Stem cell nanovesicle-polysaccharide composite functional microspheres, and preparation method and application thereof
By preparing stem cell nanovesicles and forming multilayer functional microspheres with polysaccharides and calcium alginate, the problems of low yield, high cost and short retention time in stem cell therapy for intrauterine adhesions have been solved, achieving efficient treatment of intrauterine adhesions and restoration of fertility.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANTOU UNIV MEDICAL COLLEGE
- Filing Date
- 2026-03-26
- Publication Date
- 2026-07-28
AI Technical Summary
Current stem cell therapy for intrauterine adhesions suffers from problems such as low cell survival rate, tumorigenesis risk, immune rejection, inconvenient storage and transportation, low nanovesicle yield, high cost, and short in vivo retention time. Traditional exosome preparation methods are inefficient and costly, making it difficult to achieve clinical translation.
By combining stem cell nanovesicles with Tremella fuciformis polysaccharide, Poria cocos polysaccharide, and calcium alginate to form multilayered functional microspheres, and then using freeze-drying, reconstitution, and high-voltage electrostatic microcapsule encapsulation technology, high-yield, highly active, and stably sustained-release stem cell nanovesicle-polysaccharide composite functional microspheres were prepared, forming a multilayered structure to prolong the retention time.
It significantly prolongs the residence time of stem cell nanovesicles in the uterus, enhances the therapeutic effect, promotes endometrial hyperplasia, gland regeneration and angiogenesis, inhibits fibrosis, and restores fertility, showing promising clinical translation potential.
Smart Images
Figure CN122461344A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a stem cell nanovesicle-polysaccharide composite functional microsphere, its preparation method, and its application. Background Technology
[0002] Current research on stem cell therapy for intrauterine adhesions covers a variety of stem cell types and delivery strategies, including human amniotic epithelial stem cell therapy for refractory severe intrauterine adhesions, and collagen scaffold combined with umbilical cord or autologous bone marrow-derived mesenchymal stem cells to repair thin endometrium and restore fertility.
[0003] While mesenchymal stem cells (MSCs) show therapeutic potential for treating intrauterine adhesions, MSC-based cell products suffer from drawbacks such as low cell viability, tumorigenicity risk, immune rejection, inconvenient storage and transportation, and short in vivo retention time. Furthermore, traditional exosome preparation methods (e.g., ultracentrifugation) are inherently flawed, characterized by extremely low yields, cumbersome processes, lengthy processing times, and high costs, severely hindering their clinical translation. MSC-derived nanovesicles (MSC-NVs), with their tunable particle size (10-1000 nm), simple preparation process, and high yield, have become an ideal cell-free therapeutic carrier to replace natural exosomes, exhibiting high activity and more stable quality. Currently, MSC-derived nanovesicles are primarily prepared using physical methods, such as mechanical extrusion and shearing. Extrusion-based MSC nanovesicles suffer from low yields (significant cell loss during extrusion) and high costs (requiring specialized extrusion equipment and disposable filter membranes, which are expensive). Summary of the Invention
[0004] The purpose of this invention is to provide a high-yield, highly active, stable, sustained-release stem cell nanovesicle-polysaccharide composite functional microsphere, its preparation method, and its application, to solve the problems of low yield, high cost, and short in vivo therapeutic retention time of current stem cell nanovesicles obtained by extrusion. The stem cell nanovesicle-polysaccharide composite functional microspheres of this invention can be produced on a clinical scale, and have a good multilayer structure and stable sustained-release system.
[0005] A method for preparing stem cell nanovesicle-polysaccharide composite functional microspheres includes the following steps: (1) Stem cell nanovesicles were freeze-dried in a complex polysaccharide of Tremella fuciformis polysaccharide and Poria cocos polysaccharide with a mass ratio of (1:5)-(5:1); (2) Dissolve the lyophilized powder obtained in step (1) in pure water, physiological saline or PBS buffer at pH 6.5-7.5 to obtain a suspension of stem cell nanovesicles@TFP / PCP (NV@TFP / PCP) lyophilized powder; (3) The stem cell nanovesicles@TFP / PCP lyophilized powder suspension obtained in step (2) is mixed with a solution containing 0.1%~3.0% (w / v) sodium alginate and 0.1% - 3.0% (w / v) tremella polysaccharide and poria polysaccharide complex polysaccharide in a mass ratio of (1:5)-(5:1); (4) Prepare a calcium chloride solution with a concentration of 1.0% ~ 2.5% (w / v) and use a high voltage electrostatic microcapsule embedding machine to prepare stem cell nanovesicle-polysaccharide composite functional microspheres.
