RNA interference factor loaded temperature-sensitive biomimetic material for uterine cavity repair and preparation method of RNA interference factor loaded temperature-sensitive biomimetic material

Abstract

The invention relates to an RNA interference factor loaded temperature-sensitive biomimetic material for uterine cavity repair and a preparation method of the RNA interference factor loaded temperature-sensitive biomimetic material. The temperature-sensitive biomimetic material is composed of a temperature-sensitive hydrogel carrier and an RNA interference factor loaded on the temperature-sensitive hydrogel carrier, the temperature-sensitive hydrogel carrier takes poly (N-isopropylacrylamide) as a main chain, and the main chain, chitosan and sodium hyaluronate form a copolymerization network structure through free radical polymerization; the RNA interference factor is loaded in the temperature-sensitive hydrogel carrier through a nano-particle carrier, the nano-particle carrier is PLGA-CS (poly (lactic-co-glycolic acid)-CS nano-particles, and MALAT1-siRNA and / or miRNA-21 simulants are wrapped in the PLGA-CS nano-particles. According to the application, a temperature-sensitive gel material and an RNA interference technology are combined for the first time to construct a novel uterine cavity repairing carrier, and through the synergistic effect of the physical barrier characteristic (body temperature gelling to seal a wound surface) of the temperature-sensitive gel and the gene regulation function (targeted inhibition of a fibrosis pathway) of RNA interference, the postoperative adhesion recurrence risk of the uterine cavity is remarkably reduced, and the postoperative healing of the uterine cavity is promoted. And high-quality repair of endometrial structures and functions is promoted.

Description

Technical Field

[0001] The present application relates to the technical field of medical bionic materials, and in particular to an RNA interference factor-loaded temperature-sensitive bionic material for uterine cavity repair, a preparation method thereof, and a uterine cavity repair system constructed based on the material. Background Art

[0002] In reproductive medicine, repairing uterine cavity damage is a major clinical challenge. This is particularly true in conditions such as retained embryos and chronic endometritis. Patients often experience critical issues such as incomplete endometrial repair, intrauterine adhesions, and endometrial thinning, which can severely impact fertility. While various intervention strategies have been developed for repairing uterine cavity damage, their efficacy remains limited by factors such as material properties, mechanism of action, and delivery efficiency. The specific status and limitations are as follows:

[0003] 1. Limitations of existing uterine cavity repair technologies

[0004] Intrauterine adhesion (IUA) is a major cause of female infertility. Its clinical treatment mainly includes mechanical barrier therapy, drug therapy, and biomaterial repair. However, all of them have significant shortcomings:

[0005] Mechanical barrier method (such as balloon stent): reduces the formation of intrauterine adhesions through physical barriers, but its operation is traumatic and may induce secondary infection; and it is easy to cause secondary damage to the new endometrium during the stent removal process, affecting the repair effect.

[0006] Drug treatment (such as estrogen therapy): Repair is achieved by promoting endometrial proliferation, but the sensitivity of different individuals to estrogen varies significantly. Long-term use may also have potential risks such as inducing abnormal endometrial hyperplasia.

[0007] Biomaterial repair (such as gelatin, hyaluronic acid, etc.): It promotes tissue regeneration by providing physical support, but its degradation rate is too fast (usually only a few days to a few weeks), making it difficult to maintain long-term microenvironment stability and unable to meet the continuous action cycle required for endometrial repair.

[0008] 2. Application of Thermosensitive Materials in Uterine Cavity Repair and Existing Problems

[0009] Thermosensitive polymers are temperature-responsive and can undergo a sol-gel phase transition at body temperature (approximately 37°C), making them potential carriers for localized drug delivery within the uterine cavity. Among them, poly (N-isopropylacrylamide) (PNIPAM), due to its low critical solution temperature (LCST ≈ 32°C) close to physiological temperature, can rapidly form a gel and load drugs within the uterine cavity, and has been explored for localized sustained drug release.

[0010] However, the application of existing PNIPAM-based materials in uterine cavity repair still has the following key drawbacks:

[0011] (1) Insufficient biocompatibility: PNIPAM itself lacks biological activity and easily induces mild inflammatory responses in the body, limiting the long-term retention safety of the material.

[0012] (2) Lack of anti-fibrosis function: The core pathological mechanism of intrauterine adhesions involves the abnormal activation of the transforming growth factor-β1 / Smad3 (TGF-β1 / Smad3) signaling pathway, while existing PNIPAM materials only serve as physical barriers and cannot actively regulate this pathway to inhibit the fibrosis process.

[0013] (3) Poor drug delivery stability: Although RNA interference factors (such as siRNA and miRNA) can target and regulate key factors such as TGF-β1, they are easily degraded by intrauterine nucleases and lack effective sustained-release carriers, resulting in insufficient local concentration and short duration of action, making it difficult to achieve a sustained gene silencing effect.

