A modified Phoenix Coat corneal repair material, its preparation method and application

Abstract

This invention discloses a modified *Echinochloa crus-galli* corneal repair material, its preparation method, and its application. The material efficiently removes the calcium carbonate shell layer of the eggshell using an EDTA-imidazole mixed solution, leaving no calcium residue. Immersion treatment at an optimized alkaline concentration partially denatures the main structural proteins, loosening the natural fiber network of the *Echinochloa crus-galli* and improving optical clarity while maintaining mechanical integrity. Alkaline treatment further improves the physicochemical properties of the membrane: surface hydrophilicity, equilibrium water content, and degradation rate. The modified *Echinochloa crus-galli* has a saturated water content close to that of the natural cornea, and its degradation rate matches the corneal defect regeneration and repair rate. Furthermore, quaternization imparts excellent antibacterial properties to the *Echinochloa crus-galli*. The efficacy of corneal regeneration and repair was evaluated using a New Zealand white rabbit anterior lamellar keratotomy model. Implantation of the modified *Echinochloa crus-galli* corneal repair material effectively promoted coherent epithelial closure and orderly stromal remodeling, resulting in a cornea with function identical to a natural, undamaged cornea.

Description

Technical Field

[0001] This invention relates to the field of medical repair materials technology, specifically to a modified Phoenix Coat corneal repair material, its preparation method, and its application. Background Technology

[0002] Globally, some people still suffer irreversible vision loss due to corneal diseases such as bullous keratosis, keratoconus, and cicatricial opacities. However, the availability of corneal donor tissue worldwide remains severely insufficient, and corneal transplantation surgery carries significant risks, including transplant rejection, infection, and postoperative complications.

[0003] In recent years, the rise and development of tissue-engineered cornea research has brought new hope to corneal patients, including decellularized porcine corneal matrix, polymer scaffolds, collagen, and chitosan scaffolds. Among these, decellularized porcine corneal matrix retains the natural layered collagen structure, exhibits low immunogenicity and excellent biocompatibility, making it one of the artificial scaffold materials for corneal regeneration. However, decellularized porcine corneal matrix is ​​a xenograft, and the decellularization process may degrade glycosaminoglycans, weaken mechanical strength and optical transparency. Residual cell debris may trigger an immune response and is prone to vascularization, leading to scarring and reduced transparency.

[0004] Another typical example is the amnion, but its application is limited by its rapid degradation and high cost. Therefore, there is an urgent need to develop advanced biomaterials that can overcome these limitations.

[0005] Eggshell membrane (ESM), known as "phoenix skin" in Traditional Chinese Medicine, is inexpensive and readily available. Chinese patent document CN1436544A discloses a traditional Chinese medicine eye drop for treating eye diseases. Targeting the characteristics of many stubborn eye diseases, this invention primarily utilizes traditional Chinese medicine to prepare eye drops, achieving good therapeutic effects in treating various intractable eye conditions. For a long time, phoenix skin has been used for hemostasis and anti-inflammation, and in Traditional Chinese Medicine clinical practice, it is used to treat corneal ulcers and nasal mucosal ulcers.

[0006] Studies have shown that ESM (extrapermeable membrane) acts as a semi-permeable barrier, allowing small molecules such as water to permeate while excluding large molecules such as glucose and proteins. This selective permeability is the basis for its wound management efficacy. However, natural ESM has insufficient light transmission and poor hydrophilicity, making it unsuitable for direct use as a corneal repair material. Therefore, there is an urgent need to find a modification method for ESM to give it excellent light transmittance, high water saturation content, and antibacterial properties, making it suitable for the preparation of corneal repair materials. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for preparing a modified Phoenix Coat corneal repair material. The resulting modified Phoenix Coat corneal repair material possesses excellent light transmittance, surface hydrophilicity, and high saturated water content, while also exhibiting excellent antibacterial properties, making it suitable for application in the field of corneal repair.

[0008] A method for preparing a modified Phoenix Coat corneal repair material includes the following steps:

[0009] (1) Soak the washed fresh eggs in a mixed solution of ethylenediaminetetraacetic acid (EDTA) and imidazole until the calcium carbonate shell is completely dissolved. Collect the eggshell membrane, wash it, and remove the yolk and egg white.

