A rsl3-loaded liposome-hydrogel composite delivery system, and a preparation method and application thereof

Abstract

The present application relates to a kind of RSL3 loaded liposome-hydrogel composite delivery system, which is composed of hydrogel matrix and liposome nanoparticle uniformly dispersed in it in physical embedding manner, the liposome nanoparticle is formed by the encapsulation of ferroptosis inducer RSL3 in liposome carrier, and the liposome carrier is a lipid bilayer vesicle formed by assembling DSPC, cholesterol and DSPE-PEG2000;The hydrogel matrix is a three-dimensional porous network structure formed by Schiff base cross-linking reaction of carboxymethyl chitosan and oxidized hyaluronic acid.The present application also provides the application of the composite delivery system in the preparation of contraceptive drugs.Experiments in vivo and in vitro prove that the composite delivery system can achieve complete contraception, and the uterus is normal, liver and kidney function is normal, reproductive function is completely reversible, and the development index of offspring is qualified.The present application realizes non-hormonal, reversible and locally safe contraceptive effect by inducing trophoblast cell ferroptosis, and provides a new solution for the development of contraceptive technology.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a composite hydrogel delivery system, particularly a liposome-hydrogel composite delivery system that can induce ferroptosis in trophoblast cells, as well as its preparation method and its application in the preparation of contraceptive drugs. Background Technology

[0002] Unintended pregnancy and its associated social and health burdens remain a pressing public health issue affecting people of reproductive age worldwide. An ideal contraceptive strategy should ensure high efficacy while also being convenient, having good local safety, and preserving future fertility. However, current contraceptive methods consistently fall short of achieving an ideal balance across these dimensions. Hormonal drugs require systemic administration, and long-term use may interfere with the hypothalamic-pituitary-ovarian axis, increasing the risk of thrombosis and endocrine disorders. Emergency contraceptives, as a post-coital remedy, are strictly limited to a 72-hour effective window, failing to meet the clinical need for early intervention in non-emergency situations. While intrauterine devices (IUDs) can achieve local effects, their placement is invasive, potentially inducing endometritis and perforation risks, and requires operation by qualified medical personnel.

[0003] The core issue of the aforementioned dilemma lies in the inherent and irreconcilable contradiction between contraceptive efficacy and tissue safety, and between duration of action and reproductive reversibility. Therefore, developing a drug delivery system that can respond to the local microenvironment of the uterus and achieve spatiotemporal control is of significant scientific importance for balancing contraceptive efficacy and tissue safety.

[0004] Implantation is the initial step in establishing pregnancy, and trophoblast cells, as functional cells that directly contact and invade the maternal endometrium, require normal proliferation and migration capabilities as a prerequisite for successful implantation. If trophoblast cells can be precisely eliminated before implantation begins, the pregnancy process can be terminated before a strong connection is established at the embryo-maternal interface. This strategy combines high efficiency and local controllability. However, ensuring the effectiveness of killing target cells while avoiding damage to the normal endometrium and ovarian reserve remains a significant technological bottleneck in this field.

[0005] In recent years, ferroptosis, a regulatory cell death mechanism characterized by the accumulation of lipid peroxides and inactivation of glutathione peroxidase 4 (GPX4), has emerged as a novel intervention target for the selective elimination of specific cell populations due to its unique metabolic dependence. Studies have shown that rapidly proliferating cells often exhibit particular susceptibility to this novel cell death mechanism. On the one hand, actively proliferating cells require large amounts of synthesized lipids for biomembrane construction, leading to elevated intracellular polyunsaturated fatty acid (PUFA) levels, providing abundant substrates for lipid peroxidation. On the other hand, to support rapid metabolism, these cells often maintain relatively high baseline levels of reactive oxygen species (ROS), making their redox balance more vulnerable.

[0006] The high invasiveness and proliferative properties of human chorionic trophoblast cells (HTR-8 / SVneo) during implantation may give them a unique sensitivity to ferroptosis in terms of lipid metabolism and oxidative stress, which provides an important biological basis for targeted intervention by inducing ferroptosis. Summary of the Invention

[0007] The purpose of this invention is to provide a liposome-hydrogel composite delivery system loaded with RSL3 and its preparation method, as well as its application as a non-hormonal local reversible contraceptive, which can maintain the natural physiological balance of the female reproductive system to the greatest extent while ensuring high contraceptive efficacy.

[0008] Intrauterine local administration of drugs can concentrate them at the target site, making it an ideal strategy for highly effective contraception. Injectable hydrogels, due to their excellent biocompatibility, biodegradability, and in-situ molding capabilities, have become highly promising drug reservoirs and delivery carriers. Based on the above theoretical foundation, this invention constructs a liposome-hydrogel composite delivery system loaded with the ferroptosis inducer RSL3. Through multiple synergistic mechanisms, it precisely removes trophoblast cells at key stages of embryo implantation, providing a new solution for non-hormonal, reversible, and locally safe contraceptive strategies.

[0009] To achieve the above-mentioned objectives, the present invention first provides a liposome-hydrogel composite delivery system loaded with RSL3, referred to as RSL3@Lip / CMCS-OHA, which is specifically composed of a hydrogel matrix and RSL3 liposome nanoparticles uniformly dispersed therein by physical embedding.

[0010] The RSL3 liposome nanoparticles are formed by encapsulating the ferroptosis inducer RSL3 in a liposome carrier. The average particle size of the nanoparticles is 100-130 nm, the encapsulation efficiency is ≥85%, and the drug loading is 0.5-2 wt%.

[0011] Specifically, the ferroptosis inducer RSL3, officially named (1S,3R)-RSL3, is a glutathione peroxidase 4 (GPX4) inhibitor. After entering target cells, RSL3 irreversibly inhibits GPX4 activity, blocking the cells' ability to clear lipid peroxides, leading to a large accumulation of intracellular lipid peroxides and triggering ferroptosis in the target cells. In vitro experiments of this invention have confirmed that RSL3 can dose-dependently induce ferroptosis in human villous trophoblast cells (HTR-8 / SVneo), and this effect can be reversed by the ferroptosis-specific inhibitor Ferrostatin-1 (Fer-1).

[0012] More specifically, the liposome carrier is a lipid bilayer vesicle assembled from 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and 1,2-distearyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG2000). This liposome carrier has the clear advantage of forming a highly stable lipid membrane, where DSPC, as a high phase transition temperature phospholipid, provides a robust membrane structure; the addition of cholesterol further enhances membrane rigidity and reduces drug leakage; and the modification with DSPE-PEG2000 endows the liposomes with "stealth" properties, helping to reduce in vivo clearance. When used to encapsulate RSL3, it achieves targeted delivery and intracellular sustained release to trophoblast cells by enhancing cellular uptake, reducing stimulation of surrounding normal tissues while improving the killing efficiency of trophoblast cells.

