitosan solution, which was then stored in an ice bath. A 45% sodium β-glycerophosphate solution was prepared with triple-distilled water and sterilized using a 0.2 μm filter membrane. The two solutions were mixed in a volume ratio of 4:1 in an ice bath and stirred until homogeneous to obtain a blank thermosensitive chitosan gel.

[0033] 3. Preparation of blank thermosensitive chitosan gel in acetic acid system Chitosan with a number average molecular weight of 50,000 was dissolved in 1.00 mol / L acetic acid solution, stirred and filtered to prepare a 2.5% chitosan solution, which was then stored in an ice bath. A 55% sodium β-glycerophosphate solution was prepared with triple-distilled water and sterilized using a 0.2 μm filter membrane. The two solutions were mixed in a 6:1 volume ratio in an ice bath and stirred until homogeneous to obtain a blank thermosensitive chitosan gel.

[0034] The microstructure of the prepared blank thermosensitive gel was observed by scanning electron microscopy. The results are shown in the SEM image on the left side of Figure 2. A large number of interconnected porous structures are formed inside the gel. The pores can stably encapsulate exosomes and support the continuous release of active components. The gel is a flowing liquid at low temperature and can be completely solidified after being placed in an environment of 37°C. The phase transition effect is shown in the thermosensitive comparison photo of the test tube at the bottom of Figure 2.

[0035] Example 2: Preparation of injectable chitosan-exosome composite gel Take the blank chitosan gel in the acetic acid system prepared in Example 1, and weigh 100 μg of exosomes derived from umbilical cord mesenchymal stem cells (10 μg). 8 ~ 10 10 Mix thoroughly with an equal volume of blank gel (particles / mL) and store at low temperature.

[0036] When using, place the mixture at 37°C, apply it to the site of application, and incubate for 20 minutes. The system will change from a liquid state to a solid gel, which is the method of using the injectable exosome gel formulation of this invention.

[0037] The micromorphology and particle size characteristics of the umbilical cord mesenchymal stem cell exosomes used in this embodiment were characterized by nano-tracking and transmission electron microscopy. The results are shown in Figure 1: TEM images show that the vesicles are regular circular double membrane structures with sizes concentrated within 100 nm; the NTA particle size detection results are shown in the particle size distribution curve on the right side of Figure 2. The main peak of the exosomes is concentrated in 30–150 nm, which is consistent with the standard physicochemical characteristics of exosomes.

[0038] The composite gel remains in a fluid state at low temperatures, meeting the needs of intrauterine injection or non-invasive topical application; at physiological temperatures, it is in situ solidified and implanted at the site of injury, and the exosomes are continuously released by the slow degradation of the gel.

[0039] Example 3: In vivo efficacy test for repairing endometrial damage in rats Female SD rats (250-300g) were randomly divided into a control group and an experimental group, with 8 rats in each group. Anhydrous ethanol was injected into the uterine cavity and left for 5 minutes to destroy the endometrial epithelium, stroma, and glands, causing endometrial thinning, fibrosis, and implantation failure. The rat endometrial injury model was then established by washing with PBS. After 21 days, a repair injection was performed, with 100 μL injected into each uterus once.

[0040] Experimental group: Local injection of the exosome-carrying gel prepared in Example 2 of this invention into the uterine cavity; Control group 1: No repair drug intervention was applied; only blank chitosan gel without exosomes was injected.

[0041] Control group 2: Injection of only free exosomes. Due to their high fluidity, free exosomes will flow out through the wound after injection, failing to achieve effective drug delivery, and the effect is equivalent to no drug delivery.

[0042] After the rearing period ended, males and females were kept together in cages, and the birth data for each group were collected. The results are shown in the table below:

[0043] This experiment used the classic ethanol-induced thin endometrial injury model in the field of reproduction, and used the number of rats born (fertility function) as the gold standard evaluation index for endometrial repair effect; the only variable between the two groups was whether the gel was loaded with exosomes, and the other modeling and drug administration conditions were completely the same.

