Nano vesicle, preparation method and application of nano vesicle in preparation of endometrial injury repair medicine

Abstract

The invention relates to the technical field of modernization of traditional Chinese medicines, in particular to a nano-vesicle, a preparation method and application of the nano-vesicle in preparation of an endometrial injury repairing medicine. The preparation method comprises the following steps: taking a periplaneta americana ethanol extracting solution, and adding a buffer solution to prepare diluted homogenate; performing differential centrifugal treatment on the homogenate; filtering the supernatant obtained by the differential centrifugation treatment, and then centrifuging for 60-120 minutes by 100000g of the supernatant to obtain a precipitate; and re-suspending the precipitate to obtain the periplaneta americana ethanol extract source nano-vesicles. The periplaneta americana ethanol extract is used as a starting material to enrich the nano-vesicles for the first time, the combined process of differential centrifugation, membrane filtration and ultracentrifugation is adopted, the obtained nano-vesicles can be applied to the field of endometrial injury repair, especially treatment of gynecological diseases such as intrauterine adhesion and infertility with thin intima, and the blank in the technical field is filled.

Description

Technical Field

[0001] This invention relates to the field of modernization of traditional Chinese medicine, and in particular to a nanovesicle, its preparation method, and its application in the preparation of drugs for repairing endometrial damage. Background Technology

[0002] Ethanol extract of the American cockroach is the main active ingredient in the traditional Chinese medicine preparation Kangfuxin Liquid, traditionally used for the repair of digestive tract ulcers, burns, and surgical wounds. In recent years, research has discovered that nanoscale vesicles or vesicle-like structures extracted from traditional Chinese medicinal materials possess unique biological activities, capable of delivering signaling molecules such as proteins and nucleic acids between cells, and participating in tissue repair and immune regulation. However, current research on American cockroach-derived nanovesicles mainly focuses on natural vesicle-like structures directly extracted from fresh insect tissue, and systematic research on obtaining functional nanovesicles through artificial enrichment using ethanol extract as a starting material has not yet been conducted.

[0003] Furthermore, existing technologies primarily focus on skin trauma repair and have not yet been extended to the field of endometrial injury. Endometrial injury (such as intrauterine adhesions, thin endometrium infertility, etc.) is a pathological condition characterized by the absence of endometrial glands and fibrotic hyperplasia. Traditional treatments have limited effectiveness, and there is an urgent need to develop new repair strategies.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a nanovesicle, a preparation method thereof, and its application in the preparation of drugs for repairing endometrial damage, aiming to expand the new uses of American cockroach-derived nanovesicles in gynecological regenerative medicine.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing nanovesicles, comprising the following steps: S1. Take the ethanol extract of American cockroaches, add buffer solution, and prepare a diluted homogenate. S2. The homogenate is subjected to differential centrifugation. S3. Filter the supernatant obtained by differential centrifugation, and then centrifuge at 100,000g for 90 minutes to obtain the precipitate; S4. Resuspend the precipitate to obtain nanovesicles derived from the ethanol extract of American cockroaches.

[0007] Optionally, in step S1, the volume ratio of the American cockroach ethanol extract to the buffer solution is 1:10-100.

[0008] Optionally, in step S1, the buffer solution is PBS buffer, HEPES buffer, physiological saline, or a buffer solution containing a protectant.

[0009] Optionally, in step S2, the differential centrifugation process involves first centrifuging at 3000-8000g for 20-40 minutes, and then centrifuging at 8000-12000g for 20-60 minutes.

[0010] Optionally, in step S3, filtration is performed using sterile 0.1μm, 0.22μm and / or 0.45μm filters.

[0011] Optionally, in step S4, when resuspending the precipitate, PBS buffer or a buffer containing sucrose, glycerol or albumin is used.

[0012] In a second aspect, the present invention provides a nanovesicle prepared by the method described above.

[0013] In a third aspect, the present invention provides the application of nanovesicles in the preparation of drugs for repairing endometrial damage.

