Microbubble prepared based on in-situ gas generation method and preparation method and application thereof

Abstract

The invention discloses a microbubble prepared based on an in-situ gas generation method and a preparation method and application thereof. According to the method, the gas generating agent is entrapped in the microspheres, the triggering agent is used for activating reaction under mild conditions, gas is directly generated in situ in the microspheres, form conversion from the microspheres to the microbubbles is achieved, the gas is stably entrapped in the microbubbles, the size, density and release kinetics of the microbubbles can be accurately regulated and controlled, operation is simple, complex exogenous triggering conditions are avoided, and the method is suitable for large-scale industrial production. Therefore, a microbubble system capable of generating gas on demand is constructed, and a new strategy is provided for tumor microenvironment targeting, focus specific imaging and the like. The microbubble can be simultaneously modified with an anti-VCAM-1 antibody and entrapped with an endometritis treatment drug, a novel drug carrier which has focus targeting, drug long-acting slow release and ultrasonic visual tracking functions and is safer is provided for treatment of endometritis, and a novel solution can also be provided for precise long-acting treatment of endometritis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a microbubble prepared by in-situ gas generation method, its preparation method, and its application. Background Technology

[0002] Currently, approximately 17.5% of couples of reproductive age worldwide suffer from infertility. In vitro fertilization-embryo transfer (IVF-ET) is a core treatment for infertility; however, in clinical application, 15%–20% of IVF-ET patients still face recurrent implantation failure (RIF). Research data indicates that approximately 30% of RIF patients also have endometritis. This disease is caused by ascending bacterial infection leading to a persistent inflammatory response in the endometrium. The persistent inflammatory microenvironment damages endometrial receptivity, directly hindering embryo implantation. Current routine clinical treatment for endometritis primarily involves systemic antibiotic administration (such as oral doxycycline or intravenous levofloxacin), but this treatment method has two major limitations: firstly, it requires increasing the blood concentration of the systemically administered drug to maintain an effective drug concentration in the uterine cavity; secondly, systemic administration easily causes gastrointestinal discomfort and other toxic side effects. For patients with more severe endometritis, clinical trials have attempted local treatment via intrauterine instillation of drug solutions. However, the instillation solution is prone to leakage from the vagina due to gravity, making it difficult to maintain an effective drug concentration within the uterine cavity for an extended period. Furthermore, repeated procedures exacerbate patient discomfort. In recent years, biomaterial carrier technology has provided new insights into improving drug delivery efficiency. For example, Zhang's team developed an injectable hydrogel loaded with iodine compounds for the treatment of endometritis. However, the porous structure and large pore size of the hydrogel resulted in a cumulative release rate of up to 80% of the iodine compound within 24 hours in a simulated intrauterine fluid environment, failing to maintain an effective drug concentration for an extended period. Further analysis revealed two major technical bottlenecks in existing carrier technologies: first, existing carriers lack specific adhesion to inflamed sites of endometrial inflammation, easily being rapidly discharged with intrauterine secretions, leading to insufficient residence time within the uterine cavity and an inability to maintain an effective drug concentration for a prolonged period; second, existing carriers do not integrate real-time image tracking, making it difficult to assess the carrier delivery path and its residence within the uterine cavity, thus limiting the precision of treatment plans.

[0003] Microbubbles, due to their unique physicochemical properties, show great potential in fields such as biomedical imaging, targeted drug delivery, and disease treatment. Patent CN115737589A discloses an adhesive drug microsphere comprising an outer layer, a middle layer, and an inner layer. The outer layer is an adhesive layer composed of a positively charged hydrophilic polymer. The middle and inner layers are a cross-linked shell composed of water-soluble protein or water-soluble polymer and a porous inner layer, respectively. The porous interior is filled with gas for loading drugs. When the microspheres are loaded with drugs for treating bladder diseases, a bladder instillation therapeutic microsphere formulation can be obtained. When the bladder fluid is emptied, the drug microspheres gently adhere to the bladder wall, and when the bladder fluid increases, the drug microspheres float back up without causing damage to the bladder mucosa or epithelium. The microspheres can also be used in tumor targeting or image enhancement. However, it is difficult to control the gas content in the microspheres during the microsphere preparation process to ensure that the obtained microspheres have an appropriate particle size. The adhesive drug microspheres can be instilled into the bladder via the urethra without the risk of urethral obstruction. Therefore, providing a preparation method that can control the size of microbubble synthesis is of great significance for improving the accuracy, duration of efficacy, and safety of treatment for diseases such as endometritis. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a method for preparing microbubbles.