[0006] Furthermore, the stem cell nanovesicles include MSC-NV, exosomes, and MSC-Plasma Membrane Vesicles (MSC-PMV, i.e., cell-derived vesicles, artificial nanovesicles prepared by mechanical extrusion).
[0007] Furthermore, the voltage of the high-voltage electrostatic microcapsule encapsulation machine is adjusted to 1~9 kV, the propulsion speed is set to 10~30 mL / h, and the cross-linking is allowed to stand for 10-30 minutes.
[0008] The stem cell nanovesicle-polysaccharide composite functional microspheres obtained by the above preparation method.
[0009] Furthermore, the stem cell nanovesicle-polysaccharide composite functional microspheres include a stem cell nanovesicle@TFP / PCP lyophilized powder core layer and a TFP / PCP-Ca-AGMs microsphere outer core. The stem cell nanovesicles@TFP / PCP nanovesicles are encapsulated within the TFP / PCP-Ca-AGMs microspheres, forming a multilayered functional microsphere sustained-release system.
[0010] When the stem cell nanovesicles are MSC-NV, the above-mentioned stem cell nanovesicle-polysaccharide composite functional microspheres are MSC-NV@TFP / PCP-Ca-AGMs. These are functional microspheres formed by preparing mesenchymal stem cells (MSC) nanovesicles (NVs) by hypotonic lysis, encapsulating them with Tremella fuciformis polysaccharide and Poria cocos polysaccharide, freeze-drying them into lyophilized powder, reconstituted them with physiological saline, and then encapsulating them again with Tremella fuciformis polysaccharide, Poria cocos polysaccharide and calcium alginate.
[0011] Tremella fuciformis polysaccharide (TFP) possesses excellent viscosity, moisturizing ability, and antioxidant properties. Its water-retention capacity even surpasses that of hyaluronic acid, creating an ideal "moist healing" environment for wounds and accelerating epithelial cell migration. TFP readily forms high-viscosity viscoelastic solutions, but its gel-forming ability is weak, resulting in low gel strength. Poria cocos polysaccharide (PCP) exhibits good viscosity and immunomodulatory capabilities. Under specific conditions (such as sufficient concentration and temperature changes), PCP can form a relatively rigid, thermally reversible gel. PCP consists of regular, rigid chains that form a robust three-dimensional network framework within the system, providing mechanical strength and stability. TFP, on the other hand, consists of irregular, flexible chains rich in hydrophilic groups and negative charges. It enhances the system's moisturizing properties, bioadhesion, and protection against bioactive molecules through filling network gaps, dynamic hydrogen bonding, and electrostatic interactions. This invention comprises a composite polysaccharide consisting of regularly structured β-glucan (Poria cocos polysaccharide, which is relatively rigid but lacks sufficient inter-chain entanglement and elastic support points, resulting in high brittleness) and irregularly structured acidic heteropolysaccharide (Tremella fuciformis polysaccharide, which has high aqueous viscosity but weak gel strength and is highly hydrophilic, easily swelling and disintegrating in water, leading to rapid loss). TFP and PCP work synergistically to optimize gel strength and elasticity. The rigid helical structure of PCP acts as a "physical cross-linking point," embedding and reinforcing the continuous viscoelastic network formed by TFP. TFP, in turn, connects the cross-linking points of PCP through its flexible long chains, forming a more uniform, tougher, and less prone to water separation composite gel network. The synergistic effect of TFP / PCP is optimal when the mass ratio of TFP to PCP is in the range of 1:5 to 5:1, preferably 1:2 to 2:1. In this invention, TFP / PCP acts as an "active freeze-drying protectant" and a "primary functional synergist." Its main function is to increase the viscosity of the MSC-NVs microsphere complex for "slow release." Secondly, it acts as a "hardness modifier for calcium alginate microspheres," which can increase the elasticity of the MSC-NVs microsphere complex and prevent the high hardness of calcium alginate from causing further mechanical friction damage to the endometrial wound after being applied to a damaged uterus, thus avoiding further damage to the wound.
