Butyric acid methacrylate double esterized starch, its preparation method and application in corneal defect in situ filler

Abstract

The application discloses a kind of methacrylic acid succinic acid double ester starch and its preparation method and application in corneal defect in situ filling agent, belong to the technical field of biomedical materials.The methacrylic acid succinic acid double ester starch is grafted carboxylation treatment to starch, then graft double bond is prepared.The corneal defect in situ filling agent of the application is composed of methacrylic acid succinic acid double ester starch (StacMA), methacrylated gelatin (GelMA), photo initiator and solvent.The natural biological macromolecule based corneal filling agent of starch and gelatin in the application has good biocompatibility, excellent light response adhesion characteristics and excellent mechanical properties, and the in-situ photocrosslinking of hydrogel is realized by ultraviolet irradiation, while excellent mechanical properties and strong adhesion capacity are generated, to realize the stable adhesion of in-situ forming artificial cornea on corneal implant bed, promote the regeneration of damaged corneal natural structure and the reconstruction of visual function.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a methacrylic acid succinic acid diesterized starch, its preparation method, and its application in in situ fillers for corneal defects. Background Technology

[0002] Corneal disease is one of the most common causes of vision impairment and blindness worldwide. The most effective treatment for advanced corneal disease is corneal transplantation; however, this treatment is effective only in about 5% of patients. Besides donor shortages and high costs, corneal transplantation surgery carries inherent risks of immune rejection and infection associated with allogeneic grafts. Furthermore, 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 post-suture dehiscence. Sutureless corneal defect healing, which structurally and functionally mimics the natural cornea, represents a potential method for promoting endogenous corneal regeneration.

[0003] Sutureless in situ corneal tamponade should possess the following properties: Firstly, in situ corneal reconstruction at the wound site requires the ability to tightly adhere the corneal substitute (in situ cornea or allogeneic cornea) to the implant bed without sutures, which is highly attractive for clinical surgery. Secondly, it should support corneal repair and regeneration and mechanically match the corneal stroma to provide protection. Specifically, the substitute should support the regeneration of the extracellular matrix (ECM) of the stroma and the development of functional corneal epithelium to protect intraocular contents from pathogenic invasion. Simultaneously, the substitute should be robust enough to withstand intraocular pressure (IOP) and other injuries, requiring good mechanical properties.

[0004] Currently, there are no commercially available sutureless in-situ corneal occluders because it is difficult to simultaneously achieve excellent mechanical, optical, adhesive, and biocompatibility properties. However, to address the shortcomings of surgical sutures, several commercially available bio-adhesives have been developed as alternative materials to attempt sutureless wound closure in corneal transplantation. Current commercial bio-adhesives are mainly divided into two categories: cyanoacrylate-based and fibrin glue-based. Cyanoacrylate-based bio-adhesives have high adhesive strength but significant degradation toxicity, resulting in extremely poor biocompatibility. Fibrin glue-based bio-adhesives, on the other hand, have good biocompatibility but weak adhesive strength and poor mechanical properties. Therefore, there is still a lack of sutureless in-situ corneal occluders on the market that can achieve lamellar transplantation of corneal defects. Summary of the Invention

[0005] To overcome the current shortage of human donor corneas, and the fact that sutures in corneal transplantation surgery hinder normal corneal tissue repair, causing secondary damage and easily inducing inflammation, the present invention aims to provide a methacrylic acid succinic acid diesterized starch, its preparation method, and its application in in-situ corneal defect fillers. This in-situ corneal defect filler exhibits strong adhesion, controllable mechanical properties, and excellent biocompatibility.

[0006] The objective of this invention is achieved through the following technical solutions.

[0007] A methacrylic acid succinic acid diesterized starch (StacMA) has the structure shown in Formula I:

[0008]

[0009] Formula I.

[0010] The above-described method for preparing methacrylic acid succinic acid diesterized starch includes the following steps:

[0011] (1) Gelatinize the starch solution;

[0012] (2) After gelatinization, cool the solution to below 60°C (0-60°C), then add succinic anhydride to the solution and maintain pH=8-10 for 4-24 hours.

[0013] (3) After the reaction is complete, add methacrylic anhydride dropwise to the solution to maintain pH=8-10 and react for 2-8 hours;

[0014] (4) After the reaction is completed, the starch is dialyzed, centrifuged and dried to obtain methacrylic acid succinic acid diesterized starch.

