Methacrylate, succinate-diesterified starch, and preparation method therefor and use thereof in in-situ filler for corneal defects

Abstract

The present invention belongs to the technical field of biomedical materials. Disclosed are a methacrylate, a succinate-diesterified starch, a preparation method therefor and the use thereof in an in-situ filler for corneal defects. The methacrylate, succinate-diesterified starch is prepared by means of performing graft carboxylation on starch and then grafting a double bond. The in-situ filler for corneal defects consists of the methacrylate succinate-diesterified starch (StacMA), gelatin methacryloyl (GelMA), a photoinitiator and a solvent. The corneal filler has a good biocompatibility, an excellent photoresponsive adhesion property and excellent mechanical properties, and can be used for promoting the regeneration of the natural structure of a damaged cornea and the reconstruction of visual function.

Description

A methacrylate succinate diester starch and its preparation method and application in corneal defect in situ filling agent Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and particularly relates to methacrylate succinate diester starch, a preparation method thereof, and application of the starch in a corneal defect in situ filler. Background Art

[0002] Corneal disease is one of the most common causes of visual impairment and blindness worldwide. The most effective treatment for advanced corneal disease is corneal transplantation, however, this treatment option is only effective in approximately 5% of patients. In addition to the shortage of donors and the high cost of surgery, corneal transplantation also carries the inherent risk of immune rejection and infection of allogeneic grafts. In addition, the procedure requires advanced surgical skills, and the use of sutures can lead to complications such as suture erosion, suture site infiltration, infectious keratitis, impending wound dehiscence, and wound dehiscence after suture removal. Sutureless implantation in corneal defect healing mimics the natural cornea in structure and function and is a potential method to promote endogenous corneal regeneration.

[0003] Sutureless in situ formed corneal filling materials should have the following properties: On the one hand, the scheme of in situ forming the cornea at the wound surface needs to achieve the ability to tightly adhere the corneal substitute to the implant bed (in situ formed cornea or allogeneic cornea) without sutures, which is of great appeal to clinical surgery. On the other hand, it supports corneal repair and regeneration and mechanically matches the corneal stroma to play a protective role. Specifically, the substitute should support the regeneration of the extracellular matrix (ECM) of the mesenchyme and the development of functional corneal epithelium to protect the intraocular contents from pathogenic invasion. At the same time, the substitute should be strong to withstand intraocular pressure (IOP) and other injuries, which requires the substitute to have good mechanical properties.

[0004] Currently, there are no commercially available sutureless corneal in situ filling agents. This is because it is difficult to simultaneously achieve excellent mechanical properties, optical properties, adhesion properties, and good biocompatibility. However, to address the shortcomings of surgical sutures, several commercial bioadhesives are currently available as alternative materials to sutures in an attempt to achieve sutureless wound closure in corneal transplantation. Current commercial bioadhesives are mainly divided into two categories: cyanoacrylate bioadhesives and fibrin glue bioadhesives. Cyanoacrylate bioadhesives have high adhesion ability but significant degradation toxicity, resulting in extremely poor biocompatibility. Fibrin glue bioadhesives have good biocompatibility but weak adhesion strength and poor mechanical properties. Therefore, there is still a lack of sutureless corneal in situ filling agents on the market that can be used for lamellar corneal defect transplantation.

[0005] Summary of the Invention

[0006] To overcome the current shortage of human donor corneas and the fact that corneal transplant sutures hinder normal corneal tissue repair, causing secondary damage and easily inducing inflammation, the present invention aims to provide a methacrylated succinic acid diester starch, its preparation method, and its use as an in situ filler for corneal defects. This in situ filler exhibits strong adhesion, controllable mechanical properties, and excellent biocompatibility.

[0007] The purpose of the present invention is achieved through the following technical solutions.

