An anti-inflammatory and anti-fibrotic immunomodulatory function synergistic silk fibroin drug-loaded membrane and a preparation method and application thereof

Abstract

The present application aims to solve the problem of filtering bleb scarring caused by inflammatory response and excessive proliferation of fibroblasts after glaucoma filtration surgery. The present application uses a scalable one-step electrospinning combined with a polyphenol-metal self-assembly process to load mitomycin C on silk fibroin as a matrix and deposit epigallocatechin gallate-zinc nanoparticles on the surface of the fiber membrane in situ to construct a multifunctional synergistic drug-loaded membrane. The drug-loaded membrane exerts antioxidant and anti-inflammatory effects through the rapid release of EGCG, while achieving long-term release of MMC to inhibit cell proliferation and regulating macrophage polarization to M2 type. In vitro experiments confirm that the material has excellent ROS scavenging capacity and blood compatibility; in vivo mouse subcutaneous implantation experiments show that it can effectively inhibit the expression of local inflammatory factors, and by detecting the expression of alpha-SMA to evaluate the risk of fibrosis induced by the material, the results confirm that the material does not cause significant fibrosis reaction and tissue rejection. The present application provides a safe and efficient new strategy for preventing and treating postoperative scarring.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and drug delivery technology, specifically to a silk fibroin drug-loaded membrane with synergistic anti-inflammatory, anti-fibrotic, and immunomodulatory functions, its preparation method, and its application, which is particularly suitable for the preparation of drugs to prevent scarring of filtering blebs after glaucoma filtration surgery. Background Technology

[0002] Glaucoma is the leading cause of irreversible blindness worldwide, and filtration surgeries such as trabeculectomy are currently the main treatments for glaucoma. However, postoperative scarring of the filtering bleb due to inflammation and excessive fibroblast proliferation is the leading cause of surgical failure. Currently, antimetabolites (such as mitomycin C and MMC) are commonly used clinically to inhibit scarring, but traditional administration methods (such as single intraoperative application) have problems such as difficulty in precisely controlling drug concentration and a high risk of serious complications (such as hypotension and leakage of the filtering bleb).

[0003] In recent years, ophthalmic drug delivery systems (such as solid implants, gels, and nanocarriers) have attracted much attention, but many technical bottlenecks remain. For example, while the core-shell nanofiber membrane reported by Ning et al. (2025) utilizes natural products to achieve time-sequential release, it does not involve potent antimetabolite drugs such as MMC or reactive oxygen species (ROS) scavenging mechanisms. Existing systems such as PLGA-IOL and DEX implantable particles often focus only on single anti-inflammatory or anti-fibrotic functions, lacking precise immunomodulation of macrophage polarization from M1 to M2 in the postoperative microenvironment. In addition, traditional drug-loaded membrane preparation processes are cumbersome and difficult to scale up, making large-scale production difficult. Therefore, there is an urgent need to develop a novel ophthalmic drug-loaded membrane that combines multiple synergistic mechanisms, has a simple process, and can precisely regulate the postoperative microenvironment. Summary of the Invention

[0004] To address the aforementioned technical deficiencies in existing technologies, this invention provides a silk fibroin drug-loaded membrane with synergistic anti-inflammatory, anti-fibrotic, and immunomodulatory functions, along with its preparation method and applications.

[0005] The technical solution adopted in this invention is: a silk fibroin drug-loaded membrane with synergistic anti-inflammatory, anti-fibrotic, and immunomodulatory functions, characterized in that it comprises an SF-MMC electrospun membrane obtained by electrospinning silk fibroin (SF) and mitomycin C (MMC) through electrospinning, and an epigallocatechin gallate-zinc (EGCG-Zn) polyphenol-metal self-assembled coating deposited in situ on the surface of the SF-MMC electrospun membrane.

[0006] Furthermore, during the electrostatic drug-loaded co-spinning process, the concentration of silk fibroin (SF) was 7.0 wt%.

[0007] Furthermore, during the electrostatic drug-loaded co-spinning process, the concentration of mitomycin C (MMC) was 2.5 wt%.

[0008] Furthermore, in the polyphenol-metal (EGCG-Zn) self-assembled coating, EGCG and Zn²⁺ + The molar concentration ratio is 1:1.

[0009] Furthermore, the in vitro hemolysis rate of the drug-loaded membrane is less than 5%.