[0014] In summary, existing technologies in uterine cavity repair face core problems such as insufficient biocompatibility, lack of anti-fibrosis function and unstable drug delivery. There is an urgent need to develop a new repair material that has good biocompatibility, targeted anti-fibrosis function and stable drug delivery capability. Summary of the Invention

[0015] In order to address the above problems, the present application proposes a new RNA interference factor-loaded thermosensitive bionic material for uterine cavity repair.

[0016] To address the core issues of existing uterine cavity repair materials, such as insufficient biocompatibility, lack of anti-fibrosis function, and unstable drug delivery, the present invention proposes the following technical strategies to achieve efficient and targeted uterine cavity injury repair through multi-dimensional collaborative design:

[0017] 1. Material copolymerization modification: improving biocompatibility and controllable degradation

[0018] A new composite hydrogel material was constructed by copolymerizing the thermosensitive polymer PNIPAM with chitosan (CS) and hyaluronic acid (HA). Chitosan (CS), as a natural cationic polysaccharide, has good biocompatibility and cell adhesion, which can reduce the risk of inflammatory response to PNIPAM. Hyaluronic acid (HA), as a natural component of the extracellular matrix, is rich in viscoelastic functional groups and can regulate the hydrophilicity of the material and its compatibility with tissues. The copolymerization of the two with PNIPAM not only retains the thermosensitive gel properties of PNIPAM (LCST≈32°C), but also significantly improves the biocompatibility of the material by introducing bioactive groups (such as the amino group of CS and the carboxyl group of HA). At the same time, by regulating the copolymerization ratio and molecular weight, the degradation rate of the material is optimized to match the physiological cycle of endometrial repair, avoiding microenvironmental instability caused by excessive degradation or foreign body reactions caused by excessive degradation.

[0019] 2. Construction of a Thermosensitive Controlled Release System: Achieving Uterine Injury Sealing and Slow Release of RNA Interference Factors

[0020] Based on the above-mentioned copolymer-modified material, a thermosensitive controlled-release system was further constructed. This system is in a sol state at room temperature (<32°C) and can be minimally invasively injected into the injured area through a hysteroscope or catheter. Upon exposure to body temperature (≈37°C), the material rapidly undergoes a sol-gel phase transition, forming a semisolid gel network that tightly adheres to the uterine cavity wound, acting as a physical seal and reducing the invasion of external sources of infection and the exudation of tissue fluid. Furthermore, the three-dimensional porous structure of the gel network can load RNA interference factors (such as siRNA and miRNA mimics) and achieve the encapsulation and sustained release of target molecules through intermolecular hydrogen bonds and electrostatic interactions. Compared with traditional carriers, this thermosensitive gel can continuously release RNA interference factors at the injured site, preventing their rapid degradation in the complex environment of the uterine cavity (such as nucleases and mucus), ensuring stable local drug concentration and prolonging the duration of action.

[0021] 3. Targeted gene regulation: precisely inhibiting fibrosis and promoting intimal regeneration

[0022] Targeting the abnormal activation of the TGF-β1 / Smad3 signaling pathway, the core pathological mechanism of intrauterine adhesions, the present invention uses MALAT1-siRNA (small interfering RNA for long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1) and miRNA-21 mimics as key functional molecules. MALAT1-siRNA silences MALAT1 gene expression, inhibiting TGF-β1 transcriptional activation and downstream Smad3 phosphorylation, thereby blocking the overexpression of fibrosis-related genes (such as α-SMA and collagen I / III); miRNA-21 mimics target and bind to PTEN mRNA (a negative regulator of the TGF-β1 / Smad3 pathway), upregulating PTEN expression and inversely inhibiting Smad3 phosphorylation and downstream signaling. The synergistic effect of the two can significantly inhibit the progression of endometrial fibrosis. At the same time, by regulating the balance between fibroblasts and epithelial cells, they promote the proliferation and differentiation of endometrial stem cells, ultimately achieving structural and functional regeneration of the damaged endometrium.

[0023] In summary, the present invention systematically solves the key bottlenecks of existing uterine cavity repair technology through the triple strategy of optimizing carrier performance through material copolymerization modification, ensuring drug delivery through a temperature-sensitive controlled release system, and intervening in pathological mechanisms through targeted gene regulation, providing a new, efficient, safe, and precise solution for the repair of uterine cavity injuries.