[0008] A methacrylate succinate diester starch (StacMA), the structure of which is shown in Formula VII:

[0009] The above-mentioned method for preparing methacrylic acid succinate diester starch comprises the following steps:

[0010] (1) gelatinizing the starch solution;

[0011] (2) After gelatinization, the temperature is lowered to below 60°C (0-60°C), and then succinic anhydride is added to the solution, maintaining the pH at 8-10, and reacting for 4-24 hours;

[0012] (3) After the reaction is completed, methacrylic anhydride is added dropwise to the solution, maintaining the pH at 8-10, and reacting for 2-8 hours;

[0013] (4) After the reaction is completed, the starch is dialyzed, centrifuged, and dried to obtain methacrylate succinate diester starch.

[0014] Preferably, the amount of starch, succinic anhydride and methacrylic anhydride is in a mass ratio of 1:0.5:0.5 to 1:1:1.

[0015] Preferably, the preparation process of methacrylate succinate diester starch (StacMA) is as follows:

[0016] Preferably, the preparation steps of methacrylate succinate diester starch (StacMA) are:

[0017] (1) Potato starch was dissolved in 100 ml of deionized water at a concentration of 5% (w / v) and gelatinized at 90°C at 500 rpm for 1 h.

[0018] (2) After gelatinization, the temperature was lowered to 40°C, and then succinic anhydride was added to the solution. The pH was maintained at 8-10 and the reaction was carried out at 500 rpm for 6 hours.

[0019] (3) After the reaction, methacrylic anhydride was added dropwise to the solution, and the pH was maintained at 8-10. The reaction was carried out at 500 rpm for 4 hours.

[0020] (4) After the above reaction is completed, the solution is placed in a dialysis bag (8000-14000) and stored in deionized water for 5 days (the water is changed 3 times a day).

[0021] (5) After dialysis, the product was centrifuged (8000 rpm, 2 min) and the supernatant was collected.

[0022] (6) The product was freeze-dried, and the obtained sample of methacrylate succinate diester starch was stored in a dry environment at room temperature.

[0023] In step (1), since starch is insoluble in water, it needs to be pre-gelatinized to increase the reaction efficiency.

[0024] The purpose of maintaining pH=8-10 in steps (2) and (3) is to catalyze the reaction between the anhydride and the primary hydroxyl group to accelerate the formation of an ester bond.

[0025] The purpose of dialysis in step (4) is to remove unreacted succinic anhydride, methacrylic anhydride, and methacrylic acid generated by the reaction in the solution of steps (2) and (3).

[0026] The purpose of the centrifugation in step (5) is to further purify the product and remove water-insoluble impurities.

[0027] The purpose of storing in a dry environment in step (6) is to prevent the hydrophilic methacrylate succinate diester starch from absorbing moisture.

[0028] A corneal defect in situ filling agent, comprising methacrylate succinate diester starch (StacMA), methacryloylated gelatin (GelMA), a photoinitiator and a solvent;

[0029] The structure of the methacrylate succinate diester starch is shown in Formula IX:

[0030] The structure of the methacrylated gelatin is shown in Formula XI:

[0031] Preferably, the amount of the methacrylate succinate diester starch in the in situ filling agent for corneal defects is 0.01-0.05 g / ml.

[0032] Preferably, the amount of the methacrylated gelatin used in the in situ filling agent for corneal defects is 0.1-0.15 g / ml.

[0033] Preferably, the photoinitiator is photoinitiator I2959.

[0034] Preferably, the amount of the photoinitiator in the in situ filling agent for corneal defects is 0.005 g / ml.

[0035] Preferably, the solvent is PBS buffer solution.

[0036] Preferably, the preparation process of methacrylated gelatin (GelMA) is as follows:

[0037] Preferably, the preparation steps of methacrylated gelatin (GelMA) are:

[0038] (1) Gelatin was dissolved in 100 ml of PBS buffer (1×) at a concentration of 10% (w / v) at 50°C and 500 rpm for 1 h.