[0010] The present invention also provides a method for preparing the above-mentioned silk fibroin drug-loaded membrane, characterized by comprising the following steps: (1) Preparation of SF-MMC drug-loaded membrane: Mitomycin C (MMC) was added to a 7.0 wt% silk fibroin (SF) hexafluoroisopropanol HFIP solution to make the final concentration of mitomycin C (MMC) 2.5 wt%. The mixture was stirred in the dark for 6 h, and then electrospun to obtain the SF-MMC drug-loaded membrane. (2) Preparation of SF-MMC@EGCG-Zn drug-loaded membrane: EGCG and zinc chloride (ZnCl2) were dissolved in phosphate buffer (PBS, pH 7.4) and prepared at 25°C in the dark to obtain a light yellow-green transparent coating solution; the SF-MMC drug-loaded membrane prepared in step (1) was cut into sheets and floated on the surface of the coating solution, and reacted at 25°C and 50 rpm for 2 h to allow the EGCG-Zn self-assembled coating to be deposited on the surface of the SF-MMC drug-loaded membrane, thus constructing the SF-MMC@EGCG-Zn drug-loaded membrane.

[0011] Furthermore, the electrospinning process parameters in step (1) are: voltage 12 kV, injection speed 4 mL / h. - ¹, receiving distance 12 cm, ambient humidity ≤40%RH, ambient temperature 22-25℃.

[0012] Further, the concentration of epigallocatechin gallate (EGCG) in the coating solution described in step (2) is 1 mg / mL. - ¹.

[0013] Furthermore, the concentration of ZnCl2 in the coating solution described in step (2) is 0.6 mmol / L. - ¹.

[0014] The present invention also provides the application of the above-mentioned silk fibroin drug-loaded membrane in the preparation of anti-follicular scarring drugs after glaucoma filtration surgery.

[0015] Furthermore, the antifollicular scarring drug is used to inhibit the abnormal proliferation of human Tenon's cystofibroblasts (HTFs) and regulate the polarization of macrophages from M1 to M2 types to inhibit implant-induced local scar hyperplasia, and maintain the patency of filtration follicles and aqueous humor drainage through long-acting antifibrotic effects.

[0016] The beneficial effects of this invention are as follows: This invention provides a silk fibroin drug-loaded membrane with synergistic anti-inflammatory, anti-fibrotic, and immunomodulatory functions, as well as its preparation method and application. Compared with the prior art, it has the following advantages: 1. Stable process and easy to mass-produce: It adopts commercial medical-grade SF, eliminating the cumbersome degumming-dissolving-dialysis process. Combined with one-step electrospinning and rapid self-assembly coating process, the process has high reproducibility.

[0017] 2. Synergistic effects of multiple functions: The rapid release of EGCG achieves efficient ROS clearance and anti-inflammatory effects, while the long-term sustained release of MMC inhibits fibroblast proliferation and regulates macrophage polarization towards the M2 type, thus achieving a triple effect of anti-inflammatory, anti-fibrotic and immune regulation.

[0018] 3. High safety: Zn² + The concentration was below the corneal toxicity threshold, and the EGCG-Zn complex, after dilution in the aqueous humor, yielded Zn²⁺. + The concentration rapidly decreased to a safe range. In vitro hemolysis experiments showed that the hemolysis rate of the drug-loaded membrane of this invention was less than 5%, exhibiting excellent blood compatibility and making it suitable for intraocular implantation.

[0019] 4. Significant in vivo effects: In vivo subcutaneous implantation experiments in mice showed that the material did not induce significant fibrosis or tissue rejection after implantation. The expression level of the pro-inflammatory factor TNF-α in the tissues surrounding the implantation site was extremely low, and the expression level of α-smooth muscle actin (α-SMA) was significantly reduced, proving that it can effectively inhibit local scar hyperplasia induced by the implant and maintain the patency of filtration blebs. Attached Figure Description

[0020] Figure 1 SEM images of SF film morphology were prepared for different SF solution concentrations in Example 1.

[0021] Figure 2 SEM images of the morphology of SF drug-loaded membranes prepared for different MMC concentrations in Example 2.

[0022] Figure 3 The images show SEM images of the SF drug-loaded film morphology under different coating deposition conditions in Example 3.

[0023] Figure 4 A statistical chart showing the fiber diameters of different SF membranes.

[0024] Figure 5This is the in vitro cumulative release curve of MMC.