[0024] In order to achieve the above objectives, this application provides the following technical solutions:

[0025] The present application provides an RNA interference factor-loaded thermosensitive biomimetic material for uterine cavity repair, wherein the thermosensitive biomimetic material is composed of a thermosensitive hydrogel carrier and an RNA interference factor loaded thereon, wherein:

[0026] The thermosensitive hydrogel carrier uses poly N-isopropylacrylamide (PNIPAM) as the main chain and forms a copolymer network structure with chitosan (CS) and sodium hyaluronate (HA) through free radical polymerization;

[0027] The RNA interference factor is loaded into the thermosensitive hydrogel carrier via a nanoparticle carrier, wherein the nanoparticle carrier is PLGA-CS nanoparticles encapsulated with MALAT1-siRNA and / or miRNA-21 mimics.

[0028] Furthermore, the thermosensitive bionic material of the present application forms a three-dimensional network structure by triggering thermosensitive gelation through body temperature, thereby sealing the uterine cavity wound and locally releasing RNA interference factors for repairing uterine cavity injuries.

[0029] Furthermore, in the thermosensitive bionic material of the present application, the preparation method of the thermosensitive hydrogel carrier is as follows:

[0030] (1) Dissolve chitosan and sodium hyaluronate in deionized water and adjust the pH to neutral;

[0031] (2) adding N-isopropylacrylamide monomer and cross-linking agent N,N'-methylenebisacrylamide (MBAA);

[0032] (3) After nitrogen is passed through to remove oxygen, the initiator ammonium persulfate (APS) is added;

[0033] (4) React at 4°C for 12 hours;

[0034] (5) After dialysis to remove impurities, PNIPAM-CS-HA dry powder was obtained by freeze-drying;

[0035] (6) Before use, PNIPAM-CS-HA dry powder was added to PBS buffer to re-dissolve and prepare liquid hydrogel.

[0036] Furthermore, in the thermosensitive biomimetic material of the present application, the mass ratio of PNIPAM, CS and HA in the thermosensitive hydrogel carrier is (50-80):(10-30):(5-20).

[0037] Furthermore, in the thermosensitive bionic material of the present application, the RNA interference factor is loaded as follows:

[0038] (1) PLGA-CS nanoparticles were prepared using a double emulsification-solvent evaporation method. A buffer solution (citrate buffer, pH = 4.0) containing MALAT1-siRNA and / or miRNA-21 mimics was used as the internal aqueous phase, PLGA was dissolved in an organic solvent (dichloromethane or ethyl acetate) as the oil phase, 1-2% surfactant was added, and colostrum was formed by ultrasonic emulsification.

[0039] (2) Injecting colostrum into an external aqueous phase containing 1-3% polyvinyl alcohol (PVA) for secondary emulsification to form a double emulsion;

[0040] (3) Add chitosan solution (dissolved in dilute acetic acid, pH = 5.0) to the double emulsion and stir for 4-6 hours to allow chitosan to deposit on the PLGA surface through electrostatic adsorption;

[0041] (4) RNA interference factor nanoparticles were prepared after centrifugal purification and freeze-drying;

[0042] (5) The prepared RNA interference factor nanoparticles are evenly dispersed in the PNIPAM-CS-HA hydrogel carrier to prepare an injectable thermosensitive gel.

[0043] Furthermore, in the thermosensitive biomimetic material of the present application, the particle size of the RNA interference factor nanoparticles is 150-250 nm, the Zeta potential is +20 to +35 mV, the encapsulation efficiency is >80%, and the drug loading is 2-5% w / w.

[0044] Furthermore, in the thermosensitive biomimetic material of the present application, the mass-to-volume ratio of the RNA interference factor nanoparticles to the PNIPAM-CS-HA hydrogel carrier is (1-5 mg): (1 mL), and the RNA interference factor nanoparticles are evenly dispersed in the PNIPAM-CS-HA hydrogel carrier pre-cooled at 4°C, mixed evenly by gentle magnetic stirring at 4°C for 30 minutes, and stored at low temperature in the dark.

[0045] Furthermore, in the thermosensitive bionic material of the present application, the LCST (lower critical solution temperature) of the thermosensitive hydrogel carrier is 30-34°C, and the sol-gel transition is completed within 3-5 minutes at body temperature (37°C), forming a three-dimensional network structure that fits the endometrial wound surface, and the adhesion strength is >20 kPa.

[0046] Furthermore, in the thermosensitive biomimetic material of the present application, the RNA interference factor is released through a dual controlled release mechanism: PLGA-CS nanoparticles are hydrolyzed to slowly release siRNA / miRNA, while the three-dimensional network structure of the thermosensitive hydrogel carrier delays the diffusion of PLGA-CS nanoparticles, achieving local long-term sustained release with an effective period of ≥7 days.