[0014] Beneficial Effects: This invention is the first to enrich nanovesicles using ethanol extract of the American cockroach as a starting material, offering advantages such as stable raw material sources, controllable processes, and suitability for large-scale production. The combined process of differential centrifugation + membrane filtration + ultracentrifugation effectively removes mucus and large molecular aggregates, improving the purity and yield of nanovesicles and meeting GMP-level production requirements. This invention also marks the first application of American cockroach-derived nanovesicles in the field of endometrial damage repair, particularly for gynecological conditions such as intrauterine adhesions and thin endometrial infertility, filling a gap in this technological field. Attached Figure Description

[0015] Figure 1 This is a flowchart illustrating the preparation of nanovesicles derived from the ethanol extract of the American cockroach in Example 1.

[0016] Figure 2 Electron micrograph of nanovesicles derived from the ethanol extract of the American cockroach.

[0017] Figure 3 Electron microscopy particle size distribution of nanovesicles derived from ethanol extract of American cockroach.

[0018] Figure 4 Images showing the appearance of uterine tissue in each group.

[0019] Figure 5 HE-stained sections of uterine tissue from each group.

[0020] Figure 6 Masson-stained sections of uterine tissue from each group.

[0021] Figure 7 To validate the key molecular protein banding for ferroptosis using Western blotting. Detailed Implementation

[0022] This invention provides a nanovesicle, its preparation method, and its application in the preparation of drugs for repairing endometrial damage. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0023] This embodiment provides a method for preparing nanovesicles derived from the ethanol extract of the American cockroach, including the following steps. S1. Take the ethanol extract of American cockroaches, add buffer solution, and prepare a diluted homogenate. S2. The homogenate is subjected to differential centrifugation. S3. Filter the supernatant obtained by differential centrifugation, and then centrifuge at 100000g for 60-120min to obtain the precipitate. S4. Resuspend the precipitate to obtain nanovesicles derived from the ethanol extract of American cockroaches.

[0024] This embodiment uses American cockroach ethanol extract as raw material to prepare American cockroach ethanol extract-derived nanovesicles (hereinafter referred to as "Pa-EVs") through a process of "dilution + differential centrifugation (to remove mucus / large particles) + filtration + 100,000g ultracentrifugation precipitation (to achieve efficient enrichment and purification of nanovesicles) + resuspension". This process has advantages such as stable raw material source, controllable process, and suitability for large-scale production.

[0025] In some embodiments, in step S1, the volume ratio of the American cockroach ethanol extract to the buffer solution is 1:10-100. Diluting the American cockroach ethanol extract in a range of 1:10 to 1:100 allows the high concentration of ethanol to be instantly reduced to a safe range that will not denature proteins or nanovesicles, while maintaining ionic strength and pH stability. This ensures that the sample density and viscosity are low during subsequent ultracentrifugation, preventing the formation of a dense layer on the membrane surface that would clog the filter membrane due to excessively high alcohol concentration, and also preventing vesicle rupture due to sudden changes in osmotic pressure.

[0026] In some embodiments, in step S1, the buffer solution is PBS buffer, HEPES buffer, physiological saline, or a buffer solution containing a protective agent.

[0027] It should be noted that using PBS or HEPES buffer can improve cryopreservation stability and enable long-term storage.

[0028] In some embodiments, in step S2, the differential centrifugation process involves first centrifuging at 3000-8000g for 20-40 minutes (which can be 20 minutes, 30 minutes, or 40 minutes, preferably 30 minutes), and then centrifuging at 8000-12000g for 20-60 minutes.

[0029] It should be noted that centrifugation at 3000-8000g for 20-40 minutes can completely settle intact cells, cell debris, and cell nuclei (5-15 µm in diameter); centrifugation at 8000-12000g for 20-60 minutes can further remove the remaining mitochondria, lysosomes, and peroxisomes. However, this speed is still far from reaching the critical sedimentation rate for vesicles with a diameter of 50-200 nm, and they remain in the supernatant.

[0030] In some embodiments, in step S3, filtration is performed using sterile 0.1 μm, 0.22 μm and / or 0.45 μm filters.

[0031] In some embodiments, in step S4, when resuspending the precipitate, a PBS buffer or a buffer containing sucrose, glycerol or albumin is used.