[0013] Furthermore, in the liposome carrier, the molar ratio of DSPC, cholesterol and DSPE-PEG2000 as components is preferably 58:35:7.

[0014] Specifically, the hydrogel matrix is ​​a three-dimensional porous network structure formed by a Schiff base crosslinking reaction of carboxymethyl chitosan (CMCS) and oxidized hyaluronic acid (OHA). This hydrogel matrix possesses injectability, in-situ molding capability, self-healing properties, and pH-dependent degradation behavior. It can be administered non-invasively into the uterine cavity via vagina, rapidly forming in situ under physiological conditions and maintaining structural integrity in the dynamic uterine environment. It regulates the drug release rate according to the physiological environment of the uterus, serving as a macroscopic drug reservoir to achieve loading and long-term retention of RSL3 liposome nanoparticles, thereby achieving precise coverage of the pre-implantation window (day 4 of pregnancy).

[0015] Furthermore, as a preferred embodiment, the final concentration of RSL3 in the composite delivery system of the present invention is 0.5–2 μM.

[0016] This invention successfully constructed an RSL3@Lip / CMCS-OHA composite delivery system by uniformly dispersing RSL3 liposome nanoparticles in a hydrogel matrix through physical embedding. The hydrogel matrix acts as a primary macroscopic network, delaying the diffusion of RSL3 liposome nanoparticles, while the RSL3 liposome nanoparticles serve as a secondary nanoreservoir, further regulating the molecular release of RSL3. After slow release from the hydrogel matrix, the RSL3 liposome nanoparticles enter trophoblast cells via the liposome carrier, inhibiting GPX4 activity through intracellular sustained-release of RSL3 and inducing ferroptosis in trophoblast cells, thus precisely eliminating the embryo before implantation. This "two-stage sustained-release" design enables synergistic effects in vivo, overcoming the burst release problem of traditional drug-loaded hydrogels and achieving long-lasting drug coverage. In vitro and in vivo experiments demonstrate that this composite delivery system can achieve complete contraception with normal uterine morphology, normal liver and kidney function, completely reversible reproductive function, and satisfactory offspring development indicators.

[0017] The present invention also provides a method for preparing the RSL3-loaded liposome-hydrogel composite delivery system. Specifically, a thin-film dispersion method is used, in which DSPC, cholesterol, DSPE-PEG2000 and RSL3 are dissolved in an organic solvent, and a lipid film is formed by rotary evaporation. After adding buffer solution for hydration incubation, an RSL3 liposome nanoparticle suspension is obtained. The RSL3 liposome nanoparticle suspension is then dispersed in a carboxymethyl chitosan solution, and an oxidized hyaluronic acid solution is added to perform a Schiff base crosslinking reaction to form a hydrogel matrix in which RSL3 liposome nanoparticles are uniformly dispersed by physical embedding.

[0018] The organic solvent is a good solvent for DSPC, cholesterol, DSPE-PEG2000, and RSL3, i.e., a non-aqueous solvent that can dissolve the raw materials but does not react with them. The preferred organic solvent of this invention is a mixture of chloroform and methanol, more preferably a chloroform-methanol mixture solution with a volume ratio of 2:1.

[0019] Furthermore, the present invention preferably involves rotary evaporation at 30–40°C to remove organic solvents and form a uniform lipid film.

[0020] Furthermore, the present invention preferably involves adding the lipid film to Tris-HCl buffer and incubating it at 60–70°C for 1–3 hours.

[0021] More specifically, the preparation method of the present invention further includes conventional ultrasonic, extrusion filtration and centrifugation purification treatment of the hydrated RSL3 liposome nanoparticle suspension. The liposome particle size is reduced and its distribution uniformity is improved by pulsed ultrasonication. The suspension is repeatedly extruded and filtered through 0.45 μm and 0.22 μm needle filters to remove large particle aggregates and unhydrated lipid membrane fragments. Free drugs and solvents are removed by high-speed centrifugation using ultrafiltration centrifuge tubes to obtain an RSL3 liposome nanoparticle suspension with highly uniform particle size and good purity.

[0022] The oxidized hyaluronic acid described in this invention can be prepared by conventional methods well known to those skilled in the art, such as, but not limited to, the sodium periodate oxidation method, in which hyaluronic acid reacts with sodium periodate under light-protected conditions and is then quenched with ethylene glycol.

[0023] Furthermore, the light-protected reaction time is preferably 4 to 8 hours.

[0024] Finally, the present invention also provides the application of the RSL3-loaded liposome-hydrogel composite delivery system in the preparation of contraceptive drugs.

[0025] Specifically, the composite delivery system of the present invention, as a non-hormonal, locally reversible contraceptive drug, induces ferroptosis in trophoblast cells through non-invasive vaginal administration to achieve contraception.

[0026] In vitro and in vivo experimental results show that the RSL3-loaded liposome-hydrogel composite delivery system of the present invention has the following characteristics:

[0027] 1) It achieves highly efficient, local, and completely reversible contraception. In a rat unilateral uterine horn self-control model, the composite delivery system of this invention can achieve complete contraception on the treated side at a final RSL3 concentration of 1 μM, while the structure and function of the contralateral uterus and ovary remain intact. The contraceptive effect is precise, reproductive function is fully restored after drug withdrawal, and the developmental indicators of both generations of offspring are qualified, with no long-term developmental toxicity.

[0028] 2) It possesses a multi-synergistic mechanism of "dual-stage sustained release + precise delivery". This invention uses the CMCS / OHA hydrogel matrix as the primary macroscopic reservoir to achieve in-situ shaping and long-term retention within the uterine cavity; and uses RSL3 liposome nanoparticles as the secondary nanodelivery unit to achieve precise delivery and intracellular sustained release to trophoblast cells. The two work together to overcome the burst release problem of traditional drug-loaded hydrogels and achieve precise coverage of the pre-implantation window.

[0029] 3) By inhibiting GPX4 to activate trophoblast cell ferroptosis. RSL3 irreversibly inhibits GPX4 activity, triggering the ferroptosis pathway. This mechanism can effectively induce ferroptosis in trophoblast cells, which are a key element in embryo implantation; their death can directly block implantation. This invention is the first to apply the ferroptosis mechanism to the field of contraception, achieving precise removal of the embryo before implantation.

[0030] 4) It has good local and systemic safety. No inflammation, necrosis or fibrosis was observed in the uterus on the treatment side, no pathological changes were found in the major organs, and liver and kidney function indicators were normal.