[0012] The TFP / PCP of this invention can also synergize with sodium alginate, and β-glucan and acidic heteropolysaccharides work synergistically. Tremella fuciformis polysaccharide is an acidic heteropolysaccharide (main chain is mannan, side chain contains glucuronic acid). Its aqueous solution has high viscosity, but weak gel strength and strong hydrophilicity, making it prone to swelling and disintegration in water, resulting in rapid loss. Poria cocos polysaccharide is mainly β-glucan, which has strong rigidity, but insufficient entanglement between molecular chains and a lack of elastic support points, leading to high brittleness. The combination of these three components achieves a balance of "moderate flexibility, long-lasting sustained release, and high adhesion," jointly forming a protective layer for the nanovesicles, creating a more flexible sustained-release matrix that increases viscosity and moisturizes, thereby aiding in the sustained release of MSC-NVs within the uterine cavity.
[0013] A formulation comprising the aforementioned stem cell nanovesicle-polysaccharide composite functional microspheres. These stem cell nanovesicle-polysaccharide composite functional microspheres can serve as a sustained-release drug composition capable of prolonging the intrauterine action time of stem cell nanovesicles such as MSC-NVs, forming a multilayered functional microsphere sustained-release product.
[0014] The above-mentioned stem cell nanovesicle-polysaccharide composite functional microspheres are used in the preparation of drugs for repairing endometrial damage, treating / preventing intrauterine adhesions, or restoring fertility.
[0015] The above-mentioned stem cell nanovesicle-polysaccharide composite functional microspheres are used in the preparation of drugs for tissue damage and aplastic diseases. Examples include, but are not limited to, diabetic skin wounds, myocardial injury, and osteochondral defects.
[0016] Compared with existing technologies, this invention is the first to combine stem cell nanovesicles such as MSC-NVs with biodegradable natural biomaterials (Tremella fuciformis polysaccharide and Poria cocos polysaccharide) and calcium alginate to form a multilayer functional microsphere sustained-release system. During freeze-drying, Tremella fuciformis polysaccharide and Poria cocos polysaccharide protect MSC-NVs and form a primary functional complex; upon reconstitution, the primary functional complex of MSC-NVs, along with sodium alginate, calcium ions, Tremella fuciformis polysaccharide, and Poria cocos polysaccharide, constructs a more robust microsphere sustained-release system, MSC-NV@TFP / PCP-Ca-AGMs. The stem cell nanovesicle-polysaccharide composite functional microspheres of this invention, such as MSC-NV@TFP / PCP-Ca-AGMs, solve the problem of short retention time of highly bioactive nanovesicles in the uterus and possess excellent anti-inflammatory and anti-fibrotic functions. Compared with free stem cell nanovesicles (including MSC-NV), the stem cell nanovesicle-polysaccharide composite functional microspheres of this invention have the following main advantages: (1) Sustained release and enhanced efficacy: The TFP / PCP-Ca-AGMs microsphere system prepared in this invention significantly prolongs the residence time of stem cell nanovesicles such as MSC-NVs in the uterus, thereby enhancing and prolonging the therapeutic effect.
[0017] (2) Excellent therapeutic effect: In the severe IUA mouse model, the stem cell nanovesicle-polysaccharide composite functional microspheres of the present invention, such as MSC-NV@TFP / PCP-Ca-AGMs, significantly promoted endometrial hyperplasia (increased Ki-67+ cells), gland regeneration (increased gland density), angiogenesis (enhanced CD31+ signal) and inhibited fibrosis (reduced collagen deposition) compared with free stem cell nanovesicles such as MSC-NVs.