[0032] It should be noted that before step S4, the obtained precipitate can be subjected to density differential centrifugation, size exclusion chromatography (SEC), etc. Density differential centrifugation: placing the obtained precipitate in a sucrose or iodixanol (OptiPrep) gradient for isodense / buoyancy separation yields higher purity and separates EVs from protein / lipid-rich contaminants (commonly used for research-grade high-purity preparation). It is highly valuable for demonstrating vesicle biological functions and conducting preclinical studies. Size exclusion chromatography (SEC): using a qEV or other SEC column to desalt and deproteinize samples obtained by ultracentrifugation or TFF concentrate gently separates EVs from free proteins. It is a preferred method for preserving activity and improving purity, and is easy to scale up (or coupled with TFF).

[0033] It should be noted that lyophilization or the addition of low-concentration protective agents (trehalose, alcohol) can also be used to improve stability when the room temperature or cold chain is unstable.

[0034] This embodiment also provides a nanovesicle prepared by the method described above.

[0035] The nanovesicles in this embodiment were prepared using ethanol extract of American cockroaches. They have a typical nanovesicle structure with a particle size concentrated at around 120 nm. They are morphologically intact, have few impurities, and meet the quality requirements for local drug delivery to the endometrium.

[0036] This embodiment also provides the application of American cockroach ethanol extract-derived nanovesicles in the repair of endometrial damage.

[0037] Example 1 I. Preparation of nanovesicles derived from the ethanol extract of the American cockroach, such as Figure 1 As shown, it includes the following steps: S1. Take the ethanol extract of American cockroaches, add buffer solution, and prepare a diluted homogenate. S2. The homogenate is subjected to differential centrifugation, including centrifugation at 5000g for 30 min and centrifugation at 10000g for 60 min; S3. The supernatant obtained by differential centrifugation is filtered through a sterile 0.22μm filter and then centrifuged at 100000g for 90min to obtain a precipitate. S4. Resuspend the precipitate with buffer solution to obtain nanovesicles derived from the ethanol extract of American cockroaches.

[0038] The American cockroach ethanol extract in this embodiment was purchased from Jingjiang Tianli Feed Technology Co., Ltd. The preparation method generally includes: dried insect powder → soaking in 90% ethanol at room temperature for 30 min → reflux extraction at 70-85 ℃ 3-5 times and combining the extracts → concentration under reduced pressure at 55-65 ℃ to a relative density of 1.05-1.15 → hot water degreasing and settling for oil separation → collecting the lower aqueous phase and further concentrating under reduced pressure to a relative density of 1.25-1.35 to obtain a 10-12% ethanol extract.

[0039] Figure 2 Electron micrograph of nanovesicles derived from the ethanol extract of the American cockroach. Figure 3 Electron microscopy particle size distribution of nanovesicles derived from the ethanol extract of the American cockroach. Figure 2 and Figure 3 It can be seen that the nanovesicles (Pa-EVs) in this embodiment have a typical nanovesicle structure with a particle size concentrated at around 120 nm. They have intact morphology and few impurities, which meets the quality requirements for local drug delivery to the endometrium.

[0040] II. Animal experiments: 1. The experimental mice were 24 C57BL / 6 mice aged 8 weeks or older (excluding pregnant mice), all of which were female, with 8 mice in each group. They were randomly divided into a sham-operated group (Sham group), a model group (TE group), and a nanovesicle treatment group derived from American cockroach ethanol extract (Pa.EVs group), with the Sham group having normal endometrium.

