Application of adipose-derived stem cell artificial nano-vesicle in preparation of medicine for promoting thrombogenesis, adipose-derived stem cell artificial nano-vesicle loaded with sanguisorba monomer DMAG and preparation method of adipose-derived stem cell artificial nano-vesicle loaded with sanguisorba monomer DMAG

Abstract

The invention provides application of adipose-derived stem cell artificial nano-vesicles in preparation of drugs for promoting platelet generation. The invention also provides an adipose-derived stem cell artificial nano-vesicle loaded with a sanguisorba monomer DMAG, and the adipose-derived stem cell artificial nano-vesicle is prepared by extracting a cell membrane of adipose-derived stem cells and entrapping DMAG by using a natural phospholipid bilayer structure of the cell membrane so as to form a nano-sized vesicle structure. The artificial nano-vesicle inherits good biocompatibility of adipose-derived stem cells, can effectively reduce immunological rejection of an organism, and protects DMAG from degradation effects such as in-vivo enzymolysis and the like; and a protective delivery system can be formed in vivo, so that rapid metabolism and elimination of DMAG in vivo are reduced, and the effective release time of DMAG in vivo is prolonged. The artificial nano-vesicles significantly increase the number of peripheral blood platelets, shorten the bleeding time and reduce the immunogenicity at the same time.

Description

Technical Field

[0001] This invention relates to the use of adipose-derived stem cell artificial nanovesicles in the preparation of drugs that promote platelet production, as well as an adipose-derived stem cell artificial nanovesicle loaded with DMAG, its preparation method, and its use. Background Technology

[0002] Thrombocytopenia is a common blood disorder characterized by a decrease in the number of platelets in peripheral blood, leading to an increased bleeding tendency. Chemotherapy, radiotherapy, immune diseases, and hematopoietic stem cell transplantation can all cause thrombocytopenia. Currently, clinical treatment mainly relies on platelet transfusions or TPO receptor agonists, but these methods carry risks such as donor shortages, infection risks, drug side effects, and immune responses to platelet transfusions.

[0003] 3,3'-Dimethoxyellagic acid-4'-O-β-D-glucoside (DMAG) is a polyphenolic compound extracted from Sanguisorba officinalis (CAS No. 51803-68-0). It possesses various biological activities, including anti-inflammatory, antioxidant, and cell proliferation-promoting effects. Studies have shown that DMAG can promote megakaryocyte differentiation and maturation by activating the PI3K / Akt / mTOR signaling pathway, thereby increasing platelet production. Wang Long et al. (Southwest Medical University, 2021-08-12) disclosed the platelet-promoting function of DMAG. However, its poor water solubility, short in vivo half-life, and weak bone marrow targeting greatly limit its clinical application.

[0004] Adipose-derived stem cells (ADSCs) are a type of adult stem cell with multipotent differentiation potential, and are abundant and readily available. ADSCs can secrete a variety of bioactive factors that support the proliferation and differentiation of hematopoietic cells. Furthermore, extracellular vesicles (EVs) derived from ADSCs exhibit good biocompatibility and low immunogenicity, making them suitable as drug delivery systems.

[0005] However, natural vesicles (EVs) have low yields, non-editable contents, poor targeting, and lack specific active molecules that promote platelet production. Artificial nanovesicles (AVs) are vesicle-like structures with nanoscale dimensions (typically 1-1000 nanometers) prepared through artificial synthesis or engineering. These vesicles possess a bilayer phospholipid structure or polymer membrane structure similar to a cell membrane, capable of encapsulating bioactive substances such as drugs, genes, and proteins. In the biomedical field, they are mainly used for drug delivery, disease diagnosis, and tissue engineering. This invention, by loading DMAG into artificial vesicles (AVs) derived from adipose-derived stem cells, enables targeted drug delivery, increases drug concentration in target tissues, and reduces side effects on other tissues.

[0006] Zhao Chen, Research on the efficacy and mechanism of action of adipose-derived stem cell-derived exosomes in treating knee osteoarthritis, Soochow University, March 2020, disclosed that adipose-derived stem cell-derived exosomes can be used to treat knee osteoarthritis. Liu Wenjian, Human adipose-derived stem cell extracellular vesicles accelerate diabetic wound healing by promoting PI3K-AKT-mTOR-HIF-1α-mediated angiogenesis, Nanchang University, May 2023, disclosed the use of human adipose-derived stem cell extracellular vesicles in treating diabetic wounds.

[0007] There are currently no reports of using adipose-derived stem cell vesicles to increase platelet count, nor are there any reports of combining DMAG with stem cell-derived artificial nanovesicles to increase platelet count. Summary of the Invention

[0008] This invention provides the use of adipose-derived stem cell artificial nanovesicles in the preparation of drugs that promote platelet production, as well as an adipose-derived stem cell artificial nanovesicle loaded with DMAG, its preparation method, and its use.

[0009] This invention provides the use of adipose-derived stem cell-derived artificial nanovesicles in the preparation of drugs that promote platelet production.

[0010] The drug mentioned is a medication for treating thrombocytopenia.

[0011] The adipose-derived stem cell artificial nanovesicles are derived from human or animal adipose tissue and are obtained through separation, culture and purification steps.

