A method for analyzing the effectiveness of a drug-encapsulating material for treating intrauterine adhesions

Abstract

The application discloses a method for analyzing the effectiveness of a drug loading material for treating intrauterine adhesion, comprising: performing multi-aspect research on the drug loading material and generating an effectiveness analysis result. In terms of performance characterization, the material morphology and pores are analyzed by using microscopic and specific surface area technologies, the drug and carrier compatibility is verified, and the drug physical state is detected; in vitro analysis covers drug release kinetics, and the release amount is measured in a simulated environment, and a curve is fitted; biological compatibility evaluation is cell co-culture with a material leaching liquor, and cell activity is detected; in vivo, the drug loading material is implanted in an animal model, and imaging and pathological technologies are used to evaluate the curative effect; and finally, the effectiveness analysis conclusion of the drug loading material is obtained by comprehensively analyzing the results. The method breaks through the limitation of traditional single-point testing, constructs a whole-chain evaluation model of "structural characteristics-release behavior-biological compatibility-repair efficiency", and provides a scientific basis for rational design and clinical transformation of intrauterine adhesion treatment materials.

Description

Technical Field

[0001] The invention belongs to the technical field of drug packaging material testing, and particularly relates to a method for analyzing the effectiveness of a drug packaging material for treating intrauterine adhesions. Background Art

[0002] Intrauterine adhesions are a common gynecological disease, mainly caused by intrauterine surgery, infection or trauma, which can lead to menstrual abnormalities, infertility and recurrent miscarriage, seriously affecting women's reproductive health. Current clinical treatment mainly relies on hysteroscopic surgery to loosen adhesions, but the postoperative recurrence rate is high (about 20%-30%). There is an urgent need to develop long-acting drug delivery systems to inhibit fibrosis and promote endometrial repair. Drug-encapsulated materials serve as carriers for local sustained-release drugs. Their physicochemical properties, drug release kinetics and biocompatibility directly determine the therapeutic effect and safety. However, existing analytical methods mostly focus on a single performance indicator and lack a full-dimensional correlation evaluation of the material's function-structure-efficacy, resulting in low clinical translation efficiency.

[0003] The current evaluation system for drug delivery materials has significant limitations:

[0004] Fragmented performance characterization: Traditional methods such as scanning electron microscopy (SEM) or surface area analysis only analyze material morphology or porosity in isolation, without correlating the dynamic relationship between drug loading capacity and release behavior;

[0005] Inadequate in vitro release simulation: Existing in vitro dissolution tests mostly use static media, which cannot simulate the real physiological environment such as uterine fluid flow and pH fluctuations, resulting in a disconnect between the release curve and the actual drug efficacy in vivo;

[0006] Single biocompatibility evaluation: Conventional cytotoxicity tests (such as the MTT assay) only assess acute toxicity, ignoring the chronic effects of material degradation products on endometrial cells and the risk of inflammatory factor release under long-term exposure;

[0007] Disjunction between in vitro and in vivo data: A lack of a collaborative analytical framework for efficacy validation in animal models and in vitro experiments makes it difficult to predict in vivo repair efficacy (such as angiogenesis and collagen deposition regulation) from in vitro parameters. For example, a publication reported that chitosan-loaded drug microspheres, while exhibiting a gradual in vitro release rate, failed to account for the effects of uterine contractions on material deformation, leading to a sudden in vivo release of the drug and exacerbating local inflammatory responses. These deficiencies result in a lack of systematic guidance for material optimization, extending R&D cycles and increasing the risk of clinical failure.

[0008] To address the above issues, there is an urgent need to establish an effectiveness evaluation system that integrates multi-dimensional analysis: by correlating material microstructure (such as pore connectivity) with drug release kinetics, combining dynamic physiological environment simulation with cellular-molecular level biological response analysis, and ultimately achieving a closed data loop through in vivo efficacy verification. Summary of the Invention

[0009] In view of this, the present invention proposes a method for analyzing the effectiveness of drug-encapsulated materials for the treatment of intrauterine adhesions. The present invention breaks through the limitations of traditional single-point testing through a multi-dimensional analysis method, and constructs a full-chain evaluation model of "structural characteristics-release behavior-biocompatibility-repair efficacy", which provides a scientific basis for the rational design and clinical transformation of intrauterine adhesion treatment materials, and can comprehensively evaluate the effectiveness of drug-encapsulated materials to ensure their safety and effectiveness in the treatment of intrauterine adhesions.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] The present invention provides a method for analyzing the effectiveness of a drug-encapsulated material for treating intrauterine adhesions, comprising:

[0012] Characterization of drug-encapsulated material performance: Microscopic morphology analysis is used to characterize the surface morphology and pore structure of the material. Specific surface area and pore size analysis techniques are used to determine the pore characteristics of the material. Chemical spectroscopy is used to verify the compatibility of the drug and the carrier. Thermal analysis is used to detect the physical state of the drug in the carrier.

[0013] In vitro drug release kinetics analysis: The drug-loaded material is placed in a dynamic dissolution apparatus in a dissolution medium that simulates the physiological environment of the uterine cavity. The drug release amount is measured regularly using chromatography technology. The release curve is fitted with a mathematical model to analyze the cumulative release rate, release half-life, and burst release effect.

[0014] In vitro biocompatibility evaluation: The material extract was co-cultured with endometrial cells. Cell viability and proliferation were determined using cell activity assays. The degree of cell damage was assessed using apoptosis assays. The inflammatory potential of the material was analyzed using inflammatory factor assays.

[0015] In vivo efficacy verification: The drug-loaded material is implanted in an animal model of intrauterine adhesions. Imaging techniques are used to assess the recovery of uterine cavity morphology. Histopathology techniques are used to analyze the degree of adhesion and fibrosis. Molecular biology techniques are used to detect the expression of repair-related genes and proteins.

