Preparation and application of short fibers for collecting inflammatory fluid and intelligently responding to inflammation

Abstract

This invention provides the preparation and application of short fibers that recruit inflammatory fluid and intelligently respond to inflammation, belonging to the field of biomedical technology. Based on the principle of click chemistry, this invention constructs a short fiber scaffold (PGCL) that can recruit inflammatory fluid, regulate inflammatory cells, and intelligently respond to inflammation while antagonizing estrogen through a two-step covalent grafting of levonorgestrel (LNG) onto the surface of electrospun polylactic acid / gelatin (PG) nanofibers. This scaffold, through triazole bonds, can not only recruit hydrogen ions from the inflammatory fluid but also achieve intelligent controlled release of LNG, thereby regulating inflammatory cells while simultaneously antagonizing estrogen and thoroughly controlling the acidic inflammatory microenvironment. The intelligent scaffold system developed in this invention provides a new strategy for EH treatment that combines high efficiency and fertility protection through a triple mechanism of action: spatiotemporally specific drug release, acidic microenvironment regulation, and inflammation-immune modulation, and has significant clinical translational value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the preparation and application of short fibers that recruit inflammatory fluid and intelligently respond to inflammation. Background Technology

[0002] Inflammation is an important defense mechanism of the body against infection, injury, or autoimmune abnormalities, and the formation and regulation of inflammatory fluid and exudate are core aspects of the pathophysiological process of inflammation. In the acute inflammatory phase, moderate vascular exudation helps to clear pathogens and necrotic tissue, a process strictly regulated by space and time. However, in chronic inflammation, the continuous accumulation of inflammatory fluid can lead to progressive tissue damage, pathological fibrosis, and organ dysfunction, becoming a key pathological feature of many diseases (such as endometrial hyperplasia, rheumatoid arthritis, and sepsis).

[0003] Recent studies have shown that endometrial inflammation is a core driving factor in endometrial hyperplasia and its malignant transformation, with the accumulation of inflammatory fluid leading to a severe imbalance in endometrial homeostasis. The concentration of pro-inflammatory factors in the retained inflammatory fluid is abnormally elevated, exhibiting an acidic inflammatory microenvironment (e.g., IL-6 can reach 10-20 times the physiological level). These mediators form a positive feedback loop through autocrine / paracrine mechanisms, further exacerbating the disease process. Specifically, pro-inflammatory factors (such as TNF-α and IL-6) not only activate the NF-κB signaling pathway but also cross-diagnose with the estrogen receptor (ERα), thereby synergistically promoting abnormal endometrial proliferation. Furthermore, they form a complex cross-diagnostic network with the estrogen receptor, synergistically driving abnormal endometrial hyperplasia. Therefore, developing functional intelligent responsive materials that recruit and regulate inflammatory fluid to antagonize estrogen for precise treatment of endometrial hemorrhage (EH) is crucial to overcoming current treatment bottlenecks. Although significant progress has been made in recent years in understanding inflammatory regulation mechanisms, how to precisely intervene in the generation and absorption of inflammatory fluid and balance pro-inflammatory and anti-inflammatory responses for precise treatment of EH remains a major challenge in both clinical and basic research.

[0004] The accumulation of inflammatory fluid becomes further complicated by the continued development of the chronic inflammatory microenvironment. In recent years, the pathological role of the inflammatory microenvironment in the development and progression of endometrial hyperplasia has received increasing attention. Extensive clinical evidence shows that significant dysregulation of inflammatory factors in the endometrial tissue of patients with endometrial hemorrhage (EH) promotes abnormal proliferation of endometrial cells. This further exacerbates the pathological process of EH by regulating the imbalance in the expression of cell cycle proteins (such as Cyclin D1) and apoptosis-related proteins (such as Bcl-2 / Bax). Notably, the acidic inflammatory microenvironment, primarily influenced by inflammatory cells (such as activated macrophages and abnormally proliferating endometrial cells), leads to increased H⁺ efflux and a decrease in local pH (pH 6.5-6.8) through overexpression of type V H⁺-ATPase. Furthermore, it promotes the expression of pro-inflammatory factors such as IL-1β and TNF-α by activating proton-sensing receptors (such as GPR4 and GPR65) and signaling pathways (NF-κB, NLRP3 inflammasome, etc.). Regulating this microenvironment can suppress inflammatory pathways, reduce the release of pro-inflammatory factors, and promote macrophage polarization towards the anti-inflammatory M2 type. Studies have shown that neutralizing the acidic microenvironment can reduce factors such as IL-6 and TNF-α in EH models. Therefore, effectively regulating the acidic inflammatory microenvironment and clearing intrauterine fluid accumulation may be a key strategy to block the progression of EH.

[0005] However, current clinical treatment of endometrial hyperplasia (EH) still faces many challenges. While the levonorgestrel-releasing intrauterine system (LNG-IUS) can reverse 60-80% of simple EH, it is not fully compatible with complex atypical hyperplasia and lacks the ability to completely eliminate inflammatory fluid from the uterine cavity. Surgical treatment, although curative, leads to irreversible loss of fertility. In recent years, 3D bioprinting technology has provided a new approach for constructing personalized endometrial repair scaffolds, but traditional printing materials often induce inflammatory reactions due to excessive mechanical strength and still cannot effectively eliminate and regulate uterine cavity fluid. In contrast, electrospun short fiber scaffolds, with their high specific surface area, optimized porous structure, and good biocompatibility, can not only efficiently absorb inflammatory fluid but also possess a certain shape memory function, perfectly conforming to the irregular anatomical structure of the uterus. Therefore, how to strictly design and regulate the acidic microenvironment of the inflammatory fluid, thereby regulating the expression of inflammatory factors, is the key to solving endometrial hyperplasia.

[0006] The pathogenesis of endometrial hyperplasia involves multiple factors, with long-term unantagonized estrogen stimulation being the main pathogenic factor. This stimulation continuously activates the estrogen receptor signaling pathway, leading to abnormal proliferation of endometrial glands and an imbalance in the gland / stromal ratio. In terms of treatment strategies, progestins are the core therapeutic agents, primarily inhibiting the proliferative effects of estrogen through a negative feedback mechanism. Levonorgestrel (LNG) has become the first-line drug in clinical practice due to its high selectivity (Kd=1.8±0.3 nM) and strong affinity for the progesterone receptor (PR). However, existing drug delivery systems have significant limitations. While traditional oral progestin therapy shows an 85% reversal rate for simple hyperplasia, it suffers from systemic adverse reactions such as breakthrough bleeding and abnormal liver function. Local drug delivery systems, such as LNG-IUS, can increase the treatment efficacy for complex hyperplasia to 65%, but their rigid structure makes it difficult to adapt to individual uterine cavity anatomical variations, requiring premature removal in approximately 15% of patients due to discomfort such as pain or bleeding. More importantly, the drug release curves of existing systems are not intelligently designed and cannot dynamically respond to changes in the pathological process.

[0007] To overcome these technological bottlenecks, researchers have attempted to develop various novel drug delivery systems. Injectable thermosensitive hydrogel systems have achieved long-term contraceptive effects in animal models through physical drug loading, but lack tissue targeting. Further improved injectable hydrogel microspheres have shown some potential for targeted release to lesions, but their rapid degradation characteristics (retention time in vivo <2 weeks) make it difficult to meet the standard 4-6 week treatment cycle requirement for endometrial hyperplasia.

[0008] Therefore, developing a novel treatment system with intelligent release characteristics, the ability to dynamically regulate the pathological microenvironment, and compatibility with standard treatment cycles has become a key scientific issue that urgently needs to be addressed in the current treatment of endometrial hyperplasia. Summary of the Invention

[0009] The present invention aims to solve the aforementioned technical problems by providing a method for preparing and applying short fibers that recruit inflammatory fluid and intelligently respond to inflammation. The technical objective of this invention is to develop a novel treatment system with intelligent release properties, the ability to dynamically regulate the pathological microenvironment, and compatibility with standard treatment cycles, thereby providing a promising treatment strategy for the current field of endometrial hyperplasia treatment.

[0010] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: This invention first provides a method for preparing short fibers that recruit inflammatory fluid and intelligently respond to inflammation, comprising the following steps: (1) Electrospinning was performed using a solution of polylactic acid and gelatin as the spinning solution to prepare a nanofiber membrane, which was then homogenized and freeze-dried to prepare short nanofibers. (2) The nanofibers obtained in step (1) were reacted with NHS-PEG2K-N3 to prepare azide-functionalized short fibers, and then levonorgestrel was grafted by click chemical grafting to obtain drug-loaded short fibers. (3) Disperse the drug-loaded short fibers in water to form a suspension, then mix with chitosan, add glutaraldehyde aqueous solution to carry out Schiff base crosslinking reaction, and then inject into a mold to prepare chitosan composite short fiber scaffold.

[0011] This invention, based on a strategy of regulating inflammatory cells to suppress the continuous acidification of the inflammatory microenvironment, innovatively employs click chemistry to construct a smart, responsive short-fiber scaffold system with hydrogen ion recruitment capabilities. First, a stable loading of LNG is achieved on the surface of electrospun PG nanofibers via a two-step covalent grafting method. The triazole covalent connection not only ensures targeted drug release but also specifically activates the drug in the slightly acidic environment of the lesion through a pH-responsive mechanism, thereby achieving a hydrogen ion-triggered sustained-release effect (initial burst release <25%, sustained release >28 days), perfectly matching the standard treatment cycle for endometrial hyperplasia. Furthermore, the scaffold's superabsorbency and shape memory properties ensure a perfect fit to the uterine cavity morphology, while its porous structure provides optimized conditions for nutrient exchange and inflammatory regulation of intrauterine fluid accumulation. Meanwhile, the system exhibits a multi-effect synergistic therapeutic mechanism: on the one hand, it significantly inhibits estrogen-driven cell cycle progression (G0 / G1 phase arrest rate of 68.3±5.2%) and promotes apoptosis (apoptosis rate of 35.7±4.1%); on the other hand, it improves the inflammatory acidic microenvironment by downregulating VEGF expression (inhibition rate of 72.4±6.3%) and promoting macrophage M2 polarization (M2 / M1 ratio increased by 3.2 times).

