A photoinduced bioadhesive based on dextran and gelatin, its preparation and application

Abstract

This invention discloses a photoinduced bioadhesive based on dextran and gelatin, its preparation, and its application. The bioadhesive components include an oxidation product of dextran and two modified gelatin products. The oxidized dextran component undergoes ring-opening oxidation of some structural units based on its original macromolecular structure to form aldehyde groups, while the main chains of the two modified gelatin components remain unchanged from the original macromolecular structure. This multi-component bioadhesive not only exhibits excellent adhesion and durability in wet environments but also, due to its photoresponsive adhesion, provides a superior repair-promoting effect when used as a closure material in corneal transplantation surgery, replacing surgical sutures.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a photoinduced bioadhesive based on dextran and gelatin, and its preparation and application. Background Technology

[0002] Corneal diseases caused by eye trauma, bacterial and viral infections, etc., can lead to blindness. Currently, the main clinical treatment for these diseases is corneal transplantation based on surgical sutures.

[0003] However, given the limitations of surgical sutures, their use in corneal transplantation significantly restricts the effectiveness of the treatment. Specifically, the use of sutures requires creating a new wound on the corneal tissue, and suture removal can easily lead to postoperative complications, causing secondary damage to the healed cornea. Furthermore, this surgical procedure is challenging and demands a high level of surgical skill from the surgeon. Therefore, corneal transplantation based on surgical sutures, while technically extremely difficult, offers only limited therapeutic efficacy and is not a suitable clinical treatment for blinding corneal diseases.

[0004] Currently, to address the shortcomings of surgical sutures, bio-adhesives have been developed as alternative materials to attempt sutureless wound closure in corneal transplantation. Commercially available bio-adhesives are mainly divided into two categories: cyanoacrylate-based bio-adhesives and fibrin glue-based bio-adhesives.

[0005] These two types of bioadhesives each have their own characteristics. Cyanoacrylate bioadhesives have high adhesion ability, but their significant degradation toxicity and extremely poor biocompatibility greatly limit their application in the biomedical field. On the other hand, fibrin glue bioadhesives have excellent biocompatibility, but their extremely low and weak adhesion strength also greatly limits their application in the corneal field.

[0006] Furthermore, the adhesive properties of these bio-adhesives lack spatiotemporal controllability, making their clinical use particularly difficult. Therefore, there is currently a lack of bio-adhesives on the market that can effectively replace surgical sutures for corneal transplantation. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a photoinduced bioadhesive based on dextran and gelatin, as well as its preparation and application.

[0008] This invention is specifically designed for corneal tissue engineering. It combines oxidized dextran with a gelatin-based material that has photocurability to obtain a hydrogel material with excellent light-responsive adhesion and long-lasting adhesion performance in wet environments, which is used to meet the needs of corneal defect repair.

[0009] This invention, based on natural polymers as the main scaffold, introduces functional groups with specific chemical functions into the system. This ensures excellent biocompatibility while also endowing the bio-adhesive with rapid photoresponsive adhesion and extremely strong and long-lasting tissue adhesion in humid environments. This system comprehensively overcomes the shortcomings of various bio-adhesives mentioned above, more effectively achieving the fixation of corneal donors on the corneal implant bed, promoting the regeneration of damaged corneal natural structures and the restoration of physiological functions, and has enormous application potential in various biomedical fields such as corneal tissue engineering.

[0010] This invention is achieved through the following technical solution:

[0011] A photoresponsive bioadhesive based on dextran oxidation and gelatin modification, comprising oxidized dextran, acrylic anhydride-acylated gelatin, and cysteine-acylated gelatin, with the following structural formulas:

[0012]

[0013]

[0014] The preparation based on oxidized dextran (OD), acrylic anhydride acylated gelatin (G-AA), and cysteine ​​acylated gelatin (G-SH) includes the following steps:

[0015] Preparation steps of oxidized dextran (OD):

[0016] (1) Dissolve the dextran at a concentration of 10% (w / v) in 100 ml of deionized water and dissolve at 500 rpm for 1 h.

[0017] (2) After the dextran is completely dissolved, add 8g of NaIO4 to the solution and react for 3h at 25℃ and 500rpm under light-protected conditions.

[0018] (3) After the reaction is complete, add diethylene glycol in an amount equal to that of NaIO4 to the solution and continue to maintain the above reaction conditions for 0.5-1h.