[0016] Based on the performance characterization of drug encapsulation materials, in vitro drug release kinetics analysis results, in vitro biocompatibility evaluation and in vivo efficacy verification results, the effectiveness analysis results of the encapsulation materials are generated.

[0017] Preferably, the process of characterizing the performance of the drug encapsulating material includes:

[0018] Microscopic morphology analysis technology: Scan the material surface through high-resolution microscopic imaging methods to obtain surface morphology and pore structure information of the material. The material's drug loading capacity and release kinetics are evaluated based on the pore geometry, distribution uniformity, and connectivity.

[0019] Specific surface area and pore size analysis technology: Based on the principle of gas adsorption-desorption, by setting the type of adsorbed gas and the pressure range, the specific surface area, pore size distribution and pore volume data of the material are measured to determine the pore density characteristics of the material and its influence on the drug diffusion rate;

[0020] Chemical spectroscopy: Molecular vibrational spectroscopy is used to characterize the chemical bonding state between the drug and the carrier. By comparing the characteristic absorption peaks of the drug, carrier, and composite material, the presence of hydrogen and ionic chemical interactions between the drug and the carrier can be verified, and the stability of the drug in the carrier can be evaluated.

[0021] Thermal analysis technology: Detect changes in the thermal effect of materials under programmed temperature conditions. By analyzing the peak shape, temperature range and peak area of ​​the endothermic or exothermic peak, the physical state and thermal stability of the drug in the carrier are determined, thereby predicting the phase change behavior of the material at body temperature.

[0022] Preferably, the in vitro drug release kinetics analysis of the encapsulated material specifically includes:

[0023] The drug-loaded material is placed in a dynamic dissolution device, and a dissolution medium environment simulating the physiological environment of the uterine cavity is constructed;

[0024] Dissolution medium samples were collected regularly by chromatography to quantitatively determine the drug release concentration;

[0025] The release curve was fitted by combining the zero-order kinetic model, the first-order kinetic model and the Higuchi diffusion model;

[0026] Key release parameters are calculated based on the release curve, including the cumulative release rate reflecting the total amount of drug released, the release half-life characterizing the duration of drug sustained release, and the burst effect index for evaluating the risk of initial burst release.

[0027] Preferably, the dissolution medium environment is constructed by:

[0028] preparing a dissolution medium, wherein the dissolution medium contains a basic buffer of pH 7.2-7.6 to simulate the pH of the uterine cavity, 0.5-2% w / v of mucin is added to simulate the uterine mucus layer, and the ion concentration is adjusted to simulate the electrolyte environment of the uterine fluid;

[0029] A flow cell system or reciprocating cylinder device is used to carry the dissolution medium. The reciprocating motion of the piston at a frequency of 1–2 Hz simulates uterine peristalsis, and the medium flow rate is controlled at 1–5 mL / min to simulate the renewal rate of uterine fluid.

[0030] The flow-through cell system or reciprocating cylinder apparatus carrying the dissolution medium is maintained in a constant temperature water bath environment at 37±0.5°C to obtain a dissolution medium environment.

[0031] Preferably, during the in vitro biocompatibility evaluation of the encapsulated material:

[0032] Prepare the material extract in advance, using cell culture medium or saline as the extraction medium. Set the extraction ratio to meet the ISO10993-5 standard. Set the extraction parameters to 37°C and static extraction for 24–72 hours to simulate the initial release stage of the material in the uterine cavity. After filter sterilization, the extract is diluted into a series of concentrations for gradient exposure experiments.

[0033] Construct a cell co-culture system, select primary endometrial epithelial cells or immortalized cell lines as culture cells, and select a 37°C, 5% CO2, and Special culture medium for estrogen, adding the material extract to the cell culture system, and conducting gradient exposure experiments on the cultured cells at different concentrations and exposure times;

[0034] Using the exposure experiment results, cell activity detection, cell apoptosis detection and inflammatory factor detection were performed on the selected exposure experiment results to obtain the detection results of the encapsulation material on cell survival rate and proliferation ability, cell damage degree and inflammatory activity.

[0035] Preferably, the cell activity detection process includes:

[0036] Add WST-8 reagent to the cells and incubate for 2–4 h. Wait for the mitochondrial dehydrogenase of the living cells to reduce WST-8 to orange-yellow formazan. Measure the absorbance at 450 nm using a microplate reader to calculate the cell viability.

[0037] 5-ethynyl-2'-deoxyuridine is added to cells to label proliferating DNA, and a fluorescent dye coupling reaction is used to quantify the proportion of proliferating cells using flow cytometry or fluorescence microscopy;

[0038] The process of cell apoptosis detection includes:

[0039] Annexin V-FITC reagent was used to label the externalized phosphatidylserine in apoptotic cells, and propidium iodide was used to penetrate the dead cell membrane and stain the nucleus. Flow cytometry was used to analyze necrotic cells, late apoptotic cells, live cells, and early apoptotic cells in four quadrants, and the total apoptotic rate of cells was calculated.

[0040] The cells were lysed and proteins were extracted. The fluorescent substrate Ac-DEVD-AMC was added, and the fluorescence intensity at 380 nm excitation / 460 nm emission was measured to calculate the enzyme activity.

[0041] The inflammatory factor detection process includes:

[0042] The cell supernatant was collected and added to a 96-well plate coated with antibody to capture IL-6 and Factors were detected by enzyme-labeled secondary antibody colorimetry at 450 nm, and factor concentrations were calculated using a standard curve.

[0043] Total cell RNA was extracted and reverse transcribed into cDNA, inflammatory gene primers were designed and real-time fluorescence quantification was performed using SYBR Green fluorescent dye. The relative gene expression was calculated.