[0012] Further transcriptomic analysis revealed that this system bidirectionally regulates macrophage polarization through the NF-κB signaling pathway, inhibiting the persistent acidification of the inflammatory microenvironment. Most importantly, in vivo experiments using an EH rat model demonstrated that the PGCL biomimetic estrogen ligand scaffold significantly reduced endometrial thickness and glandular density, while simultaneously decreasing microangiogenesis and the expression of key inflammatory factors, thus effectively treating endometrial hyperplasia. In summary, this study applies click chemistry to inhibit the persistent acidification of the inflammatory microenvironment and to a local intrauterine drug delivery system. By mimicking endogenous hormones to precisely antagonize estrogen, it provides a new solution to key issues in current treatments of endometrial hyperplasia, such as fertility impairment and high recurrence rates.

[0013] Furthermore, the electrospinning process in step (1) is as follows: 1-2 mL feed rate, 20-25 kV working voltage and 8-14 cm receiving distance.

[0014] Furthermore, the polylactic acid mentioned in step (1) has a molecular weight of 100,000 Da, and the weight percentage of polylactic acid to gelatin is 20%-30%.

[0015] Furthermore, in step (1), polylactic acid and gelatin are dissolved in hexafluoroisopropanol to prepare a spinning solution, the concentration of which is 10-12% (w / v).

[0016] Furthermore, the homogenization conditions described in step (1) are homogenization at ≥13,000 rpm (e.g., 14,000 rpm) for 30 min.

[0017] Furthermore, the click chemistry described in step (2) is a two-step method. First, the short fibers are reacted with NHS-PEG2K-N3 at a molar ratio of 1:3 to prepare azide-functionalized short fibers. Then, levonorgestrel is grafted onto the short fibers through a click chemistry reaction in the catalytic system of CuCl and sodium citrate to obtain drug-loaded short fibers.

[0018] Furthermore, the weight ratio of drug-loaded short fibers to chitosan in step (3) is 2:1.

[0019] Furthermore, in step (3), the molar ratio of amino to aldehyde groups in the Schiff base crosslinking reaction is controlled to be 1:0.8.

[0020] A second objective of this invention is to provide short fibers that recruit inflammatory fluid and intelligently respond to inflammation, prepared by the method described above.

[0021] A third objective of this invention is to provide the application of the short fibers described above, which recruit inflammatory fluid and intelligently respond to inflammation, in the preparation of a medicament for treating endometrial hyperplasia.

[0022] The beneficial effects of this invention are as follows: (1) Based on the principle of click chemistry, this invention innovatively constructs a short fiber scaffold (PGCL) that can recruit inflammatory fluid to regulate the intelligent response of inflammatory cells to antagonize estrogen by two-step covalent grafting of levonorgestrel (LNG) onto the surface of electrospun polylactic acid / gelatin (PG) nanofibers. This scaffold can not only recruit hydrogen ions in the inflammatory fluid through triazole bonds, but also realize the intelligent controlled release of LNG, thereby regulating inflammatory cells while antagonizing estrogen and thoroughly regulating the acidic inflammatory microenvironment.

[0023] (2) The results of this study show that in the slightly acidic microenvironment of the lesion area, hydrogen ions in the inflammatory fluid trigger the breakage of triazole bonds in short fibers, prompting LNG to target and release intelligently antagonistic estrogen for precise treatment of EH. Simultaneously, the superabsorbent and self-expanding properties of PGCL can adapt to complex uterine cavity morphologies, and its high porosity (>85%) also promotes the recruitment of inflammatory fluid. Furthermore, mechanistic studies have found that LNG promotes apoptosis by arresting the cell cycle and significantly downregulates the expression of VEGF and pro-inflammatory factors. Transcriptome sequencing reveals that it reduces the expression of type V H⁺-ATPase by inhibiting the NF-κB signaling pathway and regulating macrophage polarization, thereby reducing EH inflammation-mediated progesterone resistance. Animal experiments show that PGCL treatment reduced endometrial thickness by 56.7%, glandular density by 43.7%, and inflammatory factor expression by 52.5-79.6% in EH model rats.

[0024] (3) The intelligent stent system developed in this invention provides a new strategy for EH treatment that combines high efficiency and fertility protection through a triple mechanism of spatiotemporal specific drug release, acidic microenvironment regulation and inflammation-immune regulation, and has important clinical translational value. Attached Figure Description

[0025] Figure 1 Physicochemical properties and functional evaluation of polyglutamic acid (PG), chitosan (CS), polyglutamic acid-chitosan complex (PGC), and polyglutamic acid-chitosan crosslinked compound (PGCL) scaffolds; morphological and elemental characterization analysis: (A) macroscopic appearance of PG, CS, PGC, and PGCL scaffolds; (B) cross-sectional scanning electron microscope (SEM) images of the four scaffold groups; (C) corresponding elemental distribution maps (carbon (C), oxygen (O), and copper (Cu).

[0026] Figure 2 Physicochemical properties and functional evaluation of polyglutamic acid (PG), chitosan (CS), polyglutamic acid-chitosan complex (PGC), and polyglutamic acid-chitosan copolymer (PGCL) scaffolds were assessed. Chemical structure analysis included: (A) Fourier transform infrared (FTIR) spectroscopy; (B) X-ray diffraction (XRD) patterns; and (C) thermogravimetric analysis (TGA) curves. In vitro performance included: (DD) cumulative drug release curves of the PGCL scaffolds; and (E) degradation behavior of all scaffold groups under simulated physiological conditions. Surface and structural properties included: (F) water contact angle measurements; and (G) porosity analysis. Water absorption behavior included: (H) water absorption kinetics; and (I) cyclic water absorption capacity during 10 consecutive compression cycles. Data are presented as mean ± standard deviation. Statistical analysis was performed using two-way ANOVA combined with post-hoc tests, n = 3, *p < 0.05.

[0027] Figure 3(A) Representative images of live / dead staining of Ishikawa cell line after co-culturing PG, CS, PGC and PGCL with Ishikawa cell line for 1 day, and representative images of cytoskeleton staining of Ishikawa cells on day 3 in different groups; (B) Representative images of EdU immunofluorescence staining of Ishikawa cells after 4 days of treatment in different groups; (C) Cell proliferation of Ishikawa cells in different groups after 1, 4 and 7 days of culture by CCK-8 assay; (D) Statistical chart of EdU positivity rate of Ishikawa cells in each group on day 4. Cell proliferation was determined by EdU assay, and proliferating cells were identified by green fluorescent markers, with all cell nuclei labeled with blue fluorescent markers. (EG) MKI67 RT-qPCR (E) and cellular immunofluorescence (F) were used to analyze changes in MKI67 mRNA expression and proliferation marker protein levels in Ishkava cells across different groups on day 4. (HJ) PCNA RT-qPCR (H), statistical graphs of PCNA mRNA expression (I), and representative graphs of PCNA protein green fluorescent immunostaining in each group (J) – results of Ishkava cell proliferation marker detection on day 4. Crystal violet stained images (K) and quantification of Ishkava cell migration across different groups (L); two-way ANOVA (using a post-hoc test), n = 3, * p < 0.05.

[0028] Figure 4PGCL scaffolds inhibited the proliferation and migration of endometrial stromal cells (T-HESCs); (A) Live / dead cell staining after co-culturing T-HESCs with PG, CS, PGC, or PGCL scaffolds for 1 and 3 days (live cells: green; dead cells: red); (B) Morphological changes of T-HESCs on days 1 and 3 using phalloidin staining (nuclei: DAPI, blue); (C) Cell proliferation activity was assessed by CCK-8 assay at days 1, 4, and 7; (D) Representative image of EdU+ proliferating cells on day 4 (green); (E) Quantification of EdU-positive cells (proliferating cells: green; total nuclei: blue). MKI67 expression analysis: (F) RT-qPCR analysis of MKI67 mRNA levels (day 4); (G) Immunofluorescence staining of MKI67 protein (green); (H) Quantitative analysis of MKI67 fluorescence intensity. PCNA expression profile: (I) RT-qPCR analysis of PCNA mRNA levels (day 4); (J) Representative images of PCNA immunofluorescence (green); (K) Statistical comparison of PCNA expression; (L-M) Crystal violet staining images and scratch healing rate counts of T-HESCs scratch assay, with pairwise comparisons between different groups using Tukey's post-hoc test, n = 3; * p < 0.05.

[0029] Figure 5 To evaluate the anti-angiogenic effect of the PGCL scaffold; (A) live / dead and cytoskeleton detection of HUVECs cultured on each scaffold; (B) CCK-8 assay of HUVEC proliferation in the PG, CS, PGC and PGCL groups on days 1, 4 and 7; (C, D) RT-qPCR analysis of MKI67 and PCNA transcription levels in HUVECs on day 7; (E–G) Immunofluorescence staining of MKI67 and PCNA in HUVECs on day 7; (H) Representative images of HUVEC channel formation in each group; (I–K) Quantitative analysis of channel formation; (L) VEGF levels in culture supernatant measured by ELISA; Two-way ANOVA (including Tukey post-hoc test), n = 3; *p < 0.05.