[0012] The method for preparing the artificial nanovesicles from adipose-derived stem cells includes the following steps:

[0013] Isolation and culture of adipose-derived stem cells: Adipose-derived stem cells are extracted from adipose tissue and cultured and expanded in a culture medium containing growth factors;

[0014] Cell membrane extraction: Cultured adipose-derived stem cells are collected, and cell membrane components are extracted using physical or chemical methods. This invention also provides the use of adipose-derived stem cell artificial nanovesicles in combination with DMAG in the preparation of drugs that promote platelet production, wherein the mass ratio of adipose-derived stem cell artificial nanovesicles to DMAG is:

[0015] 3-7 parts adipose-derived stem cell artificial nanovesicles, 1 part DMAG; preferably, the mass ratio of adipose-derived stem cell artificial nanovesicles to DMAG is:

[0016] Five portions of adipose-derived stem cell artificial nanovesicles and one portion of DMAG.

[0017] The drug mentioned is a medication for treating thrombocytopenia.

[0018] This invention provides an adipose-derived stem cell artificial nanovesicle loaded with DMAG, wherein the adipose-derived stem cell artificial nanovesicle is used as a carrier to load DMAG, and the mass ratio of the adipose-derived stem cell artificial nanovesicle to DMAG is:

[0019] 3-7 parts of adipose-derived stem cell artificial nanovesicles and 1 part of DMAG.

[0020] Preferably, the mass ratio of adipose-derived stem cell artificial nanovesicles to DMAG is:

[0021] Five portions of adipose-derived stem cell artificial nanovesicles and one portion of DMAG.

[0022] The present invention also provides a method for preparing the DMAG-loaded adipose stem cell artificial nanovesicles, which includes the following steps:

[0023] a. Weigh out adipose-derived stem cell artificial nanovesicles and DMAG;

[0024] b. Mix DMAG with cell membrane components and use ultrasound-assisted methods or other suitable nanoencapsulation techniques to induce the cell membrane to encapsulate DMAG to form nanovesicles.

[0025] The present invention also provides the use of the aforementioned DMAG-loaded adipose stem cell artificial nanovesicles in the preparation of drugs that promote platelet production.

[0026] The drug mentioned is an intravenous injection drug.

[0027] The artificial nanovesicles ADSCs-AVs@DMAG of this invention consist of the following three parts:

[0028] Membrane shell: derived from the cell membrane of adipose-derived stem cells (ADSC), retaining membrane proteins of the source cell (such as CD73, CD90, CXCR4); Inner nucleus: encapsulates DMAG or its pharmaceutically acceptable salts or derivatives;

[0029] The structural parameters are as follows: average particle size: 50–200 nm; zeta potential: -10 mV to -25 mV; DMAG loading rate: 7.98%; encapsulation efficiency: 34.7% (measured by HPLC).

[0030] The constructed nanovesicles typically have a particle size between 50 and 200 nm. This particle size range allows them to pass smoothly through physiological barriers such as capillary walls to reach local areas, while also exhibiting good colloidal stability and being less prone to aggregation and precipitation.

[0031] The encapsulation efficiency of DMAG in nanovesicles is around 34.7%, and the drug loading rate is 7.98%, ensuring that there is sufficient drug to exert its effect continuously.

[0032] Because of their adipose-derived stem cell membrane origin, artificial nanovesicles exhibit good biocompatibility, failing to elicit significant immune responses in both in vitro and in vivo experiments. Furthermore, they can prolong the in vivo circulation time of DMAG, significantly improving drug bioavailability compared to free drugs.

[0033] This invention encapsulates adipose-derived stem cell artificial vesicles with DMAG. Cell experiments (flow cytometry detection of cell surface antigen CD41 / 42b expression) and animal experiments (in vivo investigation of the bioactivity of DMAG-loaded artificial vesicles on mice with thrombocytopenia) both show that the DMAG-loaded adipose-derived stem cell artificial vesicles are more effective than ADSCs-Exo alone or DMAG alone, indicating that this formulation can effectively improve the therapeutic effect and precise delivery of drugs.

[0034] The core of this invention is the construction of artificial nanovesicles loaded with DMAG from adipose-derived stem cells. By extracting the cell membrane of adipose-derived stem cells and utilizing their natural phospholipid bilayer structure, DMAG is encapsulated to form nanoscale vesicle structures. These vesicles not only inherit the biocompatibility of adipose-derived stem cells, forming a protective delivery system in vivo, but also overcome the poor water solubility of DMAG, reducing its rapid metabolism and clearance in vivo, thus achieving slow and controlled drug release. This significantly increases peripheral blood platelet count and shortens bleeding time, while simultaneously reducing immunogenicity. Attached Figure Description

[0035] Figure 1 Morphology of ADSCs-Exo and ADSCs-AVs observed under an electron microscope (A: ADSCs-Exo, B: ADSCs-AVs);

[0036] Figure 2 Western blot analysis was performed to detect the expression of two vesicle surface markers (both conforming to the characteristics of three positive and one negative exosome markers).