[0010] (2) Soak the phoenix skin obtained in step (1) in an alkaline solution, and after washing, obtain a transparent phoenix skin film;

[0011] (3) After the transparent Phoenix Coat membrane obtained in step (2) is subjected to quaternization reaction, it is washed to obtain modified Phoenix Coat corneal repair material.

[0012] In this invention, the calcium carbonate shell layer of eggs is efficiently removed using an EDTA-imidazole mixed solution, leaving no calcium residue. Furthermore, due to the insufficient light transmission of natural eggshell membranes, this invention develops an "alkaline transparency" method: immersion treatment at an optimized alkaline concentration partially denatures the main structural proteins (collagen and keratin), loosening the natural fiber network of the eggshell membrane and improving optical clarity while maintaining mechanical integrity. The alkaline treatment further improves the physicochemical properties of the membrane: surface hydrophilicity, equilibrium water content, and degradation rate. The modified eggshell membrane has a saturated water content close to that of the natural cornea, its degradation rate matches the corneal defect regeneration and repair rate, and it possesses excellent antibacterial properties through quaternization.

[0013] The modified Phoenix Coat corneal repair material prepared by the above method has excellent light transmittance, surface hydrophilicity and high saturated water content, as well as excellent antibacterial properties, and can be applied in the field of corneal repair.

[0014] Preferably, in step (1), the fresh eggs are eggs that are less than one month old.

[0015] Preferably, in step (1), the concentrations of ethylenediaminetetraacetic acid and imidazole in the mixed solution are both 5-15 wt%.

[0016] Preferably, in step (1), after removing the egg white and yolk, the phoenix skin is soaked in PBS solution for storage, and the storage time is ≤72 h.

[0017] Preferably, in step (2), the alkaline solution is an aqueous solution of sodium hydroxide, potassium hydroxide, or lithium hydroxide.

[0018] More preferably, the concentration of the alkaline solution is 0.5~2 M.

[0019] Preferably, in step (2), the temperature of the phoenix skin soaking in the alkaline solution is 15~35 ℃ and the time is 0.5~4 h.

[0020] In this invention, as the concentration of the alkali solution increases and the treatment time is extended, the contact angle of the modified Phoenix Coat corneal repair material gradually decreases and the hydrophilicity of the material gradually increases.

[0021] Preferably, in step (3), the reagent used in the quaternization reaction is an aqueous solution of 2,3-epoxypropyl-trimethylammonium chloride with a concentration of 0.01~0.05 g / mL.

[0022] In this invention, the epoxy group of 2,3-epoxypropyl-trimethylammonium chloride undergoes ring-opening under alkaline aqueous solution conditions, covalently bonding with nucleophilic amino groups (mainly from lysine residues) or hydroxyl groups on the protein molecular chain of *Echinochloa crus-galli*, thereby transferring the positively charged quaternary ammonium cation (-N... + (CH3)3) is grafted onto the phoenix coat to give it antibacterial properties.

[0023] Preferably, in step (3), the temperature of the quaternization reaction is 50~60 °C and the time is 12~24 h.

[0024] Preferably, in step (3), after the quaternization reaction, the Phoenix Coat membrane is repeatedly soaked in deionized water for 3 to 5 times, and after washing, the modified Phoenix Coat corneal repair material is obtained.

[0025] The present invention also provides a modified Phoenix Coat corneal repair material prepared by the above preparation method.

[0026] Preferably, the modified Phoenix Coat corneal repair material has a light transmittance of 70%~80%, a contact angle ≤55°, and a saturated water content ≥80%.

[0027] Preferably, the modified Phoenix Coat corneal repair material inhibits bacterial growth.

[0028] More preferably, the bacteria are Escherichia coli and Staphylococcus aureus.