[0030] Figure 6PGCL can regulate the polarization state of macrophages, inhibit pro-inflammatory signal transduction, and alter the transcriptome characteristics of epithelial cells in EH; (A) Immunofluorescence staining of RAW 264.7 macrophages to show the M1 marker CD86 and M2 marker CD206 in different groups; (B-D) Quantitative analysis of fluorescence signals; (E-G) qRT-PCR analysis of V-type H+-ATPase subunits (ATP6V1A, ATP6V1B2, ATP6V0C) in macrophages; (H-J) Expression levels of cytokines IL-1β, IL-6, and TNF-α in culture supernatant by ELISA; (K) Normalized signal intensity of PGC and PGCL-treated samples in RNA-seq dataset; (L) Principal component analysis (PCA) showing the clustering of PGC and PGCL samples; (M) Hierarchical cluster analysis showing the systematic differences between the two groups; (N) Compared with PGC Volcano plots of differentially expressed genes (DEGs) in the PGCL group compared to the previous group; (O) plots of the top 20 DEGs enriched by downregulation; (P) plots of the top 20 KEGG pathways enriched by downregulation. Two-way ANOVA (Tukey's post hoc), n = 3; *p < 0.05.

[0031] Figure 7 Uterine morphology and histopathology in rats with endometrial hyperplasia after PGCL treatment; (A) Schematic diagram of endometrial hyperplasia model induction and treatment regimen; (B) Gross morphology of the uterus in all experimental groups; (C-E) Comparison of uterine wet weight and uterine index (uterine wet weight / body weight); (F) Representative H&E stained uterine sections; (G-I) Quantitative assessment of endometrial thickness, area and glandular density based on H&E stained tissue sections; Two-way ANOVA, Tukey test, n = 6; *p < 0.05.

[0032] Figure 8 To investigate the effect of PGCL on inhibiting endometrial hyperplasia and angiogenesis in a rat model of endometriosis; (A) Immunofluorescence of MKI67 in uterine tissues of each experimental group; (B) Quantification of MKI67 expression by H score; (C, D) qRT-PCR analysis of MKI67 and PCNA mRNA levels in each experimental group; (E) Immunohistochemical visualization of PCNA expression in uterine specimens; (F) H score of PCNA expression; (G) Immunohistochemical staining of CD31-labeled blood vessels; (H) Quantification of blood vessel count; Two-way ANOVA and Tukey test were performed, n = 6; *p < 0.05.

[0033] Figure 9 PGCL can alleviate the inflammatory microenvironment remodeling in rats with endometriosis. (A-C) The expression of V-ATPase subunits (ATP6V1B2, ATP6V1A, ATP6V0C) in the endometrial tissue of four groups of rats was detected by qRT-PCR. (D-F) The protein expression of IL-1β, IL-6, and TNF-α was quantitatively detected by ELISA. (G-L) The expression of IL-1β, IL-6, and TNF-α in rat endometrial tissue was detected by immunohistochemistry, and representative images and scores are provided. Two-way ANOVA (Tukey's post hoc) was used, n = 6; *p < 0.05.

[0034] Figure 10 This is a schematic diagram of a smart responsive short-fiber scaffold for the precise treatment of endometriosis by recruiting inflammatory fluid, modulating the immune system, and counteracting the effects of estrogen; (A) Manufacturing a short-fiber scaffold capable of recruiting inflammatory fluid and intelligently modulating inflammatory cell responses to counteract the effects of estrogen; (B) Connecting LNG to the PGCL scaffold via a click chemical reaction; (C) The PGCL scaffold precisely treats endometrial hyperplasia by modulating inflammatory cell polarization and regulating the inflammatory acidic microenvironment. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention. Example

[0036] I. Experimental Methods 1. Preparation of Levonorgestrel-modified nanofibers Levonorgestrel-functionalized nanofibers (PGNL) ​​were prepared using electrospinning-click chemical coupling technology. The specific preparation process is as follows: First, polylactic acid (PLA, Mw: 100,000, Guangzhou Maipu Regenerative Medicine Co., Ltd.) and gelatin (Gelatin, Type A, Sigma-Aldrich, USA) were dissolved in hexafluoroisopropanol (HFIP, >99%, Shanghai Darui Chemical Co., Ltd.) at a mass ratio of 20%-30% to prepare a 10% (w / v) spinning solution. After obtaining a homogeneous solution by magnetic stirring for 12 h, PG nanofiber membranes were prepared by electrospinning under the conditions of a feed rate of 1-2 mL / h, a working voltage of 20-25 kV, and a receiving distance of 8-14 cm. Subsequently, PG short fiber powder was obtained by high-speed homogenization (14,000 rpm, 30 min) and freeze-drying. Further surface modification was carried out using a two-step method: first, PG short fibers were reacted with NHS-PEG2K-N3 (Shaanxi Xingbei Aike Biotechnology Co., Ltd.) at a molar ratio of 1:5 to prepare azide-functionalized short fibers (PGN), and then levonorgestrel was grafted by click chemical reaction under the CuCl / sodium citrate catalysis system to finally obtain drug-loaded short fibers (PGNL).

[0037] 2. Preparation of PGCL stent This invention constructs a hierarchical porous tissue engineering scaffold system using cryogenic casting technology, including pure chitosan scaffolds (CS), pure short fiber scaffolds (PG), and chitosan / short fiber composite scaffolds (PGC scaffolds and drug-loaded PGCL scaffolds). Under strictly controlled temperature and humidity conditions (25±1℃, 50±5% RH), a suitable amount of PG or drug-loaded PGNL short fibers are first uniformly dispersed in ultrapure water to form a stable suspension using high-speed shear emulsification (10,000 rpm, 10 min). Subsequently, a corresponding proportion of high-degree-of-deacetylation chitosan (100-200 mPa·s, degree of deacetylation ≥95%) is added, and preliminary mixing is achieved by magnetic stirring (10 min). Acetic acid is then added to dissolve the chitosan, followed by the addition of 1% glutaraldehyde aqueous solution for Schiff base crosslinking reaction. Appropriate crosslinking density is ensured by adjusting the molar ratio of amino to aldehyde groups (1:0.8). The mixed solution was injected into a mold, and the ice crystals were induced to grow in a directional manner by programmed cooling (-80℃, 24 h) to form a three-dimensional scaffold with an anisotropic pore structure. Finally, the scaffold was obtained by freeze-drying and neutralization with 1% NaHCO3 solution. By precisely controlling the total solid content (25wt%), crosslinking agent concentration (1%), and freezing parameters, a biomimetic scaffold with controllable pore size distribution (50-200 μm) and porosity >90% was successfully constructed. Among them, the pure chitosan scaffold (CS) without the addition of short fibers and the pure short fiber scaffold (PG) obtained by thermal crosslinking without the addition of chitosan were used as control groups to evaluate the synergistic effect of the composite scaffold.

[0038] 3. Characterization of PGCL stent (1) Morphological observation and elemental distribution of PGCL scaffold The microstructure and elemental distribution of the scaffolds were characterized using field emission scanning electron microscopy (FE-SEM, JEOL JSM-7800F) combined with energy dispersive X-ray spectroscopy (EDS, Oxford X-MaxN 80). First, PG, CS, PGC, and PGCL scaffolds (n=3 / group) were precisely cut into cylindrical samples with a diameter of 5 mm. After being fixed with conductive adhesive, they were sputtered with gold at a current of 5-10 mA for 60 s in a high-vacuum coating system (LeicaEM ACE600) to obtain a conductive layer with a thickness of 15±2 nm. Secondary electron images were acquired under the conditions of accelerating voltage 15 kV and working distance 10 mm. Simultaneous EDS surface scanning analysis (active time 60 s, process time 120 s) was performed to detect the characteristic X-ray distributions of carbon (C), oxygen (O), and copper (Cu). Aztec Energy 3.3 software was used for ZAF-corrected semi-quantitative analysis. All samples were vacuum dried for 24 hours before testing and the tests were conducted in a clean room with constant temperature and humidity of 23±2℃ and 45±5% RH to ensure data reliability.

[0039] (2) Fourier transform infrared (FTIR), crystal structure (XRD) and thermal stability (TGA) characterization of PGCL scaffold Multi-scale characterization techniques were employed to systematically analyze the chemical structure, crystallographic features, and thermal stability of PG, CS, PGC, and PGCL scaffolds. First, total reflectance Fourier transform infrared spectroscopy (ATR-FTIR, Nicolet iS50) was used to analyze these structures in the 600-3600 cm⁻¹ range. -1 The chemical groups of each scaffold were characterized within the wavenumber range. The test parameters were set as follows: resolution 4 cm⁻¹, 32 scans, and spectral data were acquired using a diamond ATR crystal at a constant pressure of 100 N. Subsequently, the crystallization behavior of the material was analyzed using X-ray diffraction (XRD, Bruker D8 Advance). The test conditions were: Cu Kα radiation source (λ=1.5406 Å), tube voltage 40 kV, tube current 300 mA, scan rate 6° / min, scan range 10–60° (2θ), and a zero-background silicon sample holder was used to ensure test accuracy. Thermogravimetric analysis (TGA, TA Q500) was performed under a nitrogen atmosphere (50 mL / min), with the temperature increased from room temperature to 800°C at a rate of 10°C / min, and the mass change of 5.0 ± 0.1 mg of sample was accurately recorded.

[0040] (3) Characterization of PGCL stent porosity This study established a high-precision porosity determination scheme based on the improved Archimedes principle. All scaffold samples (PG, CS, PGC, and PGCL, n=4 / group) were first dehydrated to constant weight in a 25℃ constant-temperature drying oven (ΔW<0.1% / h). Geometric dimensions were measured using an electronic digital caliper (accuracy ±0.01 mm) to calculate the apparent volume (V0), and the dry weight (W0) was recorded using a precision analytical balance (accuracy 0.01 mg). Subsequently, the samples were completely immersed in anhydrous ethanol (purity ≥99.8%, ρ=0.789 g / cm³) and ultrasonically assisted (40 kHz) for 2 hours to ensure complete penetration. Immediately after removal, residual liquid was removed using quantitative filter paper (0.22 μm), and the wet weight (W1) was determined within 30 seconds. The porosity calculation formula is: Porosity (%) = [(W1−W0) / (ρ·V0)]×100% All data were corrected for temperature compensation (±0.1℃), and each sample was measured in triplicate with the mean value. This method was validated using standard glass microspheres (with a known porosity of 35±2%), and the relative error was <1.5%. The experimental process was strictly controlled and carried out on a vibration-proof platform.

