Visualized medical hydrogel and preparation method and application thereof

Abstract

This invention provides a visualized medical hydrogel, its preparation method, and its application, belonging to the fields of biomedical materials and biomedical engineering technology. The preparation method is as follows: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added to a gum arabic solution for activation, followed by the addition of 3-aminophenylborate salt. After the reaction is complete, the product is freeze-dried to obtain borate-treated gum arabic. Fluorescent nanoparticles are dissolved in water, and gelatin, borate-treated gum arabic, and arginine solution are added. 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are then added for activation, yielding the visualized medical hydrogel. This visualized medical hydrogel exhibits excellent fluorescence, anti-inflammatory, and self-repairing functions, demonstrating good spinal cord injury repair performance and real-time monitoring performance in the application of biological tissue materials for spinal cord injury repair.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and biomedical engineering technology, and relates to a visualized medical hydrogel, its preparation method and application. Background Technology

[0002] Spinal cord injury (SCI) is devastating. In SCI, the powerful traumatic impact on the spinal cord leads to permanent loss of neurological function below the level of injury. SCI is one of the most important and fundamental problems facing the entire medical community, and in the past 20 years, these issues have attracted significant attention and have been systematically and deeply studied as an independent subject. Due to unfavorable microenvironmental conditions and limited intrinsic regenerative capacity of nerve cells, endogenous neural repair remains a challenge. Currently, the main clinical treatment for SCI is surgical decompression, but due to the complexity of the microenvironment at the injury site, it often fails to achieve effective therapeutic results. Today, various tissue engineering methods based on biomaterials have been used to treat SCI, including nanoparticles, decellularized extracellular matrix (dECM), and hydrogels. Among these, hydrogels have attracted considerable attention because they can provide a hydrated biological environment and encapsulate drugs that promote tissue regeneration. However, existing hydrogel materials rely on the drugs they load to promote nerve repair, but they cannot monitor the repair progress at the injury site in real time.

[0003] Therefore, it is necessary to develop a biomaterial that can improve and monitor the results of nerve regeneration therapy in real time to achieve spinal cord injury repair. Summary of the Invention

[0004] The purpose of this invention is to at least partially solve the aforementioned technical problems, and to provide a visual medical hydrogel, its preparation method, and its applications. The visual medical hydrogel prepared by this invention exhibits excellent in vivo fluorescence imaging performance, adhesion performance, anti-inflammatory and self-healing properties, and also possesses good biocompatibility and injectability, making it suitable for spinal cord injury repair and real-time monitoring of the repair process.

[0005] In a first aspect of the present invention, a method for preparing a visualized medical hydrogel is provided, the method comprising the following steps:

[0006] S1. Add peach gum to PBS buffer, adjust the pH to 4-6, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to carry out the activation reaction, then add 3-aminophenylborate salt to carry out the reaction. After the reaction is completed, freeze-dry the product to obtain borate-treated peach gum.

[0007] S2. Fluorescent nanoparticles are dissolved in water, and gelatin, the borate gum and arginine solution are added. After stirring evenly, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added to activate the reaction and obtain a visualized medical hydrogel.

[0008] In some embodiments of the present invention, the fluorescent nanoparticles have one or more of the following groups: carboxyl, amino, and aldehyde.

[0009] In some embodiments of the present invention, the fluorescent nanoparticles are fluorescent nanoparticles Se@ICG, which are prepared by the following method: selenium quantum dots are added to water, indocyanine green is added, and after the reaction is complete, the product is freeze-dried to obtain fluorescent nanoparticles Se@ICG. The selenium quantum dots have carboxyl and amino groups on their surface and have a maximum emission peak at the maximum excitation of 590 nm, with an emission wavelength of 650–750 nm.

[0010] In some embodiments of the present invention, the preparation method of selenium quantum dots is as follows: sodium selenite pentahydrate, citric acid, and arginine are dissolved in distilled water, the pH is adjusted to 9-11, the mixed solution is added to a reaction vessel, heated at 160℃-180℃ for 6-10 h, cooled to room temperature, dialyzed with distilled water for 3-5 days, and freeze-dried to obtain purified selenium quantum dots (SeQDs).

[0011] In some embodiments of the present invention, the mass ratio of the selenium quantum dots to indocyanine green is (10-15):(1-2).

[0012] In some embodiments of the present invention, in step S1, the mass ratio of the gum arabic, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and 3-aminophenylborate salt is (100-120):(47-50):(28-30):(28-30).