[0015] Preferably, the mass ratio of starch, succinic anhydride, and methacrylic anhydride is 1:0.5:0.5 to 1:1:1.

[0016] The preferred preparation process for succinic acid diesteryl methacrylate starch (StacMA) is as follows:

[0017]

[0018] Preferred steps for preparing succinic acid diesteryl methacrylate starch (StacMA):

[0019] (1) Dissolve potato starch at a concentration of 5% (w / v) in 100 ml of deionized water and gelatinize at 500 rpm and 90℃ for 1 h.

[0020] (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.

[0021] (3) After the reaction is complete, add methacrylic anhydride dropwise to the solution, maintain pH=8-10, and react at 500 rpm for 4 hours.

[0022] (4) After the above reaction is completed, place the solution in a dialysis bag (8000-14000) and store it in deionized water for dialysis for 5 days (the water is changed 3 times a day).

[0023] (5) After dialysis, the product is centrifuged (8000 rpm, 2 min) and the supernatant is collected.

[0024] (6) The product was freeze-dried and the resulting sample of methacrylic acid succinic acid diesterized starch was stored in a dry environment at room temperature.

[0025] Step (1) Since starch is insoluble in water, it needs to be gelatinized beforehand to increase the reaction efficiency.

[0026] The purpose of maintaining pH 8-10 in steps (2) and (3) is to catalyze the reaction between acid anhydrides and primary hydroxyl groups to accelerate the formation of ester bonds.

[0027] The purpose of step (4) dialysis is to remove unreacted succinic anhydride, methacrylic anhydride, and methacrylic acid generated in the solution from steps (2) and (3).

[0028] The purpose of centrifugation in step (5) is to further purify the product and remove impurities that are insoluble in water.

[0029] The purpose of storing in a dry environment in step (6) is to prevent the hydrophilic methacrylate succinic acid diesterized starch from absorbing moisture.

[0030] An in-situ corneal defect filler is composed of starch succinate diesteryl methacrylate (StacMA), methacrylamide gelatin (GelMA), a photoinitiator, and a solvent.

[0031] The structure of the methacrylic acid succinic acid diesterized starch is shown in Formula II:

[0032]

[0033] Formula II

[0034] The structure of the methacrylamide gelatin is shown in Formula III:

[0035]

[0036] Formula III

[0037] Preferably, the amount of methacrylic acid succinic acid diesterized starch used in the corneal defect in situ filler is 0.01-0.05 g / ml.

[0038] Preferably, the amount of the methacrylamide gelatin used in the corneal defect in situ filler is 0.1-0.15 g / ml.

[0039] Preferably, the photoinitiator is photoinitiator I2959.

[0040] Preferably, the amount of photoinitiator used in the corneal defect in situ filler is 0.005 g / ml.

[0041] Preferably, the solvent is a PBS buffer solution.

[0042] Preferably, the preparation process of methacrylated gelatin (GelMA) is as follows:

[0043]

[0044] Preferred steps for preparing methacrylated gelatin (GelMA):

[0045] (1) Dissolve gelatin at a concentration of 10% (w / v) in 100 ml PBS buffer (1×) and dissolve at 50 °C and 500 rpm for 1 h.

[0046] (2) After the gelatin is completely dissolved, add 1 ml of methacrylic anhydride (MA) dropwise into the solution at a rate of 0.2 ml / min. After the addition is complete, react at 50 °C, pH=8-10, and 500 rpm for 4 h.

[0047] (3) After the reaction is complete, dialyze in deionized water at 32 °C (8-14 kDa).

[0048] (4) Dialyze with frequent water changes for 5 days, freeze-dry the product, and store the resulting GelMA sample in a dry environment at room temperature.

[0049] Maintaining a reaction temperature of 50°C in steps (1) and (2) is to prevent the gelatin solution from gelling during the reaction, thus hindering the grafting reaction.

[0050] The purpose of maintaining pH 8-10 in step (2) is to catalyze the reaction between acid anhydride and amino group to accelerate the formation of amide bonds.

[0051] The dialysis temperature of 32°C in step (3) is similar to that in the previous two steps. The purpose is to prevent the gelatin solution from gelling during the dialysis process, which would result in the small molecules of methacrylic anhydride contained therein not being completely removed.