[0037] Figure 3 NTA analysis of particle size and particle number from a single extraction of ADSCs-Exo and ADSCs-AVs (A represents ADSCs-Exo particle size, B represents ADSCs-AVs particle size, and C represents particle number from a single extraction of ADSCs-Exo and ADSCs-AVs. For comparisons between groups, * P < 0.05, ** P < 0.01, *** P < 0.001, n = 3).

[0038] Figure 4 Drug-loaded vesicles (A is the particle size of ADSCs-AVs@DMAG, B is the intact morphology of ADSCs-AVs@DMAG as shown by TEM).

[0039] Figure 5 A is the DMAG standard curve, B is the DMAG spectrum in ADSCs-AVs@DMAG artificial vesicles, and C is the spectrum of free DMAG in ADSCs-AVs@DMAG.

[0040] Figure 6 Effects of artificial vesicles on CD41 / 42b expression in Meg-01 cells (Note: Compared with the DMAG monotherapy group, *P<0.05, **P<0.01, and ***P<0.001, n=3).

[0041] Figure 7 Effect of artificial vesicles on peripheral blood platelet count in mice with thrombocytopenia (Note: * P < 0.05, ** P < 0.01, and *** P < 0.001, n = 8).

[0042] Figure 8 Effects of artificial vesicles on platelet levels in peripheral blood of mice with thrombocytopenia (Note: * P < 0.05, ** P < 0.01, and *** P < 0.001, n = 3).

[0043] Figure 9 Effects of artificial vesicles on hematopoietic cells in peripheral blood of mice with thrombocytopenia (Note: * P < 0.05, ** P < 0.01, and *** P < 0.001, n = 3).

[0044] Figure 10 Effects of artificial vesicles on megakaryocyte differentiation in bone marrow of mice with thrombocytopenia (Note: * P < 0.05, ** P < 0.01, and *** P < 0.001, n = 3). Detailed Implementation

[0045] Example 1: A method for preparing DMAG-loaded adipose-derived stem cell artificial nanovesicles

[0046] I. Isolation and Culture of Primary Adipose-Derived Mesenchymal Stem Cells

[0047] All experimental procedures were performed inside a laminar flow hood. Before the experiment, all necessary consumables and instruments were placed inside the laminar flow hood. Surgical instruments were sterilized by autoclaving and then put into use. The laminar flow hood was sterilized by ultraviolet irradiation for 30 minutes. The thermostatic magnetic stirrer was turned on and preheated to 37°C.

[0048] After euthanizing SD rat pups by cervical dislocation, they were immediately immersed in 75% ethanol for 5 minutes for surface disinfection. After disinfection, the pups were removed, the ethanol was drained, and the pups were placed abdomen-up on a clean bench lined with sterile absorbent paper. The skin was cut along the midline of the abdomen to expose the groin area, and the groin fat tissue was separated and removed.

[0049] The obtained adipose tissue was washed three times in a culture dish containing 5 mL of PBS to remove hair and blood residue. After washing, the adipose tissue was transferred to a new sterile culture dish, and visible blood vessels and lymphatic tissue were removed and discarded using anatomical scissors and forceps. The adipose tissue was then thoroughly minced to a paste-like consistency, a process that took approximately 15 minutes, and the overall operation time was controlled within 1 hour.

[0050] Transfer the minced adipose tissue to a 50mL yellow-capped reagent bottle, add 4 times its volume of 0.2% type I collagenase solution, and add a magnetic stir bar. Seal the bottle opening with sealing film and place it on a 37℃ constant temperature magnetic stirrer for digestion at 80rpm for 1 hour and 30 minutes.

[0051] After digestion, the outer surface of the reagent bottle was disinfected by spraying with 75% ethanol and transferred to a clean bench. The digestion solution was aspirated using a Pasteur pipette and filtered through a 200-mesh nylon mesh into a 50 mL centrifuge tube. An equal volume of DMEM / F12 complete culture medium was then added to terminate the digestion reaction. The tube was centrifuged at 1500 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in an appropriate amount of culture medium. The tube was then centrifuged again at 1200 rpm for 5 min. The supernatant was discarded, and the cell pellet was resuspended in 4 mL of culture medium. The resulting cell suspension was seeded into T25 cell culture flasks and cultured at 37°C, 5% CO2, and saturated humidity.

[0052] After 24 hours of culture, observe the cell growth status under a microscope. Generally, after 2–7 days of culture, adipose-derived mesenchymal stem cells can be observed to crawl out of the tissue block and gradually adhere to the wall. Based on the cell growth status, perform a half-volume medium replacement to remove non-adherent cells.

[0053] II. Extraction of exosomes from adipose-derived stem cells

[0054] Collect the cell supernatant from the culture of adipose-derived stem cells and centrifuge at 10,000 × g for 30 min at 4°C to remove cell debris and impurities. After centrifugation, carefully aspirate the supernatant and transfer it to a new sterile centrifuge tube.

[0055] The supernatant was transferred to a 100 kD ultrafiltration tube and centrifuged at 3500 × g for 15 min at 4 °C to concentrate the supernatant. After concentration, the resulting solution was filtered through a 0.22 μm microporous membrane, and the filtrate was collected.