[0025] Figure 6 This is a statistical chart showing the DPPH free radical scavenging rate of drug-loaded membranes.

[0026] Figure 7 This is a diagram showing the in vitro hemolysis test results of the drug-loaded membrane of the present invention.

[0027] Figure 8 The graph shows the cell viability detection results at different time points (24h, 48h, 72h).

[0028] Figure 9 Immunofluorescence staining of α-SMA in the tissue surrounding the implantation site of mice 14 days after subcutaneous implantation of the drug-loaded membrane of the present invention (red fluorescence represents positive expression of α-SMA).

[0029] Figure 10 HE staining and TNF-α immunohistochemical analysis of tissue sections from mice 2 weeks after subcutaneous implantation of the drug-loaded membrane of this invention.

[0030] Figure 11 The image shows the results of detecting the expression levels of fibrosis-related markers (α-SMA, Col-1, FN) in HTFs cells. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1: Preparation of SF membrane Instruments and equipment: Electrospinning apparatus Raw materials: Water-soluble silk fibroin (EFL-SF-001, 6-10 kDa), hexafluoroisopropanol (HFIP) Technical parameters and data: SF concentrations were set at 5.0 / 7.0 / 9.0 wt%, and electrospinning was performed using a voltage of 10-15 kV, a spinning speed of 3-5 mL / h, a distance of 10-15 cm, and a humidity ≤40%. 7.0 wt% SF was considered the optimal concentration, resulting in SF membranes with fiber diameters of 0.20±0.05 μm, no beads, and a batch CV <5%. Figure 1 ).

[0033] Example 2: Preparation of SF-MMC drug-loaded membrane Instruments and equipment: Electrospinning apparatus Raw materials: 7.0 wt% SF solution, MMC (concentration 1.0 / 2.5 wt%) Technical parameters and data: Under the above electrospinning conditions, with a fixed SF content of 7.0 wt%, different concentrations of MMC were added for co-spinning. It was verified that 2.5 wt% MMC was the optimal formulation balancing drug loading and morphology, with a drug loading efficiency ≥80%. Figure 2 ).

[0034] Example 3: Preparation of a coated drug-loaded membrane Instruments and equipment: Related coating preparation apparatus Raw materials: SF (7.0 wt%)-MMC (2.5 wt%) drug-loaded membrane, EGCG, Zn² + Operation process: Using EGCG and Zn² + Through coordination, under alkaline conditions, an EGCG-Zn coating was deposited on the surface of the SF-MMC membrane to construct a series of SF-MMC@EGCG-Zn drug-loaded membranes.

[0035] Conclusion: EGCG:Zn 2+ =1:1 is the optimal coating ratio ( Figure 3 ).

[0036] Coating characterization: SEM: A continuous nano-coating was visible on the fiber surface, with a diameter increasing to 0.32 ± 0.07 μm ( Figure 4 ).

[0037] XPS: The Zn2p peak is located at 1021.7 eV. Water contact angle: decreased from 60° to 40°, indicating improved hydrophilicity.

[0038] Example 4: Evaluation of in vitro release and antioxidant properties of drug-loaded membranes 1. Drug Release Performance Evaluation: To verify the long-lasting sustained-release capability of the drug-loaded membrane of this invention in vivo, the in vitro cumulative release behavior of MMC was tested using the dialysis bag method in PBS buffer (pH 7.4, 37℃). For example... Figure 5 As shown, the SF-MMC@EGCG-Zn drug-loaded membrane exhibits an ideal biphasic release profile of "initial burst release + subsequent sustained release." Within the first 24 hours after implantation, approximately 20% of the drug is released, which helps to rapidly suppress early postoperative inflammation and fibrosis. Subsequently, a stable sustained-release phase begins, with continuous release over 14 days, achieving a release rate of over 80%. This long-lasting release kinetics perfectly meets the clinical need for anti-scarring after glaucoma filtration surgery.

[0039] 2. Antioxidant Performance Evaluation: To verify whether the surface EGCG-Zn coating retained its ROS scavenging ability, an in vitro evaluation was conducted using the DPPH free radical scavenging experiment. For example... Figure 6As shown, the pure SF-MMC membrane exhibits extremely low DPPH scavenging rate; while the SF-MMC@EGCG-Zn drug-loaded membrane modified with EGCG-Zn coating demonstrates significant antioxidant activity, with a DPPH free radical scavenging rate exceeding 85%. This confirms that during the self-assembly of the membrane, the key polyphenolic hydroxyl activity of EGCG is effectively preserved, enabling it to exert a powerful ROS scavenging and anti-inflammatory effect in the early stages of implantation.