[0018] (3) Restoration of fertility: In a severe IUA animal model, this invention has demonstrated that the MSC-NV@TFP / PCP-Ca-AGMs composition can more effectively promote endometrial regeneration and successfully restore fertility than free MSC-NVs, with a pregnancy rate of 66.7% and healthy offspring (normal embryo size and placental morphology), showing great potential for clinical translation. Attached Figure Description
[0019] Figure 1 This is a schematic diagram of the preparation process of stem cell nanovesicle-polysaccharide composite functional microspheres of the present invention; Figure 2 The images show: left image of MSC-NV@TFP / PCP-Ca-AGMs microspheres under an optical microscope; middle image of MSC-NV@TFP / PCP-Ca-AGMs microspheres under a scanning electron microscope; and right image of MSC-NV within MSC-NV@TFP / PCP-Ca-AGMs microspheres under a scanning electron microscope. Figure 3 In vivo imaging of mice after being injected into the uterine cavity with DiR-labeled free MSC-NVs and MSC-NV@TFP / PCP-Ca-AGM formulations, respectively. The mouse on the left is free MSC-NVs, and the mouse on the right is MSC-NV@TFP / PCP-Ca-AGM. Figure 4 This is an image showing the effect of MSC-NV@TFP / PCP-Ca-AGMs promoting endometrial repair in a mouse model of intrauterine adhesions. Figure 5 Immunofluorescence staining image showing the effect of MSC-NV@TFP / PCP-Ca-AGMs on promoting endometrial repair in a mouse model of intrauterine adhesions; Figure 6 This is a quantitative analysis diagram based on H&E staining and immunofluorescence, where groups 1, 2, 3, 4, and 5 represent Ctrl, IUA, TFP / PCP-Ca-AGMs, MSC-NV, and MSC-NV@TFP / PCP-Ca-AGMs groups, respectively. Figure 7 This is a diagram illustrating the therapeutic effect of MSC-NV@TFP / PCP-Ca-AGMs on fibrosis in mice with intrauterine adhesions. Figure 8 This is a graph showing the effect of MSC-NV@TFP / PCP-Ca-AGMs on the fertility of mice with intrauterine adhesions. Detailed Implementation
[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.
[0021] Example 1 A method for preparing stem cell nanovesicle-polysaccharide composite functional microspheres, such as... Figure 1 As shown, the main steps include: 1. Preparation of MSC-NV The main steps include: culturing and expanding MSCs to the required quantity; resuspending the cells in hypotonic buffer and lysing them on ice for a certain time (e.g., 30-60 minutes); purifying the nanovesicles by differential centrifugation (e.g., first removing the cell nuclei and debris at low speed, then centrifuging at high speed to obtain NV precipitate); resuspending the cells in PBS, filtering to remove bacteria, and obtaining high-yield MSC-NVs.
[0022] Specifically, this includes: when P3-P6 generation mesenchymal stem cells reach 85-90% confluence in culture dishes, cells are collected by digestion with 0.05% trypsin (Gibco, USA). Cells are washed twice with pre-cooled phosphate-buffered saline (ZSGB-BIO, China), counted, and then analyzed at 1×10⁻⁶. 5 ~8×10 7 Resuspend the cells at a density of 100 cells / mL in hypotonic solution and seal. Incubate at 0–4°C with shaking at 50–500 rpm for 1–72 hours to lyse. After lysis, perform gradient centrifugation: first, centrifuge at 50–700 × g for 2–30 minutes at 4°C to remove nuclei and cell debris; then, centrifuge at 1000–20000 × g for 5–30 minutes to remove organelles; finally, ultracentrifuge at 10,000–100,000 × g for 30–120 minutes at 4°C. Discard the supernatant, resuspend the nanovesicle precipitate, and further purify it by size exclusion chromatography or ultrafiltration.
[0023] The hypotonic solution used in this embodiment consists of water, a membrane stabilizer, and a protease inhibitor. The membrane stabilizer and the protease inhibitor each have a mass percentage of 0.1% to 5% in the hypotonic solution. The membrane stabilizer is selected from one or more of EDTA, EGTA, MgCl2, sucrose, trehalose, and mannitol; the buffer is selected from one or more of Tris, HEPES, and PBS. The pH of the hypotonic solution is 7.0-8.5, and the osmotic pressure is 20-280 mOsm / kg (preferably 50-200 mOsm / kg).
[0024] 2. MSC-NV freeze-drying The purified nanovesicles were added to a mixed solution of Tremella fuciformis polysaccharide (TFP) and Poria cocos polysaccharide (PCP) (mass ratio ranging from 1:5 to 5:1, preferably 1:2 to 2:1). After pre-cooling at -80°C, the mixture was freeze-dried for 4 to 24 hours using a freeze dryer (condenser temperature -50°C, Foring Corporation, China). The complex remained stable for more than one year.
[0025] 3. Reconstitute The MSC-NV lyophilized powder obtained in step 2 is dissolved in 1 mL of pure water, physiological saline, or PBS buffer at pH 6.5-7.5 at a concentration of 50-300 mg.