[0005] A second objective of this invention is to provide microbubbles prepared by the above-described preparation method.

[0006] The third objective of this invention is to provide a method for preparing uterine inflammation-targeting microbubbles.

[0007] The fourth objective of this invention is to provide uterine inflammation-targeting microbubbles prepared by the above-described preparation method.

[0008] The fifth objective of this invention is to provide the application of the above-mentioned uterine inflammation-targeting microbubbles in the preparation of a medicament for treating endometritis.

[0009] A sixth objective of the present invention is to provide a formulation comprising the above-mentioned uterine inflammation-targeting microvesicles.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a method for preparing microbubbles, the method comprising the following steps: S1. Mix the protein aqueous solution and the gas-generating agent evenly; S2. Add an organic solvent containing a surfactant to the mixed solution obtained in step S1, and stir rapidly to prepare droplets; S3. Add a cross-linking agent solution to the droplets obtained in step S2 to cross-link and obtain microspheres with a protein cross-linked outer shell; S4. Add a triggering agent to the microspheres obtained in step S3. The triggering agent reacts with the gas-generating agent and generates gas in situ, resulting in microbubbles with a protein cross-linked outer shell and an internal gas core structure.

[0011] This invention provides a method for preparing microbubbles based on in-situ gas generation. First, a protein aqueous solution and a gas-generating agent are mixed uniformly. A surfactant is added to prepare droplets, and then a cross-linking agent is added to obtain microspheres with a protein cross-linked outer shell. Finally, a triggering agent is added, and after mixing with the gas-generating agent, gas is generated in-situ inside the microspheres, thus obtaining microbubbles prepared by the in-situ gas generation method. The microbubbles of this invention based on the in-situ gas generation method have a two-layer structure: an outer shell structure and an internal gas core structure. The microbubble outer shell is formed by protein cross-linking, possessing certain mechanical strength and structural stability. It can encapsulate drugs and release them to the drug delivery site. Simultaneously, the presence of the cross-linked outer shell prevents excessively rapid release, maintaining an effective drug concentration at the drug delivery site for a prolonged period. The internal gas core structure is filled with a stable gas through in-situ gas generation, preventing leakage. The gas and the cross-linked protein shell form an ultrasonic impedance difference interface, enabling visual tracking under ultrasound.

[0012] This invention utilizes an in-situ gas generation method to prepare microbubbles. By pre-embedding a gas-generating agent within microspheres or an emulsion template, a trigger agent is used to initiate the reaction under mild conditions, directly generating gas at target sites within the microspheres to form microbubbles. This technology allows for precise design of microbubble size, density, and release kinetics by controlling the amounts of the gas-generating agent and trigger agent. It relies on the inherent reactions between materials to generate gas, avoiding complex external triggering conditions. Furthermore, it can be combined with smart materials (such as pH / enzyme-responsive polymers) to construct "on-demand gas-generating" microbubble systems, providing new strategies for tumor microenvironment targeting and lesion-specific imaging.

[0013] Furthermore, the proteins in step S1 include, but are not limited to, bovine serum albumin, gelatin, collagen, or complexes thereof.

[0014] Furthermore, the concentration of the protein aqueous solution in step S1 is 5–30 wt%.

[0015] Furthermore, the gas-generating agent in step S1 includes, but is not limited to, nano-calcium carbonate, sodium bicarbonate, potassium bicarbonate, or urea.

[0016] Furthermore, the concentration of the gas-generating agent is 5–50 wt%. The concentration of the gas-generating agent is related to the amount of gas produced, and the amount of gas produced determines the size of the microbubble particles. An appropriate particle size ensures that the microbubbles can be instilled into the uterine cavity through the vagina and achieve uniform dispersion and stable retention within the uterine cavity.