[0047] In addition, the present application also relates to the use of the above-mentioned thermosensitive bionic material in the preparation of a uterine cavity repair preparation, wherein the thermosensitive bionic material realizes the following functions in the uterine cavity repair preparation:

[0048] (1) Physically seal the uterine cavity wound to reduce infection and tissue fluid exudation;

[0049] (2) sustained release of MALAT1-siRNA and / or miRNA-21 mimics to target and inhibit the TGF-β1 / Smad3 signaling pathway, reducing fibroblast hyperproliferation and collagen deposition;

[0050] (3) Promote the proliferation and differentiation of endometrial stem cells and accelerate the regeneration of endometrial structure and function.

[0051] In summary, this application makes the following innovations and improvements to the prior art:

[0052] Pioneering combined application: For the first time, thermosensitive gel material is combined with RNA interference technology to construct a new carrier for uterine cavity repair, breaking through the technical limitations of traditional single material or single gene intervention.

[0053] Dual action mechanism: Through the synergistic effect of the physical barrier properties of the thermosensitive gel (gelling at body temperature to seal the wound) and the gene regulatory function of RNA interference (targeted inhibition of fibrosis pathways), the risk of postoperative adhesion recurrence is significantly reduced.

[0054] Precise temporal and spatial intervention: Based on the characteristics of the temperature-sensitive controlled release system, the drug can achieve "time-controlled release" (body temperature-triggered gelation-sustained release) and "spatial targeted enrichment" (local high concentration maintenance) at the site of uterine cavity injury, greatly improving treatment efficiency.

[0055] Therefore, compared with existing thermosensitive materials, the RNA interference factor-loaded thermosensitive bionic material for uterine cavity repair of the present invention has the following advantages:

[0056] (1) Significant therapeutic effect: It can effectively reduce the formation of postoperative adhesions in the uterine cavity and promote the high-quality repair of the endometrial structure and function.

[0057] (2) Excellent safety: It can replace traditional balloons, hyaluronic acid gels and hormone therapies, reducing the risks of foreign body reactions and hormone side effects.

[0058] (3) Strong translational potential: The technical solution is feasible and has good prospects for translation into clinical applications, as verified by in vitro experiments and animal models.

[0059] Other features and advantages of this application will be described in detail in the following description, or will be understood through the implementation of the relevant technical solutions of this application. The objectives and other advantages of this application can be achieved through the technical features and technical means clearly indicated in the description, claims, and drawings, and obtained through the implementation of these technical contents. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] To more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly describes the drawings used in describing the embodiments. It should be noted that the drawings illustrate only some of the embodiments of the present application. Those skilled in the art can deduce other relevant drawings based on these drawings without inventive effort.

[0061] Figure 1 This is a flow chart of the preparation method of RNA interference factor-loaded thermosensitive bionic material used for uterine cavity repair in this application. DETAILED DESCRIPTION

[0062] To make the objectives, technical solutions, and advantages of the embodiments of this application more clearly understood, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the accompanying drawings. It should be understood that the described embodiments are only some of the embodiments of this application, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of this application without inventive effort are intended to fall within the scope of protection of this application.

[0063] In this document, the term "including" and any variations thereof (such as "including," "comprising," etc.) are open-ended expressions and should be understood as meaning "including but not limited to," meaning that the listed contents are not exhaustive and may include other contents not explicitly mentioned. The term "based on" should be understood as meaning "based, at least in part," meaning that the basis or condition referred to may not be the only factor and may also involve other relevant factors. The term "one embodiment" should be understood as meaning "at least one embodiment," meaning that the described embodiment is not the only possible implementation method and that other similar embodiments may exist.

[0064] In this application, the terms "a" and "a plurality" are used to modify related elements or features in an illustrative, non-restrictive manner. Unless the context clearly indicates otherwise, "a" should be understood as meaning "at least one," and "a plurality" should be understood as meaning "at least two." Those skilled in the art should interpret these terms appropriately based on the semantics and logical relationships of the context to ensure that they encompass the possibility of "one or more."

[0065] Figure 1 The following is a preparation process of the RNA interference factor-loaded thermosensitive biomimetic material for uterine cavity repair provided by the present application, comprising the following steps:

[0066] S1. Preparation of PNIPAM-CS-HA thermosensitive hydrogel carrier: Dissolve CS and HA in deionized water and adjust the pH to neutral. Add NIPAM monomer and crosslinker MBAA, deoxygenate with nitrogen, and then add initiator APS. React at 4°C for 12 hours, then dialyze and lyophilize to obtain PNIPAM-CS-HA dry powder. Before use, reconstitute the PNIPAM-CS-HA dry powder in PBS buffer to prepare a liquid hydrogel.

[0067] S2. Preparation of RNAi factor-loaded PLGA-CS nanoparticles: MALAT1-siRNA and / or miRNA-21 mimics were encapsulated in PLGA-CS nanoparticles using a double emulsion-solvent evaporation method. After surface modification with CS, the particles were centrifuged and lyophilized to produce RNAi factor nanoparticles.