[0052] A method for preparing an in-situ corneal defect filler as described in any of the above claims includes the following steps:

[0053] (1) Dissolve starch succinate diesterol and photoinitiator together in PBS buffer solution (protected from light), and fully dissolve the two components by vortexing and stirring to obtain solution I;

[0054] (2) Dissolve methacrylamide gelatin and photoinitiator together in PBS buffer solution, and promote dissolution in a 37 ℃ water bath (avoiding light) to obtain solution II;

[0055] (3) Mix solution I and solution II evenly to obtain corneal defect in situ filler.

[0056] Step (1) Since the StacMA solution has a high viscosity and its dissolution conditions are different from those of the modified gelatin, a strategy of dissolving it separately and then mixing it is adopted.

[0057] In step (2), the modified gelatin has the characteristic of high dissolution temperature, so a heating dissolution strategy is adopted.

[0058] This invention employs a novel method to chemically modify natural polymers, introducing functional groups with specific properties. Specifically, the modified starch component undergoes graft carboxylation treatment on some of its structural units, aiming to introduce carboxyl groups onto the macromolecular chain to increase starch 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 macromolecules, preserving the secondary structure of starch. While ensuring biocompatibility, this system endows the biofiller with rapid photoresponsiveness, excellent mechanical properties, and strong adhesion. This system enables in-situ filling, shaping, and secure attachment of rivets to the corneal implant bed at corneal lamellar grafting sites, promoting the regeneration and reconstruction of damaged corneal structure and function. It has significant application potential in various biomedical fields, including corneal tissue engineering.

[0059] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0060] (1) The raw materials of the present invention are derived from natural substances and are easy to obtain. The method for preparing methacrylic acid succinic acid diesterized starch and methacrylated gelatin has the advantages of simple circuit and convenient operation, low reagent toxicity, simple purification method, high yield and easy product preservation.

[0061] (2) The biological filler of the present invention consists of natural polymer-based materials, which have good biocompatibility and can effectively promote the repair of damaged biological tissues without causing serious inflammatory reactions.

[0062] (3) The biological filler obtained by combining methacrylic acid succinic acid diesterized starch and methacrylamide gelatin of the present invention has unique light-responsive adhesion properties and good mechanical properties while maintaining the original good biocompatibility. It can achieve seamless filling of the wound in corneal lamellar transplantation and achieve stable adhesion of the filler material. Attached Figure Description

[0063] Figure 1a , Figure 1b These are schematic diagrams illustrating the synthetic routes of succinic acid diesterized starch (StacMA) and methacrylated gelatin (GelMA) obtained in Example 1 of this invention.

[0064] Figure 2a , Figure 2b The NMR spectra are those of succinic acid diesterized starch (StacMA) and methacrylated gelatin (GelMA) obtained in Example 1 of this invention.

[0065] Figure 3 The graph shows the mechanical properties and elongation at break of the obtained bio-adhesive in Example 3 of the present invention (the control group is GelMA).

[0066] Figure 4 The graph shows the mechanical properties and fracture strength of the obtained bio-adhesive in Example 3 of the present invention (the control group is GelMA).

[0067] Figure 5 The graph shows the test results of the photoresponsive adhesion performance of the obtained bio-adhesive in Example 2 of the present invention.

[0068] Figure 6 This is a diagram showing the biocompatibility results of the obtained bio-adhesive in Example 4 of the present invention.

[0069] Figure 7 This is a graph showing the permeability of the obtained bio-adhesive in Example 5 of the present invention.

[0070] Figure 8 The graph shows the photocuring performance of the obtained bio-adhesive in Example 6 of the present invention. Detailed Implementation

[0071] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the embodiments of the present invention are not limited thereto. The raw materials and reagents used in the following examples are all commercially available.

[0072] Figure 1a , Figure 1b These are schematic diagrams illustrating the synthetic routes of succinic acid diesterized starch (StacMA) and methacrylated gelatin (GelMA) in this invention.

[0073] In this embodiment of the invention, all adhesion experiments were conducted according to the American Society for Testing and Materials (ASTM) F2205-05 shear lap test method. Mechanical property tests were performed according to the ASTM F2150-19 guideline, using a universal testing machine (Instron 5967) for uniaxial tensile testing and DMA for unconfined compression testing to characterize the different mechanical properties of the materials.

[0074] The units for w / v below are g / ml.