[0017] Furthermore, the surfactant in step S2 includes, but is not limited to, Span 80 or Span 85; the concentration is 3 to 10 wt%.

[0018] Furthermore, the organic solvent in step S2 includes, but is not limited to, n-octane or toluene.

[0019] Furthermore, in step S2, the stirring speed is 1000-10000 rpm, and the stirring time is 1-5 min.

[0020] Furthermore, the crosslinking agent in step S3 includes, but is not limited to, terephthaloyl chloride or glutaraldehyde. Furthermore, the concentration of the crosslinking agent is 5–25 wt%, and the crosslinking time is 5–15 min.

[0021] Furthermore, the triggering agent in step S4 includes, but is not limited to, hydrochloric acid, acetic acid, or urease.

[0022] Furthermore, the concentration of the trigger is 0.1–1 M and the volume is 1–10 mL.

[0023] Furthermore, the microbubble size is 30–120 μm.

[0024] The present invention also provides microbubbles prepared by any of the above-described preparation methods.

[0025] This invention also provides a method for preparing uterine inflammation-targeting microvesicles, the preparation method comprising the following steps: S1. The uterine inflammation-targeting antibody was modified on the surface of the above microvesicles to obtain uterine inflammation-targeting microvesicles; S2. Add an endometritis treatment agent to the uterine inflammation-targeting microbubbles obtained in step S1 to obtain targeted microbubbles loaded with the endometritis treatment agent.

[0026] By modifying the surface of microbubbles with uterine inflammation-targeting antibodies, they can target the site of endometrial inflammation. The microbubbles are loaded with endometritis treatment agents and gradually release the drug into the uterine cavity. At the same time, the presence of the outer shell cross-linking layer prevents the release from being too rapid, thus maintaining an effective drug concentration in the uterine cavity for a long time.

[0027] Furthermore, the uterine inflammation-targeting antibody in step S1 includes, but is not limited to, anti-VCAM-1 antibody.

[0028] Furthermore, the endometritis treatment agent in step S2 includes, but is not limited to, anti-inflammatory drugs, growth factors, hormones, or combinations thereof.

[0029] Furthermore, the anti-inflammatory drugs include, but are not limited to, levofloxacin hydrochloride, clindamycin hydrochloride, or doxycycline hydrochloride; growth factors include, but are not limited to, granulocyte colony-stimulating factor; and hormones include, but are not limited to, human chorionic gonadotropin.

[0030] Furthermore, in step S1, the uterine inflammation-targeting antibody is modified onto the surface of microvesicles via chemical conjugation and biotin-avidin bridging.

[0031] Furthermore, the chemical coupling method is the EDC / NHS coupling method.

[0032] Preferably, the loading method of the therapeutic agent in step S2 includes, but is not limited to, active drug loading based on the principle of concentration gradient diffusion.

[0033] The present invention also provides uterine inflammation-targeting microbubbles prepared by the above preparation method.

[0034] This invention relates to uterine inflammation-targeting microbubbles based on in-situ gas generation. The microbubble surface is modified with an anti-VCAM-1 antibody, enabling the microbubbles to target inflamed areas of the endometrium with high VCAM-1 secretion levels. The microbubbles are loaded with an endometritis treatment agent, allowing for continuous drug release within the uterine cavity and maintaining an effective drug concentration for an extended period. The gas core structure within the microbubble is filled with a stable gas through in-situ gas generation, allowing for precise control of the microbubble size and ultrasound visualization to track its distribution on the endometrial surface, enabling dynamic adjustments to the perfusion procedure. These microbubbles, through their lesion targeting, drug loading and sustained-release characteristics, and ultrasound visualization capabilities, address the problems of low efficiency and significant toxic side effects associated with systemic drug administration in current clinical endometritis treatments. They also overcome key shortcomings of other drug carriers, such as rapid expulsion with uterine secretions, difficulty in assessing carrier delivery pathways and uterine cavity retention, and the risk of carrier blockage within the body.