[0056] The filtrate was transferred to an ultracentrifuge tube and centrifuged at 120,000 × g for 120 min at 4 °C. After centrifugation, the supernatant was discarded, and the precipitate was resuspended in 300 μL of pre-cooled PBS to obtain adipose-derived stem cell exosomes (ADSCs-Exo).

[0057] III. Extraction of extracellular vesicles from adipose-derived stem cells

[0058] The cultured adipose-derived stem cells were digested with trypsin digestion solution, and the cells were collected and washed twice with PBS. The cells were then placed in an ice bath for 15 min, followed by the addition of cell membrane extraction buffer and another 15 min in an ice bath. The samples were then subjected to three freeze-thaw cycles in liquid nitrogen to thoroughly disrupt the cell structure.

[0059] After freeze-thaw cycles, the sample was centrifuged at 700 g for 10 min, and the supernatant was collected and centrifuged again at 14000 g for 30 min. The supernatant was discarded, the precipitate was resuspended in PBS, and sonicated for 4 min to obtain adipose-derived stem cell artificial vesicles (ADSCs-AVs).

[0060] IV. Preparation of DMAG-loaded exosomes from adipose-derived stem cells

[0061] The exosomes obtained above were resuspended in PBS and diluted to the required concentration. DMAG was added at a mass ratio of 5:1 (exosomes to DMAG) to prepare a DMAG-extracellular vesicle suspension.

[0062] The obtained suspension was placed in an ultrasonic apparatus and treated at 28% ultrasonic intensity. A cycle of 15 seconds of sonication followed by a 2-minute pause was performed, for a total of four cycles, all on ice. After ultrasonication, the sample was allowed to stand at room temperature for 30 minutes to promote the recovery of the exosome membrane structure. It was then incubated overnight at 4°C in a shaker. The following day, the sample was processed using a 100 kD ultrafiltration tube to remove unloaded free DMAG, ultimately obtaining DMAG-loaded ADSCs-Exo@DMAG.

[0063] V. Preparation of DMAG-loaded adipose-derived stem cell vesicles

[0064] The vesicles obtained above were resuspended in PBS and diluted to the required concentration. DMAG was added at a mass ratio of 5:1 (extracellular vesicles to DMAG) to prepare a DMAG-extracellular vesicle suspension.

[0065] The obtained suspension was placed in an ultrasonic apparatus and treated at 28% ultrasonic intensity. A cycle of 15 seconds of sonication followed by a 2-minute pause was performed, for a total of four cycles, all on ice. After ultrasonication, the sample was allowed to stand at room temperature for 30 minutes to promote the recovery of the extracellular vesicle membrane structure. It was then incubated overnight at 4°C in a shaker. The following day, the sample was processed using a 100 kD ultrafiltration tube to remove unloaded free DMAG, ultimately obtaining DMAG-loaded ADSCs-AVs@DMAG.

[0066] The following experiments demonstrate the beneficial effects of the present invention.

[0067] Example 1: Characterization and efficacy test of ADSCs-AVs@DMAG of the present invention.

[0068] 1. Experimental steps:

[0069] 1.1 Morphological observation of extracellular vesicles of adipose-derived stem cells (ADSCs-Exo, ADSCs-AVs)

[0070] (1) Fixation with 4% paraformaldehyde;

[0071] (2) Add 5 μL of the resuspended precipitate to the copper mesh and dry for 20 min;

[0072] (3) Adsorption: For the prepared exosome sample, use a pipette to draw 10 μL of suspension and drop it onto the prepared Parafilm sealing film (the back of the sealing film is attached to the table). Place the copper mesh of the carrier film face down and let it naturally adsorb the suspension drop for 10-15 minutes. Then use filter paper strips to remove the excess drop and let it dry slightly.

[0073] (4) Staining: Pipette 10 μL of 2% phosphotungstic acid solution onto the sealing film, place the copper mesh with the front side facing the staining solution, and invert it to stand for 3-5 min.

[0074] (5) Drying: Use filter paper strips to absorb excess liquid droplets and air dry under an incandescent lamp;

[0075] (6) Observation and photography: Observe and photograph under a transmission electron microscope.

[0076] 1.2 Protein characterization of ADSCs-Exo and ADSCs-AVs

[0077] (1) Extraction of ADSCs-Exo and ADSCs-AVs proteins

[0078] After lysing ADSCs-Exo and ADSCs-AVs samples with exosome lysis buffer, add 5× protein loading buffer and boil in a 100℃ metal bath for 10 min to denature them.

[0079] (2) Western blot

[0080] a. Prepare the separating gel (lower layer) and stacking gel (upper layer) according to the instructions of the 10% PAGE gel rapid preparation kit. Prepare a 1.0 mm gel. Take equal volumes of the lower gel solution and lower gel buffer, as well as the upper gel solution and upper gel buffer. Add an appropriate amount of modified ammonium persulfate solution and mix well. First, pour the upper gel mixture into the gel preparation glass plate, then pour the lower gel mixture into the gel preparation glass plate. Insert the comb teeth and let it solidify at room temperature for about 20 minutes.