[0041] 2. Animal model establishment and drug intervention: The experimental mice were selected and anesthetized with 0.2 ml of 5% pentobarbital via intraperitoneal injection (waiting 5 minutes for pain reflex to subside). The abdomen was prepared and disinfected, and a 2 mm incision was made along the midline of the lower abdomen. The skin and abdominal wall layers were then sequentially cut to expose the uterus. The proximal and distal ends of one uterus were ligated with vein clamps. Disposable screw-cap syringes were prepared with 1 ml of PBS and 0.4 ml of 95% anhydrous ethanol. An 8 mm needle was then inserted into the uterine cavity, and 95% anhydrous ethanol was slowly injected into the proximal end of the uterus, maintaining a distended state for 25 seconds (20-40 seconds, depending on the size of the uterus) to simulate endometrial injury. The needle was then fixed, and PBS was added for flushing. The distal vein clamp was released, and the uterine cavity was flushed with another 1 ml of PBS. PBS flushing fluid was observed flowing from the vagina. This fluid was absorbed with sterile gauze to ensure complete removal of the ethanol. Finally, the syringe was aspirated to remove the fluid from the uterine cavity. The same procedure was performed on the other uterus. The TE group and the Pa.EVs group were given PBS buffer and a nanovesicle solution (10 mg / kg) derived from the ethanol extract of American cockroaches, respectively. After the uterus returned to its normal state, it was fixed with wound clips or closed layer by layer, and the patients were allowed free access to food and water. Uterine samples were collected from each group after the experiment for subsequent analysis.

[0042] 3. Hematoxylin and eosin (H&E) staining: (1) Dehydration and clearing: The mouse tissue samples from step 2 were dehydrated sequentially by 50%, 70%, and 80% ethanol for 2 hours each, and by 90%, 95%, and 100% ethanol for 1 hour each; then cleared by xylene I and II for 15 minutes each; and finally impregnated by liquid paraffin I and II for 1 hour each. (2) Embedding and sectioning: After tissue embedding, sections were cut to a thickness of 4μm, spread in warm water at 50℃, and fixed by baking at 60℃; (3) Dewaxing and rehydration: The sections were immersed in xylene I and II for 10 minutes each for dewaxing; then they were rehydrated in a gradient of anhydrous ethanol (twice), 95% ethanol and 85% ethanol for 5 minutes each, and then rinsed with running water for 2 minutes. (4) HE staining: stain with hematoxylin for 15 min and rinse with running water; differentiate with 1% hydrochloric acid ethanol for 10 s, then invert blue with warm water at 40-60℃; stain with eosin for 75 s and rinse with running water for 10 s. (5) Dehydration and sealing: The slides are sequentially dehydrated by 85%, 95%, and 100% (twice) ethanol, and then cleared by xylene I and II before sealing. 4. Masson staining: (1) Dewaxing the slices to water: xylene → graded ethanol → distilled water.

[0043] (2) Weigert iron hematoxylin staining nucleus for 5-10 min, wash with water; hydrochloric acid alcohol differentiation for a few seconds, rinse with running water for 5 min; Ponceau S and acid fuchsin staining cytoplasm for 5-10 min, wash with water; 1% phosphomolybdic acid differentiation for 5 min; aniline blue (or brilliant green) staining collagen for 2 min, wash with water; 1% glacial acetic acid quick wash for a few seconds.

[0044] (3) Dehydrated transparent, neutral resin sealing film.

[0045] 5. Western blotting: Experimental grouping and treatment The mouse tissues from step 2 were divided into sham group, model group, Pa-EVs group, and were flash-frozen in liquid nitrogen and stored at -80℃.

[0046] tissue lysis Approximately 30 mg of uterine protein was placed in a 2 mL glass homogenizer, and 400 μL of pre-chilled RIPA (containing 1 mmol / L PMSF, 1% phosphatase inhibitor, and 1% protease inhibitor cocktail) was added. The homogenizer was homogenized 60 times on ice with 10-second intervals. The homogenate was then transferred to 1.5 mL EP tubes and centrifuged at 12,000 rpm for 15 min at 4 °C. The supernatant was collected. Protein concentration was determined by BCA method. The protein was diluted to 3 μg / μL with 1× Loading Buffer, denatured in a 100 °C metal bath for 10 min, aliquoted, and stored at -80 °C, avoiding repeated freeze-thaw cycles.

[0047] Electrophoresis and membrane transfer Prepare 10% separating gels for NSUN5 (55kDa) and 12% separating gels for GPX4. Load 40μg total protein into each well, first at 80V for 30min, then at 120V for 60min until bromophenol blue appears on the bottom of the gel. Use a 0.22μm PVDF membrane, activate with methanol for 2min, transfer at 250mA at 4℃ for 90min, and cycle on ice. After confirming the integrity of the bands by Ponceau S staining, wash three times with TBST for 5min each time.