[0029] This invention also provides the application of the modified Phoenix Coat corneal repair material in the preparation of materials for corneal repair. The modified Phoenix Coat corneal repair material obtained by this invention possesses excellent light transmittance, surface hydrophilicity, and high saturated water content, while also exhibiting excellent antibacterial properties. The efficacy of corneal regeneration and repair was evaluated using a New Zealand white rabbit anterior lamellar keratotomy model. Implantation of the modified Phoenix Coat corneal repair material effectively promoted coherent epithelial closure and orderly stromal remodeling of the cornea, and the repaired cornea functioned identically to a natural, undamaged cornea.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] In this invention, alkaline treatment significantly improves the light transmittance of the corneal resurfacing membrane, while also enhancing its physicochemical properties: surface hydrophilicity, balanced water content, and degradation rate. The modified membrane's saturated water content is close to that of the natural cornea, and its degradation rate matches the rate of corneal defect regeneration and repair. Furthermore, quaternization imparts excellent antibacterial properties to the membrane. The efficacy of corneal regeneration and repair was evaluated using a New Zealand white rabbit anterior lamellar keratotomy model. Implantation of the modified corneal resurfacing membrane effectively promoted continuous epithelial closure and orderly stromal remodeling, resulting in a cornea with the same function as a natural, undamaged cornea. Attached Figure Description

[0032] Figure 1 A schematic diagram of the preparation process for the modified Phoenix Coat corneal repair material.

[0033] Figure 2 The light transmittance of the Phoenix Garment prepared in Example 2 before and after modification is shown.

[0034] Figure 3 The images show the contact angles of the phoenix feathers from Examples 1-3 after treatment with different alkalis and for different durations. From left to right, the images show the contact angle test results of the phoenix feathers after treatment with alkali (without alkali) and after treatment with alkali (as in Examples 1-3). ESM0, ESM0.5, ESM1, and ESM4 represent the phoenix feathers treated with alkali for 0, 0.5, 1, and 4 hours, respectively.

[0035] Figure 4 The values ​​represent the saturated water content of the phoenix shells in Examples 1-3 after treatment with different alkalis and for different times. ESM0, ESM0.5, ESM1, and ESM4 represent the phoenix shells treated with alkali for 0, 0.5, 1, and 4 hours, respectively.

[0036] Figure 5The images show the repair effect of the modified Phoenix Coat corneal repair material prepared in Example 2 in a rabbit anterior lamellar keratotomy model. In the images, A is a schematic diagram of the modeling process of the rabbit anterior lamellar keratotomy model and the operation of implanting the modified Phoenix Coat corneal repair material; B is a slit-lamp microscope image at 1, 2, 4 and 8 weeks after corneal repair; C is anterior segment optical coherence tomography (AS-OCT) image of the rabbit cornea at 1, 2, 4 and 8 weeks after surgery; and D is a topographic map of the rabbit cornea at 8 weeks after surgery.

[0037] Figure 6 This is a quantitative analysis diagram showing the change in corneal epithelial healing over time in a rabbit anterior lamellar keratotomy model using the modified Phoenix Coat corneal repair material prepared in Example 2.

[0038] Figure 7 Masson staining images of corneal sections, from left to right: undamaged natural cornea, modified Phoenix Coat corneal repair material prepared in Example 2 after 8 weeks of repair in a rabbit anterior lamellar keratotomy model, and blank control group after 8 weeks of staining in a rabbit anterior lamellar keratotomy model. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to the embodiments, but the implementation of the present invention is not limited to the following embodiments.

[0040] All raw materials used in this invention are commercially available.

[0041] Example 1

[0042] like Figure 1 The processing is as shown, and the specific steps are as follows:

[0043] (1) Wash the eggs that are less than 1 month old with deionized water, immerse them in a mixed solution of EDTA (5 wt%) and imidazole (15 wt%) at 25 ℃ for 18 hours until the calcium carbonate shell is completely dissolved, collect the eggshell membrane (ESM), and wash it thoroughly with deionized water to remove the egg white and yolk. Immerse the eggshell membrane in phosphate buffered saline (PBS) to prevent dehydration and store it at 4 ℃ for 1 hour for later use.