[0040] Example 5: Evaluation of biological function 1. Blood compatibility evaluation (hemolysis test): The blood compatibility of the material is evaluated using an in vitro hemolysis test. For example... Figure 7 As shown, the hemolysis rate of the SF-MMC@EGCG-Zn drug-loaded membrane prepared in this invention is significantly lower than 5%. This indicates that the material has excellent blood compatibility, will not cause red blood cell rupture, and is suitable for intraocular and subcutaneous implantation applications.

[0041] 2. Cytotoxicity evaluation (CCK-8 assay): The effect of the material extract on cell proliferation was detected using the CCK-8 assay. For example... Figure 8 As shown, after culturing for 24h, 48h, and 72h, the relative survival rate of the experimental group cells remained at a high level (>90%), and showed a normal increasing trend over time, with no significant difference from the control group. This demonstrates that the drug-loaded membrane has good cell biocompatibility and no obvious cytotoxicity.

[0042] 3. In vivo anti-fibrotic and biocompatibility evaluation (mouse subcutaneous implantation model): To verify the tissue compatibility and anti-scarring ability of the material in vivo, a mouse subcutaneous implantation model was established. Tissue samples were collected 14 days post-surgery, and sections were prepared from the implantation site for α-SMA immunofluorescence staining to assess myofibroblast activation and the degree of fiber encapsulation. Figure 9 As shown in the figure, the area circled in yellow indicates the implantation site of the SF-MMC@EGCG-Zn drug-loaded membrane, and the red fluorescent signal represents α-SMA positive expression (i.e., activated myofibroblasts). Observation revealed that at the tissue interface surrounding the implanted membrane, the fluorescence intensity of α-SMA did not show a significant increase or aggregation compared to normal tissue areas far from the implant. This result indicates that the drug-loaded membrane prepared in this invention has excellent tissue compatibility, did not induce a significant foreign body reaction after implantation, effectively inhibited the transformation and excessive proliferation of surrounding fibroblasts into myofibroblasts, and avoided the formation of dense fibrous capsule walls (scarring), thus demonstrating its good anti-fibrotic properties in vivo.

[0043] 4. Evaluation of in vivo anti-inflammatory properties (immunohistochemical analysis): To assess the inflammatory response in the early stages of material implantation, we performed HE staining and TNF-α immunohistochemical analysis on the implantation site tissue two weeks post-operation. Figure 10As shown: HE staining results: The normal group showed clear tissue structure; in the drug-loaded membrane implantation group (SF-MMC@EGCG-Zn), although the implant outline was visible two weeks after implantation (within the dashed box), there was no large-scale necrosis or severe inflammatory cell infiltration in the surrounding tissue, and the tissue structure remained relatively intact, indicating good tissue compatibility of the material. TNF-α immunohistochemical results: TNF-α is a key pro-inflammatory factor. Compared with the normal group, the positive expression of TNF-α (brownish-yellow granules) in the surrounding tissue of the drug-loaded membrane implantation group remained at a low level, without an explosive inflammatory storm. This is attributed to the rapid release and antioxidant effect of the EGCG-Zn coating, which effectively suppressed the foreign body reaction in the early stages of implantation, laying a good microenvironmental foundation for the long-term patency of the filtration blebs.

[0044] 5. Validation of the in vitro anti-fibrotic mechanism (HTFs cell experiment): Further validation of its anti-fibrotic mechanism at the level of human Tenon's cystofibroblasts (HTFs). For example... Figure 11 As shown, compared with the control group, the expression levels of key fibrosis markers α-SMA, type I collagen (Col-1), and fibronectin (FN) were significantly downregulated in HTFs cells treated with the drug-loaded membrane extract of this invention. This confirms at the molecular level that the material can directly inhibit the activation of ocular fibroblasts and the secretion of extracellular matrix.

[0045] In summary, this invention provides a safe and efficient silk fibroin drug-loaded membrane that precisely removes early postoperative ROS, inhibits abnormal proliferation of HTFs, and promotes macrophage polarization from M1 to M2 types through an "early burst release of EGCG-long-term sustained release of MMC" release mode, thereby maintaining bleb function in the long term and solving the problem of scarring after glaucoma filtration surgery.