[0019] (4) After the above reaction is completed, place the solution in deionized water and dialyze it with water changes 3 times a day (3500 Da).

[0020] (5) After dialysis for 5 days, the product was freeze-dried and the resulting sample of oxidized dextran was stored in a dry environment at room temperature.

[0021] In step (2) above, since NaIO4 decomposes in the light, it is necessary to use NaIO4 as a catalyst to catalyze the oxidation of dextran under light-protected conditions.

[0022] In step (3) above, diethylene glycol of the same molar amount as NaIO4 is added to the solution to react with NaIO4 and terminate the oxidation of dextran.

[0023] The purpose of dialysis in step (4) above is to remove the small molecules generated by the reaction of diethylene glycol and NaIO4 in the solution of step (3).

[0024] The purpose of storing the above step (5) in a dry environment is to prevent the hydrophilic oxidized dextran from absorbing moisture.

[0025] Preparation steps of acrylic anhydride acylated gelatin (G-AA):

[0026] (1) Dissolve gelatin at a concentration of 10% (w / v) in 100 ml of PBS buffer (1×) and dissolve at 50°C and 500 rpm for 1 h.

[0027] (2) After the gelatin is completely dissolved, add 1 ml of acrylate (AA) dropwise to the solution at a rate of 0.2 ml / min. After the addition is complete, maintain the reaction conditions at 50°C and 500 rpm for 3 h.

[0028] (3) After the reaction is complete, dialyze in deionized water at 40℃ (3500 Da).

[0029] (4) Dialyze with frequent water changes for 5 days, freeze-dry the product, and store the obtained sample G-AA in a dry environment at room temperature.

[0030] 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.

[0031] The dialysis temperature of 40°C in step (3) is similar to that in the previous two steps. The purpose is to prevent the gelatin solution from gelling during dialysis, which would result in the acrylic acid molecules contained therein not being completely removed.

[0032] Preparation steps of cysteine-acylated gelatin (G-SH):

[0033] (1). Dissolve gelatin at a concentration of 1% (w / v) in 300 ml of PBS buffer (1×) and dissolve at 50°C and 500 rpm for 0.5 h.

[0034] (2) After the gelatin has completely dissolved, add NaH2PO4 to the solution to adjust the pH to 4-6. The purpose of adjusting the pH is to prevent the cysteine ​​molecules added in step (3) from undergoing small molecule reactions during the reaction process, which would reduce the cysteine ​​grafting rate on the gelatin macromolecules.

[0035] (3) After pH adjustment, 1.38 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.282 g of N-hydroxysuccinimide, and 14.604 g of cysteine ​​were added to the solution sequentially. The reaction solution was placed at room temperature, purged with nitrogen, and reacted at 500 rpm for 8 hours. The addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide catalyzed the amide reaction between the amino group on the side chain of the gelatin macromolecule and the carboxyl group of cysteine, achieving the grafting purpose.

[0036] (4) After the reaction is complete, add a small amount of dithiothreitol to the solution and shake to dissolve it completely. The purpose of adding dithiothreitol to the solution after the reaction is to prevent the formation of disulfide bonds between the thiol groups on the cysteine ​​in the solution, which would lead to cross-linking of the gelatin macromolecules.

[0037] (5) Dialyze the reaction solution in 25°C deionized water with water changes 3 times a day (1000 Da).

[0038] (6) After 5 days of dialysis, the product was freeze-dried and the resulting G-SH sample was stored in a dry environment at room temperature.

[0039] The preparation method based on bio-adhesives includes the following steps:

[0040] (1) When using, the lyophilized OD and photoinitiator I2959 are dissolved together in PBS buffer solution. The two components are fully dissolved by vortexing and stirring to obtain solution I. Since OD reacts with the two modified gelatins as soon as they come into contact, and its dissolution conditions are different from those of the modified gelatin, a strategy of dissolving them separately and then mixing them is adopted.

[0041] (2) G-AA and G-SH were dissolved in PBS buffer solution at a mass ratio of 2:1 and dissolved in a water bath at 37°C to obtain solution II. Both modified gelatins have the characteristic of high dissolution temperature, and there is no reaction between them when they are dissolved and mixed. Therefore, the strategy of simultaneous heating and dissolution was adopted.

[0042] (3) Mix solution I and solution II in different proportions to prepare a series of bioadhesive prepolymers with fixed OD content but different concentrations of G-AA and G-SH.