[0039] (2) After the gelatin is completely dissolved, 1 ml of methacrylic anhydride (MA) is added dropwise at a rate of 0.2 ml / min. After the addition is complete, the reaction is carried out at 50°C, pH = 8-10, and 500 rpm for 4 hours.

[0040] (3) After the reaction, the product was dialyzed in deionized water at 32°C (8-14 kDa).

[0041] (4) Dialysis was performed for 5 days with frequent water changes, and the product was freeze-dried. The obtained GelMA sample was stored in a dry environment at room temperature.

[0042] The purpose of maintaining the reaction temperature at 50° C. in step (1) and step (2) is to prevent the gelatin solution from gelling during the reaction and hindering the grafting reaction.

[0043] The purpose of maintaining pH=8-10 in step (2) is to catalyze the reaction between the anhydride and the amino group to accelerate the formation of an amide bond.

[0044] The dialysis temperature of 32° C. in step (3) is similar to that in the first two steps. The purpose is to prevent the gelatin solution from gelling during the dialysis process, resulting in incomplete removal of the small molecules of methacrylic anhydride contained therein.

[0045] The method for preparing the in situ filling agent for corneal defect described in any one of the above items comprises the following steps:

[0046] (1) Dissolve methacrylate succinate diester starch and photoinitiator in PBS buffer solution (protected from light), and fully dissolve the two components by vortexing and stirring to obtain solution I;

[0047] (2) Dissolve the methacrylated gelatin and the photoinitiator in a PBS buffer solution and dissolve in a water bath at 37°C (protected from light) to obtain solution II;

[0048] (3) Mix solution I and solution II evenly to obtain an in situ filling agent for corneal defects.

[0049] In step (1), since the viscosity of StacMA solution is very high and its dissolution conditions are different from those of modified gelatin, a strategy of separate dissolution followed by mixing is adopted.

[0050] In step (2), the modified gelatin has a high dissolution temperature, so a heating dissolution strategy is adopted.

[0051] This invention utilizes a novel method to chemically modify a natural polymer, using it as a backbone, to introduce functional groups with specific properties. The modified starch component undergoes a graft carboxylation treatment on some of its structural units, based on the original macromolecular structure. The goal is to introduce carboxyl groups onto the macromolecular chain to increase the starch's water solubility. Subsequently, double bonds are grafted onto gelatin and modified starch to impart photoresponsive properties. The backbones of both modified products remain unchanged from the original macromolecular backbone, while retaining the starch's secondary structure. While ensuring the biocompatibility of the system, the biofiller is endowed with rapid photoresponsiveness, excellent mechanical properties, and strong adhesion. This system enables in situ filling of corneal lamellar transplant defects, forming and securing rivets and corneal implants, promoting the regeneration and reconstruction of damaged corneal structure and function. It has significant potential for application in various biomedical fields, including corneal tissue engineering.

[0052] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0053] (1) The raw materials of the present invention are derived from natural materials and are easy to obtain. The method for preparing methacrylic acid succinic acid diester starch and methacrylic acid acylated gelatin has the advantages of simple circuit, convenient operation, low reagent toxicity, simple purification method, high yield and easy storage of the product.

[0054] (2) The components of the biofiller of the present invention are all natural polymer-based materials, have good biocompatibility, and can effectively promote the repair of damage to biological tissue defects without causing severe inflammatory reactions.

[0055] (3) The biofiller obtained by compounding methacrylated succinate distearate starch and methacrylated gelatin of the present invention has unique photoresponsive adhesion properties and good mechanical properties while maintaining the original good biocompatibility. It can achieve sutureless filling of corneal lamellar transplant wounds and achieve stable adhesion of the filling material. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIG1a and FIG1b are schematic diagrams of the synthesis routes of methacrylate succinate diester starch (StacMA) and methacrylate acylated gelatin (GelMA) obtained in Example 1 of the present invention, respectively.