[0044] Preferably, the in vivo efficacy verification process includes:

[0045] Select an animal model of intrauterine adhesions and implant drug-loaded materials into it;

[0046] A miniature ultrasound imaging system was used to obtain transabdominal images of the animal model's uterine cavity, and three-dimensional reconstruction technology was used to determine the volume of the uterine cavity after modeling, the volume of the uterine cavity after treatment, and the volume of the healthy uterine cavity, and the uterine cavity volume recovery rate was calculated.

[0047] T2-weighted sequences were used to assess endometrial signal intensity, and diffusion tensor imaging was used to quantify the integrity of the uterine fiber bundles.

[0048] The uterine cavity of the animal model was extracted and fixed with 4% paraformaldehyde for 24 h. After dehydration with gradient ethanol and paraffin embedding, the sections were sliced ​​and subjected to HE staining to evaluate adhesion grade, Masson trichrome staining to calculate the proportion of fibrosis area, and immunohistochemical markers. , quantitative positive cell number;

[0049] The mRNA expression level of the target gene in the uterine cavity was determined by real-time fluorescence quantitative analysis, the expression level of the target protein in the uterine cavity was detected by Western Blot, and the concentration of the target secretory protein in the uterine cavity was detected by enzyme-linked immunosorbent assay (ELISA).

[0050] The present invention has achieved at least the following beneficial effects:

[0051] 1. The present invention uses a multi-dimensional analysis method to comprehensively evaluate the effectiveness of drug-encapsulated materials and ensure their safety and effectiveness in the treatment of intrauterine adhesions.

[0052] 2. It can simulate the physiological environment of the uterine cavity, accurately measure drug release concentration, fit the release curve through multiple kinetic models, calculate key release parameters, and effectively evaluate the release performance and clinical application potential of drug-encapsulated materials.

[0053] 3. The medium environment is constructed in a way that closely replicates the complex physiological conditions within the uterine cavity, improving the simulation accuracy of drug release experiments. Compared to static experiments, this dynamic simulation can make drug release data more closely aligned with actual clinical manifestations, providing a more predictive experimental platform for the development and evaluation of drug-encapsulation materials.

[0054] Other advantages, objectives and features of the present invention will be described in the following description and will be apparent to those skilled in the art to some extent, or those skilled in the art can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] In order to make the purpose, technical solutions and beneficial effects of the present invention more clear, the present invention provides the following drawings for illustration:

[0056] Figure 1 Flow chart of the steps of the method for analyzing the effectiveness of the drug-packaging material according to an embodiment of the present invention;

[0057] Figure 2 Flow chart of the steps of in vitro biocompatibility evaluation of encapsulation materials in an embodiment of the present invention. DETAILED DESCRIPTION

[0058] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0059] The present invention provides a method for analyzing the effectiveness of a drug-loaded material for treating intrauterine adhesions, referring to Figure 1 ,include:

[0060] Characterization of drug-encapsulated material performance: Microscopic morphology analysis is used to characterize the surface morphology and pore structure of the material. Specific surface area and pore size analysis techniques are used to determine the pore characteristics of the material. Chemical spectroscopy is used to verify the compatibility of the drug and the carrier. Thermal analysis is used to detect the physical state of the drug in the carrier.

[0061] In vitro drug release kinetics analysis: The drug-loaded material is placed in a dynamic dissolution apparatus in a dissolution medium that simulates the physiological environment of the uterine cavity. The drug release amount is measured regularly using chromatography technology. The release curve is fitted with a mathematical model to analyze the cumulative release rate, release half-life, and burst release effect.

[0062] In vitro biocompatibility evaluation: The material extract was co-cultured with endometrial cells. Cell viability and proliferation were determined using cell activity assays. The degree of cell damage was assessed using apoptosis assays. The inflammatory potential of the material was analyzed using inflammatory factor assays.

[0063] In vivo efficacy verification: The drug-loaded material is implanted in an animal model of intrauterine adhesions. Imaging techniques are used to assess the recovery of uterine cavity morphology. Histopathology techniques are used to analyze the degree of adhesion and fibrosis. Molecular biology techniques are used to detect the expression of repair-related genes and proteins.

[0064] Based on the performance characterization of drug encapsulation materials, in vitro drug release kinetics analysis results, in vitro biocompatibility evaluation and in vivo efficacy verification results, the effectiveness analysis results of the encapsulation materials are generated.

[0065] The working principle and beneficial effects of the above technical solution are as follows: Microscopic morphology analysis is used to characterize the surface morphology and pore structure of the material, which is crucial for understanding how drug-encapsulated materials promote cell attachment and drug release. Scanning electron microscopy (SEM) or transmission electron microscopy (TEM) can be used to observe the microscopic features and pore distribution of the material surface. Surface area and pore size analysis techniques, such as nitrogen adsorption-desorption, are used to determine the material's pore properties, including average pore size, porosity, and specific surface area. These parameters are important for predicting drug release behavior and cellular responses. Furthermore, chemical spectroscopy techniques, such as Fourier transform infrared spectroscopy (FTIR) or nuclear magnetic resonance (NMR) spectroscopy, are used to verify the compatibility and interaction between the drug and the carrier. Thermal analysis techniques, such as differential scanning calorimetry (DSC) or thermogravimetric analysis (TGA), are used to examine the physical state of the drug in the carrier, such as crystallinity and thermal stability, to assess drug encapsulation efficiency and stability. In in vitro drug release kinetics, the drug-loaded material is placed in a dynamic dissolution apparatus that simulates the physiological environment of the uterine cavity, and the drug release is measured time-dependently using high-performance liquid chromatography (HPLC) or UV-visible spectrophotometry. Release curves are fitted using mathematical models (such as zero-order, first-order, Higuchi, or Peppas equations), and the cumulative release rate, release half-life, and burst effect are analyzed to understand the drug release mechanism. In vitro biocompatibility evaluation is performed by co-culturing the material extract with endometrial cells. Cell viability and proliferation are measured using CCK-8 or MTT assays, cell damage is assessed using flow cytometry or fluorescent staining, and inflammatory cytokine levels are analyzed using enzyme-linked immunosorbent assay (ELISA). In an animal model of intrauterine adhesions, the drug-loaded material is implanted, and uterine cavity morphological recovery is assessed using ultrasound imaging or magnetic resonance imaging (MRI). The degree of adhesion and fibrosis is analyzed using histopathological sections, and the expression of repair-related genes and proteins is detected using real-time quantitative PCR (qRT-PCR) or Western blot. Combining the above results of drug encapsulation material performance characterization, in vitro drug release kinetics analysis, in vitro biocompatibility evaluation, and in vivo efficacy verification, a comprehensive effectiveness analysis was generated, providing a scientific basis for drug encapsulation materials for the treatment of intrauterine adhesions. Through this multi-dimensional analysis method, the effectiveness of drug encapsulation materials can be comprehensively evaluated to ensure their safety and effectiveness in the treatment of intrauterine adhesions.