[0075] Example 1

[0076] Starch was first dispersed in 100 ml of deionized water at a concentration of 5% (w / v) under conditions of 90 °C and 500 rpm. After complete gelatinization, the temperature was lowered to 40 °C, and succinic anhydride (mass ratio of starch, succinic anhydride, and methacrylic anhydride was 2:1:1) was added to the solution. The reaction was carried out at 500 rpm and 40 °C, with 5 M NaOH solution continuously added to maintain the pH at 8-10, thereby catalyzing the esterification of starch. After 6 h of reaction, methacrylic anhydride was added to the solution for secondary esterification of starch under the same conditions as the first esterification reaction, for 4 h. After the reaction was completed, the reaction solution was dialyzed against deionized water (8-14 kDa) for 5 days, then freeze-dried. The resulting StacMA sample was stored in a dry environment at room temperature.

[0077] Gelatin was first dissolved in 100 ml of PBS (1×) at a concentration of 10% (w / v) under environmental conditions of 50 °C and 500 rpm. After complete dissolution, 1% (v / v) of methacrylic anhydride was added to the solution, and the reaction was maintained at 50 °C and 500 rpm for 3 h. After the reaction was completed, the reaction solution was placed in deionized water (8-14 kDa) at 32 °C for 5 days and then freeze-dried. The resulting GelMA sample was stored in a dry environment at room temperature.

[0078] 10 mg of the StacMA and GelMA prepared above were dissolved in 0.6 ml of deuterated water, respectively. The NMR results of the products in this example are shown below. Figure 2a , Figure 2b .

[0079] like Figure 2a As shown: peak a is the alkyl peak of succinic anhydride, and the degree of substitution of succinic anhydride can be obtained by integration, which is 7%; peak c is the methyl peak of methacrylic acid; peak b is the characteristic peak of the double hydrogen of methacrylic acid, and the degree of substitution of methacrylic acid can be obtained by integration, which is 10%.

[0080] like Figure 2bAs shown: peak a is the characteristic peak of the double hydrogen of methacrylic acid, and peak b is the characteristic peak of the methylene group adjacent to the amino group on gelatin. By integration, the degree of substitution of the amino group of methacrylic acid is 70%.

[0081] Different groups with varying amounts of StacMA and GelMA were prepared as biofillers. The biofiller groups were designated as GS, with G10, G10S1, G10S3, G10S5, G15, G15S1, G15S3, and G15S5 having the following compositions: 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) via vortex sonication to obtain solutions I and II. The final ratio of the composite material was controlled by adjusting the concentrations of solutions I and II. When using this product, the two solutions are mixed evenly in equal proportions to explore the comprehensive performance of the biological filler, including mechanical properties, light transmittance, shear adhesion properties, and biocompatibility.

[0082] Example 2

[0083] (1) First, gelatin was heated and dissolved in PBS (1×) at 50 °C and 500 rpm to prepare a gelatin solution with a concentration of 20% (w / v). Then, the gelatin solution was dropped onto the surface of the adhesive slide and allowed to air dry to obtain an adhesive slide with a gelatin coating.

[0084] (2) Then, 50 μl of the biological filler prepared in Example 1 was dropped between two adhesive glass slides containing gelatin coating, and the sample was placed under ultraviolet light for 90 s to obtain a biological filler sample for adhesive strength test (the control group was not subjected to ultraviolet light).

[0085] (3) The adhesion strength of the biological filler was tested using a universal tensile testing machine with a strain rate of 5 mm / min. The results are shown in […]. Figure 5 Repeat 3-4 times for all groups.

[0086] like Figure 5 As shown, 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 thickening properties.

[0087] Example 3

[0088] (1) The biological filler prepared in Example 1 was dropped into a mold of 40×5×2.5mm and irradiated under ultraviolet light for 90 s to prepare a cuboid strip.

[0089] (2) Fix both ends of the sample strip to the fixture of the universal testing machine while wrapped with filter paper.

[0090] (3) Uniaxial tensile tests were conducted on biological filler 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 […]. Figure 3 , Figure 4 Repeat 3-4 times for all groups.

[0091] like Figure 3 , Figure 4 As shown, the addition of modified starch StacMA significantly improves the tensile strength and elongation at break of the material. When the mass ratio of GelMA to StacMA is 3:1, the tensile strength of the material is approximately three times that of pure GelMA, without any decrease in elongation at break.

[0092] Example 4

[0093] (1) The biological filler prepared in Example 1 was dropped into the mold and placed under ultraviolet light for 90 s to cure. Then the prepared sample was placed in a clean bench for ultraviolet exposure overnight for sterilization.