[0057] FIG2a and FIG2b are the NMR spectra of methacrylate succinate diester starch (StacMA) and methacrylate acylated gelatin (GelMA) obtained in Example 1 of the present invention, respectively.

[0058] FIG3 is a graph showing the mechanical properties-elongation at break of the bioadhesive obtained in Example 3 of the present invention (the control group is GelMA).

[0059] FIG4 is a graph showing the mechanical properties-breaking strength of the bioadhesive obtained in Example 3 of the present invention (the control group is GelMA).

[0060] FIG5 is a graph showing the test results of the light-responsive adhesion performance of the bioadhesive obtained in Example 2 of the present invention.

[0061] FIG6 is a graph showing the biocompatibility results of the bioadhesive obtained in Example 4 of the present invention.

[0062] FIG. 7 is a graph showing the permeability of the bioadhesive obtained in Example 5 of the present invention.

[0063] FIG8 is a graph showing the photocuring performance of the bioadhesive obtained in Example 6 of the present invention. DETAILED DESCRIPTION

[0064] The present invention will be described in further detail below with reference to the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto. The raw materials and reagents in the following examples can all be purchased.

[0065] FIG1a and FIG1b are schematic diagrams of the synthesis routes of methacrylated succinic acid diester starch (StacMA) and methacrylated gelatin (GelMA) in the present invention, respectively.

[0066] In the embodiments of the present invention, all adhesion tests were performed according to the American Society for Testing Materials ASTM F2205-05 shear lap test method. Mechanical property tests were performed according to the American Society for Testing Materials ASTM F2150-19 guideline using a universal materials testing machine (Instron 5967) for uniaxial tension testing and DMA for unconfined compression testing to characterize the different mechanical properties of the materials.

[0067] The unit corresponding to w / v below is g / ml.

[0068] Example 1

[0069] The starch was first dispersed in 100 ml of deionized water at a concentration of 5% (w / v) at 90°C and 500 rpm. After complete gelatinization, the solution was cooled to 40°C. Succinic anhydride (starch, succinic anhydride, and methacrylic anhydride in a mass ratio of 2:1:1) was then added to the solution at 500 rpm and 40°C. A 5M NaOH solution was continuously added dropwise to maintain a pH of 8-10 to catalyze the esterification of the starch. After 6 hours of reaction, methacrylic anhydride was added to the solution for a second esterification of the starch. The reaction conditions were the same as for the first esterification reaction, and the reaction time was 4 hours. After the reaction, the reaction solution was dialyzed against deionized water (8-14 kDa) for 5 days, freeze-dried, and the resulting StacMA sample was stored in a dry environment at room temperature.

[0070] Gelatin was first dissolved in 100 ml of 1× PBS at a concentration of 10% (w / v) at 50°C and 500 rpm. Once completely dissolved, 1% (v / v) methacrylic anhydride was added to the solution, and the reaction was maintained at 50°C and 500 rpm for 3 hours. After the reaction, the solution was placed in deionized water (8-14 kDa) at 32°C for 5 days, followed by freeze-drying. The resulting GelMA sample was stored in a dry place at room temperature.

[0071] 10 mg of the StacMA and GelMA prepared above were dissolved in 0.6 ml of deuterated water, respectively. The NMR test results of the products of this example are shown in Figures 2a and 2b.

[0072] As shown in Figure 2a: peak a is the alkyl peak of succinic anhydride, and through integration, the degree of substitution of succinic anhydride is 7%, peak c is the methyl peak of methacrylic acid, and peak b is the characteristic peak of double bond hydrogen of methacrylic acid. Through integration, the degree of substitution of methacrylic acid is 10%.

[0073] As shown in Figure 2b: peak a is the characteristic peak of double bond hydrogen of methacrylic acid, and peak b is the characteristic peak of methylene adjacent to the amino group on gelatin. Through integration, it can be obtained that the degree of substitution of the amino group of methacrylic acid is 70%.