[0066] In a preferred embodiment, the process of characterizing the performance of the drug encapsulating material includes:

[0067] Microscopic morphology analysis technology: Scan the material surface through high-resolution microscopic imaging methods to obtain surface morphology and pore structure information of the material. The material's drug loading capacity and release kinetics are evaluated based on the pore geometry, distribution uniformity, and connectivity.

[0068] Specific surface area and pore size analysis technology: Based on the principle of gas adsorption-desorption, by setting the type of adsorbed gas and the pressure range, the specific surface area, pore size distribution and pore volume data of the material are measured to determine the pore density characteristics of the material and its influence on the drug diffusion rate;

[0069] Chemical spectroscopy: Molecular vibrational spectroscopy is used to characterize the chemical bonding state between the drug and the carrier. By comparing the characteristic absorption peaks of the drug, carrier, and composite material, the presence of hydrogen and ionic chemical interactions between the drug and the carrier can be verified, and the stability of the drug in the carrier can be evaluated.

[0070] Thermal analysis technology: Detect changes in the thermal effect of materials under programmed temperature conditions. By analyzing the peak shape, temperature range and peak area of ​​the endothermic or exothermic peak, the physical state and thermal stability of the drug in the carrier are determined, thereby predicting the phase change behavior of the material at body temperature.

[0071] The working principle and beneficial effects of the above technical solution are as follows: The material surface is scanned using high-resolution microscopic imaging to obtain surface morphology and pore structure information. The material's drug loading capacity and release kinetics are evaluated based on the pore geometry, distribution uniformity, and connectivity. For example, scanning electron microscopy (SEM) observation of the material's surface microstructure reveals uniform pore distribution and good connectivity, indicating a high drug loading capacity. Furthermore, pore geometry (e.g., circular or elliptical) influences the drug release pathway and, therefore, the release rate.

[0072] Using gas adsorption-desorption technology, the specific surface area, pore size distribution, and pore volume of a material can be measured by specifying the type of adsorbed gas (such as nitrogen) and the pressure range. For example, in a nitrogen adsorption experiment, the Brunauer-Emmett-Teller (BET) method calculated the specific surface area of ​​the material to be 100 m² / g, the average pore diameter to be 10 nm, and the pore volume to be 0.5 cm³ / g. These parameters indicate that the material has a high pore density, which helps accelerate the diffusion rate of the drug.

[0073] Molecular vibrational spectroscopy (such as Fourier transform infrared spectroscopy (FTIR)) is used to characterize the chemical bonding between the drug and the carrier. Characteristic absorption peaks of the drug, carrier, and composite material are compared to verify the presence of hydrogen and ionic bonds between the drug and the carrier. For example, a shift in a characteristic absorption peak of the drug in the carrier (such as the hydroxyl stretching vibration peak) indicates hydrogen bonding. Furthermore, drug stability is assessed by monitoring the intensity changes of the characteristic absorption peaks of the drug in the carrier.

[0074] Under programmed temperature control, thermal analysis techniques (such as differential scanning calorimetry (DSC)) are used to monitor changes in the thermal response of the material. By analyzing the shape, temperature range, and peak area of ​​the endothermic or exothermic peaks, the physical state and thermal stability of the drug in the carrier can be determined. For example, a DSC curve showing a melting peak temperature of 60°C indicates good thermal stability of the drug in the carrier. Furthermore, by predicting the phase transition behavior of the material at body temperature, its in vivo release characteristics can be evaluated. For example, a phase transition near body temperature (37°C) facilitates controlled drug release.

[0075] In a preferred embodiment, the in vitro drug release kinetics analysis of the encapsulated material specifically includes:

[0076] The drug-loaded material is placed in a dynamic dissolution device, and a dissolution medium environment simulating the physiological environment of the uterine cavity is constructed;

[0077] Dissolution medium samples were collected regularly by chromatography to quantitatively determine the drug release concentration;

[0078] The release curve was fitted by combining the zero-order kinetic model, the first-order kinetic model and the Higuchi diffusion model;

[0079] Key release parameters are calculated based on the release curve, including the cumulative release rate reflecting the total amount of drug released, the release half-life characterizing the duration of drug sustained release, and the burst effect index for evaluating the risk of initial burst release.

[0080] The working principle and beneficial effects of the above technical solution are as follows: By placing the drug-loaded material in a dynamic dissolution apparatus, a dissolution medium environment simulates the physiological environment of the uterine cavity, allowing real-time monitoring of the drug release process. The dynamic dissolution apparatus simulates the temperature, pH, and fluid dynamics of the uterine cavity, ensuring that experimental conditions closely resemble those of a real physiological environment. For example, the dissolution medium temperature is set to 37°C, the pH to 7.4, and the flow rate to 1 mL / min to simulate the physiological conditions within the uterine cavity.