[0031] 5) Breaking through the time limitations of traditional emergency contraception and achieving an earlier window of opportunity. The composite delivery system of this invention can take effect with a single dose before implantation, breaking through the time limitations of traditional emergency contraception and integrating "early window of opportunity, accurate identification, and ferroptosis clearance" into a single platform.

[0032] The RSL3-loaded liposome-hydrogel composite delivery system of this invention takes ferroptosis as its starting point and utilizes liposome-hydrogel dual-stage delivery to achieve precise local contraception in the uterus, providing a complete technical solution for a non-hormonal, reversible, and safe local contraception strategy. Attached Figure Description

[0033] Figure 1 This is a particle size distribution diagram of the RSL3 liposome nanoparticles prepared in Example 1.

[0034] Figure 2 This is a comparison FTIR image of OHA prepared in Example 2 and raw material HA.

[0035] Figure 3 This is a SEM image of the CMCS-OHA hydrogel matrix prepared in Example 3.

[0036] Figure 4 These are macroscopic photographs of the self-healing process of the hydrogel matrix.

[0037] Figure 5 These are the swelling and degradation curves of the hydrogel matrix in simulated uterine fluid at different pH values.

[0038] Figure 6 The survival rates of HTR-8 / SVneo and HESCs cells treated with different concentrations of hydrogel matrix extract are shown.

[0039] Figure 7 The study investigated the survival rates of HTR-8 / SVneo cells and HESCs cells treated with different concentrations of RSL3 and the rescue effect of Fer-1.

[0040] Figure 8 These are anatomical photographs of the uterus of rats in each experimental group.

[0041] Figure 9 This is a Western blot analysis of representative bands and quantitative results of ferroptosis-related proteins in uterine tissue after RSL3@Lip / CMCS-OHA intervention.

[0042] Figure 10 The results show the H&E staining of the major organs and ovaries of rats in each experimental group, as well as the morphology and H&E staining of the local uterus in non-pregnant female rats after administration of the drug and in pregnant female rats after contraception.

[0043] Figure 11 These are the results of the detection of serum liver and kidney function biochemical indicators in rats of each experimental group.

[0044] Figure 12 This study compares the litter size of F1 and F2 generations of rats in the blank control group with that in the RSL3 liposome-hydrogel group. Implementation

[0045] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and specific examples. It should be noted that the embodiments are merely illustrative and are intended to provide a thorough understanding of the technical solutions of the present invention and to provide guidance for those skilled in the art to implement and apply the present invention. It should be understood that these descriptions do not constitute any limitation on the scope of protection of the present invention.

[0046] Unless otherwise expressly stated, the production processes, experiments, tests or analysis methods involved in the embodiments of the present invention are all considered to be conventional methods known to those skilled in the art, and only need to be implemented in accordance with conventional conditions or relevant product instructions. The steps and names involved are also generally clear and unambiguous in the art.

[0047] The instruments, equipment, raw materials, reagents, or samples used in the embodiments are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels or prepared by known methods, and their source does not have a substantial impact on the implementation results of the present invention.

[0048] Unless otherwise expressly defined, the scientific and technical terms used in this invention have the meanings commonly understood by one of ordinary skill in the art. In case of any conflict, the definitions in this specification shall prevail.

[0049] The terms “comprising,” “including,” “having,” etc., used in this invention should be understood as open-ended, meaning “including but not limited to.” The term “and / or” includes any and all combinations of one or more of the associated listed items. Quantitative terms such as “a,” “one,” etc., do not exclude multiples; “multiple” or “a variety” refers to quantities greater than or equal to two.

[0050] The terms "preferred", "better", and "exemplary" used in this invention are only used to describe specific solutions or effects and are not intended to limit the necessary scope of the solution or the scope of protection.

[0051] This invention relates to the description of numerical parameters (such as quantity, concentration, temperature, time, etc.), and it should be understood that reasonable deviations naturally exist due to measuring instruments, operational errors, statistical fluctuations, etc. The range of such deviations should be within limits acceptable to those skilled in the art based on common sense.

[0052] Intrauterine local drug delivery systems can concentrate drugs at the target site, making them an ideal platform for achieving highly effective contraceptive strategies. Among them, injectable hydrogels have become a highly promising drug reservoir and delivery carrier due to their good biocompatibility, biodegradability, and in-situ molding and filling capabilities. This invention designs and constructs a "liposome-hydrogel" two-stage sustained-release composite system. This system uses an injectable carboxymethyl chitosan / oxidized hyaluronic acid (CMCS-OHA) hydrogel as the primary macroscopic reservoir and retention platform to ensure the in-situ molding and long-term residence of the formulation in the uterus. Simultaneously, the ferroptosis-inducing drug RSL3 is pre-encapsulated in liposome nanoparticles composed of DSPC, cholesterol, and DSPE-PEG2000 to form a nanoscale secondary delivery unit.

[0053] This invention prepared a novel RSL3@Lip / CMCS-OHA composite delivery system and characterized the particle size, polydispersity index, encapsulation efficiency, and drug loading of the drug-loaded liposomes RSL3@Lip to evaluate its physical stability, nanoscale properties, and preparation efficiency. Next, the CMCS-OHA hydrogel matrix was prepared and characterized, and its microstructure, self-healing ability, injectability, and swelling degradation properties were investigated. The biocompatibility of the hydrogel was evaluated through in vitro cell experiments.

[0054] Based on the successful construction of RSL3@Lip / CMCS-OHA, this invention fully verified the feasibility of the formulation to achieve space-specific contraception by inducing local uterine ferroptosis through systematic in vivo rat experiments. It also demonstrated its good systemic safety, local uterine biocompatibility, and complete reproductive reversibility from multiple dimensions, providing key and solid experimental evidence for the clinical translation of non-hormonal contraceptive strategies. Example

[0055] Example 1: Preparation of RSL3 liposome nanoparticles

[0056] Weigh DSPC (4.58 mg), cholesterol (1.35 mg), and DSPE-PEG2000 (1.96 mg) into a 100 mL round-bottom flask. Accurately add 10 μL of 10 mM RSL3 stock solution (containing 0.044 mg of RSL3, dissolved in DMSO), and then add 3 mL of chloroform / methanol (volume ratio 2:1) mixed solvent. Gently shake to completely dissolve all solids, forming a clear and transparent drug-containing organic phase solution. The molar ratio of DSPC, cholesterol, and DSPE-PEG2000 in this solution is 58:35:7, and the total liposome concentration is 1 mM.

[0057] Connect the flask to a rotary evaporator and set the water bath temperature to 30°C. Initially, use a medium rotation speed and low vacuum for rotary evaporation. Once most of the solvent has evaporated and the flask walls appear cloudy, gradually increase the vacuum to the maximum. Continue for approximately 45–60 minutes until a uniform, translucent lipid film without any traces of flowing solvent forms on the inner wall of the flask. After film formation, place the flask in a vacuum desiccator and continue drying under high vacuum for at least 2 hours to ensure complete removal of trace organic solvents.