[0046] Conclusion: This invention provides a silk fibroin drug-loaded membrane with synergistic anti-inflammatory, anti-fibrotic, and immunomodulatory functions, along with its preparation method and application, addressing the challenge of scarring after glaucoma bleb surgery. It simultaneously possesses anti-inflammatory, anti-fibrotic, and immunomodulatory functions, effectively promoting wound healing and preventing scar formation, thus facilitating its clinical application. Through a one-step electrospinning-polyphenol / metal self-assembly process, the sequential release of mitomycin C (MMC) and epigallocatechin gallate (EGCG) is achieved, precisely scavenging reactive oxygen species (ROS), inhibiting HTF activation, and promoting macrophage polarization from M1 to M2 types. This not only prolongs the functional survival of the filtering bleb without increasing intraocular toxicity and reducing postoperative re-intervention, but also improves the long-term success rate of glaucoma microsurgery, filling the gap in high-end ophthalmic drug-device combinations with multiple mechanisms for synergistic prevention and treatment of filtering bleb scarring both domestically and internationally.

[0047] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

[0048] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. The application of a silk fibroin drug-loaded membrane with synergistic anti-inflammatory, anti-fibrotic, and immunomodulatory functions in the preparation of anti-follicular scarring drugs after glaucoma filtration surgery, characterized in that, The silk fibroin drug-loaded membrane comprises an SF-MMC electrospun membrane obtained by electrospinning silk fibroin (SF) and mitomycin C (MMC) through electrospinning, and an epigallocatechin gallate-zinc (EGCG-Zn) polyphenol-metal self-assembled coating deposited in situ on the surface of the SF-MMC electrospun membrane. In the epigallocatechin gallate-zinc (EGCG-Zn) polyphenol-metal self-assembled coating, EGCG and Zn²⁺ are present in the coating. + The molar concentration ratio is 1:

1.

2. The application according to claim 1, characterized in that, During the electrostatic drug-loaded co-spinning process, the concentration of silk fibroin (SF) was 7.0 wt%.

3. The application according to claim 1, characterized in that, During the electrostatic drug-loaded co-spinning process, the concentration of mitomycin C (MMC) was 2.5 wt%.

4. The application according to claim 1, characterized in that, The antifollicular scarring drug is used to inhibit the abnormal proliferation of human Tenon's cystofibroblasts (HTFs) and regulate the polarization of macrophages from M1 to M2 types to inhibit implant-induced local scar hyperplasia, and maintain the patency of filtration follicles and aqueous humor drainage through long-term antifibrotic effects.

5. A method for preparing a silk fibroin drug-loaded membrane with synergistic anti-inflammatory, anti-fibrotic, and immunomodulatory functions as described in any one of claims 1 to 3, characterized in that, Includes the following steps: (1) Preparation of SF-MMC drug-loaded membrane: Mitomycin C (MMC) was added to a 7.0 wt% silk fibroin (SF) hexafluoroisopropanol HFIP solution to make the final concentration of mitomycin C (MMC) 2.5 wt%. The mixture was stirred in the dark for 6 h, and then electrospun to obtain the SF-MMC drug-loaded membrane. (2) Preparation of SF-MMC@EGCG-Zn drug-loaded membrane: EGCG and zinc chloride (ZnCl2) were dissolved in phosphate buffer (PBS, pH 7.4) and prepared in the dark to obtain a light yellow-green transparent coating solution; the SF-MMC membrane was cut into pieces, floated in the coating solution, and reacted in a shaker at 50 rpm for 10 min; the EGCG-Zn coating was deposited on the surface of the SF-MMC membrane to construct the SF-MMC@EGCG-Zn drug-loaded membrane.

6. The preparation method according to claim 5, characterized in that, The electrospinning process parameters in step (1) are: voltage 12 kV, pushing speed 4 mL / h. - ¹, receiving distance 12 cm, humidity ≤40%RH, temperature 22-25℃.

7. The preparation method according to claim 5, characterized in that, The concentration of EGCG in the coating solution described in step (2) is 1 mg / mL. - ¹.

8. The preparation method according to claim 5, characterized in that, The concentration of ZnCl2 in the coating solution described in step (2) is 0.6 mmol / L. - ¹.

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