[0026] 4. MSC-NV@TFP / PCP-Ca-AGMs microsphere molding The MSC-NV@TFP / PCP lyophilized powder suspension reconstituted in step 3 was gently mixed with a solution containing 0.1%–3.0% (w / v) sodium alginate and 0.1%–3.0% (w / v) TFP / PCP complex polysaccharide to form a homogeneous mixture. First, a sodium alginate solution with a concentration of 0.1%–3.0% (w / v) and a solution containing 0.1%–3.0% (w / v) Tremella fuciformis polysaccharide (TFP) / Poria cocos polysaccharide (PCP) complex polysaccharide (TFP / PCP mass ratio range 1:5 to 5:1, preferably 1:2 to 2:1) were prepared by gentle mixing. The mixture was sterilized using a 0.22 μM filter membrane, and the MSC-NV@TFP / PCP lyophilized powder suspension reconstituted in step 3 was added. The mixture was then stored at 4°C. Prepare a 1.0%–2.5% (w / v) calcium chloride solution. Then, prepare calcium alginate microspheres using a high-voltage electrostatic microencapsulation machine. Connect all components of the machine, set the parameters (voltage 1–9 kV, feed rate 10–30 mL / h), and allow to stand for 10–30 minutes to ensure complete solidification of the microspheres. Prepare MSC-NV@TFP / PCP-Ca-AGMs composite microspheres with a size of 100–300 μm. After microsphere preparation, filter them from the calcium chloride solution using a 300-mesh cell sieve sterilized by autoclaving in a sterile laminar flow hood. Wash the microspheres twice with physiological saline or PBS buffer, then place them in 50 mL centrifuge tubes, mix with the appropriate volume of physiological saline, seal with a sealing film, and store at 4°C for later use.
[0027] Experimental data 1. Analysis of mesenchymal stem cell nanovesicle encapsulation like Figure 2 As shown in the leftmost figure, the MSC-NV prepared by this invention consists of spherical microcapsules with uniform particle size distribution and an average diameter of about 100~300μm.
[0028] This invention further utilizes sodium alginate to dissolve nanovesicles and then solidifies them in calcium chloride to successfully prepare microspheres encapsulating MSC nanovesicles, such as MSC-NV@TFP / PCP-Ca-AGM. Figure 2As shown in the middle image, scanning electron microscopy characterization reveals that nanovesicles are uniformly distributed at a high density on the surface of the microspheres. This three-dimensional spatial distribution is beneficial for the sustained release of the drug. Using a higher magnification scanning electron microscope, the specific morphology of the nanovesicles attached to the surface of the microspheres was observed; the nanovesicles still exhibit a spherical shape, as shown... Figure 2 As shown in the rightmost figure, the MSC-NV@TFP / PCP-Ca-AGM spheres prepared in this invention encapsulate nanovesicles using sodium alginate and anhydrous calcium chloride. Both materials are medical-grade and offer advantages such as safety, low cost, and easy availability. Furthermore, the prepared microspheres are of a suitable size, allowing for therapeutic replication in the mouse uterus via a syringe needle. Simultaneously, scanning electron microscopy confirmed the successful encapsulation of numerous nanovesicles within the microspheres.
[0029] 2. Comparison of retention time in the uterus To evaluate whether MSC-NV@TFP / PCP-Ca-AGM could prolong the residence time of nanovesicles in the uterus, this invention administered DiR-labeled free MSC-NV and MSC-NV@TFP / PCP-Ca-AGM formulations via intrauterine injection. In vivo imaging was performed as follows. Figure 3 As shown: On day 1 post-injection, the fluorescence intensity of the two groups was comparable (free NV group: 2.032 × 10⁻⁶). 8 The au, MSC-NV@TFP / PCP-Ca-AGM group was 2.193×10 8 However, during days 1 to 10, the fluorescence intensity of the MSC-NV@TFP / PCP-Ca-AGM group was consistently significantly higher than that of the MSC-NV group, and remained relatively stable from days 1 to 7, confirming that MSC-NV@TFP / PCP-Ca-AGM can significantly prolong the residence time of nanovesicles in the uterus compared to free MSC-NV.
[0030] The above results indicate that the controlled-release system based on MSC-NV@TFP / PCP-Ca-AGM can effectively enhance the retention of mesenchymal stem cell nanovesicles in the uterine cavity, thus providing a more promising treatment option for intrauterine adhesions.