[0041] (4) Drug release and degradation of PGCL stent in vitro This study systematically evaluated the drug release behavior of drug-loaded stents using ultraviolet spectrophotometry. First, a standard curve for levonorgestrel was established: a series of methanol-PBS (1:1, v / v) standard solutions ranging from 0.5 to 50 μg / mL were precisely prepared, and absorbance was measured at λmax = 240 nm. Precisely cut PGCL stent samples (5×5×2 mm, n=3) were placed in pre-treated dialysis bags (MWCO 12-14 kDa) and immersed in 50 mL of PBS release medium (pH 7.4 containing 0.01% Tween 80). The samples were stirred at 50 rpm under a constant temperature of 37±0.2℃. At preset time points (0.5-14 h), 3 mL samples were taken and an equal volume of pre-warmed medium was added. The samples were filtered through a 0.45 μm filter and measured using an ultraviolet spectrophotometer (scanning range 200-400 nm). Three parallel measurements were performed, and the average value was used. Background correction was performed using blank stent extract. Cumulative release rate was determined. Qn=(Cn×V+ΣCi−1×Vs) / W×100% Where: Qn - cumulative release rate, Cn - concentration measured in the nth time, V - medium volume, Vs - sampling volume, W - drug loading.

[0042] The biodegradation behavior of scaffolds was evaluated using an in vitro degradation assay system simulating physiological conditions. Before the experiment, each scaffold sample (n=3) was dehydrated to constant weight (ΔW<0.1mg / 24h) in a vacuum drying oven (40℃, 0.1MPa), and the initial dry weight (W1) was recorded using a precision analytical balance. Subsequently, the samples were placed in sterile 15mL centrifuge tubes, 10mL of sterile PBS buffer was added, and the degradation experiment was conducted in a constant temperature shaking incubator (37.0±0.5℃, 100±2rpm). Samples were taken at preset time points (0, 7, 14, 21, 28, 35, 42d), rinsed three times with ultrapure water to remove soluble degradation products, and then freeze-dried (-80℃, 24h) before measuring the residual dry weight (W2).

[0043] Residual rate (%) = (W2 / W1) × 100% (5) Characterization of the hydrophilicity and water absorption properties of the scaffold The surface hydrophilicity of the scaffold material was systematically characterized using static contact angle analysis. Experiments were conducted using an SL200A contact angle analyzer (Solon Tech., Shanghai, China) under constant temperature and humidity conditions (25±0.5℃, 50±5% RH). After precise preparation, 5 μL of ultrapure water was deposited onto the sample surface using a micro-injection system (Hamilton, accuracy ±0.1 μL), with deposition parameters strictly controlled at a flow rate of 1 μL / s and a needle height of 2 mm. A high-speed CCD camera was used to capture the droplet morphology, and the droplet profile was fitted based on the Young-Laplace equation. The average left and right contact angles were automatically calculated at a critical time point of 5 seconds after droplet deposition.

[0044] Furthermore, a precise evaluation system for the water absorption performance of the stent was established based on the ASTM D570 standard. Three parallel samples (n=3) were used in the experiment. First, the stent was placed in a vacuum drying oven (40℃, 10Pa) to dehydrate to constant weight (ΔW<0.1mg / h), and the initial dry weight (Wd) was recorded using a precision analytical balance. Water absorption kinetics testing was conducted under constant temperature conditions. The sample was completely immersed in ultrapure water, removed at preset time points (10-60min), and the surface moisture was quickly absorbed using filter paper. The wet weight (Ww) was determined within 30s. The water absorption rate was calculated using the formula: W(%) = [(Ww−Wd) / Wd] × 100% 4. Cell Culture In this study, we used several rigorously validated cell models for our experiments: human endometrial adenocarcinoma cell line (Ishikawa), primary human umbilical vein endothelial cells (HUVECs), RAW 264.7 mouse macrophage cell line, and immortalized human endometrial stromal cells (T-HESCs). Different culture systems were used for each cell line: Ishikawa and T-HESCs used DMEM / F12 basal medium (Gibco 11330032) supplemented with 2.5 mM L-glutamine; HUVECs used RPMI-1640 endothelial cell-specific medium containing 25 mM HEPES buffer; and RAW 264.7 used high-glucose DMEM (4.5 g / L D-glucose). All culture media were supplemented with 10% heat-inactivated fetal bovine serum (Gibco 16140071) and 1% penicillin-streptomycin (10,000 U / mL, Gibco 15140122). Cell culture was conducted under strictly controlled conditions: constant temperature of 37℃±0.2℃, 5% CO2, and saturated humidity (>95% RH).

[0045] 5. Research on the biocompatibility of scaffolds After sterilization, the scaffold was immersed in basal culture medium at a solid-liquid ratio of 0.1 g / mL (w / v) and extracted at 37°C for 24 hours. The extract was then filtered through a 0.22 μm filter to prepare a sterile extract. Three cell lines were selected: Ishikawa, HUVECs, and T-HESCs, and cultured at 2 × 10³ cells / well (96-well plate) and 5 × 10³ cells / well (96-well plate), respectively. 4 Cells were seeded at density per well (48-well plate) and co-cultured with different scaffold extracts for 1 / 4 / 7 days. Cell viability was assessed using a Calein-AM / PI double staining kit (Beyotime C2015M), where live cells were labeled with Calein-AM and dead cells with PI. After incubation at 37°C in the dark for 30 minutes, cells were observed under a fluorescence microscope (488 nm / 561 nm). Cell proliferation was simultaneously evaluated using the CCK-8 assay (Yeasen 40203ES) and EdU assay (Beyotime C0071S): for the CCK-8 assay, 10 μL of reagent was added to each well, and after incubation for 2 hours, the absorbance at 450 nm was measured using a microplate reader (BioTek Synergy H1); for the EdU assay, 10 μM EdU was used for labeling for 2 hours, followed by nuclear counterstaining with Hoechst 33342 (5 μg / mL) after 30 minutes, and observation was performed under a fluorescence microscope.

[0046] 6. Cell migration and tube formation assays This study used Transwell migration assays and scratch assays to evaluate the effects of scaffold extract on the migration behavior of Ishikawa cells and T-HESCs. In the Transwell migration assay, Ishikawa cells (5 × 10⁶ cells / year) were... 4 Cells / 100μL were suspended in serum-free medium and seeded into the upper chamber of a Corning® Transwell® polycarbonate membrane. Different groups of scaffold extracts were added to the lower chamber. After incubation at 37°C and 5% CO2 for 24 h, unmigrated cells in the upper chamber were removed with a cotton swab. Migrated cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and photographed under a light microscope. Scratch assays were performed using T-HESCs, seeded in 6-well plates (5×10⁻⁶ cells / 100μL). 5 Cells were cultured in wells to 80-90% confluence. After synchronization with serum-free medium for 12 h, vertical scratches (approximately 500 μm wide) were created using a 200 μL sterile pipette tip. The extraction medium was changed to different scaffold groups, and the cells were cultured for another 12 h. After washing with PBS, the cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and their migration was recorded under an optical microscope. The scratch closure area was quantitatively analyzed using ImageJ software.

[0047] Tube forming test: 5×10 4 HUVECs were seeded at a density of 1 cell / well into 24-well plates containing Matrigel (Corning, 356234) and cultured in different scaffold material extraction media. After 6 hours of incubation in a thermostatic incubator, cellular actin was stained with Alexa Fluor 555-labeled globular proteins (Servicebio, Cat# G1249). Tube formation was observed using a fluorescence microscope (Axio Vert. A1, Zeiss, Germany), and the number of connections was analyzed using ImageJ software.

[0048] 7. Cell immunofluorescence staining The effects of extracts from four scaffold materials—PG, CS, PGC, and PGCL—on the biological behavior of Ishikawa, HUVECs, and T-HESCs were systematically evaluated using immunofluorescence staining. The three cell lines were cultured at 1.0 × 10⁻⁶ cells / mL. 4Cells were seeded at specific densities in 24-well plates and cultured in DMEM / F12 complete medium containing 10% FBS. Equal amounts of extracts from each scaffold material were added for 4 days of intervention. After fixation with 4% paraformaldehyde for 30 min, cells were permeabilized with 0.1% Triton X-100 for 15 min, blocked with 2% BSA for 30 min, and then incubated overnight at 4°C with a specific primary antibody (see Appendix 1, Table S2). Cells were then incubated with fluorescent secondary antibody (Alexa Fluor 488 / 555, 1:500) for 1 h and DAPI nuclear staining for 5 min under dark conditions at room temperature. When RAW 264.7 cell confluence reached 70%, LPS (100 ng / mL) combined with IFN-γ (20 ng / mL) was used to induce M1 polarization. Cells were then treated with different scaffold extracts for 24 hours, and the expression of CD86 and CD206 was detected by standard immunofluorescence staining. Images were acquired using a fluorescence microscope (Zeiss-Axio Vert.A1; LeicaThunder DMi8), and the average fluorescence intensity of the target protein was quantitatively analyzed using ImageJ software.

[0049] 8. qRT-PCR and RNA sequencing analysis qRT-PCR Analysis: Total RNA was extracted from the samples using the Total RNA Isolation Kit (Vazyme, Cat#RC112-01) according to the supplier's instructions. Reverse transcription was performed using Hiscript II reverse transcriptase. qRT-PCR was performed using the SYBR qPCRMaster Mix (Vazyme, Cat# Q711-02). Relative gene expression levels were assessed using the 2-ΔΔCT method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene. Supplementary Table 1 lists all primers used.

