Preparation method of exosome-enriched silkworm chrysalis chitosan magnetic microspheres and application of exosome-enriched silkworm chrysalis chitosan magnetic microspheres in treatment of intrauterine adhesion
By preparing silkworm pupa chitosan magnetic microspheres using droplet microfluidic technology, the problems of poor targeting and low delivery efficiency in existing methods for treating intrauterine adhesions were solved. Stable in vitro enrichment of exosomes and targeted delivery into the intrauterine cavity were achieved, enhancing the therapeutic effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-03
- Publication Date
- 2026-03-10
AI Technical Summary
Existing methods for treating intrauterine adhesions suffer from problems such as high postoperative recurrence rate, difficulty in endometrial regeneration, poor targeting, low delivery efficiency, and high preparation costs. Traditional exosome drug delivery systems are difficult to meet the requirements for precise controlled release.
Using silkworm pupa chitosan magnetic microspheres as a novel delivery system, stem cell exosomes are efficiently enriched through surface charge and targeted in the uterine cavity by magnetic guidance. The preparation method includes droplet microfluidic technology to prepare silkworm pupa chitosan magnetic microspheres with an average particle size of 300-500 µm, which are used for loading and delivering mesenchymal stem cell exosomes.
It achieves stable in vitro enrichment and intrauterine targeted delivery of exosomes, with minimally invasive, safe, and targeted drug delivery effects, enhancing the targeting and therapeutic efficacy of intrauterine adhesion treatment.
Smart Images
Figure CN121622889A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a method for preparing silkworm pupa chitosan magnetic microspheres enriched with exosomes and their application in the treatment of intrauterine adhesions. Background Technology
[0002] Intrauterine adhesions (IUA) are a fibrotic disease caused by damage to the basal layer of the endometrium, clinically manifesting as menstrual abnormalities, infertility, and recurrent miscarriages. Traditional treatments suffer from high postoperative recurrence rates and difficulties in endometrial regeneration. While mesenchymal stem cells (MSCs) and their exosomes can improve endometrial repair through anti-fibrotic and angiogenesis mechanisms, their clinical application is limited by bottlenecks such as poor targeting, low delivery efficiency, and high preparation costs. Existing exosome drug delivery systems (such as liposomes and hyaluronic acid gels) are unable to meet the requirements for precise controlled release due to issues such as low drug loading rates, burst release effects, and insufficient targeting.
[0003] Silkworm pupa chitosan magnetic microspheres, as a novel delivery system, can efficiently enrich stem cell exosomes through surface charge, achieve intrauterine targeted localization through magnetic guidance, and possess good biocompatibility and biodegradability, enabling in vivo drug delivery of stem cell exosomes. They also support antibody / enzyme modification to enhance targeting and therapeutic efficacy. Magnetic microspheres are typically composed of polymers and magnetic nanoparticles. Silkworm pupa-derived chitosan exhibits higher bioactivity and raw material sustainability compared to traditional crustacean chitosan, and is more environmentally friendly. The system overcomes technological limitations through the following characteristics: 1. Precise controlled release: The positive surface charge efficiently enriches negatively charged exosomes, achieving intrauterine targeted localization through magnetic guidance, reducing non-specific distribution; 2. Functional enhancement: Supports antibody / enzyme modification to enhance targeting (e.g., binding to endometrial-specific markers). Current preparation technologies for silkworm pupa chitosan magnetic microspheres include suspension cross-linking, one-step embedding, precipitation polymerization, membrane emulsification-in-situ method, high-voltage electrostatic method, ultrasound-assisted chemical co-precipitation method, and a combination of sol-gel and reverse microemulsion methods.
[0004] In recent years, with the deepening research on silkworm pupa chitosan magnetic microspheres, their preparation technology has been continuously optimized. For example, patent CN20138005234.5 provides a method for preparing silkworm pupa chitosan magnetic microspheres by changing the pH value, which can significantly improve drug loading and sustained release effect while maintaining good magnetic targeting performance. In addition, patent CN20151009876.2 provides a multifunctional surface modification technology that covalently couples antibody proteins or enzymes to the surface of silkworm pupa chitosan magnetic microspheres through amino activation, which improves the targeting efficiency of microspheres for endometrial cells. Enzyme modification can degrade the uterine mucus barrier and promote the transmembrane penetration of exosomes. Superparamagnetic nanocomposite microspheres (CN20211023456.7) use PEG-4000 to modify the surface of Fe3O4, reducing the tendency of magnetic nucleus aggregation. Under the action of an external magnetic field, they can quickly achieve local enrichment in the uterine cavity. After the magnetic field is removed, the dispersibility of the microspheres is restored, avoiding tissue embolism. Among them, droplet microfluidics has become a widely used magnetic microsphere preparation technology in recent years due to its precise and controllable preparation process, safety and stability, and high efficiency and low consumption.