[0043] (4) The prepolymer of the bioadhesive is dropped onto the application site, and then photocuring and viscosity are generated by ultraviolet light irradiation.

[0044] (5) Subsequently, the performance of the bio-adhesive was comprehensively evaluated through characterization tests such as adhesion properties to obtain the optimal bio-adhesive concentration composition. The determination of the optimal bio-adhesive concentration composition mainly depends on the initial adhesion strength of the bio-adhesive and its long-term adhesion in a wet environment.

[0045] The bioadhesive obtained by combining oxidized dextran with two types of modified gelatin in this invention maintains its original good biocompatibility while exhibiting unique photoresponsive adhesion properties and long-lasting adhesion in wet conditions. It can effectively seal tissue wounds and perform adhesion functions in aqueous environments, offering unique advantages in applications on tissues with high water content, such as the cornea. Therefore, in the field of biomedical materials, it is particularly suitable for sealing and adhering tissue wounds in wet environments.

[0046] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0047] (1) The raw materials of the present invention are derived from natural substances, which are easy to obtain. The preparation method has the advantages of simple circuit and convenient operation, low reagent toxicity, simple purification method, high yield and easy product preservation.

[0048] (2) The components of the bioadhesive of the present invention are all natural polymer-based materials, which have good biocompatibility and can effectively promote the repair of damaged biological tissues without causing severe inflammatory reactions.

[0049] (3) The bio-adhesive of the present invention has unique photoresponsive adhesion characteristics and can exhibit unique adhesion performance after light exposure. It can greatly reduce the operational difficulty of corneal transplantation surgery and has broad application prospects in clinical practice.

[0050] (4) The bioadhesive of the present invention has unique long-lasting adhesion properties in wet environments and can perform the functions of sealing wounds and adhesion in water environments for a long time. It has significant advantages in the application of biological tissues with high water content such as cornea. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the synthetic routes of oxidized dextran (OD), acrylic anhydride acylated gelatin (G-AA), and cysteine ​​acylated gelatin (G-SH) in this invention, wherein a is a schematic diagram of the OD preparation reaction; b is a schematic diagram of the G-AA preparation reaction; and c is a schematic diagram of the G-SH preparation reaction.

[0052] Figure 2 The images show the NMR spectra of the three natural macromolecules obtained according to specific embodiments of the present invention. The left image is the NMR spectrum of oxidized dextran, and the right image is the NMR spectrum of acrylic anhydride acylated gelatin and cysteine ​​acylated gelatin.

[0053] Figure 3 The graph shows the test results of the photoresponsive adhesion performance of the obtained bio-adhesive according to Example 1 of the present invention (adhesion strength compared with fibrin glue);

[0054] Figure 4 The image shows the adhesion longevity of the obtained bio-adhesive under wet conditions according to Example 2 of the present invention (adhesion durability compared with fibrin glue);

[0055] Figure 5 The diagram shows the biocompatibility results of the obtained bio-adhesive according to Example 3 of the present invention.

[0056] Figure 6 This is a schematic diagram illustrating the application of the photoresponsive bioadhesive of the present invention. Detailed Implementation

[0057] 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.

[0058] This invention provides a bio-adhesive with photoresponsive adhesion properties and long-lasting adhesion in wet environments, its preparation method, and its application. The bio-adhesive is composed of oxidized dextran, acrylic anhydride-acylated gelatin, and cysteine-acylated gelatin.

[0059] Figure 1 This is a schematic diagram of the synthetic routes of oxidized dextran (OD), acrylic anhydride acylated gelatin (G-AA), and cysteine ​​acylated gelatin (G-SH) in this invention, where a is a schematic diagram of the OD preparation reaction; b is a schematic diagram of the G-AA preparation reaction; and c is a schematic diagram of the G-SH preparation reaction.

[0060] In this embodiment of the invention, dextran was first dissolved in 100 ml of deionized water at a concentration of 10% (w / v) under ambient conditions of 25°C and 500 rpm. After complete dissolution, 8 g of NaIO4 was added to the solution, and the oxidation of dextran was catalyzed under light-protected conditions at room temperature and 500 rpm. After 3 hours of reaction, diethylene glycol of an equimolar amount to NaIO4 was added to terminate the dextran oxidation reaction. The termination conditions were the same as those for the oxidation reaction stage, and the time was 0.5-1 hours. After the reaction was completed, the reaction solution was dialyzed in deionized water (3500 Da) for 5 days, freeze-dried, and the resulting sample OD was stored in a dry environment at room temperature.