[0031] 3. The in vivo therapeutic potential of MSC-NV@TFP / PCP-Ca-AGMs for intrauterine adhesions To evaluate the in vivo therapeutic potential of MSC-NV@TFP / PCP-Ca-AGMs for intrauterine adhesions (IUA), this invention established a mouse model exhibiting both endometrial injury and fibrosis, two key pathological features of IUA. The IUA model was induced by intrauterine injection of 95% ethanol into the right uterus of mice (the left side served as a normal control). Histological analysis showed extensive disruption of endometrial epithelial continuity and significant fibrosis 14 days post-injection, confirming successful induction of endometrial injury and fibrosis. Therefore, day 14 was chosen as the time point for subsequent treatment intervention.
[0032] To evaluate the therapeutic effect of MSC-NV@TFP / PCP-Ca-AGM on endometrial repair, mice were randomly divided into five groups: control group (untreated), IUA group (ethanol-induced injury without intervention), TFP / PCP-Ca-AGMs group (IUA model + TFP / PCP-Ca-AGMs treatment), MSC-NVs group (IUA model + MSC-NVs treatment), and MSC-NV@TFP / PCP-Ca-AGMs group (IUA model + MSC-NV@TFP / PCP-Ca-AGMs treatment). Figure 4 As shown, 14 days after ethanol injury, the uterine horns of mice in the IUA group and the TFP / PCP-Ca-AGMs group were shortened, hardened, and lost elasticity, with significant edema on the injured side (right side). In contrast, the edema in the MSC-NVs group and the MSC-NV@TFP / PCP-Ca-AGMs group was reduced, the uterine horn morphology was improved, and the texture was closer to normal. H&E staining showed that the endometrial lining in the IUA group and the TFP / PCP-Ca-AGMs group was discontinuous, while the MSC-NVs group and the MSC-NV@TFP / PCP-Ca-AGMs group showed a nearly continuous endometrial structure, indicating enhanced endometrial regeneration. The effect of the MSC-NV@TFP / PCP-Ca-AGMs group was significantly better than that of the MSC-NVs group.
[0033] Figure 5 Further immunofluorescence staining revealed that, compared with the control group, the expression of Ki-67 and CD31 was significantly reduced in the IUA group and the TFP / PCP-Ca-AGM group, indicating impaired cell proliferation and angiogenesis. In contrast, the expression of Ki-67 and CD31 was significantly increased in the MSC-NV@TFP / PCP-Ca-AGMs group and the MSC-NVs group compared with the IUA group. The MSC-NV@TFP / PCP-Ca-AGMs group showed better results, with the highest increase in the cell proliferation marker Ki-67 and the vascular marker CD31, indicating that cell regeneration and angiogenesis were significantly improved after MSC-NV@TFP / PCP-Ca-AGM treatment.
[0034] Figure 6Quantitative analysis based on H&E staining showed that the number of endometrial glands in the MSC-NV@TFP / PCP group and the MSC-NV@TFP / PCP-Ca-AGMs group was significantly increased compared with the IUA group, and the endometrial thickness was also significantly increased in all treatment groups. Quantitative analysis of Ki-67 fluorescence intensity in ImageJ confirmed that the TFP / PCP-Ca-AGMs group, the MSC-NVs group, and the MSC-NV@TFP / PCP-Ca-AGMs group all showed significant increases compared with the IUA group, consistent with improved cell proliferation. Similarly, the CD31 fluorescence intensity was significantly increased in all treatment groups, indicating enhanced angiogenesis, with the MSC-NV@TFP / PCP-Ca-AGMs group showing the best effect. Figure 7 The therapeutic effect of MSC-NV@TFP / PCP-Ca-AGMs on fibrosis in mice with intrauterine adhesions. Animal experiments showed that MSC-NV@TFP / PCP-Ca-AGMs were more effective than MSC-NVs in promoting endometrial regeneration and reducing fibrosis, demonstrating potential for treating intrauterine adhesions (IUA), and the effect of the MSC-NV@TFP / PCP-Ca-AGMs group was significantly better than that of the MSC-NVs group.
[0035] As can be seen, in the severe IUA mouse model, the MSC-NV@TFP / PCP-Ca-AGMs of the present invention significantly promoted endometrial hyperplasia (increased Ki-67+ cells), gland regeneration (increased gland density), angiogenesis (enhanced CD31+ signaling), and inhibited fibrosis (reduced collagen deposition) compared with free MSC-NVs.