[0058] Add 5 mL of Tris-HCl buffer preheated to 65 °C to the flask, reconnect it to the rotary evaporator, do not turn on the vacuum, and incubate in a 65 °C water bath for 2 h to allow the lipid film to be completely hydrated and peeled off from the flask wall to obtain a crude liposome suspension.

[0059] The crude liposome suspension was subjected to pulsed sonication in an ice-water bath with a pulse pattern of 2 seconds of sonication followed by a 5-second pause. After 5 minutes of sonication, the suspension was pre-filtered through a 0.45 μm syringe to remove large particle aggregates. Then, using a new syringe, the filtrate was filtered through a 0.22 μm syringe. This process was repeated three times to obtain a liposome suspension with uniform particle size.

[0060] The liposome suspension was transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa and centrifuged at 4000 × g for 15 min to remove unencapsulated drug and DMSO. A small amount of pre-cooled Tris-HCl buffer was added, and the liposomes were gently resuspended by pipetting. The mixture was centrifuged again for 15 min. The washing was repeated three times, and the volume was adjusted to 10 mL to obtain a purified RSL3 liposome nanoparticle suspension.

[0061] The particle size of RSL3 liposome nanoparticles was determined using dynamic light scattering, and the results are as follows: Figure 1The results showed that the average particle size of the liposomes was 112.3±1.45 nm and the polydispersity index (PDI) was 0.1022±0.009, indicating that the liposome particle size distribution was very concentrated and the system was highly uniform. This particle size range allows the liposomes to be uniformly encapsulated in the hydrogel network to achieve efficient loading, laying the foundation for constructing a controllable sustained-release liposome-hydrogel composite delivery system.

[0062] After adding methanol to the purified RSL3 liposome nanoparticle suspension to demulsify, the total drug concentration was determined by ultraviolet spectrophotometry. Then, the concentration of unencapsulated free drug was determined by taking the filtrate after ultrafiltration and centrifugation. The encapsulation rate of the RSL3 liposome nanoparticle suspension was calculated to be 88.15±1.69%, and the drug loading was 0.52±0.03%.

[0063] Example 2: Preparation of oxidized hyaluronic acid

[0064] Accurately weigh 2.00 g of hyaluronic acid (HA) and dissolve it in 100 mL of distilled water. Stir at 37 °C until completely dissolved. After cooling to room temperature, add 2.14 g of sodium periodate and stir at 25 °C in the dark for 6 h. Add 1 mL of ethylene glycol to quench the reaction and continue stirring for 1 h. Transfer the mixture to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze in distilled water for 72 h, changing the water 3 times a day. Freeze-dry for 48 h to obtain oxidized hyaluronic acid (OHA) as a white solid product.

[0065] The structure was identified using Fourier transform infrared spectroscopy (FTIR). Figure 2 In the spectrum of the oxidation product OHA, compared with the raw material HA, the oxidation product OHA has a higher concentration at 1726 cm⁻¹. -1 A significant characteristic absorption peak appeared at the point, which is attributed to the C=O stretching vibration of the aldehyde group (-CHO), proving that the specific hydroxyl group on the HA molecular chain was successfully oxidized to the aldehyde group, and the OHA was successfully prepared.

[0066] Example 3: Preparation of hydrogel matrix CMCS-OHA

[0067] Weigh 0.3g of carboxymethyl chitosan (CMCS), dissolve it in 10mL of sterile PBS, and prepare a 3wt% CMCS solution.

[0068] Weigh 0.3g of oxidized hyaluronic acid (OHA), dissolve it in 10mL of sterile PBS, and prepare a 3wt% OHA solution.

[0069] Equal volumes of the above CMCS solution and OHA solution were mixed and stirred evenly to obtain a hydrogel precursor solution, which was then allowed to stand in a water bath at 37°C to form a gel, thus preparing a 3% CMCS-OHA hydrogel matrix.

[0070] After freeze-drying, the microstructure of the hydrogel matrix was observed using a scanning electron microscope as follows: Figure 3 It exhibits a typical porous network structure with a relatively uniform pore size distribution and clearly visible pore walls. It not only demonstrates good swelling behavior but also provides ample space for drug loading and controlled release.

[0071] Two circular hydrogel discs, each 20 mm in diameter and 3 mm thick, were prepared beforehand. One disc was stained blue with a small amount of methylene blue. Both hydrogel discs were cut in half lengthwise, and the cut surfaces were brought into close contact with each other and gently pressed to align them. The natural healing process was observed after incubation at room temperature. Figure 4 In the study, the fresh cross-sections refused into a whole after 1 hour of contact, and it was observed that the dye diffused across boundaries from the stained part to the unstained part. After 24 hours, the color diffusion was relatively uniform, proving that the dynamic imine bonds in the hydrogel network can spontaneously reform after being broken.

[0072] The hydrogel precursor solution was drawn into a syringe and injected into PBS solution or written directly on paper. The injection process was observed to be smooth, with continuous and stable lines being extruded and rapid in-situ gelation. This indicates that the material can quickly solidify and form at the target location after injection, possessing the key characteristics of an in-situ molding scaffold.

[0073] After the hydrogel matrix CMCS-OHA was freeze-dried to constant weight, it was placed in simulated uterine fluid (SUF) test groups with different pH values ​​(6.8, 7.2, 7.6) and incubated at 37°C. The swelling rate and degradation rate were determined by weighing.

[0074] Figure 5 In the in vitro swelling and degradation behavior of the hydrogels exhibited a clear pH dependence and time-dependent phases. Initially, the mass loss and swelling rate of the hydrogel in the pH=6.8 group were significantly higher than the other groups, indicating that the acidic environment strongly catalyzed the disintegration of the cross-linked network. With prolonged time, the degradation curve slope of the pH=7.2 group gradually increased, surpassing that of the pH=6.8 group in the later stages, reflecting its "continuous degradation" characteristic under mildly acidic conditions. In contrast, the degradation curve of the pH=7.6 group remained consistently flat, confirming the stability of imine bonds and the long-term structural retention of the gel in an alkaline environment. These results clearly demonstrate that by adjusting the environmental pH, the degradation mode of the hydrogel can be effectively controlled, thereby matching the release or support needs under different physiological states of the uterus.

[0075] The hydrogel matrix CMCS-OHA prepared under sterile conditions was such that the ratio of material surface area to extraction medium volume was ≥1.25 cm². 2 The standard was added to the complete culture medium at 37°C and 5% CO2 for 24 hours. The extract was filtered to obtain 100% original concentration extract, which was then serially diluted with the complete culture medium to obtain 75%, 50% and 25% extracts.