[0035] The present invention also provides the application of the above-mentioned uterine inflammation-targeting microbubbles in the preparation of a drug for treating endometrial inflammation.

[0036] The present invention also provides a formulation comprising the above-mentioned uterine inflammation-targeting microvesicles.

[0037] This invention also provides the specific application of the above-mentioned uterine inflammation-targeting microbubble preparation based on in-situ gas generation in the perfusion treatment of endometritis, including the following steps: S1. The above microbubbles are injected into the uterine cavity via the vagina, so that they are targeted to the site of endometrial inflammation; S2. During the perfusion process, a routine clinical diagnostic ultrasound instrument is used to track the distribution of microbubbles on the surface of the endometrium in real time, and the perfusion operation is dynamically adjusted. S3. Microbubbles continuously release drugs at the site of endometritis, maintaining an effective drug concentration in the uterine cavity for a long time, thus achieving long-term treatment of endometritis.

[0038] Furthermore, during the perfusion process, the diagnostic ultrasound instrument used to track the distribution of microbubbles in real time is a high-frequency ultrasound of 7–15 MHz with a mechanical index (MI) of 0.3–0.7.

[0039] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a microbubble prepared by in-situ gas generation, its preparation method, and its application. By encapsulating a gas-generating agent inside a microsphere and activating the reaction under mild conditions using a trigger, gas is directly generated in-situ inside the microsphere, achieving the morphological transformation from microsphere to microbubble. The gas is stably encapsulated inside the microbubble, allowing for precise control of microbubble size, density, and release kinetics. The operation is simple and avoids complex external triggering conditions, thus constructing a "gas-generating on demand" microbubble system. This microbubble can be simultaneously modified with anti-VCAM-1 antibodies and encapsulated endometritis treatment drugs. The microbubble acts as a drug carrier, targeting the site of inflammation and remaining in the uterine cavity for an extended period. This overcomes the problem of rapid leakage of drug solutions from the vagina in existing intrauterine perfusion methods. The microbubble is internally loaded with anti-inflammatory drugs, growth factors, and hormones, allowing for continuous drug release at the site of inflammation and maintaining an effective drug concentration within the uterine cavity for a prolonged period. This addresses the issue of low drug efficiency in clinical oral or intravenous treatments. The inner gas core structure allows for visualized tracking under ultrasound. During perfusion, ultrasound imaging can be used to track the distribution and residence of microbubbles on the endometrial surface in real time, dynamically adjusting the perfusion operation and improving the precision and duration of efficacy in endometritis treatment. The microbubble, formed using an in-situ gas generation method, contains stable gas. Precise control of the microbubble size avoids the risks of excessively large gas cores caused by difficulty in controlling the inner gas content in conventional preparation methods, which could lead to blockages during in vivo use. Therefore, this invention provides a novel drug carrier that combines lesion targeting, long-term drug release, ultrasound visualization and tracking, and greater safety by developing an in-situ gas generation method to prepare microbubbles, which can provide a new solution for the precise and long-term treatment of endometritis. Attached Figure Description

[0040] Figure 1 Optical microscope images of uterine inflammation-targeting microbubbles prepared with different concentrations of gas-generating agents as described in Examples 1 and 2.

[0041] Figure 2 This is an optical microscopic comparison of the uterine inflammation-targeting microbubbles and ordinary microbubbles targeted to the site of endometrial inflammation as described in Example 3.

[0042] Figure 3This is a drug release curve of levofloxacin hydrochloride in physiological saline as described in Example 4.

[0043] Figure 4 This is an in vitro ultrasound imaging image of the uterine inflammation-targeting microbubbles described in Example 5.