[0050] RNA sequencing analysis: For transcriptome analysis, total RNA was extracted from cells co-cultured with PGC or PGCL scaffold extracts for 4 days using Trizol reagent and sequenced on the Illμmina NovaSeq6000 platform (performed by Genentech Ltd.). Raw data were aligned to the Mus musculus GRCm38 reference genome using Hisat2 (v2.0.5). Differentially expressed genes (DEGs, |log2FC| > 0.5 and P < 0.05) were screened using the limma algorithm. Expression profiles were visualized using volcano plots (Hiplot platform) and hierarchical clustering, and GO / KEGG enrichment analysis was performed using the hypergeometric distribution test (significance threshold P < 0.05).

[0051] 9. Animal experiments (1) Endometrial hyperplasia model in female rats Eight-week-old female Sprague-Dawley (SD) rats (RRID: MGI:565113, Beijing Life River Laboratory Animal Technology Co., Ltd. Shanghai Branch) were selected and housed in an SPF-grade barrier environment (temperature 25±2 °C, relative humidity 45±5%, 12h / 12h light-dark cycle), with free access to standard rodent feed and sterilized water. All animal experiments followed the international guidelines for the care and use of laboratory animals (ARRIVE 2.0) and were approved by the Animal Research Committee of Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (Approval No.: RJ2023040). First, a female rat endometrial hyperplasia model was established. The Sham group received intramuscular injections of olive oil (carrier; 1.5 mL / kg body weight) every other day for 42 consecutive days. The other three groups received intramuscular injections of estradiol benzoate (EB) (0.6 mg / 1.5 mL / kg body weight) every other day. On day 14 of the injections, all rats underwent different surgical procedures under isoflurane anesthesia in a sterile environment. All rats with endometrial hyperplasia were randomly divided into four groups (n = 6 per group): the Sham group and the EH group: the abdomen was incised and sutured, without removal of the ovaries and uterus. The EH + PGC group (stent therapy group): a 1 mm incision was made proximal to both uterine horns, and 100 mg of PGC stent material was implanted. The EH + PGCL group (drug-release therapy group): the surgical procedure was the same as the EH + PGC group, but an equivalent mass of PGCL composite stent was implanted, and the wound was sutured. Postoperatively, antibiotics (penicillin, 100,000 units intramuscularly daily) and analgesics (caspofen, 1 mg jelly daily) were administered for 3 days to prevent postoperative infection and wound licking. On day 28 postoperatively, the rats were weighed and euthanized by carbon dioxide irradiation. The uterus was then removed, trimmed, and weighed (uterine wet weight). The uterine index was defined as the ratio of uterine mass to body weight.

[0052] (2) Histological analysis The morphological characteristics of the endometrium were quantitatively assessed using standard histopathological techniques. All uterine tissue samples were fixed in 10% neutral buffered formalin for 72 hours, followed by graded ethanol dehydration, xylene clearing, and paraffin embedding. 5μm serial sections were prepared using a rotary microtome, baked in a 60°C oven for 2 hours, and then stained with hematoxylin and eosin (H&E) for routine histological examination. Microscopic photographs were taken. Endometrial thickness (ET), endometrial area (EA), and glandular density (glandular density) were used as indicators of cell hypertrophy in each rat, calculated by averaging measurements from three different locations in three different sections within each group.

[0053] (3) Immunohistochemical analysis Standard immunohistochemistry was used to detect the expression, localization, and quantification of specific proteins in endometrial tissue. Paraffin-embedded 5μm uterine tissue sections were baked at 60℃ for 2 hours, followed by dewaxing with xylene and graded ethanol dehydration. Antigen retrieval was performed using 0.01M sodium citrate buffer (pH 6.0) in a microwave incubation at 98℃ for 15 minutes to block endogenous peroxidase. Sections were blocked with 5% BSA for 30 minutes, then incubated overnight at 4℃ with specific primary antibody (antibody information is detailed in Appendix 1, Table S2). After washing with PBS, sections were incubated with HRP-labeled secondary antibody at room temperature for 1 hour. DAB staining (Agilent) was performed with strict time control, followed by hematoxylin (Servicebio) counterstaining for 30 seconds. Protein expression levels were assessed using a semi-quantitative tissue scoring system (Histoscore): staining intensity was categorized as 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive). Five high-power fields (400×) were randomly selected to calculate the percentage of positive cells. The comprehensive scoring formula was Histoscore = Σ (staining intensity score × corresponding percentage of positive cells).

[0054] II. Experimental Results and Discussion 1. Preparation and characterization of PGCL stents This study successfully constructed a three-dimensional PGCL scaffold with biomimetic estrogen ligand binding capacity and superabsorbent properties by using advanced electrospinning technology combined with efficient click chemistry. Figure 1 a). The specific preparation process is as follows: First, based on our previously established method [XX], PG composite nanofiber membranes were prepared by optimizing electrospinning parameters. After liquid nitrogen brittle fracture treatment, the PG membrane was dispersed for 30 seconds using a high-speed homogenizer to obtain short fibers with a length of 50-200 μm, which were then freeze-dried to obtain short fiber powder. Subsequently, we utilized the abundant ε-amino groups on the gelatin molecular chain (0.3 mmol -NH2 per gram of gelatin) to conduct a highly efficient amidation reaction (reaction efficiency > 90%) between the N-hydroxysuccinimide ester (NHS ester) at the end of the NHS-PEG2K-N3 molecule and the primary amino groups on the surface of the short fibers at room temperature in a borate buffer system at pH 8.5. This reaction can achieve an average grafting of 8-10 azide groups (-N3) per gelatin molecule within 24 hours, providing sufficient active sites for subsequent click chemistry reactions.

[0055] Levonorgestrel (LNG), a synthetic progestin derived from 19-nortestosterone, is widely used in the clinical treatment of endometrial hyperplasia due to its high selectivity and strong affinity for progesterone receptors (binding constant Kd = 1.8 ± 0.3 nM). However, traditional oral administration has significant limitations, including suppression of the hypothalamic-pituitary-adrenal axis (up to 30%), glucose metabolism disorders (manifested as a 15-20% decrease in insulin sensitivity), and a significantly increased risk of thrombosis (relative risk 1.5-2.0). Intrauterine application of progestins is becoming increasingly popular, thus the development of LNG functional materials has become a research hotspot. Based on the terminal alkyne (-C≡CH) characteristic of LNG molecules, a novel copper-catalyzed click chemistry reaction was innovatively employed to induce a [3+2] cycloaddition reaction between the LNG molecules and the azide groups (-N3) modified on the surface of nanofibers, forming a 1,4-disubstituted triazole bond (bond energy ≈290 kJ / mol), thus constructing a novel drug delivery system with dual functional properties. Figure 1 (b) On the one hand, the high affinity binding characteristics of LNG to progesterone receptors are fully preserved, achieving high drug concentrations locally in the endometrium; on the other hand, click chemigrafting imparts pH-responsive release properties to the system, enabling it to intelligently trigger sustained release of LNG in the slightly acidic environment of the endometrium (pH approximately 6.5), significantly prolonging the drug's duration of action. To further optimize the performance of this drug delivery system, we used chitosan as a binder for drug-loaded short fibers, constructing them into a superabsorbent scaffold (PGCL) with a three-dimensional porous structure through a solution-mixing crosslinking process. The unique three-dimensional porous fiber structure of the PGCL scaffold possesses superior water absorption; and its excellent shape memory function allows it to adapt to the irregular anatomical shape of the uterine cavity. These properties not only effectively promote the drainage of uterine fluid but also provide an ideal carrier for the uniform distribution and long-term retention of drugs within the uterine cavity, thereby significantly improving the local therapeutic effect of endometrial hyperplasia.

[0056] First, the morphology of the four scaffolds was comprehensively characterized using macroscopic digital imaging and scanning electron microscopy (SEM). Macroscopic observations showed that ( Figure 1 The PG scaffold exhibits a typical white three-dimensional porous fiber structure with randomly oriented fibers; in contrast, the CS scaffold displays a three-dimensional sheet-like morphology; while the PGC scaffold maintains its three-dimensional porous fiber structure and achieves effective cross-linking between fibers through chitosan. Notably, the LNG-modified PGCL scaffold shows a significant darkening of color macroscopically, and its internal structure transforms into a unique three-dimensional porous sheet-like stacked configuration. SEM analysis further reveals ( Figure 1The PGCL scaffold possesses a biomimetic porous three-dimensional structure composed of randomly oriented fibers and a chitosan matrix, with morphological characteristics highly similar to the natural extracellular matrix (ECM). Compared to the PGC scaffold, the PGCL scaffold exhibits a more loose and rougher microstructure on its fiber surface, indicating that the LNG modification process significantly enhances the scaffold's porosity while maintaining the overall structural integrity of the fibers. Energy dispersive spectroscopy (EDS) analysis results confirm (…). Figure 1 The basic elements such as carbon and oxygen are uniformly distributed on the surface of the scaffold. Of particular note is the detection of a characteristic signal of copper on the surface of the PGCL scaffold. Figure 1 This may be due to residual copper catalyst from the click chemistry process. Given that copper has been shown to regulate endometrial inflammation by inhibiting the expression of key inflammatory factors such as IL-6 and TNF-α, this finding provides a new perspective on the biological function of the PGCL scaffold.