[0068] S3. Disperse RNA interference factor nanoparticles in a PNIPAM-CS-HA hydrogel carrier precooled at 4°C and stir at 4°C for 30 minutes to prepare an injectable thermosensitive gel.

[0069] In order to clearly illustrate the technical solution of the present application, the following will be described in detail through embodiments of specific scenarios.

[0070] An RNA interference factor-loaded thermosensitive biomimetic material for uterine cavity repair

[0071] (1) Composition and preparation of thermosensitive bionic materials

[0072] 1. Construction of thermosensitive hydrogel carrier: PNIPAM is used as the main chain, and CS and HA are added to form a copolymer network through free radical polymerization.

[0073] Main ingredients:

[0074] N-isopropylacrylamide (NIPAM)

[0075] Chitosan (CS)

[0076] Sodium hyaluronate (HA)

[0077] Initiator: ammonium persulfate (APS)

[0078] Cross-linking agent: N,N'-methylenebisacrylamide (MBAA)

[0079] Synthesis method (free radical polymerization)

[0080] (1) Dissolve CS and HA in deionized water and adjust the pH to neutral.

[0081] Dissolve CS (chitosan) and HA (sodium hyaluronate) in deionized water. Because CS is a naturally occurring polysaccharide and is difficult to dissolve, it can be dissolved in slightly acidic conditions (such as dilute acetic acid). The pH is then adjusted to neutral (pH 6.8–7.2) to facilitate the subsequent polymerization reaction. This step creates a uniform polysaccharide solution while preventing degradation caused by high pH.

[0082] (2) Add NIPAM monomer and crosslinker MBAA

[0083] NIPAM monomer is added to the above solution. MBAA is used to create a crosslinked structure, giving the polymer a three-dimensional network that is insoluble in water but swells upon absorption. MBAA contains two double bonds and copolymerizes with NIPAM during polymerization to form a crosslinked structure.

[0084] (3) Nitrogen is passed to remove oxygen and APS is added to start the polymerization reaction

[0085] Nitrogen (or argon) is introduced into the reaction system to remove oxygen from the solution. Oxygen will capture free radicals and inhibit free radical polymerization.

[0086] (4) React at 4°C for 12 hours

[0087] Adding APS (ammonium persulfate) as an initiator generates free radicals at low temperatures, initiating the free radical polymerization of NIPAM, CS, and HA. A low temperature reaction (4°C) for 12 hours can control the polymerization rate, reduce side reactions, improve polymerization uniformity, and enhance the controllability of the molecular structure.

[0088] (5) Dialysis to remove impurities and freeze-dry to obtain PNIPAM-CS-HA dry powder

[0089] After the polymerization reaction, unreacted small molecule impurities (monomers and initiator residues) are removed through a dialysis bag (e.g., MWCO 8,000–14,000). Lyophilization is then performed to obtain a dry, porous PNIPAM-CS-HA powder.

[0090] (6) Add PBS buffer to dissolve before use to make an injectable liquid hydrogel

[0091] Before use, PNIPAM-CS-HA dry powder is added to PBS buffer to re-dissolve to form a liquid hydrogel with good fluidity; it is liquid at room temperature and quickly gels after injection into the body (body temperature 37°C), playing the role of local film formation, sustained drug release, and promoting tissue repair.

[0092] 2. Construction of RNA interference factor loading and controlled release system: MALAT1-siRNA (hereinafter referred to as siRNA) and miRNA-21 mimic (hereinafter referred to as miRNA) were encapsulated using PLGA-CS nanoparticle carriers. The nanoparticles were then evenly dispersed in the PNIPAM-CS-HA hydrogel prepared in the previous step to form an injectable thermosensitive gel, thereby constructing a dual delivery system with the following double-layer structure:

[0093] Nanocarrier layer: PLGA-CS nanoparticles encapsulate nucleic acids (siRNA / miRNA), protecting them from degradation and enhancing cellular uptake.

[0094] Hydrogel layer: Thermosensitive PNIPAM-CS-HA hydrogel acts as a reservoir for nanoparticles, providing in situ injection capability and sustained release, enabling localized and long-lasting gene regulation.