[0081] Dissolution medium samples are collected periodically using high-performance liquid chromatography (HPLC) or UV-visible spectrophotometry to quantitatively measure released drug concentration. For example, samples may be collected every 30 minutes and analyzed by HPLC to measure changes in drug concentration. HPLC methods offer high sensitivity and specificity, enabling accurate determination of released drug with detection limits down to the nanogram level.

[0082] The release curves were fitted using a combination of zero-order, first-order, and Higuchi diffusion models to analyze the drug release mechanism. For example, the zero-order kinetic model is suitable for describing constant-rate release, the first-order model is suitable for describing constant-ratio release, and the Higuchi model is suitable for describing diffusion-controlled release. By comparing the goodness of fit of different models, the primary mechanism of drug release can be determined.

[0083] Key release parameters are calculated based on the release curve, including cumulative release rate, release half-life and burst effect index. The cumulative release rate reflects the total amount of drug released. For example, a cumulative release rate of 85% within 24 hours indicates that the material has good drug loading and release capabilities. The release half-life characterizes the duration of sustained drug release. For example, a drug release half-life of 6 hours indicates that the material can achieve sustained drug release. The burst effect index assesses the risk of initial burst release. For example, a burst effect index of 0.2 indicates that the risk of burst release of the drug in the initial stage is low.

[0084] This technical solution allows for comprehensive analysis of the release characteristics of drug-encapsulated materials, providing a scientific basis for optimizing material formulations and formulation processes. This method simulates the physiological environment of the uterine cavity, accurately measures drug release concentrations, and uses multiple kinetic models to fit release curves and calculate key release parameters, effectively evaluating the release performance and clinical application potential of drug-encapsulated materials.

[0085] In a preferred embodiment, the dissolution medium environment is constructed by:

[0086] preparing a dissolution medium, wherein the dissolution medium contains a basic buffer of pH 7.2-7.6 to simulate the pH of the uterine cavity, 0.5-2% w / v of mucin is added to simulate the uterine mucus layer, and the ion concentration is adjusted to simulate the electrolyte environment of the uterine fluid;

[0087] A flow cell system or reciprocating cylinder device is used to carry the dissolution medium. The reciprocating motion of the piston at a frequency of 1–2 Hz simulates uterine peristalsis, and the medium flow rate is controlled at 1–5 mL / min to simulate the renewal rate of uterine fluid.

[0088] The flow-through cell system or reciprocating cylinder apparatus carrying the dissolution medium is maintained in a constant temperature water bath environment at 37±0.5°C to obtain a dissolution medium environment.

[0089] The working principle and beneficial effects of this technical solution are as follows: A highly simulated physiological environment is achieved by precisely configuring the dissolution medium, including a base buffer solution at pH 7.2–7.6 to simulate the physiological pH of the uterine cavity, adding 0.5–2% w / v mucin to simulate the uterine mucus layer, and adjusting the ion concentration to match the electrolyte environment of the uterine fluid. A flow cell system or reciprocating cylinder device is used to carry the dissolution medium, using a piston reciprocating motion at 1–2 Hz to simulate uterine peristalsis. The medium flow rate is precisely controlled at 1–5 mL / min to mimic the natural turnover rate of the uterine fluid. The entire device is placed in a constant temperature water bath at 37 ± 0.5°C to ensure temperature stability during the experiment. This dissolution medium environment closely replicates the complex physiological conditions of the uterine cavity, improving the accuracy of drug release simulations. Compared to static experiments, this dynamic simulation can provide drug release data that more closely resembles actual clinical performance, providing a more predictive experimental platform for the development and evaluation of drug delivery materials.

[0090] In a preferred embodiment, referring to Figure 2 During the in vitro biocompatibility evaluation of the encapsulation material:

[0091] Prepare the material extract in advance, using cell culture medium or saline as the extraction medium. Set the extraction ratio to meet the ISO10993-5 standard. Set the extraction parameters to 37°C and static extraction for 24–72 hours to simulate the initial release stage of the material in the uterine cavity. After filter sterilization, the extract is diluted into a series of concentrations for gradient exposure experiments.

[0092] Construct a cell co-culture system, select primary endometrial epithelial cells or immortalized cell lines as culture cells, and select a 37°C, 5% CO2, and Special culture medium for estrogen, adding the material extract to the cell culture system, and conducting gradient exposure experiments on the cultured cells at different concentrations and exposure times;

[0093] Using the exposure experiment results, cell activity detection, cell apoptosis detection and inflammatory factor detection were performed on the selected exposure experiment results to obtain the detection results of the encapsulation material on cell survival rate and proliferation ability, cell damage degree and inflammatory activity.