[0005] Droplet microfluidics technology, through the regulation of parameters such as flow rate, inlet pressure, and phase viscosity using microfluidic chips, precisely controls droplet size, velocity, and generation frequency. This overcomes the limitations of traditional batch methods, such as poor monodispersity and insufficient stability, enabling the efficient preparation of homogeneous drug carriers such as lipid / polymer microparticles, microcapsules, and microspheres, and achieving dynamic regulation of drug release behavior. Compared to continuous microfluidic systems, it simplifies complex fluid control processes and provides precise manipulation capabilities for droplet transport, mixing, and separation, offering a high-throughput solution for the large-scale production of monodisperse particles. In recent years, the preparation technology of magnetic microspheres based on droplet microfluidic systems has achieved numerous innovative breakthroughs. Summary of the Invention
[0006] To improve and balance the efficacy and safety stability of exosome-based in vivo drug delivery, this invention provides a method for preparing and applying silkworm pupa chitosan magnetic microspheres for exosome enrichment. Specifically, based on a droplet microfluidic system, a silkworm pupa chitosan magnetic microsphere capable of enriching exosomes was designed. Extensive in vitro and in vivo experiments verified that the synthesized silkworm pupa chitosan magnetic microspheres have good biocompatibility and directional mobility. At the same time, their drug loading and release performance are stable, and their safety meets the requirements for in vivo injection, providing a new approach for the treatment of intrauterine adhesions.
[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for preparing silkworm pupa chitosan magnetic microspheres for exosome enrichment includes the following steps: (1) Preparation of magnetic nanoparticle chitosan suspension Chitosan powder was dissolved in an acid solution to prepare a chitosan solution with a mass fraction of 1.5-2.5%. An ethanol dispersion of superparamagnetic Fe3O4 nanoparticles was added to the chitosan solution at a ratio of 20 μg: 1 mL. The mixture was then uniformly dispersed by high-speed shaking at 1500 rpm to obtain a suspension. (2) Preparation of silkworm pupa chitosan magnetic microspheres The flow rate of the suspension was controlled at 2-4 mL / h using a droplet injection pump, and a 5-5.5 kV DC high voltage was applied. The suspension was dropped into a 10 wt% sodium hydroxide solution from a height of 8-12 cm. The droplets were rapidly solidified into spheres by the high voltage electric field. The solidified silkworm pupa chitosan magnetic microspheres were washed repeatedly with deionized water until the pH of the washing solution reached neutral. Finally, the washed microspheres were air-dried at 25°C or dried using a vacuum freeze-drying device.
[0008] As an improvement, the acid solution is at least one selected from acetic acid, formic acid, dilute hydrochloric acid, dilute sulfuric acid, dilute nitric acid, and citric acid, and the concentration of the acid solution is 0.1-15% (v / v).
[0009] As an improvement, the acid solution is acetic acid with a concentration of 2.0%.
[0010] As an improvement, the amount of magnetic nanoparticles added is 0.10-0.12% of the chitosan mass.
[0011] As an improvement, the flow rate in step (2) is 4 mL / h, the DC high voltage is 5.5 kV, and the height is 10 cm. The particle size of the above-mentioned silkworm pupa chitosan magnetic microspheres can be changed by adjusting the droplet falling height and the DC high voltage. Increasing or decreasing the droplet falling height will correspondingly increase or decrease the particle size of the prepared silkworm pupa chitosan magnetic microspheres; decreasing or increasing the DC high voltage will correspondingly decrease or increase the average particle size of the prepared silkworm pupa chitosan magnetic microspheres.
[0012] The silkworm pupa chitosan magnetic microspheres prepared by any of the above methods have an average particle size of 300-500 µm; the silkworm pupa chitosan magnetic microspheres are enriched with exosomes.
[0013] The above-mentioned application of silkworm pupa chitosan magnetic microspheres in the preparation of drugs for treating intrauterine adhesions.
[0014] A drug for treating intrauterine adhesions, comprising the above-mentioned silkworm pupa chitosan magnetic microspheres.
[0015] Preferably, the drug is in the form of a gel, which can be directly injected into the uterine cavity, making the operation simple and the risk low.
[0016] Beneficial effects: Compared with existing technologies, this invention provides a method for preparing silkworm pupa chitosan magnetic microspheres enriched with exosomes and their application in the treatment of intrauterine adhesions. The prepared silkworm pupa chitosan magnetic microspheres can be used to enrich and load mesenchymal stem cell exosomes, achieving precise delivery through the application of an external magnetic field. This enables safe, stable, precise, efficient, and non-invasive or minimally invasive targeted delivery of mesenchymal stem cell exosomes into the uterine cavity. Specific advantages are as follows: (1) The present invention prepares magnetic hydrogel microspheres with magnetic chitosan suspension as the main structure. The method is simple, easy to operate, low in cost, and the morphology of the microspheres can be easily controlled.