[0095] Main ingredients:

[0096] PLGA-CS nanoparticle carrier

[0097] RNA interference factors: MALAT1-siRNA and miRNA-21 mimics

[0098] 1) MALAT1-siRNA (human)

[0099] Human MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) is a key lncRNA regulating the fibrosis process. The following siRNA sequences have been verified to have significant silencing effects:

[0100] (a) MALAT1-siRNA-1

[0101] Sense strand: 5′-GGAUGAGGAGGAUAG UAAATT-3′ (SEQ ID NO: 1);

[0102] Antisense strand: 5'-UUU ACU AUC CUC CUC AUC CTT-3' (SEQ ID NO: 2);

[0103] (b) MALAT1-siRNA-2

[0104] Sense strand: 5′-GCAUUG AGA UGA GGAAUA UUTT-3′ (SEQ ID NO: 3);

[0105] Antisense strand: 5′-AAU AUA UUC CUC AUC UCAAUG CTT-3′ (SEQ ID NO: 4);

[0106] 2) miR-21 mimics

[0107] miRNA-21 is believed to have anti-fibrotic effects and can be used in combination with MALAT1-siRNA. Sense strand: 5'-UAGCUU AUC AGACUG AUG UUG A-3' (SEQ ID NO: 5);

[0108] Antisense strand: 5′-CAACAU CAG UCU GAU AAG CUA U-3′ (SEQ ID NO: 6).

[0109] 3) RNA interference factor delivery structure

[0110] (a) Loading method: using polycations such as PEI or CS to form siRNA / miRNA nanoparticles;

[0111] (b) Encapsulation form: Encapsulated in the PNIPAM-CS-HA three-dimensional network, it is liquid at room temperature and gels to form a local controlled release structure at body temperature.

[0112] Preparation method:

[0113] (1) Preparation of PLGA-CS nanoparticles using double emulsion-solvent evaporation method

[0114] (a) Formulation:

[0115] Inner aqueous phase (W1): siRNA / miRNA-containing buffer (e.g., citrate buffer, pH = 4.0, to prevent nucleic acid degradation);

[0116] Oil phase (O): PLGA dissolved in organic solvent (dichloromethane (DCM) or ethyl acetate (EA);

[0117] Surfactant: Add 1-2% span-80 (lipophilic surfactant).

[0118] (b) Operation:

[0119] 1) Slowly inject the W1 phase into the O phase;

[0120] 2) Ultrasonic emulsification (under ice bath conditions): 100-200W short-time pulse (5-10 seconds) to form W1 / O colostrum.

[0121] Key point: Ultrasonic time / power control to avoid nucleic acid shearing and inactivation.

[0122] (2) Introducing siRNA during encapsulation (W / O / W structure)

[0123] (a) Formulation:

[0124] External aqueous phase (W2): aqueous solution containing stabilizer PVA (polyvinyl alcohol, 1-3% w / v).

[0125] (b) Operation:

[0126] 1) Inject W1 / O colostrum into W2 phase;

[0127] 2) Secondary emulsification: High-speed homogenization (8,000-12,000 rpm, 1-2 minutes) or probe sonication to form a W1 / O / W2 double emulsion.

[0128] Key Points:

[0129] siRNA / miRNA is encapsulated in the aqueous cavity of the PLGA core (avoiding direct contact with organic solvents);

[0130] PVA stabilizes the oil-water interface and prevents particle aggregation.

[0131] (3) Surface modification of chitosan to improve hydrophilicity and adhesion

[0132] Neutralize the negative charge on the PLGA surface, enhance hydrophilicity, cell adhesion and lysosomal escape capabilities.

[0133] (a) Cationic chitosan solution (dissolved in dilute acetic acid, pH = 5.0) was added to the W2 phase.

[0134] (b) CS was deposited on the PLGA surface by electrostatic adsorption after magnetic stirring for 4–6 h.

[0135] (c) Mechanism of action:

[0136] CS-NH3 + -COO of PLGA - Binds to form a positively charged shell → Enhances interaction with negatively charged cell membranes;

[0137] The mucin-adsorbing properties of CS → prolong the retention time in the endometrium of the uterine cavity.

[0138] (4) Centrifugation, freeze-drying, and resuspension to prepare siRNA / miRNA nanoparticles

[0139] (a) The solvent is evaporated and stirred for 12-24 hours to allow the organic solvent to evaporate and the PLGA to solidify to form solid nanoparticles.

[0140] (b) Purification by centrifugation: Free PVA and unencapsulated nucleic acids were removed by ultracentrifugation (20,000 g, 30 min), and the cells were washed three times with deionized water.

[0141] (c) freeze-dried for storage, adding a freeze-protectant (such as 5% mannitol), pre-freezing and then vacuum freeze-drying to obtain white nanoparticle powder.

[0142] (d) Resuspend the lyophilized powder in sterile PBS or deionized water and disperse it by ultrasonication.

[0143] Table 1 Quality control parameters

[0144] parameter Target value Detection method Particle size 150-250 nm Dynamic Light Scattering (DLS) Zeta potential +20 to +35 mV (after CS modification) Zeta Potential Instrument Encapsulation efficiency >80％ Ultrafiltration centrifugation-HPLC detection Drug loading 2-5% w / w Nucleic acid quantification kit

[0145] (5) Evenly mix the siRNA / miRNA nanoparticles into the PNIPAM-CS-HA gel and store at low temperature for later use

[0146] (a) Preparation of hydrogel precursor: freeze-dried PNIPAM-CS-HA powder was reconstituted with 4° C. pre-cooled PBS to form an injectable sol (concentration of 10-15% w / v).