[0057] To further verify the chemical structural characteristics of the materials, we conducted a systematic analysis using Fourier transform infrared spectroscopy (FTIR). The results showed a significant shift in the amide I band of gelatin in the PGC complex (from 1633 cm⁻¹ to 1531 cm⁻¹), while the glycosidic bond peak intensity decreased by 35%, fully confirming that gelatin and chitosan formed a stable composite system through intermolecular hydrogen bonds. In the PGCL scaffold, the characteristic peak of the triazole ring (C=N stretching vibration) at 2108 cm⁻¹ and the significant decrease in the intensity of the amino peak (62%) clearly confirmed the successful grafting of LNG via click chemistry. Furthermore, the newly appearing carbonyl peak at 1755 cm⁻¹ further validated this conclusion. The FTIR results not only confirmed the chemical stability of the drug-loaded system, but the revealed hydrogen bond network reconstruction mechanism (broad peak at 1412 cm⁻¹) can also provide theoretical guidance for the subsequent optimization of drug-controlled release performance. X-ray diffraction (XRD) analysis revealed the evolution of the crystal structure of the drug-loaded scaffold system. The LNG feedstock exhibited typical crystalline diffraction peaks at 2θ = 12.08°, 15.14°, and 18.34° (crystallinity 78.3%), while the composite PGCL scaffold showed only a single broad peak at 23.68° (crystallinity 9.8%), confirming that the drug was uniformly dispersed in the matrix in an amorphous state. Particularly important was the significant reduction in crystallinity of the system after drug loading (from 78.3% to 9.8%); this amorphous transformation provides a new regulatory approach for optimizing drug release kinetics.

[0058] Finally, the thermal stability of the materials was evaluated by thermogravimetric analysis (TGA). The results showed that the thermal decomposition temperature of the PGC composite was 25 °C higher than that of PG, indicating a significant enhancement in the thermal stability of the composite material. In the PGCL sample, a novel decomposition peak at 250 °C further confirmed the successful drug loading.

[0059] Furthermore, the drug release characteristics and degradation behavior of the PGCL drug-loaded scaffold were systematically characterized, revealing its advantageous mechanism as a therapeutic carrier for endometrial hyperplasia. Regarding drug release kinetics ( Figure 2 The system exhibits a typical three-stage release pattern: an initial stage (0-2 days) with a 22.6% burst release due to rapid desorption of the drug from the surface; followed by a sustained release stage (2-6 days) with ideal zero-order release kinetics, accumulating a release of 38.3%, primarily regulated by polymer swelling and pH-sensitive bond hydrolysis; and finally, a gradual slowdown in the later stage (>6 days). Notably, compared to conventional formulations, this system significantly extends drug action time by 5 times while reducing the burst release effect by 37.5%. This optimized release behavior stems from the synergistic effect of the drug reservoir built by the three-dimensional porous structure, the intelligent dissociation characteristics of pH-responsive chemical bonds, and the controllable polymer degradation kinetics. Particularly noteworthy is that this release curve perfectly matches the clinical treatment needs of endometrial hyperplasia, providing technical support for achieving the ideal "one-dose-per-week" dosing regimen through a rapid initial establishment of effective blood drug concentration, a stable maintenance of the therapeutic window in the middle stage, and a sustained extension of efficacy in the later stage. Regarding material degradation behavior (… Figure 2 Four types of stents (C1-C2) exhibited significantly different degradation characteristics in simulated physiological environments. The CS stent degraded the fastest, with its mass retention dropping to 53% after 28 days. In contrast, the drug-loaded PGCL stent demonstrated the best stability, maintaining 70% of its mass after 28 days. In-depth mechanistic studies revealed that the PGCL stent's longer degradation half-life is primarily due to three mechanisms: first, the interaction between drug molecules and the matrix significantly enhances the material's structural stability; second, the dense cross-linked network effectively inhibits the penetration of hydrolytic enzymes; and third, the introduction of hydrophobic drugs reduces the overall hydrophilicity of the material. Importantly, the controllable degradation characteristics of the PGCL stent closely match the standard 4-6 week treatment cycle for endometrial hyperplasia, and its degradation rate exhibits a good synergistic effect with drug release kinetics. This spatiotemporally consistent design provides dual protection for maintaining stable local drug concentrations.

[0060] The wettability and water absorption of scaffolds, as key performance parameters of biomaterials, have a decisive impact on cell behavior regulation and the maintenance of microenvironment homeostasis. By systematically characterizing the hydrophilic properties and water absorption kinetics of four scaffolds (PG, CS, PGC, and PGCL), the potential mechanisms by which they regulate the intrauterine microenvironment were revealed. Contact angle test results confirmed ( Figure 2(D) All scaffolds exhibit significant hydrophilic properties, with contact angles all less than 60 degrees. Among them, the PGCL scaffold demonstrates the best surface wettability, which is closely related to its chemical composition and microstructure characteristics. From a structural perspective ( Figure 2 (D) Except for the CS scaffold, the other three scaffolds all exhibit high porosity exceeding 90%, and this porous architecture provides a structural basis for fluid control. Specifically, the three-dimensional interconnected pore network of the PGCL scaffold generates a significant capillary effect, while the abundant polar groups on the surface, such as hydroxyl and amino groups, enhance the binding capacity of water molecules through hydrogen bonding.

[0061] In terms of water absorption dynamics ( Figure 2 The PGCL stent (D), PGC, and PGCL stents exhibited good fluid regulation capabilities. Cyclic compression water absorption tests revealed that, unlike the CS stent which showed structural collapse under mechanical stress (its water absorption rate decreased by 42% after the fifth cycle), the PG, PGC, and PGCL stents all exhibited stable and reversible water absorption characteristics, with a coefficient of variation of less than 5% after ten cycles. To determine whether pH changes in the pathological microenvironment affected the stent's fluid regulation performance, we evaluated the water absorption behavior of the PGCL stent under weakly acidic conditions (pH 6.8). As shown in the figure, there was no significant difference in water absorption kinetics between the two pH conditions. This indicates that the stent's superabsorbency is mainly attributed to its high porosity, hydrophilic composition, and stable three-dimensional fibrous network structure, rather than being dependent on pH interactions. This pH-independent characteristic ensures the stent's ability to stably recruit inflammatory exudate under different pathological conditions. This unique fluid regulation capability enables it to effectively maintain the homeostasis of the uterine cavity microenvironment, reducing mechanical stimulation by rapidly absorbing uterine fluid and inhibiting the expression of inflammatory factors by regulating local humidity.

[0062] 2. PGCL inhibits the proliferation and migration of endometrial effector cells. (1) PGCL inhibits the proliferation and migration of endometrial epithelial cells. Epithelial cells are one of the most important components of the endometrium, playing a vital role in its biological functions. During the development and progression of endometrial hyperplasia, the pathological changes in epithelial cells are most significant. Therefore, we first investigated the impact of biomaterials on this process in the endometrial epithelial cell line (Ishikawa). To assess the biocompatibility of the materials, we examined the survival and cytoskeleton of Ishikawa cells after co-culturing with PG, CS, PGC, and PGCL for 1 and 3 days. This is the foundation and prerequisite for verifying the non-toxicity and harmlessness of the materials. (e.g., live / dead staining...) Figure 2As shown in Figure A), with increasing culture days, more viable cells were observed in each group. The co-cultured cells all exhibited good cell shape and viability, with only a small number of dead cells. Compared to cells stained with the cytoskeleton at 3 days (…),… Figure 2 Cells stained with PGCL (CCK-8) showed good extensibility after 1 day, and Ishikawa cells were round and spherical. After 3 days of culture, clear cell morphology was visible, with cells almost covering the entire field of view. Notably, the PGCL group showed a decreasing trend in cell number, although there was no significant difference among the four groups. In summary, Ishikawa maintained normal cell viability in different materials, indicating that all four materials are non-toxic biological materials, a prerequisite for their in vivo application. To investigate the effect of PGCL on the in vitro biological function of Ishikawa, we conducted proliferation and migration assays. Figure 2 The results (C) showed that Ishikawa cells continued to proliferate on all scaffolds, and there was no significant difference between scaffolds on day 1. On day 4, the cell proliferation capacity of the PGCL group slowed down, and the OD value was lower than that of the other three groups, with statistically significant differences, which was consistent with the live / dead ratio and scaffold immunofluorescence staining trends. On day 7, this difference further widened. There was no difference in the effect of PG, CS, and PGC materials on cell proliferation activity, but the cell proliferation capacity of the PGCL group was significantly reduced, which is consistent with the in vitro effects of Dienoges drug and also consistent with the theoretical basis for treating endometrial hyperplasia. To further verify, PG, CS, PGC, and PGCL were co-cultured with Ishikawa for 4 days, and EdU staining (… Figure 2 The results showed that PG, CS, and PGC materials had no significant difference in their effects on cell proliferation activity, while the proportion of EdU-positive cells in the PGC@Dienoges group was significantly reduced, suggesting that PGCL significantly inhibited the proliferation of Ishikawa. Subsequently, we examined the expression changes of two proliferation markers (MKI67 and PCNA) in the co-cultured cells. PCR ( Figure 2 China E and Figure 2 (G) and cell immunofluorescence ( Figure 2 China F and Figure 2 The results (in vitro) indicated that the expression levels of MKI67 and PCNA mRNA and protein in the PGCL group were significantly lower than those in the other three groups. Although some results did not show statistical differences, the trend was consistent. These results suggest that the material itself does not inhibit cell proliferation, but the material group loaded with Dienogest significantly inhibited cell proliferation, indirectly reflecting that the PGCL material can normally release Dienogest and exert its biological function through Dienogest. Furthermore, studies have shown that Dienogest can also exert a therapeutic effect by inhibiting cell migration and reversing endometrial proliferation. In in vitro migration experiments (… Figure 2 In the PGCL group (I), the migration rate of Ishikawa cells was significantly reduced. These results confirm that PGCL has no cytotoxicity and can inhibit the proliferation and migration ability of Ishikawa.