[0017] (2) The present invention designs a silkworm pupa chitosan magnetic microsphere for exosome enrichment, which can easily achieve in vitro enrichment of exosomes, is safe and stable, realizes stable in vitro enrichment of exosomes and targeted delivery in the uterine cavity, and has strong in vivo and in vitro antibacterial efficacy, and is highly practical.
[0018] (3) The silkworm pupa chitosan magnetic microspheres prepared by the present invention can be used for in vitro and in vivo drug delivery, which can achieve minimally invasive, safe and targeted drug delivery. Attached Figure Description
[0019] Figure 1 The images show the products of each step in the extraction of chitosan from silkworm pupae; a) whole dried silkworm pupae, b) silkworm pupae shell powder after impurity removal, drying and pulverization, c) dried powder obtained after protein removal, d) chitin powder obtained after inorganic salt removal and drying, e) dried chitosan powder obtained after decolorization and deacetylation, and f) chitosan solution obtained by dissolving the powder in figure e in pure water.
[0020] Figure 2 The equipment used for preparing silkworm pupa chitosan magnetic microspheres includes: 1 is an injection pump, 2 is a high-voltage DC power supply, 3 is a capillary connection device, and 4 is a curing dish.
[0021] Figure 3 This is a statistical chart of the particle size of silkworm pupa chitosan magnetic microspheres. In chart a, the average particle size of silkworm pupa chitosan magnetic microspheres prepared with chitosan solutions of concentrations of 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, and 3.0 wt% at a DC voltage of 5.5 kV, with a droplet drop height of 10 cm and a flow rate controlled at 4 mL / h. Chart b shows the average particle size of silkworm pupa chitosan magnetic microspheres prepared with a 2 wt% chitosan solution at voltages of 3.5 kV, 4.0 kV, 4.5 kV, 5.0 kV, 5.5 kV, and 6.0 kV, with a droplet drop height of 10 cm and a flow rate controlled at 4 mL / h.
[0022] Figure 4Images of the silkworm pupa chitosan magnetic microspheres prepared according to this invention are shown below; a) shows magnetic nanoparticles, b) shows a comparison of actual images of chitosan microspheres and silkworm pupa chitosan magnetic microspheres, and c) and d) show chitosan microspheres and silkworm pupa chitosan magnetic microspheres observed under an optical microscope, respectively.
[0023] Figure 5 This study demonstrates the directional movement of silkworm pupa chitosan magnetic microspheres under the influence of a magnetic field. By applying magnetic fields in different directions, the silkworm pupa chitosan magnetic microspheres are controlled to move along complex trajectories.
[0024] Figure 6 The cell compatibility and proliferation activity of silkworm pupa chitosan magnetic microspheres are statistically analyzed. a) is the cell compatibility and proliferation activity of 3T3 cells, b) is the cell compatibility and proliferation activity of HUVEC cells, and c) is the cell compatibility and proliferation activity of BMSC cells.
[0025] Figure 7 The results of organ compatibility experiments with silkworm pupa chitosan magnetic microspheres are shown. Representative cuts of HE-stained sections of heart, liver, spleen, lung, and kidney from rats that had not undergone any treatment and rats that had received uterine injections of exosome-loaded silkworm pupa chitosan magnetic microspheres are compared.
[0026] Figure 8 This study presents the results and statistics of the blood compatibility experiment of silkworm pupa chitosan magnetic microspheres. The study included four groups: a pure water treatment group, a PBS control group, a microsphere PBS soaking solution treatment group, an exosome PBS soaking solution treatment group, and an exosome-loaded microsphere PBS soaking solution treatment group. 500 μL of pure water, PBS, microsphere PBS soaking solution, exosome PBS soaking solution, and exosome-loaded microsphere PBS soaking solution were added to 1 mL of PBS containing 20 μL of blood cell precipitate, respectively. The mixture was then incubated at 37 ℃ for 1 h, photographed, and its OD value was measured to calculate the hemolysis rate.
[0027] Figure 9 Characterization of the extracted exosomes: a) is a representative NTA scan of exosomes, and b) is the particle size distribution of the extracted exosomes.