[0044] Figure 5 Macroscopic images of the uterine morphology of rats in each treatment group as described in Example 6. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0046] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0047] Example 1: Uterine Inflammation-Targeting Microbubbles Constructed by In-situ Gas Generation Method A 20 wt% bovine serum albumin aqueous solution was prepared, and nano-calcium carbonate particles were added and mixed thoroughly to obtain a bovine serum albumin mixed solution containing 5 wt% calcium carbonate. 2 mL of the mixed solution was added to a 20 mL solution of toluene containing 6 wt% Span 80, and the mixture was rapidly stirred using a homogenizer (5000 rpm, 2 min) to form water-in-oil droplets. Then, 5 wt% terephthaloyl chloride (5 mL) was added to the droplets, and homogenization and cross-linking were continued (5000 rpm, 5 min) to form microspheres with a protein cross-linked outer shell. The microspheres were collected by centrifugation (1000 rpm, 2 min), washed three times with anhydrous ethanol to remove surface toluene, and then 0.1 M dilute hydrochloric acid (2 mL) was added and shaken for 10 min to generate carbon dioxide gas in situ inside the microspheres, thus preparing microbubbles. The microbubbles were washed with pure water until neutral pH. Microbubbles modified with anti-VCAM-1 antibody were prepared using the EDC / NHS coupling method. 5 mg of anti-VCAM-1 antibody was weighed and added to a 2 mL microbubble aqueous suspension containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. The mixture was incubated at room temperature with shaking for 30 min. The microbubbles were collected by centrifugation and washed with purified water. Then, 5 mg of levofloxacin hydrochloride was added to the microbubble aqueous suspension (2 mL), and the mixture was incubated at room temperature with shaking for 30 min. After washing with purified water, a uterine inflammation-targeting microbubble formulation modified with anti-VCAM-1 antibody and loaded with levofloxacin hydrochloride was obtained.

[0048] Figure 1The left image in the image is an optical micrograph of a uterine inflammation-targeting microbubble formulation modified with anti-VCAM-1 antibody and loaded with levofloxacin hydrochloride. The microbubble size is 30-50 μm, and the hollow structure inside the microbubble is filled with carbon dioxide gas.

[0049] Example 2: Uterine Inflammation-Targeting Microbubbles Constructed by In-situ Gas Generation Method Prepare a 10 wt% gelatin aqueous solution, add sodium bicarbonate and mix well to obtain a gelatin mixed solution containing 50 wt% sodium bicarbonate. Take 2 mL of the mixed solution and add it to a 20 mL solution containing 10 wt% Span 85 in n-octane. Use a homogenizer (10000 rpm, 2 min) to rapidly stir to form water-in-oil droplets. Then add 10 wt% glutaraldehyde (5 mL) as a crosslinking agent to the droplets and continue homogenization and crosslinking (10000 rpm, 5 min) to form microspheres with a protein crosslinked shell. Centrifuge (1000 rpm, 2 min) to collect the microspheres, wash three times with anhydrous ethanol to remove surface n-octane, then add 1 M acetic acid (10 mL) and shake for 10 min to generate carbon dioxide gas in situ inside the microspheres to prepare microbubbles. Wash with pure water until neutral pH. 4 mg of streptavidin was added to a 2 mL microbubble aqueous suspension containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, and incubated with shaking at room temperature for 2 h. The microbubbles were collected by centrifugation and washed with PBS. 1 mg of biotinylated anti-VCAM-1 antibody was added, and the mixture was incubated with shaking at room temperature for 1 h to prepare a uterine inflammation-targeting microbubble formulation modified with anti-VCAM-1 antibody. 5 mg of clindamycin hydrochloride was added to a 2 mL microbubble aqueous suspension and incubated with shaking at room temperature for 30 min. After washing with pure water, a uterine inflammation-targeting microbubble formulation modified with anti-VCAM-1 antibody and loaded with clindamycin hydrochloride was obtained.

[0050] Figure 1 The right image in the image is an optical micrograph of a uterine inflammation-targeting microbubble formulation modified with anti-VCAM-1 antibody and loaded with clindamycin hydrochloride. The microbubble size is 90–120 μm, and the hollow structure inside the microbubble is filled with carbon dioxide gas.

[0051] from Figure 1 As can be seen, by using different concentrations of gas-generating agents to react with triggers to prepare microbubbles, the particle size and density of the microbubbles can be significantly controlled. The higher the concentration of the gas-generating agent used, the larger the particle size of the prepared microbubbles, the larger the gas nuclei, and the smaller the density of the microbubbles. When the microbubble particle size increases, the time for the encapsulated drug to diffuse into the external environment based on the concentration gradient becomes relatively longer, that is, its release kinetics are also adjusted accordingly.