[0013] In some embodiments of the present invention, in step S1, the activation reaction takes 15 to 30 minutes.

[0014] In some embodiments of the present invention, in step S1, the reaction time after adding 3-aminophenylborate acid salt is 10-12 h.

[0015] In some embodiments of the present invention, in step S2, the ratio of fluorescent nanoparticles, gelatin, borate gum, arginine solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is (20-25) mg: (42-50) mg: (50-60) mg: (8-10) μL: (8-10) mg: (3-5) mg, and the concentration of the arginine solution is 0.1 g / mL.

[0016] In some embodiments of the present invention, the activation reaction time in step S2 is 4 to 5 hours.

[0017] In a second aspect, the present invention provides a visual medical hydrogel prepared by the above-described preparation method.

[0018] A third aspect of the present invention provides an application of the above-described visualized medical hydrogel in medical materials, wherein the medical materials are used for spinal cord injury repair or for real-time monitoring of the spinal cord injury repair process.

[0019] The visualized medical hydrogel, its preparation method, and its application according to embodiments of the present invention have at least one of the following advantages:

[0020] (1) This invention uses natural peach gum polysaccharide as the main component and adds fluorescent nanoparticles. Based on covalent and reversible non-covalent bonds, a visual adhesion self-healing hydrogel is prepared, which can realize in vivo imaging, delay the fluorescence quenching time of fluorescent ions, realize in situ injection, and has good biocompatibility.

[0021] (2) By adding fluorescent nanoparticles with carboxyl, amino or aldehyde groups, this invention not only enables in vivo imaging, but also delays the fluorescence quenching time of fluorescent ions, enables in situ injection, and enables the hydrogel to have self-healing and adhesion properties.

[0022] (3) The preparation method provided by the present invention is mild and simple.

[0023] (4) This hydrogel can realize the visual repair of spinal cord injury. Attached Figure Description

[0024] These and / or other aspects and advantages of the present invention will become apparent and readily understood from the following description of preferred embodiments taken in conjunction with the accompanying drawings, in which:

[0025] Figure 1 This is a diagram illustrating the self-healing effect test process of Test Example 1;

[0026] Figure 2 This is the fluorescence imaging result of test example 2;

[0027] Figure 3This is an in vivo imaging test result of selenium quantum dots in test example 3;

[0028] Figure 4 This is a schematic diagram of the adhesion of the medical hydrogel prepared in Example 1. Detailed Implementation

[0029] The technical solution of the present invention will be further described in detail below through embodiments and in conjunction with the accompanying drawings. In this specification, the same or similar reference numerals indicate the same or similar components. The following description of the embodiments of the present invention with reference to the accompanying drawings is intended to explain the overall inventive concept of the present invention and should not be construed as a limitation thereof.

[0030] Example 1

[0031] 1) Dissolve 5.26 mg sodium selenite pentahydrate, 38.42 mg citric acid, and 139.36 mg arginine in 40 mL of distilled water, adjust the pH to 11, add the mixed solution to a reaction vessel, heat at 180 ℃ for 6 h, cool to room temperature, dialyze against distilled water for 3-5 days, and freeze-dry to obtain purified selenium quantum dots (SeQDs). These selenium quantum dots exhibit a maximum emission peak at 590 nm of maximum excitation, with an emission wavelength of 683 nm.

[0032] 2) Add 20 mg of purified selenium quantum dots to 50 mL of distilled water, add 2.05 mg of indocyanine green (ICG), and react for 10–12 h. Wash with water, centrifuge three times, and freeze-dry to obtain Se@ICG.

[0033] 3) Weigh 1000 mg of purified peach gum and dissolve it in 100 mL of calcium and magnesium-free PBS buffer. Adjust the pH to 5 with 0.004 mol / mL hydrochloric acid. Add 470 mg of EDC•HCl and 280 mg of NHS to activate the peach gum solution for 15 min. Add 280 mg of 3-aminophenylborate salt and react for 12 h. Dialyze for 3 days and freeze-dry to obtain borate-treated peach gum (PR-3APBA).

[0034] 4) Weigh 20 mg of Se@ICG and dissolve it in 1 mL of distilled water. Add 50 mg of gelatin and 60 mg of borate gum to the Se@ICG solution.

[0035] 5) After dissolving, add 10 μL of 0.1 g / mL arginine solution, stir well, then add 10 mg EDC•HCl and 5 mg NHS, and let stand for 4-5 h to obtain boronized peach gum gelatin selenium quantum dot indocyanine green hydrogel, which is a visual medical hydrogel.