[0028] Figure 10 Images obtained using a confocal electron fluorescence microscope for the enrichment of exosomes by silkworm pupa chitosan magnetic microspheres. Red fluorescence indicates PKH26-stained exosomes, while green fluorescence indicates silkworm pupa chitosan magnetic microspheres labeled with monomolecular polystyrene nanoparticles. Detailed Implementation
[0029] The above-described solution will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All equivalent transformations or modifications made based on the substantial content of this invention should be covered within the protection scope of this invention. Implementation conditions not specified in the embodiments are generally conditions found in conventional experiments.
[0030] Example 1: Chitosan Extract from Silkworm Pupae Fresh silkworm pupae were cleaned, dried, and pulverized. They were then added to a 10% (w / v) NaOH solution at a ratio of 1g:10ml and digested at 95℃. After stirring at 600 rpm for 1 h, the mixture was filtered through double-layered gauze. Sufficient 10% (w / v) NaOH solution was added to the precipitate, and the residue was digested at 85℃ for another 9 h. The mixture was then filtered again, and the residue was washed with deionized water until the pH value was neutral.
[0031] Add sufficient 5% HCl (v / v) to the residue and treat at 25℃ for 24 hours to remove minerals. Filter to obtain a demineralized sample and wash repeatedly with deionized water until the pH value is neutral. Then dry at 60℃ for 6 hours to obtain chitin powder. Finally, add sufficient 50% (w / v) NaOH and stir at 250 rpm in a 90℃ water bath for 9 hours. Finally, filter out the alkaline solution with filter paper, wash the residue with deionized water until the pH value is neutral, and dry at 37℃ to obtain chitosan powder.
[0032] Figure 1 Images of the products from each step of chitosan extraction from silkworm pupae.
[0033] Example 2 Preparation of Magnetic Nanoparticles (FO NPs) Dissolve 1.6 mmol FeCl3-6H2O, 8.8 mmol Na3CT, and 1.8 mmol NH4Ac in 16 mL of ethylene glycol, then add 6 mmol NaOH solid and stir at 37 °C for 30 min. Transfer to a 20 mL Teflon stainless steel autoclave and heat in a muffle furnace at 190 °C for 9 h. After heating, allow to cool naturally to room temperature. Collect the black precipitate using a neodymium magnet. Finally, wash the precipitate three times with water and ethanol, and store the FO NPs in 20 mL of ethanol for later use.
[0034] Example 3: Optimization of the preparation process of silkworm pupa chitosan magnetic microspheres Figure 2 This is a physical diagram of the apparatus for preparing silkworm pupa chitosan magnetic microspheres. In the diagram, 1 is a droplet microfluidic injection pump, 2 is a high-voltage direct current (VDC) power supply, 3 is a capillary connection device, and 4 is a curing dish. The capillary connection device 3 is connected to the droplet microfluidic injection pump 1. The flow rate is controlled by the flow control chip of the droplet microfluidic injection pump 1. Simultaneously, it is connected to the VDC power supply 2 to introduce direct current, achieving controllable droplet dispersion. The dispersed droplets are collected at a certain height through the curing dish 4, and the droplets are then solidified. The specific connection method is conventional technology in this field and is not particularly special; therefore, it will not be described in detail in this embodiment.
[0035] 1. Effect of chitosan solution concentration on the preparation of silkworm pupa chitosan magnetic microspheres The preparation steps of silkworm pupa chitosan magnetic microspheres are as follows: Step 1: Dissolve chitosan powder in an acid solution to prepare a chitosan solution with a mass fraction of 1-3%; add the ethanol dispersion of superparamagnetic Fe3O4 nanoparticles to the chitosan solution at a ratio of 20 μg: 1 mL, and disperse it evenly by high-speed shaking at 1500 rpm to obtain a suspension. Step 2: Using a droplet injection pump, the flow rate of the suspension is controlled at 4 mL / h, and a 5.5 kV DC high voltage is applied. The suspension is dropped from a height of 10 cm into a 10 wt% sodium hydroxide solution. The droplets are rapidly solidified into spheres by the action of the high voltage electric field. The solidified silkworm pupa chitosan magnetic microspheres are washed repeatedly with deionized water until the pH of the washing solution reaches neutral. Finally, the washed microspheres are air-dried at 25°C to obtain silkworm pupa chitosan magnetic microspheres.
[0036] To test the effect of different concentrations of chitosan solution on microspheres, the concentrations prepared in step 1 were 1.0 wt%, 1.5 wt%, 2.0 wt%, 2.5 wt%, and 3.0 wt%, respectively, and the remaining steps were the same.
[0037] Experimental results are as follows Figure 3 As shown in (a), the microspheres prepared with chitosan solution concentrations in the range of 1.5-2.5 wt% exhibited relatively stable particle sizes, while those prepared with a 3.0 wt% chitosan solution had larger particle sizes, making in vivo drug delivery difficult. Furthermore, the microspheres prepared with a 1.0 wt% chitosan solution had extremely low strength, making it difficult to maintain their morphology and ensuring drug loading stability.