[0052] Example 3: Targeting Study of Microbubbles Targeting Uterine Inflammation By perfusion of 1×10 into the bilateral uterine cavities of female SD rats 9 A rat model of endometritis was established by perfusing CFU / mL *E. coli* bacterial suspension (100 μL / side). After successful model establishment, two rats were selected and randomly divided into an experimental group and a control group. The experimental group rats were perfused bilaterally with the uterus containing the uterine inflammation-targeting microbubble preparation-physiological saline suspension (10 mg / mL, 100 μL / side) prepared in Example 1. The control group rats were perfused bilaterally with the uterus containing ordinary microbubbles (microbubbles obtained by the preparation method of Example 1 without "VCAM-1 antibody and levofloxacin hydrochloride")-physiological saline suspension (10 mg / mL, 100 μL / side) prepared in Example 1. After perfusion for 30 min, the left uterus of both groups of rats was flushed: 100 μL of physiological saline was slowly injected into the left uterine cavity of each rat using a 1 mL syringe, and after standing for 10 s, the fluid was aspirated. The perfusion-aspiration cycle was repeated 3 times; the right uterus was not treated. After euthanizing the rats, the bilateral uterine tissues were completely dissected, cut along the longitudinal axis of the uterus, and laid flat on a glass slide. The slides were gently rinsed three times with physiological saline along the direction of the uterine cavity to remove any untargeted microbubbles. The slides were then placed under an optical microscope to observe the presence of residual microbubbles on the endometrial surface.

[0053] Comparative optical micrographs of uterine inflammation-targeted microbubble formulations and ordinary microbubbles targeting inflamed areas of the endometrium are shown below. Figure 2 As shown, the results indicate that the uterine inflammation-targeting microbubble preparation in the experimental group showed significant aggregation at the site of endometrial inflammation, while the ordinary microbubble residue in the control group was extremely low, indicating that the microbubbles described in this invention can effectively target the site of endometrial inflammation.

[0054] Example 4: Investigation of drug release rate of uterine inflammation-targeting microvesicles The uterine inflammation-targeting microbubble formulation prepared in Example 1 was uniformly dispersed in preheated physiological saline at a mass-to-volume ratio of 1:50 to form a microbubble-physiological saline suspension. The suspension was transferred to a 37°C constant temperature water bath for incubation, and physiological saline was aspirated at different time points to determine the concentration of levofloxacin hydrochloride in the solution.

[0055] The results are as follows Figure 3As shown, in physiological saline simulating the uterine cavity environment, the levofloxacin hydrochloride loaded on the microbubbles exhibited a biphasic release characteristic. The first 3 hours were a burst release phase, with a cumulative drug release rate of approximately 32%. Subsequently, a sustained release phase began, reaching a cumulative release rate of 71% after 120 hours. In the early stage of drug release, levofloxacin hydrochloride was rapidly released into the physiological saline and reached an effective drug concentration. In the later stage, the drug was released slowly, maintaining the drug concentration in the physiological saline within the effective range. This indicates that the controlled-release behavior of this uterine inflammation-targeting microbubble preparation can prolong the drug's action time at the site of endometrial inflammation, maintain an effective drug concentration in the uterine cavity for a longer period, and improve the treatment effect of endometritis.

[0056] Example 5: Exploring the Visualization Function of Targeted Microbubbles in Uterine Inflammation A 1% w / v agarose solution was poured into a container, and a silicone tube was embedded in the container. After the agarose solidified, a tissue prosthesis with a channel was obtained. The prosthesis channel was filled with the uterine inflammation-targeting microbubble preparation-physiological saline suspension solution prepared in Example 1, and then ultrasound imaging was performed using a portable ultrasound imaging device.

[0057] The results are as follows Figure 4 As shown, after the microbubble-saline suspension was injected, a strong ultrasound signal was generated in the channel, indicating that the uterine inflammation-targeting microbubble preparation has ultrasound visualization function.