[0063] (2) PGCL inhibits the proliferation and migration of endometrial stromal cells. Another important component of the endometrium is the endometrial stromal cell, so we then investigated the biocompatibility and biological function of the material in an immortalized endometrial stromal cell line (T-HESCs). Live / dead staining ( Figure 3 (A) and cytoskeleton staining ( Figure 3 The results (B) indicated that with increasing culture days, more viable cells were observed in each group, with only a small number of dead cells. T-HESCs exhibited a spindle shape, good extensibility, and normal morphology, maintaining normal cell viability in different materials. Consistent with epithelial cells, the PGCL group showed a decreasing trend in cell number starting from day three, but this difference was not statistically significant. CCK-8 ( Figure 3 The results (C) showed that T-HESCs continued to proliferate on all scaffolds. On day 4, there was no difference in the effect of PG, CS, and PGC materials on cell proliferation activity. Cell proliferation in the PGCL group was slower, and the OD value was lower than the other three groups, consistent with live / dead cell count and scaffold immunofluorescence staining. On day 7, this difference widened further, with a significant decrease in cell proliferation in the PGCL group. EdU staining (… Figure 3 The results (D) showed that the proportion of EdU-positive cells in the PGCL group was significantly lower than that in the PG, CS, and PGC groups, suggesting that PGCL significantly inhibited the proliferation of T-HESCs. The proliferation marker MKI67 ( Figure 3 (Middle E and 3 Middle F) and PCNA ( Figure 3 Similar results were obtained from changes in the expression of G and H in 3. Furthermore, in in vitro scratch experiments ( Figure 3 In the PGCL group, the scar healing rate of T-HESCs was significantly reduced, suggesting that PGCL inhibits the migration of T-HESCs. These results fully demonstrate that PGCL has no cytotoxicity and can inhibit the proliferation and migration of T-HESCs.

[0064] 3. Evaluation of PGCL stent's inhibition of angiogenesis Angiogenesis plays a crucial role in the development and progression of endometrial hyperplasia, and inhibiting angiogenesis is a key therapeutic target for endometrial hyperplasia. Therefore, we investigated the biological functions of materials on blood vessels in vascular endothelial cell lines (HUVECs). First, we used live / dead staining (… Figure 4 (A) and cytoskeleton staining ( Figure 4(B) Verification of the biocompatibility of the material. The results showed that with increasing culture days, more live cells were observed in each group, with only a small number of dead cells. The co-cultured cells all exhibited good cell morphology and viability. CCK-8 ( Figure 4 The results (C) showed that HUVECs continued to proliferate on all scaffolds. Unlike the effects of PGCL on Ishikawa and T-HESCs, the proliferation capacity of HUVECs did not differ significantly between scaffolds on day 4. It wasn't until day 7 that the cell proliferation capacity in the PGCL group significantly slowed down, with a statistically significant difference. This indicates that a longer co-culture time is required for PGCL to exert its effect on vascular endothelial cells. Therefore, after co-culturing PG, CS, PGC, and PGCL with HUVECs for 7 days, EdU staining (…) Figure 4 The results of the PGCL (dihydrotesting) study showed that the proportion of EdU-positive cells was significantly reduced in the PGCL group, suggesting that PGCL significantly inhibited the proliferation of HUVECs. Subsequently, PCR (… Figure 4 (Middle E and 4 Middle G) and cell immunofluorescence ( Figure 4 Results from (F and H in group 4) indicated that the expression levels of the proliferation markers MKI67 and PCNA in the PGCL group were significantly lower than those in the other three groups. In the angiogenesis assay ( Figure 4 In the study, ImageJ software was used to count the number of nodes. Compared with the other three material groups, the PGCL group formed a sparser vascular network with a significantly reduced number of nodes, indicating that the PGCL extract significantly inhibited angiogenesis.

[0065] 4. Evaluation of in vitro inflammation regulation of PGCL stent In recent years, alterations in the local inflammatory microenvironment and the role of cytokines have become increasingly important in the etiology of endometrial hyperplasia. In our in vitro experiments, we used the classic LPS-induced mouse macrophage line (RAW 264.7) model to study inflammation. Immunofluorescence results of CD86 / CD206 cells showed that, compared to the control group, LPS stimulation of RAW 264.7 cells increased CD86 fluorescence intensity, decreased CD206 fluorescence intensity, and increased the CD86 / CD206 fluorescence intensity ratio, suggesting macrophage differentiation towards the pro-inflammatory M1 type, confirming the successful construction of the inflammatory cell model. However, after adding PGCL extract, the increased CD86 fluorescence intensity significantly decreased, while the decreased CD206 fluorescence intensity increased, and the ratio of the two fluorescence intensities decreased, suggesting macrophage differentiation towards the anti-inflammatory M2 type. These results indicate that PGCL can inhibit inflammation.

[0066] Studies have shown that macrophage infiltration and endometrial proliferation are significantly greater in macrophages than in normally proliferating endometrium. Activated macrophages release various cytokines, including IL-1β, IL-6, and TNF-α, which promote disease progression. To simulate this state, we used the classic LPS-induced mouse macrophage cell line (RAW 264.7) as a model for inflammation studies in vitro. PCR ( Figure 4 J, K and L) and ELISA ( Figure 4 The results (M, N, and O) indicated that, compared to the control group, LPS stimulation of RAW264.7 cells significantly increased the expression levels of IL-1β, IL-6, and TNF-α, demonstrating the successful establishment of the inflammatory cell model. However, the addition of PGCL extract significantly reduced the elevated levels of IL-1β, IL-6, and TNF-α, inhibiting the secretion of inflammatory factors by macrophages. These results suggest that PGCL can inhibit angiogenesis and inflammation.

[0067] To understand the potential molecular mechanisms involved in PGCL treatment of endometrial hyperplasia, we performed high-throughput RNA-seq on Ishikawa cells treated with PGC and PGCL scaffolds, respectively. Figure 5 Figure A shows the normalized signal intensities of the two sets of samples after PGC and PGCL processing, exhibiting good homogeneity. Principal component analysis (PCA) of the two sets of samples... Figure 5 (B) This indicates that samples from the same group are clustered closely on the PCA plot, suggesting similar gene expression patterns, good reproducibility, and high data quality. Conversely, samples from two groups are far apart on the PCA plot, implying significant differences in gene expression between the two groups and different biological significance. Hierarchical clustering analysis ( Figure 5 (C) shows a systemic change in mRNA expression between the two groups. To better illustrate the results of differentially expressed genes, we performed a volcano plot of the differentially expressed genes. Figure 5 The data was visualized using a GO enrichment analysis (D) method, which included 153 upregulated genes and 431 downregulated genes. We then performed GO enrichment analysis on the downregulated genes. Figure 5 The study found that the downregulated pathways were mainly concentrated in epithelial cell proliferation, indicating that the proliferation capacity of Ishikawa cells treated with PGCL decreased compared to the PGC group, which is consistent with the previous cell experiment results.

[0068] We also performed a KEGG pathway enrichment analysis on the downregulated genes. Figure 5To determine the potential biological roles of differentially expressed genes, we screened the top 20 pathways with more than two differentially expressed genes, including cellular senescence, the p53 signaling pathway, cell cycle, and DNA replication. The most significantly downregulated pathway was the phosphatidylinositol 3-kinase / protein kinase B (PI3K / AKT) signaling pathway, one of the core pathways regulating cell survival, proliferation, metabolism, and immune responses. Recent studies have found that this pathway is closely related to the occurrence, development, and resolution of inflammation, especially playing a key role in immune cell activation, inflammatory factor release, and inflammation-related diseases. In macrophages, PI3K / AKT phosphorylates and activates the IKK complex (IκB kinase), promoting NF-κB nuclear translocation and driving the expression of pro-inflammatory factors such as TNF-α, IL-6, and IL-1β. Based on previous cell experiments, we hypothesize that PGCL may exert its anti-inflammatory effect by inhibiting the transduction of the PI3K / AKT / NF-κB signaling pathway. The mitogen-activated protein kinase (MAPK) signaling pathway is one of the core pathways regulating intracellular inflammatory responses. Through cascade phosphorylation, it transmits signals, activates transcription factors (such as c-Fos and c-Jun members of the AP-1 complex), and induces the expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. MAPK plays a crucial role in the initiation, amplification, and resolution of inflammation and is closely related to various inflammatory diseases. The MAPK signaling pathway was downregulated in Ishikawa cells treated with PGCL, suggesting that PGCL may also exert an anti-inflammatory effect by inhibiting the MAPK / c-Fos / c-Jun signaling pathway.