[0094] The working principle and beneficial effects of the above technical solution are as follows: During the in vitro biocompatibility evaluation of the encapsulated material, an extract must first be prepared. According to the ISO 10993-5 standard, an appropriate extraction medium is selected, typically cell culture medium or saline. The material is cut into small pieces of a specific size and placed in the extraction medium, ensuring that the contact area between the material and the medium meets the standard extraction ratio, for example, 1 cm² of material corresponds to 1 mL of extraction medium. The mixed system is then placed in a constant temperature environment at 37°C for 24–72 hours of static extraction. This process simulates the initial release phase of the material in the uterine cavity and allows for the full release of soluble components into the extract. Following the extraction, the extract is sterilized using a filter membrane to remove possible bacterial and other impurities. The extract is then diluted into a series of concentration gradients in preparation for subsequent gradient exposure experiments. Next, a cell co-culture system is established, using either primary endometrial epithelial cells or immortalized cell lines as the culture medium. Cells are seeded into culture dishes or plates and cultured in a 37°C, 5% CO2 incubator in a specialized estrogen-containing culture medium. Once the cells have reached an appropriate growth state, different concentrations of the material extract are added to the cell culture system. A gradient exposure experiment is performed with varying exposure durations to assess the cell response under different conditions. The exposure results are used to assess cell viability, apoptosis, and inflammatory factors in the selected experimental groups. Cell viability assays typically use cell counting kits such as CCK-8, which measure the cell's ability to reduce the reagents to reflect cell survival and proliferation. Apoptosis assays utilize Annexin V-FITC and PI double staining, followed by flow cytometry analysis of cell apoptosis, thereby assessing the degree of cell damage caused by the material. Inflammatory factor assays primarily measure the secretion levels of specific inflammatory factors, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), in the culture supernatant using methods such as enzyme-linked immunosorbent assays (ELISAs) to analyze the inflammatory potential of the material.

[0095] Through the above series of detailed experimental steps and detection methods, the effects of the encapsulated material on endometrial cells can be comprehensively and deeply evaluated from multiple dimensions such as cell survival, proliferation, damage and inflammatory response, providing data support for the safety and effectiveness of the material and ensuring its reliability in clinical applications.

[0096] In a preferred embodiment, the cell activity detection process includes:

[0097] Add WST-8 reagent to the cells and incubate for 2–4 h. Wait for the mitochondrial dehydrogenase of the living cells to reduce WST-8 to orange-yellow formazan. Measure the absorbance at 450 nm using a microplate reader to calculate the cell viability.

[0098] 5-ethynyl-2'-deoxyuridine is added to cells to label proliferating DNA, and a fluorescent dye coupling reaction is used to quantify the proportion of proliferating cells using flow cytometry or fluorescence microscopy;

[0099] The process of cell apoptosis detection includes:

[0100] Annexin V-FITC reagent was used to label the externalized phosphatidylserine in apoptotic cells, and propidium iodide was used to penetrate the dead cell membrane and stain the nucleus. Flow cytometry was used to analyze necrotic cells, late apoptotic cells, live cells, and early apoptotic cells in four quadrants, and the total apoptotic rate of cells was calculated.

[0101] The cells were lysed and proteins were extracted. The fluorescent substrate Ac-DEVD-AMC was added, and the fluorescence intensity at 380 nm excitation / 460 nm emission was measured to calculate the enzyme activity.

[0102] The inflammatory factor detection process includes:

[0103] The cell supernatant was collected and added to a 96-well plate coated with antibody to capture IL-6 and Factors were detected by enzyme-labeled secondary antibody colorimetry at 450 nm, and factor concentrations were calculated using a standard curve.

[0104] Total cell RNA was extracted and reverse transcribed into cDNA, inflammatory gene primers were designed and real-time fluorescence quantification was performed using SYBR Green fluorescent dye. The relative gene expression was calculated.

[0105] The working principle and beneficial effects of the above technical solution are as follows: By adding the WST-8 reagent to cells and incubating them for 2–4 hours, mitochondrial dehydrogenases within the living cells reduce WST-8 to an orange-yellow formazan product. Subsequently, absorbance is measured at 450 nm using a microplate reader, and cell viability is calculated based on a standard curve to assess the material's effect on cell viability. Simultaneously, 5-ethynyl-2'-deoxyuridine (EdU), a reagent that incorporates into proliferating DNA, is added to the cells. Through a fluorescent dye coupling reaction and detection using flow cytometry or fluorescence microscopy, the proportion of proliferating cells can be quantitatively analyzed, reflecting the material's effect on cell proliferation.

[0106] In the apoptosis assay, Annexin V-FITC reagent was used to label phosphatidylserine externalized in apoptotic cells, while propidium iodide (PI) was used to penetrate the dead cell membrane and stain the nuclei. Flow cytometry was performed using a four-quadrant analysis to distinguish necrotic cells, late apoptotic cells, live cells, and early apoptotic cells, and the total apoptotic rate was calculated. Furthermore, cells were lysed and protein was extracted. The fluorescent substrate Ac-DEVD-AMC was added, and the fluorescence intensity at 380 nm excitation and 460 nm emission was measured to calculate enzyme activity to evaluate the effect of the material on the activity of apoptosis-related enzymes.

[0107] For inflammatory cytokine detection, cell supernatants were first collected and added to 96-well plates coated with specific antibodies to capture inflammatory factors such as IL-6. Absorbance was measured at 450 nm using an enzyme-linked secondary antibody chromogenic assay, and inflammatory cytokine concentrations were calculated using a standard curve. Furthermore, total cellular RNA was extracted and reverse-transcribed into cDNA. Specific primers targeting inflammation-related genes were designed, and real-time quantitative PCR was performed using SYBR Green fluorescent dye. The relative expression of genes was calculated using the 2-ΔΔCt method to assess the effects of the materials on the inflammatory response.

[0108] This technical solution comprehensively evaluates the biocompatibility of encapsulated materials from multiple perspectives, including cell survival, proliferation, apoptosis, and inflammatory response, through a variety of biological detection methods. WST-8 and EdU detection reflect the effects of materials on cell function from the perspectives of cell viability and proliferation capacity, respectively; Annexin V-FITC and PI staining combined with flow cytometry accurately analyze cell apoptosis; and protein and gene level detection of inflammatory factors reveals the potential inflammatory properties of the materials. These methods complement each other, providing rich data support to ensure a comprehensive evaluation of the biocompatibility of the materials. Compared with a single detection method, a multi-dimensional evaluation strategy can more accurately predict the biological response of materials in the in vivo environment, provide a scientific basis for the screening and optimization of biomaterials, and thus improve the safety and effectiveness of encapsulated materials in clinical applications.