[0044] (2) Immerse the phoenix skin obtained in step (1) in 0.5 M NaOH solution at 15 °C for 0.5 hours, then quickly remove and rinse thoroughly to remove residual alkali and obtain a transparent phoenix skin film;

[0045] (3) Weigh 0.2 g of 2,3-epoxypropyl-trimethylammonium chloride, dissolve it in 20 mL of deionized water to obtain an aqueous solution of 2,3-epoxypropyl-trimethylammonium chloride, heat it to 50 °C, and then put it into the transparent-treated phoenix coat membrane obtained in step (2), and react for 24 hours; after the reaction, soak the phoenix coat membrane in deionized water for 30 min, take out the modified phoenix coat membrane, soak it in deionized water again for 30 min, repeat this 3 times to obtain the modified phoenix coat corneal repair material. Place the obtained modified phoenix coat corneal repair material on a piece of paper with "Seeking Truth and Innovation" written on it, and the text can still be clearly observed, indicating that the modified phoenix coat has good transparency (e.g., Figure 1 (As shown).

[0046] The modified phoenix coat corneal repair material prepared above has a light transmittance of 70%, and its contact angle and saturated water content are as follows: Figure 3 and Figure 4 As shown, with a saturated water content of 80% and a contact angle of 52.2°, it can inhibit 99.9% of Escherichia coli and Staphylococcus aureus.

[0047] Example 2

[0048] (1) Wash the eggs that are less than 1 month old with deionized water, immerse them in a mixed solution of EDTA (10 wt%) and imidazole (10 wt%) at 40 ℃ for 20 hours until the calcium carbonate shell is completely dissolved, collect the eggshell membrane (ESM), and wash it thoroughly with deionized water to remove the egg white and yolk. Immerse the eggshell membrane in phosphate buffered saline (PBS) to prevent dehydration and store it at 4 ℃ for 48 hours for later use.

[0049] (2) Immerse the phoenix skin obtained in step (1) in 1.5 M KOH solution at 25 °C for 1 hour, then quickly remove and rinse thoroughly to remove residual alkali and obtain a transparent phoenix skin film;

[0050] (3) Weigh 0.6 g of 2,3-epoxypropyl-trimethylammonium chloride, dissolve it in 20 mL of deionized water to obtain an aqueous solution of 2,3-epoxypropyl-trimethylammonium chloride, heat it to 55 °C, and then put it into the transparent treated phoenix coat membrane obtained in step (2) and react for 18 hours. After the reaction is completed, soak the phoenix coat membrane in deionized water for 30 min, take out the modified phoenix coat membrane, soak it in deionized water again for 30 min, repeat this process 3 times to obtain the modified phoenix coat corneal repair material.

[0051] The modified Phoenix Coat corneal repair material prepared above has a light transmittance of 72%, and its contact angle and saturated water content are as follows: Figure 3 and Figure 4As shown, with a saturated water content of 80% and a contact angle of 39.6°, it can inhibit 99.9% of Escherichia coli and Staphylococcus aureus.

[0052] Example 3

[0053] (1) Wash the eggs that are less than 1 month old with deionized water, immerse them in a mixed solution of EDTA (15 wt%) and imidazole (5 wt%) at 50 ℃ for 24 hours until the calcium carbonate shell is completely dissolved, collect the eggshell membrane (ESM), and wash it thoroughly with deionized water to remove the egg white and yolk. Immerse the eggshell membrane in phosphate buffered saline (PBS) to prevent dehydration and store it at 4 ℃ for 72 hours for later use.

[0054] (2) Immerse the phoenix skin obtained in step (1) in 2 M KOH solution at 35 °C for 4 hours, then quickly remove and rinse thoroughly to remove residual alkali and obtain transparent phoenix skin film;

[0055] (3) Weigh 1 g of 2,3-epoxypropyl-trimethylammonium chloride, dissolve it in 20 mL of deionized water to obtain an aqueous solution of 2,3-epoxypropyl-trimethylammonium chloride, heat it to 60 °C, and then put it into the transparent treated phoenix coat membrane obtained in step (2) and react for 12 hours. After the reaction is completed, soak the phoenix coat membrane in deionized water for 30 min, take out the modified phoenix coat membrane, soak it in deionized water again for 30 min, repeat this process 3 times to obtain the modified phoenix coat corneal repair material.