[0076] Human chorionic trophoblast cells (HTR-8 / Svneo) and human endometrial stromal cells (HESCs) were cultured at a density of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 96-well plates and pre-cultured for 24 hours. Different concentrations of extraction buffer were then added, and the cells were cultured for another 24 hours. Cell viability was assessed using the CCK-8 assay. Figure 6 The results showed that after treatment with extracts at concentrations of 25–100% for 24 hours, the survival rate of both cell types remained above 90%, indicating that the hydrogel extracts did not exhibit significant cytotoxicity to either type of uterine-related cell.

[0077] Example 4: Preparation of RSL3@Lip / CMCS-OHA composite delivery system

[0078] The RSL3 liposome nanoparticle suspension prepared in Example 1 was diluted with sterile PBS to a concentration of 2 μM to serve as the diluted liposome working solution.

[0079] Weigh 0.3g of carboxymethyl chitosan (CMCS) and add it to 10mL of the above diluted liposome working solution. Stir well to obtain a 3wt% CMCS precursor solution containing RSL3 liposomes.

[0080] Weigh 0.3g of oxidized hyaluronic acid (OHA), dissolve it in 10mL of sterile PBS, and prepare a 3wt% OHA solution.

[0081] Equal volumes of the above CMCS precursor solution and OHA solution were mixed and stirred evenly in a water bath at 37°C, and allowed to stand to form a gel to prepare the RSL3@Lip / CMCS-OHA composite delivery system, wherein the final concentration of RSL3 was 1 μM.

[0082] Example 5: Preparation of RSL3@Lip / CMCS-OHA composite delivery system

[0083] The RSL3 liposome nanoparticle suspension prepared in Example 1 was diluted with sterile PBS to a concentration of 2 μM to serve as the diluted liposome working solution.

[0084] Weigh 0.2 g of carboxymethyl chitosan (CMCS) and add it to 10 mL of the above diluted liposome working solution. Stir well to obtain a 2 wt% CMCS precursor solution containing RSL3 liposomes.

[0085] Weigh 0.2g of oxidized hyaluronic acid (OHA), dissolve it in 10mL of sterile PBS, and prepare a 2wt% OHA solution.

[0086] Equal volumes of the above CMCS precursor solution and OHA solution were mixed and stirred evenly in a water bath at 37°C, and allowed to stand to form a gel to prepare the RSL3@Lip / CMCS-OHA composite delivery system, wherein the final concentration of RSL3 was 1 μM.

[0087] Example 6: Preparation of RSL3@Lip / CMCS-OHA composite delivery system

[0088] The RSL3 liposome nanoparticle suspension prepared in Example 1 was diluted with sterile PBS to a concentration of 4 μM to serve as the working solution for the diluted liposomes.

[0089] Weigh 0.4 g of carboxymethyl chitosan (CMCS) and add it to 10 mL of the above diluted liposome working solution. Stir well to obtain a 4 wt% CMCS precursor solution containing RSL3 liposomes.

[0090] Weigh 0.4g of oxidized hyaluronic acid (OHA), dissolve it in 10mL of sterile PBS, and prepare a 4wt% OHA solution.

[0091] Equal volumes of the above CMCS precursor solution and OHA solution were mixed and stirred evenly in a 37°C water bath, and allowed to stand to form a gel, thus preparing the RSL3@Lip / CMCS-OHA composite delivery system, wherein the final concentration of RSL3 was 2 μM.

[0092] Comparative Example 1: Preparation of Lip / CMCS-OHA composite delivery system

[0093] Weigh DSPC (4.58 mg), cholesterol (1.35 mg), and DSPE-PEG2000 (1.96 mg) into a 100 mL round-bottom flask. Add 3 mL of chloroform / methanol (2:1 volume ratio) mixed solvent and gently shake to dissolve and form a clear, transparent organic phase solution. Connect the flask to a rotary evaporator and evaporate at 30 °C to remove the solvent until a uniform, translucent lipid film forms on the inner wall of the flask. Place the flask in a vacuum desiccator and continue drying for at least 2 hours to ensure that trace amounts of organic solvent are completely removed.

[0094] Add 5 mL of Tris-HCl buffer preheated to 65 °C to a flask, and incubate in a 65 °C water bath for 2 h to completely hydrate the lipid membrane, obtaining a crude liposome suspension. The liposome suspension is then sonicated and filtered sequentially through 0.45 μm and 0.22 μm syringe filters, repeated three times. The suspension is then transferred to an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa, centrifuged at 4000 × g, washed three times, and brought to a final volume of 10 mL to obtain a purified blank liposome nanoparticle suspension.

[0095] Take the above blank liposome nanoparticle suspension and dilute it with sterile PBS to the same volume multiple as in Example 4. Weigh 0.3 g of carboxymethyl chitosan (CMCS) and add it to 10 mL of the diluent. Stir well to obtain a 3 wt% CMCS precursor solution containing blank liposomes.

[0096] Equal volumes of the above-mentioned CMCS precursor solution and the OHA solution of Example 4 were mixed and stirred evenly in a water bath at 37°C, and allowed to stand to form a gel, thus preparing the Lip / CMCS-OHA composite delivery system.

[0097] Comparative Example 2: Preparation of RSL3 / CMCS-OHA drug-loaded hydrogel matrix

[0098] Take a 10mM RSL3 stock solution and dilute it to 2μM with sterile PBS.

[0099] Weigh 0.3g of carboxymethyl chitosan (CMCS) and add it to 10mL of the above diluted RSL3 solution. Stir well to obtain a 3wt% CMCS precursor solution containing free RSL3.

[0100] Weigh 0.3g of oxidized hyaluronic acid (OHA), dissolve it in 10mL of sterile PBS, and prepare a 3wt% OHA solution.

[0101] Equal volumes of the above CMCS precursor solution and OHA solution were mixed and stirred evenly in a 37°C water bath, and allowed to stand to form a gel, thus preparing the RSL3 / CMCS-OHA drug-loaded hydrogel matrix.

[0102] Example 7: Selective toxicity of RSL3 to trophoblast cells

[0103] To assess the cell-selective toxicity of the ferroptosis inducer RSL3, its effect on the viability of human chorionic trophoblast cells (HTR-8 / SVneo) and human endometrial stromal cells (HESCs) was detected using the CCK-8 assay.

[0104] HTR-8 / SVneo cells and HESCs cells were seeded into 96-well plates and treated with different concentrations of RSL3 (0, 0.01, 0.1, 1, 2, 5, 10 μM) for 24 h. Cell viability was detected by CCK-8 assay.