[0036] 4. Effects of MSC-NV@TFP / PCP-Ca-AGMs on reproductive function To assess the impact of different treatments on reproductive function, female mice treated with 95% ethanol injection to induce IUA were co-cultured with male mice at a 2:1 ratio for 14 days, and then the embryo implantation status on the damaged side (right) and the undamaged side (left) of the uterus was examined.
[0037] Table 1. Comparison of the effects of different treatments on reproductive function
[0038] like Figure 8As shown in Table 1, no embryos were observed in the damaged uterus in the IUA group, TFP / PCP-Ca-AGMs group, and MSC-NVs group, while embryo implantation was observed in the undamaged uterus in all groups. In contrast, embryo implantation was found in both uterine horns of mice in the MSC-NV@TFP / PCP-Ca-AGMs group: 1-4 embryos were observed in 4 out of 6 mice in the damaged uterus, while no implantation was observed in the damaged uterus in the remaining 2 mice, resulting in a pregnancy rate of 66.7% in the damaged uterus. Therefore, the MSC-NV@TFP / PCP-Ca-AGMs composition obtained after the coating treatment of this invention can effectively promote endometrial regeneration and successfully restore fertility, increasing the pregnancy rate to 66.7% and producing healthy offspring (normal embryo size and placental morphology), demonstrating great potential for clinical translation.
[0039] In the above embodiments, MSC-NV can be replaced with exosomes and MSC plasma membrane vesicles, and stem cell nanovesicle-polysaccharide composite functional microspheres can still be prepared. The performance of the obtained exosome nanovesicle-polysaccharide composite functional microspheres and MSC plasma membrane nanovesicle-polysaccharide composite functional microspheres is also far superior to that of free exosomes and MSC plasma membrane vesicles.
Claims
1. A method for preparing stem cell nanovesicle-polysaccharide composite functional microspheres, characterized in that, Includes the following steps: (1) Stem cell nanovesicles were freeze-dried in a complex polysaccharide of Tremella fuciformis polysaccharide and Poria cocos polysaccharide with a mass ratio of (1:5)-(5:1); (2) Dissolve the lyophilized powder obtained in step (1) in pure water, physiological saline or PBS buffer with pH 6.5-7.5 to obtain a suspension of stem cell nanovesicles@TFP / PCP lyophilized powder; (3) The stem cell nanovesicles@TFP / PCP lyophilized powder suspension obtained in step (2) is mixed with a solution containing 0.1%~3.0% (w / v) sodium alginate and 0.1% - 3.0% (w / v) tremella polysaccharide and poria polysaccharide complex polysaccharide in a mass ratio of (1:5)-(5:1); (4) The mixture obtained in step (3) is mixed with a calcium chloride solution with a concentration of 1.0% ~ 2.5% (w / v) and a high voltage electrostatic microcapsule embedding machine to prepare stem cell nanovesicle-polysaccharide composite functional microspheres.
2. The preparation method according to claim 1, characterized in that, The stem cell nanovesicles include MSC-NV, exosomes, and MSC plasma membrane vesicles.
3. The preparation method according to claim 1, characterized in that, The voltage of the high-voltage electrostatic microcapsule encapsulation machine is adjusted to 1~9 kV, the propulsion speed is set to 10~30 mL / h, and the cross-linking is allowed to stand for 10-30 minutes.
4. Stem cell nanovesicle-polysaccharide composite functional microspheres obtained by the preparation method according to any one of claims 1-3.
5. The stem cell nanovesicle-polysaccharide composite functional microspheres according to claim 4, characterized in that, It includes a stem cell nanovesicle @TFP / PCP lyophilized powder core layer and a TFP / PCP-Ca-AGMs microsphere outer core.
6. A formulation comprising the stem cell nanovesicle-polysaccharide composite functional microspheres of claim 4.
7. The use of the stem cell nanovesicle-polysaccharide composite functional microspheres according to claim 4 in the preparation of formulations for repairing endometrial damage, treating / preventing intrauterine adhesions, or restoring fertility.
8. The use of the stem cell nanovesicle-polysaccharide composite functional microspheres according to claim 4 in the preparation of formulations for tissue damage and aplastic diseases.