[0056] The modified Phoenix Coat corneal repair material prepared above has a light transmittance of 80%, and its contact angle and saturated water content are as follows: Figure 3 and Figure 4 As shown, with a saturated water content of 82% and a contact angle of 26.1°, it can inhibit 99.9% of Escherichia coli and Staphylococcus aureus.

[0057] Application example: Verifying the repair capabilities of modified Phoenix Coat corneal repair materials

[0058] 1) All animal experiments were conducted in accordance with the protocol approved by the Animal Care and Use Committee of Zhejiang Clinical Medical Research Institute (No.: ZJCLA-IACUC-20011060). Male New Zealand white rabbits aged 10-12 weeks were used in the experiments. Corneal laminar surgery was performed under aseptic conditions to simulate corneal injury. General anesthesia was induced by intramuscular injection of xylazine hydrochloride at a dose of 1-2 mg / kg body weight.

[0059] 2) A 200-micrometer-deep defect area was prepared in the center of the cornea using a corneal trephine with a diameter of 5 mm, followed by lamellar keratotomy at the same depth.

[0060] 3) Remove the modified Phoenix Coat disc (7 mm in diameter) and fix it to the corneal defect with 10-0 absorbable sutures. Figures 5-7 The "ESM" group was included. Additionally, a corneal defect was created without implanting any material, serving as a blank control group. Figures 5-7 The "Control" group.

[0061] 4) Slit-lamp and anterior optical coherence tomography examinations were performed at 1, 2, 4 and 8 weeks postoperatively.

[0062] Eight weeks post-surgery, corneal topography and tissue section staining were performed.

[0063] Figure 5 The image shows the repair effect of the modified Phoenix Coat corneal repair material prepared in Example 2 in a rabbit anterior lamellar keratotomy model. A corneal defect area with a diameter of 5 mm and a depth of 200 μm was created on the left eye of a male New Zealand white rabbit using a corneal trephine. Then, a Phoenix Coat disc with a diameter of 7 mm was sutured and fixed with 10-0 absorbable sutures. Figure 5 (A in the middle). Figure 5 Figure B shows slit-lamp microscopic images taken at 1, 2, 4, and 8 weeks post-corneal repair surgery, allowing observation of tissue response and epithelialization after material implantation. The modified Phoenix Coat group exhibited an initial immune response within the first two weeks, manifesting as mild haze. However, from weeks 4 to 8, corneal stromal regeneration accompanied by degradation of the Phoenix Coat material resulted in significantly better corneal transparency compared to the blank control group. At 8 weeks post-surgery, the blank control group showed obvious white lesion margins, while the Phoenix Coat-treated corneas showed seamless tissue integration, demonstrating complete epithelial and stromal recovery. Furthermore, at 4 weeks post-surgery, slit-lamp observation showed no significant edema, scarring, or neovascularization in the Phoenix Coat group, and the stromal maintained uniform thickness and optical transparency. Figure 5 Image C in the figure represents anterior segment optical coherence tomography (AS-OCT) images of the rabbit cornea at 1, 2, 4, and 8 weeks post-surgery. At 1 week post-surgery, the Phoenix Coat-treated cornea showed a clear defect boundary and an ablation zone with approximately 40% thickness loss in the stromal layer; however, the intact low-reflectivity upper stromal belt indicated complete epithelialization. By week 2, the defect boundary became blurred, and by week 4, the defect edge was indistinguishable. At week 8, the thickness and reflectivity of the regenerated stromal were identical to that of the natural cornea, with a smooth surface, regular contours, and a remarkably similar appearance to healthy tissue, indicating complete corneal recovery.