[0105] Figure 7 The results showed that RSL3 exhibited significant cytotoxicity against HTR-8 / SVneo cells, with an IC50 value of 1.5%. 50The optimal concentration was 0.1739 μM; when the concentration increased to 2 μM, cell viability was almost completely inhibited. However, RSL3 at the same concentration range did not cause significant toxicity to HESCs, and cell viability remained above 80%. These results indicate that endometrial stromal cells have strong tolerance to RSL3, suggesting that RSL3 can selectively kill trophoblast cells at appropriate concentrations without significantly affecting endometrial stromal cell viability, providing a basis for its application in intrauterine local administration.

[0106] To further clarify the toxic mechanism of RSL3, a rescue experiment was conducted using the ferroptosis-specific inhibitor Ferrostatin-1 (Fer-1). Cell viability was detected by CCK-8 assay after co-treatment with 1 μM Fer-1 and 2 μM RSL3. Figure 7 The results showed that Fer-1 could significantly reverse the decrease in cell viability induced by RSL3 in HTR-8 / SVneo and HESCs, confirming that the cell death induced by RSL3 in both cell types was mainly carried out through the ferroptosis pathway, indicating that although the two cell types had significant differences in sensitivity to RSL3, their lethal mechanisms both depended on ferroptosis.

[0107] Example 8: Evaluation of the in vivo contraceptive efficacy of the RSL3@Lip / CMCS-OHA combined delivery system

[0108] Healthy female SD rats aged 8–10 weeks were selected and sexually matured, and were housed together with male rats of the same strain aged 12–14 weeks at a ratio of 2:1. All animals were acclimatized for one week in an SPF environment (temperature 22±2℃, humidity 50±10%, 12h light-dark cycle) at the Animal Center of Shanxi Medical University.

[0109] The vaginas of female mice were observed approximately every 12 hours, both in the morning and evening, using two methods to aid in determining pregnancy:

[0110] Vaginal plug examination: Visually inspect the vaginal opening. A positive result shows a milky white or pale yellow gelatinous plug blocking the vaginal opening. A negative result shows a clear vaginal opening without any foreign objects.

[0111] Vaginal smear microscopy: A smear of vaginal secretions is taken and observed under an optical microscope. A positive result shows a large number of clearly morphologically distinct sperm with flagella, while a negative result shows only a small number of epithelial cells and white blood cells.

[0112] A positive result using any of the above methods confirms a successful pregnancy, and the date of detection is counted as day 0.5 of pregnancy (GD0.5). Those without a positive result continue to be observed in the same cage. Female mice that do not conceive after three consecutive days should rest for a few days before being replaced with a male mouse and re-entered into the same cage.

[0113] Female mice were randomly divided into the following groups: 1) Blank control group (no intervention, serving as a control for normal non-pregnant uterine morphology); 2) Blank liposome-hydrogel group (injected with the drug-free Lip / CMCS-OHA composite delivery system of Comparative Example 1 to evaluate the biocompatibility of the carrier material); 3) Free RSL3 hydrogel group (injected with the drug-loaded hydrogel matrix of RSL3 / CMCS-OHA of Comparative Example 2); 4) RSL3 liposome-hydrogel group (injected with the RSL3@Lip / CMCS-OHA composite delivery system of Example 4). The administration volume for each group was 30 μL.

[0114] Except for the blank control group, which did not receive pregnancy treatment, all other groups confirmed pregnancy.

[0115] The rat blastocyst begins implantation around day 4.5 of gestation, and the trophoblast cells then rapidly differentiate and invade the endometrium. The ferroptosis inducer RSL3 is designed to target these rapidly proliferating trophoblast cells. Local administration on day 4 of gestation allows the drug delivery system to be pre-positioned in the uterine cavity and form a sustained-release reservoir before the target cells become active, thus achieving a precise and efficient contraceptive effect at the onset of implantation.

[0116] On day 4 of gestation (GD4), rats were anesthetized with inhaled isoflurane and placed in a supine position with their heads lowered and tails elevated. A disposable, sterile, blunt-tipped, side-opening gavage syringe (0.5 mm outer diameter) was inserted through the vaginal opening and advanced along the reproductive tract to the left uterine horn. 30 μL of the corresponding formulation was slowly injected via vaginal administration. The contralateral (right) uterine horn was kept undisturbed throughout the process as a strict self-control to exclude individual differences and verify the local specificity of the effect.

[0117] At the end of the experiment on day 10 of gestation (GD10), rats in each group were first deeply anesthetized by intraperitoneal injection of sodium pentobarbital, and blood was collected through the abdominal aorta. The upper serum layer was separated for subsequent serum biochemical index detection. All rats were then euthanized by carbon dioxide inhalation. Bilateral uterine horns, bilateral ovaries, and major organs such as heart, liver, spleen, lungs, and kidneys were then harvested for observation. Some tissues were fixed with 4% paraformaldehyde for subsequent pathological evaluation.

[0118] Figure 8 The anatomical observation results of the uterus on day 10 of pregnancy are presented. The results in the figure show that both uterine horns in the blank liposome-hydrogel group showed typical pregnancy status, with full uterine cavity and orderly arranged normal gestational sacs.

[0119] Simultaneously, it can be seen that both the free RSL3 hydrogel group and the RSL3 liposome-hydrogel group achieved complete contraception of the left uterine horn, with no visible normal gestational sac in the uterine cavity. However, their effects on the local uterine health were drastically different. Although the left uterine horn in the free RSL3 hydrogel group lacked a gestational sac, it appeared dark purple and showed a significant loss of normal elasticity, indicating some local irritation or toxicity. In stark contrast, the left uterine horn in the RSL3 liposome-hydrogel group, while achieving complete contraception, exhibited the same size, color, appearance, and tissue elasticity as the normal uterine horn in the non-pregnant control group, demonstrating good biocompatibility.

[0120] Furthermore, it can be seen that all right uterine horns used as their own control groups developed normally, confirming the strict locality and specificity of the drug's effect.

[0121] The above results collectively demonstrate that the liposome-hydrogel delivery system can significantly improve local tissue adverse reactions caused by free drugs, and maintain the health of target organs to the greatest extent while ensuring contraceptive efficacy.

[0122] Example 9: Effects of the RSL3@Lip / CMCS-OHA composite delivery system on the expression of ferroptosis-related proteins in uterine tissue

[0123] The critical window period for RSL3-induced collapse of the intracellular antioxidant defense system and the accumulation of lipid peroxidation reaching detectable levels is 24–48 hours after intervention, which is conducive to capturing early and well-defined molecular events. Therefore, pregnant mice in each group were euthanized 48 hours after contraceptive intervention (day 6 of pregnancy, GD6), and the left uterine horn was dissected and removed, then placed in sterile, enzyme-free EP tubes and frozen at -80°C.