[0094] (2) After sterilization, the prepared biological filler was soaked in a complete culture medium at a rate of 0.2 g / ml for 72 h at an environment of 37 ℃ to prepare the biological filler extract.

[0095] (3) After soaking, the prepared extract was filtered through a 0.22 μm filter membrane and sterilized twice before being used to culture rabbit corneal epithelial cells. Cell viability and proliferation were tested using CCK-8 reagent on days 1, 3, and 5. The results are shown in the figure. Figure 6 .

[0096] like Figure 6 As shown, the material extract was non-cytotoxic, and the G10S3 group (GS) exhibited a significant effect in promoting cell proliferation. This demonstrates the excellent biocompatibility of the material.

[0097] Example 5

[0098] (1) The biological filler prepared in Example 1 was dropped into a mold with a diameter of 10 mm and a height of 100 μm and then cured under ultraviolet light for 90 s;

[0099] (2) The prepared 100 μm thick hydrogel film was spread into a quartz cuvette and the transmittance was tested on a UV-Vis spectrometer. The results are shown in the figure. Figure 7 .

[0100] like Figure 7 As shown, although the transmittance of all groups in the visible light region was lower than that of the pure GelMA group, the transmittance was still greater than 80%, indicating that the material has excellent transparency and meets the application scenarios in the field of corneal repair.

[0101] Example 6

[0102] The bio-filler prepared in Example 1 was subjected to photocuring performance testing on the test stage of a rheometer, with the optimal component G10S3. Specific parameters were as follows: 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 / cm². 2 See results Figure 8 .

[0103] like Figure 8 As shown: Group G10S3 under extremely low ultraviolet light energy irradiation (1.5 mW / cm²) 2 It still exhibits excellent gelling properties (gelling within 30 seconds, reaching 80% of maximum strength within 60 seconds). The low UV intensity is significant in clinical use, as it minimizes tissue damage. Rheological data indicate that the material possesses excellent gelling properties and broad clinical application prospects.

[0104] The above specific embodiments are all feasible implementations of the present invention, and further describe in detail the purpose, technical solution and beneficial effects of the present invention. However, the implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A methacrylic acid succinic acid diesterized starch, characterized in that, The structure is shown in Equation I: Formula I.

2. The method for preparing succinic acid diesterized starch according to claim 1, characterized in that, Includes the following steps: (1) Gelatinize the starch solution; (2) After gelatinization, cool the solution to below 60°C, then add succinic anhydride to the solution and maintain pH=8-10 for 4-24 h. (3) After the reaction is complete, add methacrylic anhydride dropwise to the solution to maintain pH=8-10 and react for 2-8 hours; (4) After the reaction is completed, the starch is dialyzed, centrifuged and dried to obtain methacrylic acid succinic acid diesterized starch.

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

1.

4. A corneal defect in situ filler, characterized in that, It is composed of succinic acid diesterized starch, methacrylamide gelatin, photoinitiator and solvent; The structure of the methacrylic acid succinic acid diesterized starch is shown in Formula II: Formula II The structure of the methacrylamide gelatin is shown in Formula III: Formula III.

5. The corneal defect in-situ filler according to claim 4, characterized in that, The amount of the methacrylic acid succinic acid diesterized starch used in the corneal defect in-situ filler is 0.01-0.05 g / ml.

6. The corneal defect in-situ filler according to claim 4, characterized in that, The amount of the methacrylamide gelatin used in the corneal defect in-situ filler is 0.1-0.15 g / ml.

7. The corneal defect in-situ filler according to claim 4, characterized in that, The photoinitiator is photoinitiator I2959.

8. The corneal defect in-situ filler according to claim 4, characterized in that, The amount of photoinitiator used in the corneal defect in-situ filler is 0.005 g / ml.

9. The corneal defect in-situ filler according to claim 4, characterized in that, The solvent is PBS buffer solution.

10. A method for preparing an in-situ corneal defect filler according to any one of claims 4-9, characterized in that, Includes the following steps: (1) Dissolve methacrylic acid succinic acid diesterized starch and photoinitiator together in PBS buffer solution to obtain solution I; (2) Dissolve methacrylamide gelatin and photoinitiator together in PBS buffer solution to obtain solution II; (3) Mix solution I and solution II evenly to obtain corneal defect in situ filler.

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