[0147] (b) Nanoparticle dispersion: PLGA-CS / siRNA / miRNA lyophilized powder was added to the hydrogel precursor at the target ratio (e.g., 1-5 mg NPs / mL gel) and stirred gently with magnetic stirring at 4°C for 30 min to ensure uniform dispersion of the nanoparticles and avoid aggregation.

[0148] (c) Storage conditions: Store in the dark and at low temperature (4°C) to prevent nucleic acid degradation and hydrogel pregelation.

[0149] (6) Control mechanisms and advantages

[0150] Table 2 Description of controlled release mechanism

[0151]

[0152] (2) Application methods and operation procedures of thermosensitive bionic materials

[0153] 1. Application method: Inject the liquid hydrogel thermosensitive bionic material into the damaged area of ​​the uterine cavity through the hysteroscopic catheter.

[0154] Advantages: Precise targeting, with injection under direct hysteroscopic guidance, ensures complete coverage of the gel over damaged areas of the endometrium (such as the anterior and posterior uterine walls, uterine cornu, and other areas prone to adhesions). Supporting evidence: Clinical studies have shown that hysteroscopically guided biomaterial injection can achieve drug coverage exceeding 95% (*Fertil Steril. 2018*), significantly superior to traditional intrauterine balloon placement (coverage <70%).

[0155] 2. Gelation process: At body temperature (37°C), the PNIPAM structure undergoes a phase transition, and the hydrogel forms a three-dimensional network structure, which solidifies rapidly and firmly adheres to the endothelial wound surface.

[0156] (1) Gelation time: Sol-gel transition is completed within 3-5 minutes (Biomacromolecules 2020).

[0157] (2) Adhesion strength: The interfacial adhesion between the gel and the moist endometrium is >20 kPa (10-15 kPa higher than the uterine contraction pressure), which resists uterine contraction and shedding.

[0158] (3) Tissue compatibility evidence: Rabbit uterine cavity model experiments showed that there was no acute inflammatory response after PNIPAM-CS-HA gel implantation, and the macrophage infiltration level was <5% within 7 days (the collagen membrane in the control group was >30%).

[0159] 3. RNA sustained release process: Over time, RNA interference factors are slowly released from the nanoparticles, and the effective period can reach 7 to 10 days.

[0160] 4. Uterine cavity barrier function: forming the triple functions of "drug-barrier-stent", preventing uterine cavity adhesion again and promoting endometrial regeneration.

[0161] (3) In vitro experimental verification

[0162] Cell models: human endometrial fibroblasts (hEMFs) and human decidual mesenchymal stem cells (hDMSCs).

[0163] Experimental content and methods:

[0164] 1. MTT / CCK-8 assay: evaluate the effect of gel on cell proliferation activity;

[0165] 2. Transwell assay: detect cell migration / invasion ability and evaluate healing promotion effect;

[0166] 3. qRT-PCR: Detect the expression of key mRNAs such as MALAT1, TGF-β1, COL1A1, and α-SMA;

[0167] 4. Western blot: detect the expression changes of TGF-β1 / Smad3 pathway-related proteins;

[0168] 5. ELISA: Detect the secretion levels of fibrosis factors such as TGF-β1 and VEGF.

[0169] (IV) Animal Experiment Verification

[0170] Model establishment: IUA model mice (mechanical injury + lipopolysaccharide injection)

[0171] Group treatment: blank group, PNIPAM group, RNAi group, combined group

[0172] Treatment period: 7 to 14 days

[0173] Detection indicators:

[0174] HE staining: observe the thickness of the endometrium and the number of glands;

[0175] Masson staining: assesses the degree of fibrosis (blue collagen deposition);

[0176] Immunohistochemistry: Detect the expression of Ki67 (proliferation), α-SMA (fibrosis), and CD31 (angiogenesis).

[0177] Although the above discussion contains several specific implementation details, these details should not be construed as limiting the scope of this application. The above description is merely a preferred embodiment of this application and an illustration of the technical principles employed. Those skilled in the art should understand that the scope of disclosure involved in this application is not limited to the technical solutions formed by the specific combination of the above technical features. At the same time, this application should also cover other technical solutions formed by any combination of the above technical features or their equivalents, without departing from the above disclosed concepts.