[0109] In a preferred embodiment, the in vivo efficacy verification process includes:

[0110] Select an animal model of intrauterine adhesions and implant drug-loaded materials into it;

[0111] A miniature ultrasound imaging system was used to obtain transabdominal images of the animal model's uterine cavity, and three-dimensional reconstruction technology was used to determine the volume of the uterine cavity after modeling, the volume of the uterine cavity after treatment, and the volume of the healthy uterine cavity, and the uterine cavity volume recovery rate was calculated.

[0112] T2-weighted sequences were used to assess endometrial signal intensity, and diffusion tensor imaging was used to quantify the integrity of the uterine fiber bundles.

[0113] The uterine cavity of the animal model was extracted and fixed with 4% paraformaldehyde for 24 h. After dehydration with gradient ethanol and paraffin embedding, the sections were sliced ​​and subjected to HE staining to evaluate adhesion grade, Masson trichrome staining to calculate the proportion of fibrosis area, and immunohistochemical markers. , quantitative positive cell number;

[0114] The mRNA expression level of the target gene in the uterine cavity was determined by real-time fluorescence quantitative analysis, the expression level of the target protein in the uterine cavity was detected by Western Blot, and the concentration of the target secretory protein in the uterine cavity was detected by enzyme-linked immunosorbent assay (ELISA).

[0115] The working principle and beneficial effects of the above-mentioned technical solution are as follows: By implanting a drug-loaded material into an animal model of intrauterine adhesions, transverse and longitudinal sections of the uterine cavity were acquired through transabdominal scanning using a miniature ultrasound imaging system. Three-dimensional reconstruction techniques were used to determine the volume of the modeled, treated, and healthy uterine cavity, and the uterine cavity volume recovery rate was calculated to visually assess the material's effect on uterine morphology restoration. Furthermore, T2-weighted sequences were used to assess endometrial signal intensity, and diffusion tensor imaging was used to quantify the integrity of the uterine fiber bundles, comprehensively analyzing the repair of the uterine cavity structure. The uterine cavity of the animal model was extracted, fixed with 4% paraformaldehyde, dehydrated with graded ethanol, and embedded in paraffin. Sections were then sectioned and stained with hematoxylin and eosin to assess adhesion grade. Masson's trichrome staining was used to calculate the percentage of fibrosis area, and immunohistochemical staining was used to quantify the number of positive cells, further exploring the histopathological repair effect. Furthermore, real-time fluorescence quantitative analysis was used to measure the mRNA expression level of the target gene, combined with Western blotting to detect the expression level of the target protein, and ELISA to measure the concentration of the target secreted protein, to reveal the molecular biological mechanism by which the material promotes uterine cavity repair. This technical solution comprehensively utilizes imaging, histopathology, and molecular biology methods to comprehensively and multi-levelly evaluate the therapeutic effect of drug-loaded materials on intrauterine adhesions in vivo, ensuring the comprehensiveness and accuracy of the data and providing a scientific basis for clinical application.

[0116] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for analyzing the effectiveness of a drug-encapsulated material for treating intrauterine adhesions, characterized in that: include: Characterization of drug-encapsulated material performance: Microscopic morphology analysis is used to characterize the surface morphology and pore structure of the material. Specific surface area and pore size analysis techniques are used to determine the pore characteristics of the material. Chemical spectroscopy is used to verify the compatibility of the drug and the carrier. Thermal analysis is used to detect the physical state of the drug in the carrier. In vitro drug release kinetics analysis: The drug-loaded material is placed in a dynamic dissolution apparatus in a dissolution medium that simulates the physiological environment of the uterine cavity. The drug release amount is measured regularly using chromatography technology. The release curve is fitted with a mathematical model to analyze the cumulative release rate, release half-life, and burst release effect. In vitro biocompatibility evaluation: The material extract was co-cultured with endometrial cells. Cell viability and proliferation were determined using cell activity assays. The degree of cell damage was assessed using apoptosis assays. The inflammatory potential of the material was analyzed using inflammatory factor assays. In vivo efficacy verification: The drug-loaded material is implanted in an animal model of intrauterine adhesions. Imaging techniques are used to assess the recovery of uterine cavity morphology. Histopathology techniques are used to analyze the degree of adhesion and fibrosis. Molecular biology techniques are used to detect the expression of repair-related genes and proteins. Based on the performance characterization of drug encapsulation materials, in vitro drug release kinetics analysis results, in vitro biocompatibility evaluation and in vivo efficacy verification results, the effectiveness analysis results of the encapsulation materials are generated.

2. The method for analyzing the effectiveness of a drug-loaded material for treating intrauterine adhesions according to claim 1, characterized in that: The process of characterizing the performance of drug encapsulation materials includes: Microscopic morphology analysis technology: Scan the material surface through high-resolution microscopic imaging methods to obtain surface morphology and pore structure information of the material. The material's drug loading capacity and release kinetics are evaluated based on the pore geometry, distribution uniformity, and connectivity. Specific surface area and pore size analysis technology: Based on the principle of gas adsorption-desorption, by setting the type of adsorbed gas and the pressure range, the specific surface area, pore size distribution and pore volume data of the material are measured to determine the pore density characteristics of the material and its influence on the drug diffusion rate; Chemical spectroscopy: Molecular vibrational spectroscopy is used to characterize the chemical bonding state between the drug and the carrier. By comparing the characteristic absorption peaks of the drug, carrier, and composite material, the presence of hydrogen and ionic chemical interactions between the drug and the carrier can be verified, and the stability of the drug in the carrier can be evaluated. Thermal analysis technology: Detect changes in the thermal effect of materials under programmed temperature conditions. By analyzing the peak shape, temperature range and peak area of ​​the endothermic or exothermic peak, the physical state and thermal stability of the drug in the carrier are determined, thereby predicting the phase change behavior of the material at body temperature.