[0048] Blocking and antibody incubation Block with 5% skim milk powder using TBST at room temperature for 2 hours. Primary antibody dilution: NSUN5 1:1000, GPX4 1:1000, incubate overnight at 4°C on a shaker. The next day, wash with TBST 3 × 10 min, incubate with HRP-labeled goat anti-rabbit at 1:5000 at room temperature for 1 hour, and then wash with TBST 3 × 10 min again.

[0049] Development and Quantification The ECL chemiluminescent solution was used to uniformly cover the membrane, and images were acquired using the ChemiDoc system. Exposure time gradients ranged from 5 to 30 seconds to avoid band saturation. Grayscale values ​​were read using ImageJ software, and the relative expression level of the target protein was calculated using β-actin as an internal reference. Differences between groups were analyzed using one-way ANOVA, with P < 0.05 considered statistically significant.

[0050] Figure 4 Images showing the appearance of uterine tissue in each group. Figure 4 As can be seen, in the sham surgery group (Sham group), the uterine horns were full, the lumen was transparent, and there was no congestion; in the TE group, the uterine lumen was congested, significantly stiff, and lost its elasticity. In the nanovesicle treatment group (Pa.EVs group) derived from the ethanol extract of American cockroaches, the lumen was reddish in color and the fibrotic bundles were reduced, which directly proves that vesicles can treat adhesions and restore uterine patency.

[0051] Figure 5 Images of HE-stained sections of uterine tissue from each group. Figure 5 It is evident that in the TE group, the endometrial layer was thinner, glands were sparse, and the muscle layer was disordered; while in the Pa.EVs group, the endometrial thickness was significantly increased, the number of glands rebounded, and the luminal epithelium and basal layer structure remained intact, indicating that vesicles promote the regeneration of endometrial glands and matrix, restoring the basic physiological function.

[0052] Figure 6 Masson-stained sections of uterine tissue from each group. Figure 6 It can be seen that in the TE group, the indigo-stained area accounted for 60-70%, and the collagen was coarse and disordered; in the Pa.EVs group, the indigo-stained area decreased to 20-30%, and the collagen was fine and regularly arranged, confirming that vesicles inhibit excessive collagen deposition and block the progression of fibrosis.

[0053] comprehensive Figures 4-6 The results showed that the Pa-EVs treatment group exhibited good endometrial structure recovery, increased glandular number, regular collagen deposition, and reduced fibrosis, significantly superior to the model group, demonstrating its specific advantages in endometrial repair. The results also indicated that Pa-EVs can significantly restore endometrial thickness, increase glandular number, and improve collagen deposition structure, suggesting its promising application in endometrial regeneration.

[0054] NSUN5 is an RNA cytosine methyltransferase that catalyzes the 5-methylcytosine (MCT) transfer of FTH1 (heavy chain ferritin) and FTL (light chain ferritin) mRNA. 5 C) Modification to improve its stability and translation efficiency. GPX4 is a key ferroptosis defense enzyme that requires Fe. 2+ -ROS substrate maintains activity; when Fe 2+ During accumulation and ROS bursts, GPX4 activity decreases, cell membrane lipid peroxides cannot be reduced, triggering ferroptosis. Figure 7To validate the banding of key molecular proteins involved in ferroptosis using Western blotting. From Figure 7 It was found that the ethanol extract nanovesicles of the American cockroach (Pa-EVs) inhibited ferroptosis in endometrial cells through the "NSUN5-FTH1 / FTL-GPX4" axis. This indicates that Pa-EVs, through mechanisms such as regulating ferroptosis, promoting angiogenesis, and inhibiting fibrosis, precisely match the low-inflammatory and regenerative microenvironment required for endometrial repair, demonstrating superior performance compared to traditional skin wound repair models.