[0124] Frozen uterine tissue was harvested, and total protein was extracted using RIPA lysis buffer containing protease inhibitors. Protein quantification was performed using the BCA method. β-actin was used as an internal control. Expression levels of GPX4, ACSL4, HO-1, and Nrf2 were detected using conventional Western blot methods. Protein bands were obtained by ECL chemiluminescence imaging. ImageJ software was used for quantitative analysis of band grayscale values. Statistical analysis was performed to create bar charts. Results are shown below. Figure 9 As shown.

[0125] Western blot results showed that, compared with the blank control group and the blank liposome-hydrogel group, the RSL3 liposome-hydrogel group had significantly downregulated GPX4, significantly upregulated ACSL4, and significantly increased HO-1 and Nrf2.

[0126] It can be seen that the expression level of glutathione peroxidase 4 (GPX4), an important intracellular antioxidant enzyme and a core negative regulator of ferroptosis, was significantly downregulated in the experimental group. Simultaneously, the expression of acyl-CoA synthase long chain member 4 (ACSL4), a key enzyme responsible for catalyzing the esterification of polyunsaturated fatty acids and promoting lipid peroxidation, was significantly upregulated. This molecular combination of GPX4 inhibition and ACSL4 activation constitutes the classic pathway driving ferroptosis. As a response to oxidative stress, the expression of heme oxygenase-1 (HO-1) was also significantly increased in the experimental group. Furthermore, the expression of nuclear factor E2-related factor 2 (Nrf2), a key transcription factor regulating cellular antioxidant responses, was also significantly increased. This may be a compensatory defense response of cells in the face of strong oxidative stress induced by RSL3.

[0127] In summary, these parallel molecular changes—namely, the breakdown of the antioxidant defense system (GPX4) and the enhancement of lipid peroxidation driver (ACSL4), coupled with responses to oxidative stress markers (HO-1) and defense pathway activation (Nrf2)—form a complete chain of evidence that confirms at the protein level that the RSL3@Lip / CMCS-OHA complex delivery system can specifically activate the ferroptosis signaling pathway locally in the uterus. This provides a fundamental mechanistic explanation for its targeted contraceptive effect.

[0128] Example 10: Comprehensive Safety Evaluation of the RSL3@Lip / CMCS-OHA Composite Delivery System

[0129] 1. Serum liver and kidney function tests

[0130] Serum samples collected in Example 8 were analyzed using a fully automated biochemical analyzer to detect the levels of liver function-related enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and kidney function-related metabolites blood urea nitrogen (BUN) and creatinine (CREA). Specific results are as follows: Figure 10 .

[0131] There were no significant differences in serum ALT, AST, ALT / AST, ALP, BUN, and CREA levels among the blank control group, the blank liposome-hydrogel group, and the RSL3 liposome-hydrogel group. All levels were within the normal physiological reference range, indicating that neither the blank carrier nor the drug-loaded formulation caused damage to hepatocyte membrane integrity, mitochondrial damage, or bile excretion dysfunction, nor did it cause detectable damage to glomerular filtration function. This demonstrates that the RSL3@Lip / CMCS-OHA composite delivery did not cause any observable liver or kidney dysfunction at an effective dose, confirming the good in vivo safety of this formulation from a systemic metabolic perspective.

[0132] 2. Histopathological evaluation

[0133] Uterus, ovary, and major organs such as heart, liver, spleen, lung, and kidney of gestational rats collected on day 10 of pregnancy (GD10) in Example 8 were fixed in paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E. Their morphology and structure were then observed under a light microscope. The results are as follows: Figure 11 .

[0134] Figure 11 In (A), no significant toxicity was observed in any of the major organs of the blank control group, the blank liposome-hydrogel group, and the RSL3 liposome-hydrogel group. No significant differences were observed in the microstructure of the organs among the groups. Specifically, the myocardial fibers were neatly arranged with clear striations; the liver lobule structure was intact, with hepatocyte cords arranged radially, and no fatty degeneration or necrosis was observed; the red and white pulp boundaries of the spleen were clear; the alveolar structure was clear, without inflammatory exudate or consolidation; and the glomeruli and renal tubules had normal morphology.

[0135] Regarding ovarian tissue, the ovaries in the blank control group exhibited typical physiological characteristics of non-pregnancy, with follicles at different developmental stages, including primordial follicles, primary follicles, secondary follicles, and a small number of atretic follicles. The stromal structure was normal, and corpora lutea were rare, consistent with the non-pregnant state during the estrous cycle. The ovaries in the blank liposome-hydrogel group exhibited typical physiological characteristics of pregnancy, with well-developed, full, and abundant corpora lutea. Follicles at all stages had normal morphology, and no abnormal increase in follicular atresia or signs of corpus luteum insufficiency were observed. This indicates that the carrier material itself does not interfere with the ovarian endocrine function of the pregnant mother, and the normal maintenance of the embryo indirectly confirms the integrity of the ovarian progesterone secretion function. The left ovary structure in the RSL3 liposome-hydrogel group was completely consistent with that in the blank control group, with no pathological changes such as degenerative changes, inflammatory infiltration, or fibrosis. This confirms that even when ferroptosis is successfully induced locally to achieve a contraceptive effect, the preparation still has good ovarian safety and does not cause observable damage to the maternal reproductive endocrine reserves.

[0136] The above results consistently indicate that the RSL3@Lip / CMCS-OHA composite delivery system, when administered locally, did not cause significant systemic toxicity or ovarian dysfunction.

[0137] Figure 11(B) The safety of uterine tissue was assessed from two dimensions. First, tissue samples were taken from the uterine horn of the treated uterus on days 2, 4, and 6 after administration of RSL3@Lip / CMCS-OHA to a non-pregnant woman. H&E staining showed that the treated uterus maintained an intact tissue structure at all time points, with intact endometrial epithelium, tightly packed stromal cells, and normal glandular morphology. The smooth muscle cell bundles of the myometrium were arranged in an orderly manner, and no abnormal inflammatory cell infiltration, hemorrhage, or fibrosis was observed, indicating that the preparation did not cause persistent or progressive damage to the normal non-pregnant uterus, and its local effects had reversible potential. Second, in a pregnant rat model, the safety of the treated uterus (left side, where contraception was in place) was observed on day 10 of pregnancy. Macroscopic anatomical diagrams showed that although there was no gestational sac in the treated uterine horn, its size and color were not abnormally atrophied, congested, or adhered compared to the uterine horn of the blank control group. The corresponding H&E staining further confirmed that the decidualization reaction of the treated uterus had subsided, the endometrium was undergoing physiological repair, the myometrial structure was intact, and no necrosis or pathological scar formation was observed. The control uterus on the other side showed a normal mid-pregnancy morphology, with the embryo developing well.