[0061] In this embodiment of the invention, 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) acrylic anhydride was added to the solution, and the environmental conditions during the gelatin dissolution stage were maintained for 3 hours. After the reaction was completed, the reaction solution was placed in deionized water (3500 Da) at 40°C for 5 days, then freeze-dried. The resulting sample G-AA was stored in a dry environment at room temperature.

[0062] In this embodiment of the invention, gelatin was first dissolved in 300 ml of PBS (1×) at a concentration of 1% (w / v) under environmental conditions of 50°C and 500 rpm. After complete dissolution, 2.25 g of sodium dihydrogen phosphate was added to the solution to adjust the pH value. Subsequently, 1.38 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.282 g of N-hydroxysuccinimide, and 14.604 g of cysteine ​​were added to the solution sequentially. The reaction was carried out at room temperature and 500 rpm under nitrogen purging for 8 h. After the reaction was completed, 0.225 g of dithiothreitol was added to the solution and shaken to dissolve it. After dissolution, the reaction solution was placed in deionized water (1000 Da) for 5 days, then freeze-dried. The resulting sample G-SH was stored in a dry environment at room temperature.

[0063] In this embodiment of the invention, 6 mg of the OD prepared above was dissolved in 0.6 ml of deuterated dimethyl sulfoxide; 6 mg of the G-AA and G-SH prepared above were dissolved in 0.6 ml of deuterated water, respectively, for 1H NMR spectroscopy. The NMR test results of the products in this embodiment are shown below. Figure 2 .

[0064] In this embodiment of the invention, groups with the same OD content but different G-AA and G-SH contents (the mass ratio of G-AA to G-SH was fixed at 2:1) were set up to prepare bio-adhesives. ASO was used to represent the bio-adhesive groups, where ASOⅠ consisted of 8% G-AA, 4% G-SH, and 5% OD; ASOⅡ consisted of 10% G-AA, 5% G-SH, and 5% OD; and ASOⅢ consisted of 12% G-AA, 6% G-SH, and 5% OD. OD and photoinitiator I2959 were dissolved together in PBS (1×) using vortex sonication to obtain solution I, and G-AA and G-SH were dissolved together in PBS (1×) by heating to obtain solution II. The two solutions were mixed thoroughly before use to explore the photoresponsive adhesion performance, long-term wet adhesion, and biocompatibility of the bio-adhesives.

[0065] In this embodiment of the invention, all adhesion tests shown in the figure were performed according to the American Society for Testing and Materials (ASTM) shear overlap test method.

[0066] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0067] Example 1:

[0068] (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.

[0069] (2) Then, 100 μl of the above-prepared bio-adhesive was dropped between two adhesive glass slides containing gelatin coating, and the sample was placed under ultraviolet light for 90 s to obtain a bio-adhesive sample for adhesion strength test (the control group was not subjected to ultraviolet light irradiation).

[0070] (3) The adhesion strength of the bio-adhesive was tested using a universal tensile testing machine with a strain rate of 5 mm / min. The results are shown in [Figure number missing]. Figure 3 Repeat 3-4 times for all groups.

[0071] Example 2:

[0072] (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.

[0073] (2) Then, 100 μl of the above-prepared bioadhesive was dropped between two adhesive glass slides containing gelatin coating, and the sample was placed under ultraviolet light for 90 s to obtain a bioadhesive sample for adhesion strength test. The prepared bioadhesive sample was then immersed in PBS (1×) solution.

[0074] (3) The adhesion strength of different concentrations of bio-adhesives was tested using a universal tensile testing machine at different immersion times. The strain rate of the universal tensile testing machine was 5 mm / min. The results are shown in […]. Figure 4 Repeat 3-4 times for all groups.

[0075] Example 3:

[0076] (1) The above-prepared bioadhesive was dropped into the mold and placed under ultraviolet light for 90s. Then it was sterilized by immersing in 75% ethanol, PBS (1×) and complete culture medium in sequence.

[0077] (2) After sterilization, the prepared bioadhesive was soaked in a complete culture medium at a rate of 0.2 g / ml for 72 h at an environment of 37°C to prepare a bioadhesive extract.

[0078] (3) After soaking, the prepared extract was used for L929 cell culture. Cell viability and proliferation were tested using CCK-8 reagent on days 1, 3, and 5. The results are shown in the figure. Figure 5 .