[0074] Different groups of StacMA and GelMA were set up to prepare biofillers. GS was used to represent the biofiller group, where the compositions of G10, G10S1, G10S3, G10S5, G15, G15S1, G15S3, and G15S5 were 10% GelMA, 0% StacMA; 10% GelMA, 1% StacMA; 10% GelMA, 3% StacMA; 10% GelMA, 5% StacMA; 15% GelMA, 0% StacMA; 15% GelMA, 1% StacMA; 15% GelMA, 3% StacMA; and 15% GelMA, 5% StacMA. StacMA and GelMA were dissolved in PBS (1×) with photoinitiator I2959 (0.5% w / v) by vortex sonication to obtain solutions I and II, respectively. The final ratio of the composites was controlled by controlling the concentrations of I and II. When used, the two solutions are mixed evenly in equal proportions to explore the comprehensive properties of the biofiller, including mechanical properties, transmittance, shear adhesion properties, and biocompatibility.

[0075] Example 2

[0076] (1) First, gelatin was dissolved in PBS (1×) at 50°C and 500 rpm to prepare a 20% (w / v) gelatin solution. The gelatin solution was then added dropwise to the surface of an adhesive glass slide and allowed to air-dry to obtain an adhesive glass slide with a gelatin coating.

[0077] (2) 50 μl of the biofiller prepared in Example 1 was then added dropwise between two adhesive glass slides containing gelatin coatings, and the sample was irradiated under ultraviolet light for 90 seconds to obtain a biofiller sample for adhesion strength testing (the control group was not irradiated with ultraviolet light).

[0078] (3) The adhesion strength of the biofiller was tested using a universal tensile testing machine with a strain rate of 5 mm / min. The results are shown in Figure 5. All groups were repeated 3-4 times.

[0079] As shown in Figure 5: With the addition of modified starch StacMA, the overall adhesion of the material is significantly enhanced, and the adhesion performance is positively correlated with the amount of StacMA, indicating that StacMA has excellent viscosity-increasing properties.

[0080] Example 3

[0081] (1) The biofiller prepared in Example 1 was added dropwise to a 40×5×2.5 mm mold and irradiated under ultraviolet light for 90 seconds to prepare a rectangular parallelepiped strip.

[0082] (2) Wrap the two ends of the sample strip with filter paper and fix them on the fixture of the universal material testing machine.

[0083] (3) Uniaxial tensile tests were performed on biofiller specimens with different compositions using a universal tensile testing machine. The strain rate of the universal tensile testing machine was 100% / min. The results are shown in Figures 3 and 4. All groups were repeated 3-4 times.

[0084] As shown in Figures 3 and 4, the addition of modified starch StacMA significantly improves the material's breaking strength and elongation. When the mass ratio of GelMA to StacMA is 3:1, the material's breaking strength is approximately three times that of pure GelMA, while the elongation at break remains unchanged.

[0085] Example 4

[0086] (1) The biofiller prepared in Example 1 was added dropwise to a mold and placed under ultraviolet light for 90 seconds to cure. The prepared sample was then placed in a clean bench for ultraviolet exposure overnight for sterilization.

[0087] (2) After sterilization, the prepared biofiller was immersed in a complete culture medium at a concentration of 0.2 g / ml at 37° C. for 72 h to prepare a biofiller extract.

[0088] (3) After the immersion, the obtained extract was filtered through a 0.22 μm filter membrane for secondary sterilization and then used to culture rabbit corneal epithelial cells. Cell survival and proliferation were tested using CCK-8 reagent on days 1, 3, and 5, respectively. The results are shown in FIG6 .

[0089] As shown in Figure 6, the material extract showed no cytotoxicity, and the G10S3 group (GS) showed a significant effect in promoting cell proliferation, demonstrating the excellent biocompatibility of the material.