[0036] Example 2

[0037] 1) Dissolve 26.3 mg sodium selenite pentahydrate, 192.1 mg citric acid, and 696.8 mg arginine in 200 mL of distilled water, adjust the pH to 11, add the mixed solution to the reaction vessel, heat at 180 °C for 6 h, cool to room temperature, dialyze against distilled water for 3-5 days, and freeze dry to obtain purified selenium quantum dots (SeQDs).

[0038] 2) Add 39.98 mg of purified selenium quantum dots to 50 mL of distilled water, add 4.01 mg of indocyanine green (ICG), and react for 10–12 h. Wash with water, centrifuge three times, and freeze-dry to obtain Se@ICG.

[0039] 3) Weigh 987 mg of purified peach gum and dissolve it in 100 mL of calcium and magnesium-free PBS buffer. Adjust the pH to 5 with 0.004 mol / mL hydrochloric acid. Add 466 mg of EDC•HCl and 274 mg of NHS to activate the peach gum solution for 15 min. Add 291 mg of 3-aminophenylborate salt and react for 12 h. Dialyze for 3 days and freeze-dry to obtain borate-treated peach gum (PR-3APBA).

[0040] 4) Weigh 19.88 mg of Se@ICG and dissolve it in 1 mL of distilled water. Add 50.11 mg of gelatin and 59.76 mg of borate gum to the Se@ICG solution.

[0041] 5) After dissolving, add 10 μL of 0.1 g / mL arginine solution, stir well, then add 10.02 mg EDC•HCl and 4.86 mg NHS, and let stand for 4-5 h to obtain boronized peach gum gelatin selenium quantum dot indocyanine green hydrogel, which is a visual medical hydrogel.

[0042] Example 3

[0043] 1) Dissolve 26.5 mg sodium selenite pentahydrate, 191.8 mg citric acid, and 700 mg arginine in 200 mL of distilled water, adjust the pH to 11, add the mixed solution to the reaction vessel, heat at 180 °C for 6 h, cool to room temperature, dialyze with distilled water for 3-5 days, and freeze dry to obtain purified selenium quantum dots (SeQDs).

[0044] 2) 19.68 mg of purified selenium quantum dots were added to 50 mL of distilled water, followed by 1.97 mg of indocyanine green (ICG). The reaction was allowed to proceed for 10–12 h. The mixture was washed with water, centrifuged three times, and freeze-dried to obtain Se@ICG.

[0045] 3) Weigh 1024 mg of purified peach gum and dissolve it in 100 mL of calcium and magnesium-free PBS buffer. Adjust the pH to 5 with 0.004 mol / mL hydrochloric acid. Add 468 mg of EDC•HCl and 277 mg of NHS to activate the peach gum solution for 15 min. Add 287 mg of 3-aminophenylborate salt and react for 12 h. Dialyze for 3 days and freeze-dry to obtain borate-treated peach gum (PR-3APBA).

[0046] 4) Weigh 20.05 mg of Se@ICG and dissolve it in 1 mL of distilled water. Add 49.76 mg of gelatin and 60.12 mg of borate gum to the Se@ICG solution.

[0047] 5) After dissolving, add 10 μL of 0.1 g / mL arginine solution, stir well, then add 9.98 mg EDC•HCl and 5.06 mg NHS, and let stand for 4–5 h to obtain boronized peach gum gelatin selenium quantum dot indocyanine green hydrogel, which is a visual medical hydrogel.

[0048] Comparative Example 1: Gel without selenium fluorescent particles

[0049] 1) Weigh 1000 mg of purified peach gum and dissolve it in 100 mL of calcium and magnesium-free PBS buffer. Adjust the pH to 5 with 0.004 mol / mL hydrochloric acid. Add 470 mg of EDC•HCl and 280 mg of NHS to activate the peach gum solution for 15 min. Add 280 mg of 3-aminophenylborate salt and react for 12 h. Dialyze for 3 days and freeze-dry to obtain borate-treated peach gum (PR-3APBA).

[0050] 2) Weigh 50 mg of gelatin and dissolve it in 1 mL of distilled water, then add 60 mg of borate-treated peach gum.

[0051] 3) After dissolving, add 10 μL of 0.1 g / mL arginine solution, stir well, then add 10 mg EDC•HCl and 5 mg NHS, and let stand for 4–5 h to obtain a hydrogel without selenium fluorescent particles.