[0058] Example 6: Exploring the therapeutic effect of targeted microbubbles for uterine inflammation The concentration of 1×10 was injected into the bilateral uterine cavities of female SD rats. 9 A rat model of endometritis was established using 100 μL of CFU / mL Escherichia coli bacterial suspension. After successful model establishment, the rats were randomly divided into three groups of six rats each: a saline infusion group, a drug administration group, and a drug-loaded microbubble infusion group. During treatment, the drug administration group received 200 μL of levofloxacin hydrochloride solution (containing 10 mg / kg of the active ingredient) via gavage injection daily for 14 consecutive days. The drug-loaded microbubble infusion group received 200 μL of drug-loaded microbubble-saline suspension (10 mg / mL of microbubble concentration, 10% w / w) vaginally into the uterine cavity weekly for two weeks (administration was performed on days 1 and 8 after successful model establishment). The saline infusion group received an equal volume of saline solution vaginally into the uterine cavity once a week for two weeks. After the final intervention, the rats were euthanized by overdose anesthesia, and the bilateral uterine tissues were completely removed. Histological sections were prepared and H&E staining was performed to observe histopathological changes.

[0059] Macroscopic images of the uterine morphology of rats in each group are shown below. Figure 5As shown, the therapeutic effect of intrauterine instillation of drug-loaded microbubbles is better than that of oral administration, and better than that of intrauterine instillation of normal saline, indicating that the targeted microbubble preparation for uterine inflammation can improve the therapeutic effect of endometritis.

[0060] The descriptions of the exemplary embodiments presented above are merely illustrative of the technical solutions of the present invention and are not intended to be exhaustive or to limit the invention to the precise forms described. Obviously, those skilled in the art can make many changes and variations based on the above teachings. The exemplary embodiments were chosen and described to explain the specific principles of the invention and its practical applications, thereby enabling others skilled in the art to understand, implement, and utilize the various exemplary embodiments of the invention and their various alternatives and modifications.

Claims

1. A method for preparing microbubbles, characterized in that, The preparation method includes the following steps: S1. Mix the protein aqueous solution and the gas-generating agent evenly; S2. Add an organic solvent containing a surfactant to the mixed solution obtained in step S1, and stir rapidly to prepare droplets; S3. Add a cross-linking agent solution to the droplets obtained in step S2 to cross-link and obtain microspheres with a protein cross-linked outer shell; S4. Add a triggering agent to the microspheres obtained in step S3. The triggering agent reacts with the gas-generating agent and generates gas in situ, resulting in microbubbles with a protein cross-linked outer shell and an internal gas core structure.

2. The preparation method according to claim 1, characterized in that, The gas-generating agent in step S1 is nano-calcium carbonate, sodium bicarbonate, potassium bicarbonate, or urea.

3. The preparation method according to claim 2, characterized in that, The concentration of the gas-generating agent is 5–50 wt%.

4. The preparation method according to claim 1, characterized in that, The trigger in step S4 is hydrochloric acid, acetic acid, or urease.

5. Microbubbles prepared by any of the preparation methods described in claims 1 to 4.

6. A method for preparing uterine inflammation-targeting microbubbles, characterized in that, The preparation method includes the following steps: S1. Modify the surface of the microbubbles described in claim 5 with a uterine inflammation-targeting antibody to obtain uterine inflammation-targeting microbubbles; S2. Add an endometritis treatment agent to the uterine inflammation-targeting microbubbles obtained in step S1 to obtain targeted microbubbles loaded with the endometritis treatment agent.

7. The preparation method according to claim 6, characterized in that, In step S2, the endometritis treatment agent is an anti-inflammatory drug, growth factor, hormone, or a combination thereof.

8. Uterine inflammation-targeting microbubbles prepared by the preparation method according to any one of claims 6 or 7.

9. The use of the uterine inflammation-targeting microbubbles of claim 8 in the preparation of a medicament for treating endometritis.

10. A formulation, characterized in that, The formulation comprises the uterine inflammation-targeting microbubbles of claim 8.

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