[0081] b. Based on the BCA protein concentration, add an appropriate volume of protein sample, and add 4 μL of protein marker per well;

[0082] c. First, adjust the voltage to a constant 80 V. When the bromophenol blue indicator of the electrophoresis sample reaches the separating gel, adjust the voltage to a constant 120 V and continue electrophoresis until the bromophenol blue indicator reaches the bottom of the separating gel. Soak a PVDF membrane of appropriate size in methanol for 5 min. Soak the materials in the transfer apparatus in pre-cooled transfer solution and place them in the following order from negative to positive: sponge pad - filter paper - PAGE gel - PVDF membrane - filter paper - sponge pad. Try to avoid the generation of air bubbles during the process. Transfer the membrane at a constant current of 250 mA. The transfer time is determined according to the molecular weight of the target protein band.

[0083] d. After the transfer is complete, remove the PVDF membrane and place it in an antibody incubation box containing PBST. After washing once, block with blocking buffer at room temperature for 1 h, and then incubate slowly in a shaker.

[0084] e. Dilute the antibody with the corresponding antibody diluent, add it to the antibody incubation box with the membrane facing up, and incubate overnight at 4°C. After the primary antibody incubation is complete, wash the membrane 3 times for 5 min with PBST.

[0085] f. After washing, add the corresponding secondary antibody, incubate at room temperature for 1 h, and wash the membrane 3 times × 5 min with PBST;

[0086] (3) Development

[0087] Prepare the ECL luminescent solution (mix solution A and solution B in a 1:1 ratio, prepare fresh before use), drop it onto the front side of the PVDF film, and develop it in a darkroom.

[0088] 1.3 Determination of particle size and concentration of ADSCs-Exo and ADSCs-AVs

[0089] ADSCs-Exo and ADSCs-AVs were diluted 10 times with PBS, and their particle size and concentration were determined by nanoparticle tracking analysis (NTA).

[0090] 1.4 Morphological observation of extracellular vesicles of adipose-derived stem cells encapsulating DMAG (ADSCs-AVs@DMAG)

[0091] After loading ADSCs-AVs with DMAG suspension, the morphology of ADSCs-AVs@DMAG drug-loaded vesicles was observed by electron microscopy, and the particle size of ADSCs-AVs@DMAG was detected by Malvern particle size analyzer.

[0092] 1.5 Determination of drug loading and encapsulation efficiency of ADSCs-AVs@DMAG artificial vesicles

[0093] The prepared ADSCs-AVs@DMAG was placed in an ultrafiltration tube and centrifuged at 3500×g for 15 min to obtain free DMAG and drug-loaded artificial vesicles. The volumes of the upper and lower layers were recorded. The membranes were ultrasonically ruptured using 0.1% Triton X-100 and transferred to high-performance liquid chromatography (HPLC) vials. The retention time and peak area were obtained by HPLC analysis. The values ​​were substituted into the DMAG standard curve to obtain the DMAG concentration in ADSCs-AVs@DMAG. The drug loading and encapsulation efficiency of ADSCs-AVs@DMAG were calculated according to the following formulas. The peak areas obtained were substituted into the regression equation of the DMAG standard curve to calculate the actual concentration of DMAG in the ADSCs-AVs@DMAG sample. The drug loading (DL) and encapsulation efficiency (EE) of ADSCs-AVs@DMAG were further calculated according to relevant formulas.

[0094] Calculation formula:

[0095] DL (%) = (Total drug content - Free drug content) / (Total carrier content + Total drug content - Free drug content)

[0096] EE (%) = (Total drug content - Free drug content) / Total drug content

[0097] 1.6 Flow cytometry detection of cell surface antigens CD41 / CD42b expression

[0098] On day 4 of drug intervention, cell suspension was collected and transferred to 2 mL EP tubes, centrifuged at 1500 r / min for 5 min, the supernatant was discarded, and the pellet was resuspended in 1 mL of pre-chilled PBS. The pellet was centrifuged again under the same conditions for 5 min, and the supernatant was discarded. Cell counting was performed on the cell suspension resuspended in pre-chilled PBS, and the density was adjusted to 3 × 10⁻⁶ cells / mL. 5 Cells / mL. Take 100 μL of cell suspension from each group and transfer it to a 1.5 mL EP tube. Add 5 μL of FITC-CD41 and 5 μL of PE-CD42b antibodies in the dark. Incubate at 4°C for 15 min. After incubation, add 400 μL of pre-chilled PBS to stop staining. Then filter the cell suspension through a 200-mesh nylon mesh and transfer it to a flow cytometry tube. Gently tap the tube to distribute the cells evenly. Detect using a flow cytometer. Samples should be detected within 1 h in the dark.

[0099] 1.7 Effect of artificial vesicles on peripheral blood platelet count in mice with thrombocytopenia

[0100] All drugs were administered via tail vein injection. The normal control and model groups received 0.1 mL of saline per 10 g of body weight via tail vein injection. The IL-11 positive drug group received 0.1 mL of saline containing 3 μg IL-11 (equivalent to 300 μg / kg) per 10 g of body weight via tail vein injection. The DMAG group received 0.1 mL of saline containing 50 μg DMAG (equivalent to 5 mg / kg) per 10 g of body weight via tail vein injection. The ADSCs-Exo and ADSCs-AVs groups received 0.2 mL of saline containing 200 μg ADSCs-Exo and 200 μg ADSCs-AVs per mouse via tail vein injection, respectively. The ADSCs-Exo@DMAG and ADSCs-AVs@DMAG groups received 0.2 mL of saline containing 200 μg ADSCs-Exo / ADSCs-AVs encapsulated in artificial vesicles at a DMAG dosage ratio of 5 mg / kg via tail vein injection per mouse. Administer the medication at a fixed time every other morning for 14 consecutive days (days 1, 3, 5, 7, 9, 11, and 13).