[0038] 2. Effect of DC voltage on the preparation of silkworm pupa chitosan magnetic microspheres The preparation steps of silkworm pupa chitosan magnetic microspheres are as follows: Step 1: Dissolve chitosan powder in an acid solution to prepare a 2 wt% chitosan solution; add the ethanol dispersion of superparamagnetic Fe3O4 nanoparticles to the chitosan solution at a ratio of 20 μg: 1 mL, and disperse it evenly by high-speed shaking at 1500 rpm to obtain a suspension. Step 2: Using a droplet injection pump, the flow rate of the suspension is controlled at 4 mL / h, and a DC high voltage is applied at different voltages. The suspension is dropped from a height of 10 cm into a 10 wt% sodium hydroxide solution. The droplets are rapidly solidified into spheres by the action of the high voltage electric field. The solidified silkworm pupa chitosan magnetic microspheres are washed repeatedly with deionized water until the pH value of the washing solution reaches neutral. Finally, the washed microspheres are air-dried at 25°C to obtain silkworm pupa chitosan magnetic microspheres.
[0039] To test the effect of different DC voltages on the preparation of microspheres, the concentrations prepared in step 1 were 3.5 kV, 4.0 kV, 4.5 kV, 5.0 kV, 5.5 kV, and 6.0 kV, respectively, and the remaining steps were the same.
[0040] Experimental results are as follows Figure 3 As shown in (b), within the range of 3.5 kV to 5.5 kV, the microsphere size is positively correlated with the applied DC voltage, and the decrease gradually diminishes. The droplets formed at a DC voltage of 6.0 kV exhibit poor stability, and the microspheres are irregularly shaped, making statistical analysis difficult. Therefore, the optimal DC voltage is 5–5.5 kV.
[0041] 3. Effect of droplet fall height on silkworm pupa chitosan magnetic microspheres The preparation steps of silkworm pupa chitosan magnetic microspheres are as follows: Step 1: Dissolve chitosan powder in an acid solution to prepare a 2 wt% chitosan solution; add the ethanol dispersion of superparamagnetic Fe3O4 nanoparticles to the chitosan solution at a ratio of 20 μg: 1 mL, and disperse it evenly by high-speed shaking at 1500 rpm to obtain a suspension. Step 2: Using a droplet injection pump, the flow rate of the suspension is controlled at 4 mL / h, and a DC high voltage is applied at different voltages. The suspension is dropped into a 10 wt% sodium hydroxide solution from a height of 4-12 cm. The droplets are rapidly solidified into spheres by the action of the high voltage electric field. The solidified silkworm pupa chitosan magnetic microspheres are washed repeatedly with deionized water until the pH value of the washing solution reaches neutral. Finally, the washed microspheres are air-dried at 25℃ to obtain silkworm pupa chitosan magnetic microspheres.
[0042] The same steps described above were used to test the effect of different droplet drop heights on the preparation of microspheres. The concentrations prepared in step 1 were 4 cm, 6 cm, 8 cm, 10 cm, and 12 cm, with the remaining steps being identical. No significant difference was found in the microsphere preparations prepared with different droplet drop heights in this test. Considering the ease of droplet collection and operation during the preparation process, 8-12 cm was selected as the optimal range for droplet collection.
[0043] Example 4: Preparation and Characterization of Silkworm Pupa Chitosan Magnetic Microspheres The preparation steps of silkworm pupa chitosan magnetic microspheres are as follows: The chitosan powder prepared in Example 1 was added to a freshly prepared 2% (v / v) glacial acetic acid solution and heated in a water bath at 37°C for 1 h to form a 2wt% chitosan solution. The magnetic nanoparticles prepared in Example 2 were added to the chitosan solution at a ratio of 20 μg:1 mL, and the solution was shaken at 1500 rpm for 5 min to uniformly disperse them, resulting in a magnetic nanoparticle-chitosan suspension. 1 mL of the magnetic nanoparticle-chitosan suspension was drawn using a 1 mL syringe, which was then attached to a droplet microfluidic injection pump. The parameters were adjusted to control the flow rate at 4 mL / h. A 5.5 kV DC high voltage was connected to the needle, which was connected to a capillary tube and a capillary glass tube with the capillary glass tube opening vertically downwards and its bottom 10 cm away from a curing dish containing 10 wt% NaOH solution. The suspension was dropped into the 10 wt% NaOH solution, and the droplets were separated and rapidly solidified into hydrogel microspheres by the high-voltage electric field. After the entire suspension solidified into microspheres, the solidified silkworm pupa chitosan magnetic microspheres were washed repeatedly with deionized water until the pH of the washing solution reached neutral. Finally, the washed microspheres were air-dried at room temperature to obtain silkworm pupa chitosan magnetic microspheres with an average particle size of approximately 350 μm.