[0090] Example 5

[0091] (1) The biofiller prepared in Example 1 was added dropwise to a mold with a diameter of 10 mm and a height of 100 μm and then placed under ultraviolet light for 90 seconds to cure;

[0092] (2) The prepared 100 μm thick hydrogel film was spread into a quartz cuvette and the transmittance was tested on a UV-visible spectrometer. The results are shown in Figure 7.

[0093] As shown in Figure 7: Although the transmittance of all groups in the visible light region is lower than that of the pure GelMA group, the transmittance is greater than 80%, indicating that the material has excellent transparency and meets the application scenarios in the field of corneal repair.

[0094] Example 6

[0095] The optimal component G10S3 of the biofiller prepared in Example 1 was selected and placed on the test bench of the rheometer for light curing performance testing. The specific parameters were: scanning distance 0.5 mm, before 0 s: shear strain 800%, angular frequency 10 rad / s, no UV light; after 0 s: shear strain 10%, angular frequency 10 rad / s, UV light intensity 1.5 mW / cm 2 ,The results are shown in Figure 8.

[0096] As shown in Figure 8: G10S3 group under the irradiation of ultraviolet light energy of 1.5mW / cm 2 The material also exhibits excellent gelling properties (gelling within 30 seconds and reaching 80% of its maximum strength within 60 seconds). Low UV light intensity is crucial for clinical use, as it minimizes tissue damage. Rheological data demonstrates the material's excellent gelling properties and broad clinical application prospects.

[0097] The above specific embodiments are all feasible implementation methods of the present invention, and further explain the objectives, technical solutions, and beneficial effects of the present invention in detail. However, the implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention shall be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A methacrylate succinate diester starch, characterized in that: The structure is shown in Formula I:

2. The method for preparing methacrylate succinate diester starch according to claim 1, characterized in that: The following steps are involved: (1) gelatinizing the starch solution; (2) After gelatinization, the temperature is lowered to below 60°C, and then succinic anhydride is added to the solution, maintaining the pH at 8-10, and reacting for 4-24 hours; (3) After the reaction is completed, methacrylic anhydride is added dropwise to the solution, the pH is maintained at 8-10, and the reaction is continued for 2-8 hours; (4) After the reaction is completed, the starch is dialyzed, centrifuged, and dried to obtain methacrylate succinate diester starch.

3. The method for preparing methacrylate succinate diester starch according to claim 2, characterized in that: The mass ratio of the starch, succinic anhydride and methacrylic anhydride is 1:0.5:0.5-1:1:

1.

4. An in situ filling material for corneal defect, characterized in that: It is composed of methacrylic acid succinic acid diester starch, methacrylic acid acylated gelatin, a photoinitiator and a solvent; The structure of the methacrylate succinate diester starch is shown in Formula III: The structure of the methacryloyl gelatin is shown in Formula V:

5. The in situ filling material for corneal defect according to claim 4, characterized in that: The dosage of the methacrylate succinate diester starch in the corneal defect in situ filling agent is 0.01-0.05 g / ml.

6. The in situ filling material for corneal defect according to claim 4, characterized in that: The amount of the methacryloyl gelatin used in the corneal defect in situ filling agent is 0.1-0.15 g / ml.

7. The in situ filling material for corneal defect according to claim 4, characterized in that: The photoinitiator is photoinitiator I2959.

8. The in situ filling material for corneal defect according to claim 4, characterized in that: The amount of the photoinitiator used in the corneal defect in situ filling agent is 0.005 g / ml.

9. The in situ filling material for corneal defect according to claim 4, characterized in that: The solvent is PBS buffer solution.

10. The method for preparing the in situ filling agent for corneal defect according to any one of claims 4 to 9, characterized in that: The following steps are involved: (1) dissolving methacrylate succinate diester starch and a photoinitiator in a PBS buffer solution to obtain solution I; (2) dissolving methacrylated gelatin and a photoinitiator in a PBS buffer solution to obtain solution II; (3) Mix solution I and solution II evenly to obtain an in situ filling agent for corneal defects.

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