[0101] Peripheral blood cell counts in mice were analyzed on days 0, 3, 7, 10, and 14 after whole-body irradiation and drug administration. The specific method was as follows: 40 μL of orbital blood was drawn from the retinal venous plexus using a capillary tube and aspirated into an EP tube containing anticoagulant. Peripheral blood cell counts were detected using a fully automated hematology analyzer, and the measured parameters included platelet count (PLT), red blood cell count (RBC), and white blood cell count (WBC).

[0102] 1.8 Effect of artificial vesicles on platelet levels in peripheral blood of mice with thrombocytopenia

[0103] Peripheral blood cell collection method: On day 14 of drug administration, collect 50 μL of fundus venous blood and add it to a 2 mL EP tube pre-filled with 900 μL of sodium citrate. Mix well and store at 4°C. Count the cells using a hematology analyzer immediately before use. Based on the counting results, adjust the cell density of each group to 100 × 10⁻⁶ cells / mL. 4 Platelet count / mL. Take 100 μL, add the corresponding antibody, 1.25 μL CD41 and 1.25 μL CD61, mix well and incubate on ice for 15 min. Filter through a 200-mesh nylon mesh and transfer to a flow cytometry tube pre-filled with 500 μL PBS for detection and analysis of platelet production in peripheral blood cells.

[0104] 1.9 Effects of artificial vesicles on hematopoietic cells in peripheral blood of mice with thrombocytopenia

[0105] Peripheral blood cells were obtained and processed in the same manner as above. 1.25 μL of CD41 and 1.25 μL of CD117 were added, mixed well, and incubated on ice for 15 min. The mixture was then filtered through a 200-mesh nylon mesh and transferred to a flow cytometry tube pre-filled with 500 μL of PBS. The hematopoietic cells in the peripheral blood cells were then detected and analyzed.

[0106] 1.10 Effects of artificial vesicles on megakaryocyte differentiation in the bone marrow of mice with thrombocytopenia

[0107] Bone marrow cell acquisition method: On day 14 of drug administration, three mice were randomly selected from each group. After anesthesia, the mice were euthanized by cervical dislocation. The femur was dissected and excess attached tissue was removed. Using a 5 mL disposable sterile syringe filled with physiological saline, the needle was slowly inserted from one end of the femur. Bone marrow cells were slowly flushed out with physiological saline until the femur turned white. The cells were centrifuged at 1200 r / min for 5 min, the supernatant was discarded, and 1 mL of erythrocyte lysis buffer was added. The cells were lysed on ice for 5 min, centrifuged at 1200 r / min for 5 min, and the bone marrow pellet was obtained. The supernatant was discarded, and the cells were resuspended in 1 mL of pre-cooled PBS. The cells were counted using a hematology analyzer. Based on the counting results, the cell density of each group was adjusted to 100 × 10⁶ cells / mL. 4 / mL. Take 100 μL, add the corresponding antibody, 1.25 μL CD41 and 1.25 μL CD117, mix well and incubate on ice for 15 min. Filter through a 200-mesh nylon mesh and transfer to a flow cytometry tube pre-filled with 500 μL PBS for detection and analysis of bone marrow megakaryocyte generation and differentiation.

[0108] 2. Experimental Results and Analysis

[0109] 2.1 Morphological observation of extracellular vesicles of adipose-derived stem cells (ADSCs-Exo, ADSCs-AVs)

[0110] After processing, observation under a transmission electron microscope revealed that ADSCs-Exo and ADSCs-AVs exhibited typical bilayer membrane structures. ADSCs-AVs, consistent with ADSCs-Exo, were mostly circular or elliptical in shape, with some displaying a "coffee saucer" appearance. They were uniform in size, with clear boundaries, and intact membrane structures, showing no obvious ruptures or aggregation. Slight overlap was observed between some vesicles, and the interiors of the vesicles contained homogeneous electron-transparent regions (such as...). Figure 1 (As shown).

[0111] 2.2 Protein characterization of ADSCs-Exo and ADSCs-AVs

[0112] To further verify the proteomic characteristics of ADSCs-AVs, their characteristic marker proteins were detected. Western blot results showed positive bands for typical marker proteins CD9, CD81, and TSG101 in ADSCs-AVs, while Calnexin was not detected. These results indicate that the extracted ADSCs-AVs are rich in typical exosome-related proteins, consistent with the protein characteristics of exosome-like vesicles, and are consistent with ADSCs-Exo, demonstrating that ADSCs-AVs can, to some extent, replace ADSCs-Exo. Figure 2 As shown.