[0064] In contrast, at 1 week post-surgery, the blank control group showed significant edema and discontinuous low-reflectivity areas, indicating incomplete epithelial recovery. By week 2, the edema had subsided and epithelialization was largely complete, but nearly half of the matrix thickness remained missing; by week 4, the defect edges were still clearly visible. After 8 weeks, the repaired area showed uneven reflectivity and an irregular surface, consistent with scar-mediated healing. Figure 5D in the figure represents the corneal topography of the rabbit at 8 weeks post-surgery. Corneal surface regularity was assessed using corneal topography at 8 weeks post-surgery. The corneas treated with the Phoenix Coat closely resembled healthy eyes, exhibiting a broad green positive area indicating near-spherical curvature and a smooth morphology. In contrast, the blank control group failed to produce reliable height maps, with large areas deviating from the reference sphere by more than ±50 µm, indicating significant irregularities. Figure 6 This is a quantitative analysis of corneal epithelial healing over time. The cornea treated with Phoenix Coat achieved complete epithelial closure within one week after implantation, while the negative control group still had about one-quarter of the residual defects at this time, which did not completely disappear until the fourth week of fluorescein staining.

[0065] Figure 7 Masson staining images of corneal sections are shown from top to bottom: undamaged natural cornea (i.e., the "normal cornea" group in the image), the modified Phoenix Coat corneal repair material prepared in Example 2 after 8 weeks of repair in a rabbit anterior lamellar keratotomy model, and the blank control group after 8 weeks of repair in a rabbit anterior lamellar keratotomy model. In the Phoenix Coat repair group, the newly deposited stromal collagen exhibits a regular parallel arrangement, without an increase in scar-like cells, and is tightly connected to the overlying epithelium. In contrast, the blank control group shows obvious epithelial-stromal fissures, and the repair area is severely thinned. The central corneal stromal thickness in the Phoenix Coat repair group is 361±35 µm, very close to the thickness of a healthy rabbit cornea (364±29 µm). In contrast, the untreated blank control group shows incomplete repair, with the thickness significantly reduced to 301±43 µm.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a modified Phoenix Coat corneal repair material, characterized in that, Includes the following steps: (1) Soak the washed fresh eggs in a mixed solution of ethylenediaminetetraacetic acid and imidazole until the calcium carbonate shell is completely dissolved. Collect the eggshell membrane, wash it, and remove the yolk and egg white. (2) Soak the phoenix skin obtained in step (1) in an alkaline solution, and after washing, obtain a transparent phoenix skin film; (3) After the transparent Phoenix Coat membrane obtained in step (2) is subjected to quaternization reaction, it is washed to obtain modified Phoenix Coat corneal repair material.

2. The method for preparing the modified Phoenix Coat corneal repair material according to claim 1, characterized in that, In step (1), the concentrations of ethylenediaminetetraacetic acid and imidazole in the mixed solution are both 5-15 wt%.

3. The method for preparing the modified Phoenix Coat corneal repair material according to claim 1, characterized in that, In step (2), the alkaline solution is an aqueous solution of sodium hydroxide, potassium hydroxide or lithium hydroxide.

4. The method for preparing the modified Phoenix Coat corneal repair material according to claim 3, characterized in that, The concentration of the alkaline solution is 0.5~2 M.

5. The method for preparing the modified Phoenix Coat corneal repair material according to claim 1, characterized in that, In step (2), the temperature of the phoenix skin soaked in the alkaline solution is 15~35 ℃ and the time is 0.5~4 h.

6. The method for preparing the modified Phoenix Coat corneal repair material according to claim 1, characterized in that, In step (3), the reagent used in the quaternization reaction is an aqueous solution of 2,3-epoxypropyltrimethylammonium chloride with a concentration of 0.01~0.05 g / mL.

7. The method for preparing the modified Phoenix Coat corneal repair material according to claim 1, characterized in that, In step (3), the quaternization reaction is carried out at a temperature of 50-60 °C for 12-24 h.

8. The modified Phoenix Coat corneal repair material prepared by the preparation method according to any one of claims 1 to 7.

9. The modified Phoenix Coat corneal repair material according to claim 8, characterized in that, The modified Phoenix Coat corneal repair material has a light transmittance of 70%~80%, a contact angle ≤55°, and a saturated water content ≥80%. The modified Phoenix Coat corneal repair material inhibits bacterial growth.

10. The use of the modified Phoenix Coat corneal repair material according to claim 8 or 9 in the preparation of materials for corneal repair.

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