[0069] 5. Efficacy evaluation of PGCL stent in vivo treatment for endometrial hyperplasia Next, we established an animal model of endometrial hyperplasia (EH) in rats and tested the therapeutic effect of PGCL in vivo. Figure 5 (A). An estradiol-mediated hemorrhage (EH) model was established in rats by administering estradiol benzoate. Simultaneously, rats were treated with different regimens, as detailed in "Experimental Methods - Animal Models and Treatments". In short, the experiment was divided into four groups: a sham-operated group (Sham group), an EH model group, an EH + PGC group, and an EH + PGCL group, lasting for four weeks. Then, gross images and stained tissue sections of the uterus were collected and analyzed. Gross images ( Figure 5In the study (B), it was found that the uterus of rats in the Sham group had a uniform texture and a bright, pink color. The uterus of rats in the EH model group was pale red, with significant morphological changes and increased volume, confirming the successful establishment of the EH model. The uterus of rats in the EH+ PGC group had an appearance similar to that of the EH model group, suggesting that PGC alone had no therapeutic effect. The uterus of rats in the EH+ PGCL group was significantly smaller than that of the EH model group and the EH+ PGC group, and its shape was similar to that of the Sham group. (Rat uterine wet weight (…)) Figure 5 (C) and uterine coefficient ( Figure 5 The changes in uterine wet weight and uterine coefficient in the EH model group more specifically reflect similar conclusions. The uterine wet weight and uterine coefficient in the Sham group were 0.28±0.09g and 0.10±0.04%, respectively. In the EH model group, the uterine wet weight and uterine coefficient significantly increased, reaching 0.78±0.26g and 0.33±0.11%, respectively, with statistically significant differences (P<0.01), proving the successful establishment of the EH model. The data for the EH + PGC group were similar to those in the EH model. Encouragingly, the uterine wet weight (0.41±0.15g) and uterine coefficient (0.16±0.07%) in the EH + PGCL group were significantly lower than those in the EH model group, decreasing by approximately 50%, approaching the normal uterine data in the Sham group. This suggests that PGCL partially restored the uterine morphology of EH rats, reduced uterine weight, and reversed endometrial hyperplasia. HE staining ( Figure 5 The results (E) showed that the endometrial morphology of rats in the Sham group was normal, with uniform lumen, tall columnar luminal and glandular epithelial cells, densely packed glands, and varying glandular lumen sizes. In the EH model group, the endometrial morphology was abnormal, with significantly dilated lumen, low columnar luminal and glandular epithelial cells exhibiting pseudostratification, a significantly increased number of endometrial glands, with adjacent glands even showing a "back-to-back" phenomenon, and significantly dilated glandular lumens. We measured endometrial thickness (… Figure 5 (middle F), endometrial area ( Figure 5 G) and gland density ( Figure 5 Measurements and statistical analysis were performed on the endometrial tissue in the EH model group. The results showed that, compared with the Sham group, the endometrial thickness in the EH model group was increased (342.8±143.7μm vs. 563.0±116.8μm, P<0.05), and the endometrial area was significantly increased (1.16±0.64mm). 2 vs. 2.56±0.97mm 2 The endometrial gland density increased (9.01±2.98 vs. 15.22±4.68 glands / HP, P<0.05), indicating successful model establishment. The statistical results of the EH + PGC group were similar to those of the EH model group, while the endometrial thickness (380.3±80.63 μm) and endometrial area (1.44±0.41 mm) of the EH + PGCL group were significantly different.2 The number of endometrial glands and the density of endometrial glands (8.94±3.11 glands / HP, P<0.05) decreased, approaching the normal uterus of the Sham group. These results indicate that PGCL reduced the wet weight and uterine index of the uterus in EH rats, decreased endometrial thickness, area, and gland density, and alleviated endometrial hyperplasia.

[0070] 6. Histological analysis of PGCL stent in vivo treatment of endometrial hyperplasia Through in vitro cell experiments, we observed that PGCL inhibited the proliferation of endometrial cells. To further evaluate the proliferation of rat endometrial cells, we performed tissue PCR detection and tissue staining on rat endometrial tissue. PCR results ( Figure 6 (Figures A and B) showed that the expression levels of endometrial proliferation markers MKI67 and PCNA were significantly increased in the EH model group, while the expression levels of MKI67 and PCNA were decreased in the PGCL group. Immunofluorescence staining of rat endometrial MKI67 (Figures A and B) Figure 6 The results (C) showed that the positive rate in the EH model group was 3.13±1.58%, which was 3.6 times that of the Sham group (0.87±0.48%). The positive rate in the EH + PGC group was 2.70±1.40%, while the positive rate in the PGCL group was 0.99±0.39%, which was nearly 70% lower than that in the EH model group, almost returning to the level of the Sham group. PCNA immunohistochemical staining ( Figure 6 Similar conclusions were reached in the EH model group (D), where the number of PCNA-positive endometrial cells was significantly increased, indicating enhanced endometrial proliferation, while the number of PCNA-positive cells was significantly reduced in the PGCL group. These findings collectively demonstrate that PGCL plays a role in treating endometrial hyperplasia by inhibiting endometrial cell proliferation. To assess the effect of PGCL treatment on endometrial angiogenesis, we observed angiogenesis in the endometrium by evaluating CD31 (a marker of blood vessels). Results ( Figure 6 The results showed that angiogenesis increased in the EH model group and the EH + PGC group, while angiogenesis decreased in the PGCL group, indicating that PGCL can inhibit abnormal angiogenesis during endometrial proliferation and thus inhibit endometrial proliferation.

[0071] 7. PGCL inhibits the local inflammatory environment of the endometrium in vivo. Further animal experiments were conducted to evaluate the expression of genes involved in the formation of the local inflammatory microenvironment, which can influence the occurrence and development of endometrial hemorrhage (EH) by regulating the local inflammatory state. qRT-PCR of rat endometrial tissue was performed. Figure 7 (A, B, and C) and ELISA ( Figure 7Results (D, E, and F) showed that, compared with the control group, the levels of IL-1β, IL-6, and TNF-α mRNA and protein levels in the endometrium of the EH model group and the EH + PGC group were significantly increased, while those in the PGCL group were significantly decreased, approaching the levels of the Sham group. To further confirm this, we also performed immunohistochemical staining of endometrial tissue (…). Figure 7 In the G, H, and I groups, the staining scores of IL-1β, IL-6, and TNF-α in the endometrium of the EH model group and the EH+ PGC group were significantly increased, indicating increased secretion of IL-1β, IL-6, and TNF-α. Conversely, the staining scores in the PGCL group were significantly decreased, suggesting that PGCL inhibits the secretion of inflammatory factors, thereby suppressing the local inflammatory environment of the endometrium and exerting a therapeutic effect on endometrial hyperplasia. In summary, the PGCL system, through self-expansion covering the entire uterus, slowly releases dienogest, prolonging the time that local dienogest remains in the uterine cavity, and achieves a therapeutic effect on endometrial hyperplasia by inhibiting endometrial cell proliferation, angiogenesis, and inflammation.

[0072] III. Conclusion This invention innovatively constructs a smartly responsive PGCL short-fiber scaffold based on click chemistry principles, designed to target inflammation-mediated progesterone resistance and the acidified microenvironment of endometrial hyperplasia (EH). This scaffold achieves precise proton-triggered release through a two-step covalent grafting of LNG and the introduction of triazole bonds. In a pathologically acidic microenvironment, it effectively removes protons from inflammatory exudates and releases the drug accordingly, thereby simultaneously inhibiting estrogen-driven endometrial proliferation and abnormal angiogenesis. Figure 10The superhydrophilicity, self-expansion ability, and highly porous structure of the PGCL scaffold ensure its adaptability to complex uterine cavities and efficient isolation of inflammatory fluid, providing optimized conditions for local inflammation regulation. Mechanistic studies show that this system downregulates macrophage V-type H+-ATPase expression, promotes M2 polarization, and simultaneously inhibits the PI3K / AKT / NF-κB and MAPK signaling pathways, thereby achieving bidirectional regulation of the inflammatory microenvironment from epithelial cells to immune cells and disrupting the acidification-inflammation positive feedback loop. Animal experiments further confirmed that PGCL can significantly reduce endometrial thickness and glandular density in EH model rats, while also reducing microvascular formation and inflammatory cytokine expression, demonstrating a strong therapeutic effect. In summary, this study, by integrating inflammatory microenvironment regulation, precise local drug release, and immune remodeling, provides a new strategy for the precise treatment of EH that combines efficacy with fertility preservation, laying a solid theoretical and practical foundation for future interventions targeting V-type H+-ATPase to alleviate local inflammatory acidification and reduce recurrence. Future research will focus on optimizing the degradation kinetics and drug release profile of the PGCL stent to achieve complete synchronization with the endometrial repair cycle. Systematic evaluation will be conducted in large animal models that more closely simulate human pathophysiology, thus advancing the clinical translation of this stent. This will provide a new generation of precision intervention solutions for endometrial hemorrhage (EH) and related conditions.

Claims

1. A method for preparing short fibers that recruit inflammatory fluid and intelligently respond to inflammation, characterized in that, Includes the following steps: (1) Electrospinning was performed using a solution of polylactic acid and gelatin as the spinning solution to prepare a nanofiber membrane, which was then homogenized and freeze-dried to prepare short nanofibers. (2) The nanofibers obtained in step (1) were reacted with NHS-PEG2K-N3 to prepare azide-functionalized short fibers, and then levonorgestrel was grafted by click chemical grafting to obtain drug-loaded short fibers. (3) Disperse the drug-loaded short fibers in water to form a suspension, then mix with chitosan, add glutaraldehyde aqueous solution to carry out Schiff base crosslinking reaction, and then inject into a mold to prepare chitosan composite short fiber scaffold.

2. The method according to claim 1, characterized in that, The polylactic acid mentioned in step (1) has a molecular weight of 100,000 Da and a weight percentage of 20%-30% for polylactic acid and gelatin.

3. The method according to claim 1, characterized in that, The electrospinning process described in step (1) is as follows: a feed rate of 1-2 mL / h, a working voltage of 10-25 kV, and a receiving distance of 8-14 cm.

4. The method according to claim 1, characterized in that, In step (1), polylactic acid and gelatin are dissolved in hexafluoroisopropanol to prepare a spinning solution with a concentration of 10-12% (w / v).

5. The method according to claim 4, characterized in that, The homogenization conditions described in step (1) are homogenization at ≥13,000 rpm for 30 min.

6. The method according to claim 1, characterized in that, The click chemistry described in step (2) is a two-step method. First, the short fibers are reacted with NHS-PEG2K-N3 at a molar ratio of 1:3 to prepare azide-functionalized short fibers. Then, levonorgestrel is grafted onto the short fibers through a click chemistry reaction in the catalytic system of CuCl and sodium citrate to obtain drug-loaded short fibers.

7. The method according to claim 1, characterized in that, The weight ratio of drug-loaded short fibers to chitosan in step (3) is 2:

1.

8. The method according to claim 1, characterized in that, In step (3), the molar ratio of amino to aldehyde groups in the Schiff base crosslinking reaction is controlled to be 1:0.

8.

9. Short fibers that recruit inflammatory fluid and intelligently respond to inflammation, prepared by the method of any one of claims 1-8.

10. The use of the short fibers of claim 9, which recruit inflammatory fluid and intelligently respond to inflammation, in the preparation of a medicament for treating endometrial hyperplasia.

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