[0044] Magnetic guidance tests were conducted on the microspheres: a magnet was placed below the MCM and moved according to a preset pattern to guide its movement along a specific trajectory. The movement path was captured and recorded using a high-resolution camera. By applying magnetic fields in different directions, the silkworm pupa chitosan magnetic microspheres were controlled to move directionally along different complex trajectories.
[0045] Figure 4 Images of the silkworm pupa chitosan magnetic microspheres prepared according to this invention are shown below; a) shows magnetic nanoparticles, b) shows a comparison of actual images of chitosan microspheres and silkworm pupa chitosan magnetic microspheres, and c) and d) show chitosan microspheres and silkworm pupa chitosan magnetic microspheres observed under an optical microscope, respectively.
[0046] Figure 5 The trajectory formed by the silkworm pupa chitosan magnetic microspheres moving with the magnetic field demonstrates the excellent directional movement ability of the silkworm pupa chitosan magnetic microspheres.
[0047] Example 5: Safety evaluation of silkworm pupa chitosan magnetic microspheres 1. Statistical analysis of cell compatibility and proliferation activity of silkworm pupa chitosan magnetic microspheres: In vitro biocompatibility testing was performed using 3T3 cells, HUVEC cells, and BMSC cells (all sourced from Cyagen Biosciences). The experiment was divided into a control group and a microsphere group. In the control group, 1 ml of sterile PBS was soaked in 50 ml of 10% serum-concentrated culture medium for 24 h, and the extract was obtained by filtration through a 0.22 μm filter. In the microsphere group, an equal volume of sterile silkworm pupa chitosan magnetic microspheres soaked in PBS was soaked in 50 ml of 10% serum-concentrated culture medium for 24 h, and the extract was obtained by filtration through a 0.22 μm filter. Cells were digested at 37°C for 1 min and counted. Subsequently, 90 μL of the cell suspension was prepared in each well of a 96-well cell culture plate. The culture plates were pre-cultured in an incubator for 24 h (37℃, 5% CO2). Then, 10 μL of the corresponding extract was added to each well of the culture plate, and the plates were incubated for another 24 h. Next, 10 μL of CCK8 solution was added to each well, and the plates were incubated at 37℃ for 1 h. The absorbance at 450 nm was then measured using a microplate reader. Cell viability was calculated as follows: Cell viability (%) = (OD-treated group - OD cell-free group) / (ODPBS group - OD cell-free group) × 100%.
[0048] Figure 6 The cell compatibility and proliferation activity of silkworm pupa chitosan magnetic microspheres were statistically analyzed. a) shows the compatibility and proliferation activity of 3T3 cells, b) shows the compatibility and proliferation activity of HUVEC cells, and c) shows the compatibility and proliferation activity of BMSC cells. Calcein-AM staining and CCK8 incubation absorbance assays showed that the cells in each group exhibited good morphological characteristics and high activity, demonstrating that the silkworm pupa chitosan magnetic microspheres possess good cell compatibility, are non-cytotoxic, and do not significantly affect cell proliferation.
[0049] (1) Organ compatibility statistics of silkworm pupa chitosan magnetic microspheres: The experiment was divided into a control group and a treatment group. The control group consisted of healthy female Sprague-Dawley (SD) rats weighing 180-220 g and weighing 6-8 weeks old, which received no treatment. The treatment group consisted of healthy female Sprague-Dawley (SD) rats weighing 180-220 g and weighing 6-8 weeks old, which received bilateral uterine injections of 1 mL of strictly aseptically treated silkworm pupa chitosan magnetic microspheres loaded with 100 ng / mL Exo. Heart, liver, spleen, lung, and kidney tissues were collected from both the control and treatment groups, fixed, and prepared for HE staining (the section preparation procedure was a routine operation in animal experiments). The obtained results are shown below. Figure 7 Slices.
[0050] from Figure 7The organ compatibility of silkworm pupa chitosan magnetic microspheres showed that there were no significant differences in HE-stained sections of the heart, liver, spleen, lungs, and kidneys of rats in the control and treatment groups. The intrauterine implantation of silkworm pupa chitosan magnetic microspheres had no significant effect on the heart, liver, spleen, lungs, and kidneys of rats.