[0044] The control group was injected with only blank chitosan gel, and the average number of offspring was extremely low.

[0045] After the drug administration cycle, rats were sacrificed, and bilateral intact uterine tissue was isolated for macroscopic morphological observation. The results of the isolated uterus comparison are shown in Figure 3: In the control group, which was injected with only blank gel, the endometrium was atrophied, the uterine cavity was narrowed, and tissue fibrosis was obvious; In the experimental group, which was treated with the exosome-loaded composite gel of this invention, the uterine cavity was filled, the endometrial tissue thickness was significantly restored, and embryo formation was visible. The macroscopic morphology directly confirms that this preparation has excellent repair capabilities for damaged endometrium. Combined with the results of the farrowing test, it is proved that chitosan thermosensitive gel alone has no significant repair or fertility-promoting effect on damaged endometrium; After the experimental group was injected with the exosome-loaded composite gel, the average number of offspring increased to more than 4 times that of the control group, with a significant difference.

[0046] The results confirm that the chitosan thermosensitive gel loaded with exosomes of the present invention can effectively repair ethanol-induced endometrial damage, restore the physiological functions of endometrial embryo implantation and pregnancy, and significantly improve the ability of rats to conceive and give birth after injury. Exosomes are the core effective component for endometrial repair, and the thermosensitive gel carrier can achieve the repair effect through in-situ gelation and long-term slow release of large exosomes.

Claims

1. An exosome in situ gel for damage repair, comprising a mixture of thermosensitive chitosan hydrogel and exosomes; characterized in that, The chitosan hydrogel was prepared by mixing an acidic chitosan solution with an aqueous solution of sodium β-glycerophosphate in an ice bath; the acidic chitosan solution had a mass fraction of 1.5-3 wt%, the sodium β-glycerophosphate solution had a mass fraction of 40-60 wt%, and the volume ratio of the two was 3-9:

1. The exosomes are exosomes derived from umbilical cord mesenchymal stem cells.

2. The exosome in situ gel according to claim 1, characterized in that, The chitosan acidic solution is prepared by dispersing chitosan in an acidic solution.

3. The exosome in situ gel according to claim 1, characterized in that, The number average molecular weight of the chitosan is 40,000-60,000, preferably 45,000-55,000.

4. The exosome in situ gel according to claim 1, characterized in that, The acidic solution is an acetic acid solution or a hydrochloric acid solution; preferably, the concentration of the acidic solution is 0.06 to 0.12 M.

5. The exosome in situ gel according to claim 1, characterized in that, The chitosan acidic solution contains 1.5–3 wt% chitosan.

6. The exosome in situ gel according to claim 1, characterized in that, The thermosensitive chitosan hydrogel is mixed with exosomes at a volume ratio of 1:10-10:1, preferably 1:5-5:

1.

7. The exosome in situ gel according to claim 1, characterized in that, The exosome particle concentration was 10. 8 ~10 10 per mL.

8. A method for preparing exosome in situ gel for damage repair according to any one of claims 1-7, comprising the following steps: S1. Dissolve chitosan in an acidic solution, stir and filter, store in an ice bath, and prepare an acidic chitosan solution. S2. Prepare an aqueous solution of sodium β-glycerophosphate; S3. Under ice bath conditions, add sodium β-glycerophosphate solution to chitosan solution at a volume ratio of 3-9:1 and mix well to obtain blank thermosensitive chitosan gel. S4. Take exosomes and mix them with an equal volume of blank thermosensitive gel, incubate to form a gel, and obtain the target gel.

9. The use of the exosome in situ gel for damage repair according to any one of claims 1-7 in the preparation of a drug for repairing endometrial damage, improving fertility due to endometrial damage, and improving the embryo implantation ability of the endometrium; Preferably, the exosomes are derived from human / animal mesenchymal stem cells.

10. A pharmaceutical composition for endometrial repair, comprising the exosome in situ gel for damage repair as described in any one of claims 1-7; Preferably, the pharmaceutical composition is formulated as an injection.

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