[0079] In this invention, the oxidized dextran component undergoes oxidative ring-opening treatment on some of its structural units based on the original macromolecular structure to form aldehyde groups, while the main chains of the two modified gelatin components remain unchanged from the original macromolecular structure.

[0080] The natural biomolecular-based bioadhesive based on dextran and gelatin in this invention has good biocompatibility, excellent photoresponsive adhesion characteristics, and long-lasting adhesion ability in humid environments. In situ photocrosslinking of the bioadhesive and the generation of its long-lasting adhesion ability can be achieved by ultraviolet light irradiation, thereby enabling stable adhesion of the donor cornea to the corneal implant bed, promoting the regeneration of the damaged corneal natural structure and the restoration of visual function.

[0081] The multi-component bioadhesive of this invention not only has excellent adhesion and durability in wet environments, but also, due to its photoresponsive adhesion, it has a better healing effect as a wound closure material in corneal transplantation surgery when used as a substitute for surgical sutures.

[0082] 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 photoresponsive bioadhesive based on dextran oxidation and gelatin modification, characterized in that, The photoresponsive bioadhesive consists of oxidized dextran (OD), acrylic anhydride-acylated gelatin (G-AA), and cysteine-acylated gelatin (G-SH), with the following structural formulas: ； ； ； The preparation method of the photoresponsive bioadhesive includes the following steps: (1) When using, the lyophilized OD and photoinitiator I2959 are dissolved together in PBS buffer solution, and the two components are fully dissolved by vortexing and stirring to obtain solution I; (2) Dissolve G-AA and G-SH in PBS buffer solution at a mass ratio of 2:1 and incubate in a water bath at 37 °C to obtain solution II; (3) Mix solution I and solution II in the required ratio to prepare a bioadhesive prepolymer with a fixed OD content but different concentrations of G-AA and G-SH; (4) The prepolymer of the bioadhesive is dropped onto the application site, and then photocuring and viscosity are generated by ultraviolet light irradiation.

2. The photoresponsive bioadhesive based on dextran oxidation and gelatin modification according to claim 1, characterized in that, The preparation process of the oxidized dextran OD, acrylic anhydride acylated gelatin G-AA, and cysteine ​​acylated gelatin G-SH includes the following steps: Preparation steps of oxidized dextran OD: Dissolve the dextran at a concentration of 10% w / v in 100 ml of deionized water and dissolve at 500 rpm for 1 h. After the dextran is completely dissolved, 8 g of NaIO4 is added to the solution, and the reaction is carried out at 25 °C and 500 rpm for 3 h under light-protected conditions. After the reaction was completed, diethylene glycol of the same molar amount as NaIO4 was added to the solution, and the reaction was carried out at 25 °C and 500 rpm for 0.5-1 h under light-protected conditions. After the reaction was completed, the solution was dialyzed in deionized water for 3500 Da. After dialysis, the product was freeze-dried, and the resulting oxidized dextran was stored in a dry environment at room temperature. Preparation steps of acrylic anhydride acylated gelatin G-AA: Dissolve gelatin at a concentration of 10% w / v in 100 ml of PBS buffer 1× and dissolve at 50 °C and 500 rpm for 1 h. After the gelatin is completely dissolved, 1 ml of acrylic anhydride AA is added dropwise to the solution at a rate of 0.2 ml / min. After the addition is complete, the reaction is maintained at 50 °C and 500 rpm for 3 h. After the reaction was completed, the solution was dialyzed in deionized water at 40 °C for 3500 Da. The freeze-dried product was then stored in a dry environment at room temperature. Preparation steps of cysteine-acylated gelatin G-SH: Dissolve gelatin at a concentration of 1% w / v in 300 ml of PBS buffer 1× and dissolve at 50 °C and 500 rpm for 0.5 h. After the gelatin has completely dissolved, add NaH2PO4 to the solution to adjust the pH to 4-6; After pH adjustment, 1.38 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 0.282 g of N-hydroxysuccinimide and 14.604 g of cysteine ​​were added to the solution in sequence. The reaction solution was placed at room temperature, and nitrogen gas was introduced. The reaction was carried out at 500 rpm for 8 h. After the reaction is complete, add a small amount of dithiothreitol to the solution and shake to dissolve it completely. The reaction solution was dialyzed in deionized water at 25 °C for 1000 Da. Five days after dialysis, the product was freeze-dried, and the resulting G-SH sample was stored in a dry environment at room temperature.

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