3. The method for analyzing the effectiveness of a drug-loaded material for treating intrauterine adhesions according to claim 1, wherein: The in vitro drug release kinetics analysis of the encapsulated material includes the following steps: The drug-loaded material is placed in a dynamic dissolution device, and a dissolution medium environment simulating the physiological environment of the uterine cavity is constructed; Dissolution medium samples were collected regularly by chromatography to quantitatively determine the drug release concentration; The release curve was fitted by combining the zero-order kinetic model, the first-order kinetic model and the Higuchi diffusion model; Key release parameters are calculated based on the release curve, including the cumulative release rate reflecting the total amount of drug released, the release half-life characterizing the duration of drug sustained release, and the burst effect index for evaluating the risk of initial burst release.

4. The method for analyzing the effectiveness of a drug-loaded material for treating intrauterine adhesions according to claim 3, wherein: The dissolution medium environment is constructed by: preparing a dissolution medium, wherein the dissolution medium contains a basic buffer of pH 7.2-7.6 to simulate the pH of the uterine cavity, 0.5-2% w / v of mucin is added to simulate the uterine mucus layer, and the ion concentration is adjusted to simulate the electrolyte environment of the uterine fluid; A flow cell system or reciprocating cylinder device is used to carry the dissolution medium. The reciprocating motion of the piston at a frequency of 1–2 Hz simulates uterine peristalsis, and the medium flow rate is controlled at 1–5 mL / min to simulate the renewal rate of uterine fluid. The flow-through cell system or reciprocating cylinder apparatus carrying the dissolution medium is maintained in a constant temperature water bath environment at 37±0.5°C to obtain a dissolution medium environment.

5. The method for analyzing the effectiveness of a drug-loaded material for treating intrauterine adhesions according to claim 1, wherein: During the in vitro biocompatibility evaluation of the encapsulation material: Prepare the material extract in advance, using cell culture medium or saline as the extraction medium. Set the extraction ratio to meet the ISO 10993-5 standard. Set the extraction parameters to 37°C and static extraction for 24–72 hours to simulate the initial release stage of the material in the uterine cavity. After filter sterilization, the extract is diluted into a series of concentrations for gradient exposure experiments. Construct a cell co-culture system, select primary endometrial epithelial cells or immortalized cell lines as culture cells, and select a 37°C, 5% CO2, and Special culture medium for estrogen, adding the material extract to the cell culture system, and conducting gradient exposure experiments on the cultured cells at different concentrations and exposure times; Using the exposure experiment results, cell activity detection, cell apoptosis detection and inflammatory factor detection were performed on the selected exposure experiment results to obtain the detection results of the encapsulation material on cell survival rate and proliferation ability, cell damage degree and inflammatory activity.

6. The method for analyzing the effectiveness of a drug-loaded material for treating intrauterine adhesions according to claim 5, characterized in that: The cell viability assay process includes: Add WST-8 reagent to the cells and incubate for 2–4 h. Wait for the mitochondrial dehydrogenase of the living cells to reduce WST-8 to orange-yellow formazan. Measure the absorbance at 450 nm using a microplate reader to calculate the cell viability. 5-ethynyl-2'-deoxyuridine is added to cells to label proliferating DNA, and a fluorescent dye coupling reaction is used to quantify the proportion of proliferating cells using flow cytometry or fluorescence microscopy; The process of cell apoptosis detection includes: Annexin V-FITC reagent was used to label the externalized phosphatidylserine in apoptotic cells, and propidium iodide was used to penetrate the dead cell membrane and stain the nucleus. Flow cytometry was used to analyze necrotic cells, late apoptotic cells, live cells, and early apoptotic cells in four quadrants, and the total apoptotic rate of cells was calculated. The cells were lysed and proteins were extracted. The fluorescent substrate Ac-DEVD-AMC was added, and the fluorescence intensity at 380 nm excitation / 460 nm emission was measured to calculate the enzyme activity. The inflammatory factor detection process includes: The cell supernatant was collected and added to a 96-well plate coated with antibody to capture IL-6 and Factors were detected by enzyme-labeled secondary antibody colorimetry at 450 nm, and factor concentrations were calculated using a standard curve. Total cell RNA was extracted and reverse transcribed into cDNA, inflammatory gene primers were designed and real-time fluorescence quantification was performed using SYBR Green fluorescent dye. The relative gene expression was calculated.

7. The method for analyzing the effectiveness of a drug-loaded material for treating intrauterine adhesions according to claim 1, wherein: The in vivo efficacy validation process includes: Select an animal model of intrauterine adhesions and implant drug-loaded materials into it; A miniature ultrasound imaging system was used to obtain transabdominal images of the animal model's uterine cavity, and three-dimensional reconstruction technology was used to determine the volume of the uterine cavity after modeling, the volume of the uterine cavity after treatment, and the volume of the healthy uterine cavity, and the uterine cavity volume recovery rate was calculated. T2-weighted sequences were used to assess endometrial signal intensity, and diffusion tensor imaging was used to quantify the integrity of the uterine fiber bundles. The uterine cavity of the animal model was extracted and fixed with 4% paraformaldehyde for 24 h. After dehydration with gradient ethanol and paraffin embedding, the sections were sliced ​​and subjected to HE staining to evaluate adhesion grade, Masson trichrome staining to calculate the proportion of fibrosis area, and immunohistochemical markers. , quantitative positive cell number; The mRNA expression level of the target gene in the uterine cavity was determined by real-time fluorescence quantitative analysis, the expression level of the target protein in the uterine cavity was detected by Western Blot, and the concentration of the target secretory protein in the uterine cavity was detected by enzyme-linked immunosorbent assay (ELISA).

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