[0138] The above results strongly demonstrate that the contraceptive effect achieved by the RSL3@Lip / CMCS-OHA combined delivery system has a high degree of spatial specificity. While effectively removing the pregnancy on the target side, it maximizes the protection of the tissue integrity of the uterine wall itself, providing key histological evidence for its safety as a reversible contraceptive strategy.

[0139] Example 11: Assessment of reproductive reversibility and offspring developmental safety of the RSL3@Lip / CMCS-OHA combined delivery system

[0140] To comprehensively evaluate whether the RSL3@Lip / CMCS-OHA combined delivery system has adverse effects on maternal fertility and offspring development while achieving targeted contraception, a reproductive reversibility experiment and neonatal fetal development index detection were designed and implemented. The re-fertility of the mice and the physiological and neurodevelopmental indicators of F1 and F2 generation neonatal fetuses were systematically detected.

[0141] Pregnant mice that achieved unilateral contraception in the left uterine horn through RSL3@Lip / CMCS-OHA intervention in Example 8 were selected and included in the contraception group. A normal control group was also established, consisting of healthy pregnant mice that conceived concurrently without any intervention.

[0142] Both groups of pregnant mice gave birth naturally on days 21-22 of gestation; this litter is recorded as the first litter (F1). After birth, all F1 pups were nursed normally by their mothers. On day 21 postpartum (weaning day), the mothers and pups were separated. The mothers rested in a quiet environment for 7 days to allow for the restoration of reproductive endocrine homeostasis and uterine physiological structure. After the rest period, the contraceptive group mothers and males were housed together at a 2:1 ratio for a second mating. Vaginal plugs were checked daily or vaginal smears were examined under a microscope to confirm pregnancy. Successfully pregnant mothers continued to be raised until natural delivery; these pups are recorded as the second litter (F2).

[0143] Assessment of reproductive reversibility in female mice: Figure 12 (A) Comparison of fetal mouse numbers shows that the number of F1 generation fetal mice in the contraceptive group was indeed reduced, approximately half that of the normal control group. After 7 days of weaning and rest, the mice were placed in cages with male mice, and all achieved successful secondary pregnancies, with a pregnancy rate consistent with the normal control group. The average litter size of the F2 generation fetal mice was not significantly different from that of the normal control group's F1 generation. This confirms that the reproductive organ structure and function of the female mice fully recovered after a single intrauterine drug administration, possessing normal ovulation, conception, and embryo implantation capabilities.

[0144] Physical development assessment of offspring: Body weight, body length, and tail length were measured in F1 and F2 generation newborn rats within 24 hours of birth. Results showed no statistically significant differences in any indicators between the contraceptive group F1 generation and the normal control group F1 generation; similarly, there were no significant differences between the contraceptive group F2 generation and its F1 generation. All measurements were within the normal physiological reference range for newborn SD rats of this strain. Figure 12 (B~D) indicates that transient intrauterine exposure to RSL3@Lip / CMCS-OHA did not cause persistent or transgenerational adverse effects on the physical development of fetal mice.

[0145] Neurobehavioral function assessment of offspring: Neurobehavioral function was assessed in newborn fetal mice on day 4 after birth, and the latency of righting reflex and negative geotaxis was measured. Results showed that the latency of righting reflex and negative geotaxis in all groups of newborn fetal mice was within the normal physiological range. Figure 12 (E-F). Successful completion of the righting reflex signifies the maturation of the sensorimotor integration pathway, while the negative geotactic reflex reflects the integrity of vestibular function and spatial orientation ability. The complete success of both sets of tests indicates that RSL3@Lip / CMCS-OHA did not produce detectable toxicity or developmental delay effects on the early development of the central nervous system in fetal rats.

[0146] In conclusion, the RSL3@Lip / CMCS-OHA combined delivery system does not affect the secondary fertility of female mice at effective doses, and has no adverse effects on the physical development and neurobehavioral function of F1 and F2 offspring, confirming that this contraceptive strategy has good reproductive reversibility and offspring development safety.

[0147] The above embodiments of the present invention do not describe all details exhaustively, nor do they limit the present invention to the embodiments described above. Various changes, modifications, substitutions, and variations made by those skilled in the art to these embodiments without departing from the principles and spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A liposome-hydrogel composite delivery system loaded with RSL3, comprising a hydrogel matrix and RSL3 liposome nanoparticles uniformly dispersed therein by physical embedding; The RSL3 liposome nanoparticles are formed by encapsulating the ferroptosis inducer RSL3 in a liposome carrier, wherein the liposome carrier is a lipid bilayer vesicle assembled from DSPC, cholesterol and DSPE-PEG2000. The hydrogel matrix is ​​a three-dimensional porous network structure formed by the cross-linking reaction of carboxymethyl chitosan and oxidized hyaluronic acid through a Schiff base.

2. The composite delivery system according to claim 1, characterized in that: The final concentration of RSL3 in the composite delivery system is 0.5–2 μM.

3. The method for preparing the RSL3-loaded liposome-hydrogel composite delivery system according to claim 1, characterized in that: Using a thin-film dispersion method, DSPC, cholesterol, DSPE-PEG2000, and RSL3 were dissolved in an organic solvent, rotary evaporated to form a film, and then hydrated with buffer solution to obtain an RSL3 liposome nanoparticle suspension. The RSL3 liposome nanoparticle suspension was then dispersed in a carboxymethyl chitosan solution, and an oxidized hyaluronic acid solution was added to perform a Schiff base crosslinking reaction to form a hydrogel matrix in which RSL3 liposome nanoparticles were uniformly dispersed by physical embedding.

4. The preparation method according to claim 3, characterized in that: The organic solvent is a chloroform-methanol mixture with a volume ratio of 2:

1.

5. The preparation method according to claim 3, characterized in that: Add Tris-HCl buffer and hydrate at 60-70°C for 1-3 hours.

6. The preparation method according to claim 3, characterized in that: It also includes purifying the RSL3 liposome nanoparticle suspension obtained after hydration by sonication, extrusion filtration, and centrifugation.

7. The preparation method according to claim 3, characterized in that the oxidized hyaluronic acid is prepared by reacting hyaluronic acid with sodium periodate under light-protected conditions and then quenching with ethylene glycol.

8. The preparation method according to claim 7, characterized in that: The light-protected reaction time is 4–8 hours.

9. The use of the RSL3-loaded liposome-hydrogel composite delivery system of claim 1 in the preparation of contraceptive drugs.

10. The application according to claim 9, characterized in that: The contraceptive drug is a non-hormonal, locally reversible contraceptive drug that is administered non-invasively through the vagina, inducing ferroptosis in trophoblast cells to achieve contraception.

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