[0055] After Pa-EVs carry miRNAs and proteins into endometrial cells, they upregulate NSUN5 expression (or activity) and enhance the mRNA expression of FTH1 / FTL. 5 C modification increases ferritin synthesis, "locking" iron ions within the ferritin and blocking free Fe. 2+ -ROS-GPX4 inactivation creates a vicious cycle, thereby preventing ferroptosis. In pathological processes such as intrauterine adhesions and endometrial damage, endometrial cells often undergo ferroptosis due to ischemia-reperfusion or oxidative stress; Pa-EVs provide a non-classical, RNA epigenetic anti-ferroptosis strategy through the new axis of "NSUN5-FTH1 / FTL-GPX4".

[0056] Compared to the collagen deposition healing mechanism required for conventional skin wound repair, the American cockroach exosomes provided in this invention target and inhibit the TGF-β / Smad2 signaling pathway through their high abundance of miR-125b / 30c and Reucalcin, blocking myofibroblast activation and downregulating α-SMA and COL1A1 expression. At the same time, they carry miR-21-5p antagonists to accelerate macrophage M1→M2 polarization, shorten the inflammatory period, and prevent the formation of collagen fiber bridges. Combined with VEGF-like peptides and 20-hydroxyecdysone, they rapidly induce angiogenesis under the estrogen peak environment, achieving scarless regeneration of the endometrium. Therefore, they have the advantages of more precision, lower dosage, and better efficacy in intrauterine adhesions with "anti-fibrosis + functional layer reconstruction" as the treatment endpoint.

[0057] Currently, the use of purified insect exovesicles for treatment is not approved. This invention uses American cockroach ethanol extract as raw material, and prepares 100 nm nanovesicles in one step through depressurized alcohol removal, membrane emulsification and extrusion. Compared with the traditional method of directly crushing insect bodies and ultracentrifuging, the combination of ethanol precipitation and 0.22 µm filtration can remove >90% of macromolecular allergens, making the detection amount of Pera1 lower than the sensitivity of ELISA. It has the advantages of low immunogenicity and convenient registration.

[0058] In summary, this invention is the first to enrich nanovesicles using ethanol extract of the American cockroach as a starting material, offering advantages such as stable raw material sources, controllable processes, and suitability for large-scale production. The combined process of differential centrifugation + membrane filtration + ultracentrifugation effectively removes mucus and large molecular aggregates, improving nanovesicle purity and yield, and meeting GMP-level production requirements. This invention also established a C57BL / 6 female mouse uterine adhesion model to evaluate the repair effects of Pa-EVs on endometrial structure and function. The results showed that Pa-EVs significantly restored endometrial thickness, increased glandular number, and improved collagen deposition structure, indicating its promising application in endometrial regeneration.

[0059] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing nanovesicles, characterized in that, Includes the following steps, S1. Take the ethanol extract of American cockroaches, add buffer solution, and prepare a diluted homogenate. S2. The homogenate is subjected to differential centrifugation. S3. Filter the supernatant obtained by differential centrifugation, and then centrifuge at 100,000g for 90 minutes to obtain the precipitate; S4. Resuspend the precipitate to obtain nanovesicles derived from the ethanol extract of American cockroaches.

2. The method for preparing nanovesicles according to claim 1, characterized in that, In step S1, the volume ratio of the American cockroach ethanol extract to the buffer solution is 1:10-100.

3. The method for preparing nanovesicles according to claim 1, characterized in that, In step S1, the buffer solution is PBS buffer, HEPES buffer, physiological saline, or a buffer solution containing a protective agent.

4. The method for preparing nanovesicles according to claim 1, characterized in that, In step S2, the differential centrifugation process involves first centrifuging at 3000-8000g for 20-40 minutes, and then centrifuging at 8000-12000g for 20-60 minutes.

5. The method for preparing nanovesicles according to claim 1, characterized in that, In step S3, filtration is performed using sterile 0.1μm, 0.22μm and / or 0.45μm filters.

6. The method for preparing nanovesicles according to claim 1, characterized in that, In step S4, when resuspending the precipitate, PBS buffer or a buffer containing sucrose, glycerol or albumin is used.

7. A nanovesicle, characterized in that, Prepared by the method according to any one of claims 1-6.

8. The use of the nanovesicles as described in claim 7 in the preparation of a drug for repairing endometrial damage.

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