[0113] 2.3 Determination of particle size and concentration of ADSCs-Exo and ADSCs-AVs

[0114] The particle size and concentration of ADSCs-Exo and ADSCs-AVs were determined using NTA. The results showed that the average particle size of ADSCs-Exo was approximately 64 nm, and that of ADSCs-AVs was approximately 60 nm. The particle size distribution was concentrated and showed a unimodal distribution, with the main particle size range between 50 and 150 nm. This particle size is consistent with the typical particle size characteristics of exosome-like vesicles, indicating that the extracted ADSCs-Exo and ADSCs-AVs have uniform particle size and good dispersibility.

[0115] Table 1. Results Figure 3 Although nanoparticle tracking analysis (NTA) showed that the particle concentration per unit volume of ADSCs-AVs was lower than that of ADSCs-Exo, ADSCs-AVs achieved a significantly larger extraction volume of approximately 4.8 mL in a single preparation, compared to only about 1.8 mL for ADSCs-Exo. Therefore, by comprehensively considering both extraction volume and particle concentration, calculating the total number of nanoparticles obtained in a single preparation reveals that the total yield of ADSCs-AVs in a single preparation is higher than that of ADSCs-Exo, suggesting a significant advantage in yield.

[0116] Compared to the biogenetic mechanism of exosomes, which is highly dependent on the endosome pathway, the ultrasonic cavitation effect generates strong physical shear force and microjets, which can efficiently break down multilayer liposomes or membrane structures and reassemble them into a large number of small vesicles, thereby achieving higher overall yield in a single preparation. This characteristic gives ADSCs-AVs a potential advantage in large-scale preparation and drug delivery applications.

[0117] Calculation formula: Total number of particles in a single extraction = Concentration (particles / mL) × Extraction volume (mL)

[0118] Table 1. Particle number analysis of single extractions of ADSCs-Exo and ADSCs-AVs

[0119]

[0120] 2.4 Morphological observation of extracellular vesicles of adipose-derived stem cells encapsulating DMAG (ADSCs-AVs@DMAG)

[0121] The morphology and particle size of ADSCs-AVs@DMAG after drug loading were compared and analyzed. The results showed that after DMAG loading, the morphology of ADSCs-AVs@DMAG remained good. Under transmission electron microscopy, it still showed a typical "coffee tray" shape, with a round shape and no obvious breakage. The particle size did not change significantly, but there were some vesicle fragments, so the number of particles showed an upward trend. After DMAG loading, most of the particle sizes were within 150 nm. The electron microscopy results indicated that the DMAG loading process did not destroy the membrane structure and morphological integrity of ADSCs-AVs, and it had good structural stability and uniform particle size. Through transmission electron microscopy, clear spherical vesicle structures were visible, and DMAG was encapsulated inside the vesicles (e.g., Figure 4 (As shown).

[0122] 2.5 Determination of drug loading and encapsulation efficiency of ADSCs-AVs@DMAG artificial vesicles

[0123] Figure 5 To prepare adipose-derived stem cell-loaded DMAG artificial vesicles (ADSCs-AVs@DMAG) via ultrasonic co-incubation, according to Figure 5 The peak area in the spectrum and the calculation using the DMAG standard curve showed that the drug loading rate of ADSCs-AVs@DMAG was approximately 7.98% and the encapsulation efficiency was approximately 34.7% (see Table 2). This reveals that our method has good DMAG encapsulation capacity, which means that, at the same dose, the drug delivery system can carry more drug, thereby improving the therapeutic effect on thrombocytopenia, and is expected to improve the efficiency of precise delivery and reduce the distribution in non-target tissues.

[0124] Table 2. Results of drug loading and encapsulation efficiency of ADSCs-AVs@DMAG

[0125] ADSCs-AVs@DMAG Drug loading rate (%) 7.98% Encapsulation rate (%) 34.7%

[0126] 2.6 Flow cytometry detection of cell surface antigens CD41 / CD42b expression

[0127] like Figure 6 During megakaryocyte differentiation, cell volume often increases, accompanied by the expression of specific surface markers CD41 and CD42b. To further verify the pro-differentiation effects of ADSCs-AVs@DMAG and the positive control drug PMA (0.8 nM) on Meg-01 cells, flow cytometry was used to detect the expression of Meg-01 cell surface antigens CD41 and CD42b on day 4 of intervention. The results showed that, compared with the control group, ADSCs-AVs@DMAG and the PMA positive control group had the same effect, both significantly increasing the proportion of CD41+ / CD42b+ cells in Meg-01 cells. The effect was significantly greater than that of DMAG, ADSCs-Exo, and ADSCs-AVs alone, indicating that ADSCs-AVs@DMAG can promote megakaryocyte differentiation.

[0128] 2.7 Effect of artificial vesicles on peripheral blood platelet count in mice with thrombocytopenia

[0129] like Figure 7 Whole-body radiation exposure can damage hematopoietic stem cells in the bone marrow of mice, leading to disordered levels of various blood cells. Therefore, we used a hematology analyzer to count peripheral blood cells in mice on days 0, 4, 7, 10, and 14 after irradiation. The results showed that there were no significant differences in platelet counts among the groups from day 0 to day 3 after modeling. From day 3 to day 7, platelet counts were significantly lower in all irradiated groups compared to the normal group. On day 10, platelet counts gradually increased in all irradiated groups. However, by day 14, platelet counts in the model group had not fully recovered compared to the normal group and remained at a low to normal level. In contrast, platelet levels in the positive control group and the ADSCs-AVs@DMAG group were at normal levels and significantly different from the model group, indicating that ADSCs-AVs@DMAG can promote platelet recovery to some extent.