[0051] (2) Blood compatibility statistics of silkworm pupa chitosan magnetic microspheres: Rat blood was collected for an in vitro hemolysis test. Red blood cells collected by centrifugation were resuspended in PBS to prepare a red blood cell solution. Then, 500 μL of dH2O and PBS were added to 1 mL of 10% red blood cell solution as positive and negative control groups, respectively. Simultaneously, 500 μL of silkworm pupa chitosan magnetic microspheres (MS), exosomes (Exo), and exosome-loaded silkworm pupa chitosan magnetic microspheres (MS@Exo) were added from the exudate and cultured at 37 °C for 1 h in 1000 μL of 2% red blood cell solution. After centrifugation, the hemolysis status of each group was observed. Figure 8 The hemolysis rate of silkworm pupa chitosan magnetic microspheres was statistically analyzed. Compared with the control group, the hemolysis rate of both silkworm pupa chitosan magnetic microspheres and exosomes was less than 5%.
[0052] Example 6 Characterization of exosomes enriched in vitro by silkworm pupa chitosan magnetic microspheres BMSCs were extracted from the tibia and femur of 2-week-old rats and cultured, and exosomes were extracted from the BMSC culture supernatant. The specific steps were as follows: The bone marrow cavities of the tibia and femur of 2-week-old rats were washed with sterile 10% serum cell culture medium to obtain BMSC-containing medium. The collected cells were identified, and cells conforming to the characteristics of BMSCs were selected for further culture. BMSCs from passage 2 to passage 6 were cultured in low-glucose DMEM (Gibco) supplemented with 10% Exo-free FBS (Gibco). After cell confluence reached 50-60%, the supernatant was harvested and ultracentrifuged. First, the supernatant was centrifuged at 300×g, 3000×g, and 10000×g for 10 min, 20 min, and 30 min, respectively, to separate live / dead cells, cell debris, and large vesicles. Then, after centrifugation at 100000×g for 70 min, the Exo at the bottom of the centrifuge tube was resuspended in phosphate-buffered saline (PBS) and stored at -80 °C. All of the above centrifugation processes were carried out at 4 °C.
[0053] Figure 9 To obtain exosome characterization, a is a representative NTA scan of exosomes, and b is the particle size distribution of the extracted exosomes.
[0054] Prepare 1 mL of 20 µg / mL exosome solution, wash repeatedly with PBS buffer to remove impurities, stain with red PKH26 dye for 10 min, then centrifuge at ultrahigh speed to remove the dye, and resuspend in 1 mL of sterile PBS. Mix 1 mL of the magnetic nanoparticle-chitosan suspension prepared in Example 4 thoroughly with a small amount of green fluorescent nanoparticles, and prepare fluorescently labeled silkworm pupa chitosan magnetic microspheres according to the optimal process in Example 4. Neutralize the microspheres with sterile PBS and add them to the above exosome solution. Incubate at 37°C for 0.5 h to allow the chitosan microspheres to fully bind with the exosomes. After low-speed centrifugation, separate the silkworm pupa chitosan magnetic microspheres from the supernatant using a neodymium magnet. Collect the magnetic microspheres enriched with exosomes, wash three times with phosphate buffer for 5 min each time, and discard the supernatant. Observe the collected magnetic microspheres using a confocal electron fluorescence microscope.
[0055] Figure 10 Representative images obtained using confocal electron fluorescence microscopy for the experiment of enriching exosomes on silkworm pupa chitosan magnetic microspheres. Red fluorescence indicates PKH26-stained exosomes, while green fluorescence indicates silkworm pupa chitosan magnetic microspheres labeled with single-molecule polyethylene nanoparticles.
[0056] Example 7: Experiment on the treatment of intrauterine adhesions in SD rats Following the preparation method in Example 6, silkworm pupa chitosan magnetic microspheres loaded with rat mesenchymal stem cell exosomes were prepared. Using rats as experimental subjects, the therapeutic effect of these silkworm pupa chitosan magnetic microspheres loaded with rat mesenchymal stem cell exosomes was investigated as follows: Healthy female Sprague-Dawley (SD) rats weighing 180-220 g, aged 4-6 weeks, were selected and housed in an SPF-grade environment. An endometrial injury model was established using the mechanical scraping method.
[0057] The rat uterus was randomly divided into five groups (n=6 / group): sham operation group (only abdominal surgery to expose the uterus, no mechanical damage); injury group (abdominal surgery to expose the uterus, bilateral mechanical damage to the uterus); microsphere group (abdominal surgery to expose the uterus, unilateral mechanical damage to the uterus followed by injection of 1 mL of strictly sterile silkworm pupa chitosan magnetic microspheres); exosome treatment group (abdominal surgery to expose the uterus, unilateral mechanical damage to the uterus followed by injection of 1 mL of strictly sterile 100 ng / mL exosomes); and microsphere-loaded exosome group (abdominal surgery to expose the uterus, unilateral mechanical damage to the uterus followed by injection of 1 mL of strictly sterile silkworm pupa chitosan magnetic microspheres loaded with 100 ng / mL Exo).