[0178] Those skilled in the art should also understand that they may modify the technical solutions described in the aforementioned embodiments, or replace some of the technical features therein with equivalents, without departing from the spirit and scope of the technical solutions of the embodiments of the present application. Such modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the core spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An RNA interference factor-loaded thermosensitive bionic material for uterine cavity repair, characterized in that: The thermosensitive bionic material is composed of a thermosensitive hydrogel carrier and an RNA interference factor loaded thereon, wherein: The thermosensitive hydrogel carrier has poly N-isopropylacrylamide as the main chain, and forms a copolymer network structure with chitosan and sodium hyaluronate through free radical polymerization; The RNA interference factor is loaded into the thermosensitive hydrogel carrier via a nanoparticle carrier, wherein the nanoparticle carrier is PLGA-CS nanoparticles encapsulated with MALAT1-siRNA and / or miRNA-21 mimics.

2. The thermosensitive bionic material according to claim 1, characterized in that: The thermosensitive bionic material forms a three-dimensional network structure by triggering thermosensitive gelation through body temperature, thereby sealing the uterine cavity wound surface and locally releasing RNA interference factors for repairing uterine cavity injuries.

3. The thermosensitive bionic material according to claim 1, characterized in that The preparation method of the thermosensitive hydrogel carrier is as follows: (1) Dissolve chitosan and sodium hyaluronate in deionized water and adjust the pH to neutral; (2) adding N-isopropylacrylamide monomer and cross-linking agent N,N'-methylenebisacrylamide; (3) After nitrogen is passed through to remove oxygen, the initiator ammonium persulfate is added; (4) react at 4°C for 12 hours; (5) After dialysis to remove impurities, PNIPAM-CS-HA dry powder was obtained by freeze-drying; (6) Before use, PNIPAM-CS-HA dry powder was added to PBS buffer to re-dissolve and prepare liquid hydrogel.

4. The thermosensitive bionic material according to claim 3, characterized in that: The mass ratio of PNIPAM, CS and HA in the thermosensitive hydrogel carrier is (50-80):(10-30):(5-20).

5. The thermosensitive bionic material according to claim 1, characterized in that: The loading method of the RNA interference factor is as follows: (1) PLGA-CS nanoparticles were prepared using a double emulsification-solvent evaporation method. A buffer containing MALAT1-siRNA and / or miRNA-21 mimics was used as the inner aqueous phase, PLGA was dissolved in an organic solvent as the oil phase, 1-2% surfactant was added, and colostrum was formed by ultrasonic emulsification. (2) injecting colostrum into an external aqueous phase containing 1-3% polyvinyl alcohol for secondary emulsification to form a double emulsion; (3) adding chitosan solution to the double emulsion and stirring for 4-6 hours to allow chitosan to be deposited on the PLGA surface by electrostatic adsorption; (4) RNA interference factor nanoparticles were prepared after centrifugal purification and freeze-drying; (5) The prepared RNA interference factor nanoparticles are evenly dispersed in the PNIPAM-CS-HA hydrogel carrier to prepare an injectable thermosensitive gel.

6. The thermosensitive bionic material according to claim 5, characterized in that: The RNA interference factor nanoparticles have a particle size of 150-250 nm, a Zeta potential of +20-+35 mV, an encapsulation rate of >80%, and a drug loading of 2-5% w / w.

7. The thermosensitive bionic material according to claim 5, characterized in that: The mass volume ratio of the RNA interference factor nanoparticles to the PNIPAM-CS-HA hydrogel carrier is (1-5 mg): (1 mL). The RNA interference factor nanoparticles are evenly dispersed in the PNIPAM-CS-HA hydrogel carrier pre-cooled at 4°C, mixed evenly by gentle magnetic stirring at 4°C for 30 minutes, and stored at low temperature in the dark.

8. The thermosensitive bionic material according to claim 1, characterized in that: The thermosensitive hydrogel carrier has an LCST of 30-34° C. and completes a sol-gel transition within 3-5 minutes at body temperature, forming a three-dimensional network structure that fits the endometrial wound surface, with an adhesion strength of >20 kPa.

9. The thermosensitive bionic material according to claim 1, characterized in that: The RNA interference factor is released through a dual controlled release mechanism: PLGA-CS nanoparticles hydrolyze and slowly release siRNA / miRNA, while the three-dimensional network structure of the thermosensitive hydrogel carrier delays the diffusion of PLGA-CS nanoparticles, achieving local long-term sustained release with an effective period of ≥7 days.

10. Use of the thermosensitive bionic material according to any one of claims 1 to 9 in preparing a uterine cavity repair preparation, characterized in that: The thermosensitive bionic material achieves the following functions in the uterine cavity repair preparation: (1) Physically seal the uterine cavity wound to reduce infection and tissue fluid exudation; (2) sustained release of MALAT1-siRNA and / or miRNA-21 mimics to target and inhibit the TGF-β1 / Smad3 signaling pathway, reducing fibroblast hyperproliferation and collagen deposition; (3) Promote the proliferation and differentiation of endometrial stem cells and accelerate the regeneration of endometrial structure and function.

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