[0130] 2.8 Effect of artificial vesicles on platelet levels in peripheral blood of mice with thrombocytopenia

[0131] like Figure 8We used flow cytometry to detect the levels of CD41 and CD61 labeled platelets in peripheral blood. The results showed that, compared with the model group, the CD41+ / CD61+ platelet content in the ADSCs-AVs@DMAG and IL-11 positive drug groups was significantly increased, basically consistent with the level in the normal group. This result is consistent with the peripheral blood platelet count results. At the same time, the effect of ADSCs-AVs@DMAG was better than that of DMAG, ADSCs-Exo and ADSCs-AVs alone, which also indicates that the encapsulation of drug molecules can play a synergistic role.

[0132] 2.9 Effects of artificial vesicles on hematopoietic cells in peripheral blood of mice with thrombocytopenia

[0133] like Figure 9 In addition, we used flow cytometry to assess the platelet levels in peripheral blood marked by CD41 and CD117. The results showed that compared with the normal group, the expression of CD41 / CD117 in the model group was sharply reduced, indicating that radiation caused severe bone marrow suppression. In the ADSCs-AVs@DMAG and IL-11 positive drug groups, the percentage of CD41+ / CD117+ was significantly higher than that in the model group, and the effect was better than that of DMAG, ADSCs-Exo and ADSCs-AVs alone, suggesting that platelet production was effectively restored, and the results were consistent with the results of peripheral blood platelet count.

[0134] 2.10 Effects of artificial vesicles on megakaryocyte differentiation in the bone marrow of mice with thrombocytopenia

[0135] Bone marrow is the primary site of hematopoiesis. When bone marrow hematopoietic stem cells are damaged, extramedullary hematopoiesis is induced, such as... Figure 10 We analyzed the expression of CD41 and CD117 in bone marrow using flow cytometry to investigate the effect of ADSCs-AVs@DMAG on the differentiation and maturation of megakaryocytes in the bone marrow of hematopoietic organs. The results showed that compared with the model group, the proportion of CD41+ / CD117+ representing megakaryotic progenitors in the ADSCs-AVs@DMAG group was significantly increased, which was better than the use of DMAG, ADSCs-Exo and ADSCs-AVs alone. This indicates that it promoted the maturation and differentiation of bone marrow hematopoietic stem cells and effectively restored the hematopoietic function of irradiated mice.

Claims

1. The use of adipose-derived stem cell-derived artificial nanovesicles in the preparation of drugs that promote platelet production.

2. The use according to claim 1, characterized in that: The medication described is for the treatment of thrombocytopenia.

3. The use according to claim 1, characterized in that: The aforementioned adipose-derived stem cell artificial nanovesicles are derived from human or animal adipose tissue and are obtained through separation, culture, and purification steps.

4. The use according to claim 3, characterized in that: The method for preparing the artificial nanovesicles from adipose-derived stem cells includes the following steps: Isolation and culture of adipose-derived stem cells: Adipose-derived stem cells are extracted from adipose tissue and cultured and expanded in a culture medium containing growth factors; Cell membrane extraction: Collect cultured adipose-derived stem cells and extract cell membrane components using physical or chemical methods.

5. The application of adipose-derived stem cell artificial nanovesicles combined with DMAG in the preparation of drugs that promote platelet production; among which, The mass ratio of adipose-derived stem cell artificial nanovesicles to DMAG is: 3-7 parts adipose-derived stem cell artificial nanovesicles, 1 part DMAG; preferably, the mass ratio of adipose-derived stem cell artificial nanovesicles to DMAG is: Five portions of adipose-derived stem cell artificial nanovesicles and one portion of DMAG.

6. The use according to claim 5, characterized in that: The medication described is for the treatment of thrombocytopenia.

7. An artificial nanovesicle for adipose-derived stem cells loaded with DMAG, characterized in that: It uses adipose-derived stem cell artificial nanovesicles as a carrier to load DMAG, wherein the mass ratio of adipose-derived stem cell artificial nanovesicles to DMAG is: 3-7 parts of adipose-derived stem cell artificial nanovesicles and 1 part of DMAG.

8. The DMAG-loaded adipose-derived stem cell artificial nanovesicles according to claim 7, characterized in that: The mass ratio of adipose-derived stem cell artificial nanovesicles to DMAG is: Five portions of adipose-derived stem cell artificial nanovesicles and one portion of DMAG.

9. A method for preparing DMAG-loaded adipose-derived stem cell artificial nanovesicles as described in claim 7 or 8, characterized in that: It includes the following steps: a. Weigh out adipose-derived stem cell artificial nanovesicles and DMAG; b. Mix DMAG with cell membrane components and use ultrasound-assisted method to promote the formation of nanovesicles by encapsulating DMAG in the cell membrane.

10. Use of the DMAG-loaded adipose-derived stem cell artificial nanovesicles of claim 7 or 8 in the preparation of a medicament for promoting platelet production, wherein the medicament is an intravenously administered medicament.

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