[0058] Fourteen days post-surgery (covering a complete estrous cycle), rats were euthanized, and bilateral uterine tissue was completely dissected, fixed in 4% paraformaldehyde for 24 h, dehydrated with graded ethanol, and then embedded in paraffin. The tissues were stained with hematoxylin and eosin (H&E) and Masson's trichrome staining, and then observed and photographed under a microscope. Representative images were selected to measure endometrial thickness (vertical distance from the basement membrane to the luminal epithelium), glandular density (number of glands / high-power field), and fibrosis area ratio (percentage of fibrotic area to total endometrial area), and the data were statistically analyzed. The results are shown in Table 1 and... Figure 1 As shown.
[0059] Table 1 shows the average endometrial thickness, collagen deposition fraction, and glandular number in rats from the blank group, control group, and each experimental group.
[0060] The statistical results above show that the endometrium in the injury group was significantly thinner, with numerous areas of collagen deposition and severe glandular damage, confirming the effectiveness of our established IUA animal model. However, the microsphere group showed no significant improvement in endometrial thickness, collagen deposition, or glandular number compared to the injury group, suggesting that the microspheres themselves have little therapeutic or repairing effect on IUA in rats. The exosome treatment group and the microsphere + exosome treatment group showed a significant increase in average uterine thickness and a significant reduction in collagen deposition. Two weeks after treatment, the number of endometrial glands in the exosome treatment group and the microsphere + exosome treatment group was significantly lower than that in the control group, but significantly higher than that in the injury group and the microsphere treatment group. The microsphere + exosome treatment group had a higher average endometrial thickness and less collagen deposition compared to the exosome treatment group alone.
[0061] Based on the above analysis, it can be seen that the Exo treatment of the present invention significantly increases the thickness and number of glands in the endometrium, while significantly reducing the deposition of collagen in the uterus to reduce endometrial fibrosis, and administration via the MS@CS-Exo complex will bring better therapeutic effects.
Claims
1. A method for preparing exosome-enriched silkworm chrysalis chitosan magnetic microspheres, characterized in that, The method comprises the following steps: (1) preparing a magnetic nanoparticle chitosan suspension Chitosan powder is dissolved in an acid solution to prepare a chitosan solution with a mass fraction of 1.5-2.5%; the ethanol dispersion of superparamagnetic Fe3O4 nanoparticles is added to the chitosan solution at a ratio of 20 μg of magnetic nanoparticles to 1 mL of chitosan solution, and the mixture is uniformly dispersed by high-speed oscillation at 1500 rpm to obtain the suspension; (2) preparing silkworm chrysalis chitosan magnetic microspheres The flow rate of the suspension is controlled by a liquid drop injection pump at 2-4 mL / h, and a direct current high voltage of 5-5.5 kV is connected, the suspension is dropped into a 10 wt% sodium hydroxide solution from a height of 8-12 cm, and the liquid drops are quickly solidified into spherical shape by the action of the high-voltage electric field, the solidified silkworm chrysalis chitosan magnetic microspheres are washed with deionized water for multiple times until the pH value of the washing liquid reaches neutral, and finally, the washed microspheres are air-dried at 25°C or dried by using a vacuum freeze-drying device.
2. The method of claim 1, wherein the method is characterized by, The acid solution is at least one of acetic acid, formic acid, dilute hydrochloric acid, dilute sulfuric acid, dilute nitric acid, and citric acid, and the concentration of the acid solution is 0.1-15% (v / v).
3. The method of claim 2, wherein the method is characterized by, The acid solution is acetic acid with a concentration of 2.0%.
4. The method of claim 1, wherein the method is characterized by, The amount of the magnetic nanoparticles added is 0.1-0.12% of the mass of the chitosan.
5. The method of claim 1, wherein the method is characterized by, In step (2), the flow rate is 4 mL / h, the direct current high voltage is 5.5 kV, and the height is 10 cm.
6. The magnetic chitosan microspheres prepared according to the method of any one of claims 1-5, characterized in that, The average particle size of the silkworm chrysalis chitosan magnetic microspheres is 300-500 μm, and the silkworm chrysalis chitosan magnetic microspheres are rich in exosomes.
7. Use of the silkworm chrysalis chitosan magnetic microspheres according to claim 6 in the preparation of a drug for treating intrauterine adhesion.
8. A drug for treating intrauterine adhesion, characterized by, The drug comprises the silkworm chrysalis chitosan magnetic microspheres according to claim 6.
9. The drug for treating intrauterine adhesion according to claim 8, wherein the drug is a drug for treating intrauterine adhesion. The dosage form of the drug is a gel preparation.