[0052] Test Example 1

[0053] The self-healing effect test process of the visualized medical hydrogel prepared in Example 1 is as follows: Figure 1As shown in the diagram. The specific process is as follows: Take 0.5 mL of the visualized medical hydrogel (A) prepared in Example 1 and cut it evenly into four small pieces (B). Gently bring the four small pieces of visualized adhesive self-healing hydrogel close together and wait for 5 minutes. After a period of time, the four pieces of bio-gel form a whole (C), and it will not break when lifted by holding one end with tweezers (D). It can be seen that the visualized adhesive self-healing hydrogel provided by the present invention forms a whole after a period of time when the four pieces of bio-gel are cut, and it does not break when stretched, proving that the bio-gel has good self-healing properties.

[0054] The hydrogel without selenium fluorescent particles prepared in Comparative Example 1 was uniformly cut into four small pieces and gently brought together. After waiting for 5 minutes, it was found that it did not form a whole. This shows that the gel without selenium fluorescent particles does not have self-healing properties. This further proves that adding selenium fluorescent particles can make the hydrogel have self-healing and adhesion properties.

[0055] Test Example 2

[0056] The Se@ICG prepared in Example 1 was dissolved in distilled water to prepare a 20 mg / mL Se@ICG solution.

[0057] 0.1 mL of the visualization medical hydrogel or Se@ICG solution (20 mg / mL) prepared in Example 1 was injected into the back of rats. In vivo imaging was performed on the rats 24 hours after injection to observe whether there was a fluorescent signal on the rat's back. The results are as follows: Figure 2 As shown.

[0058] As can be seen, mice injected subcutaneously with the visualized medical hydrogel prepared in Example 1 still showed strong fluorescence signals on their backs after 24 hours, while mice injected with Se@ICG solution (20 mg / mL) showed no fluorescence signals on their backs after 24 hours. This proves that the visualized medical hydrogel provided by the present invention can achieve in situ injection and improve aggregation.

[0059] Test Example 3

[0060] Inoculate 1x10⁻⁶ cells into a 24-well plate. 5 Fibroblasts were cultured adherently, and 10 μL of selenium quantum dot solution (20 mg / mL) was added to each well. After 24 hours of co-culture, the cells were observed under a fluorescence microscope to check for fluorescence signals. The cell imaging results are as follows: Figure 3 As shown in the left figure, the results indicate that selenium quantum dot solution can induce red fluorescence signals in cells.

[0061] 1 mL of selenium quantum dot solution (20 mg / mL) was subcutaneously injected into the back of a rat. Immediately after injection, in vivo imaging was performed to observe for any fluorescent signals in the back. The animal in vivo imaging results are as follows: Figure 3As shown in the right figure, the results indicate that selenium quantum dot solution cannot be used for subcutaneous imaging on the back of rats. This demonstrates that although selenium quantum dots possess fluorescence properties, they cannot achieve in vivo imaging.

[0062] Figure 4 This is a visualization of the adhesion effect of the medical hydrogel prepared in Example 1. From Figure 4 It can be seen that the severed spinal cord can stick together after applying the bio-adhesive, proving that the bio-adhesive has good adhesion.

[0063] While some embodiments of the present general inventive concept have been shown and described, those skilled in the art will understand that changes may be made to these embodiments without departing from the principles and spirit of the present general inventive concept, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for preparing a visualized medical hydrogel, characterized in that, The preparation method includes the following steps: S1. Add peach gum to PBS buffer, adjust the pH to 4-6, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to carry out the activation reaction, then add 3-aminophenylborate salt to carry out the reaction. After the reaction is completed, freeze-dry the product to obtain borate-treated peach gum. S2. Dissolve fluorescent nanoparticles in water, add gelatin, the borate gum and arginine solution, stir evenly, then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to activate the reaction and obtain the visualized medical hydrogel; The fluorescent nanoparticles have one or more of the following groups on their surface: carboxyl, amino, and aldehyde.

2. The preparation method according to claim 1, characterized in that, The fluorescent nanoparticles are Se@ICG fluorescent nanoparticles, which are prepared by the following method: Selenium quantum dots were added to water, indocyanine green was added, and after the reaction was complete, the product was freeze-dried to obtain the fluorescent nanoparticles. The selenium quantum dots have carboxyl and amino groups on their surface and have a maximum emission peak at 590 nm with a wavelength between 650 and 750 nm.

3. The preparation method according to claim 2, characterized in that, The mass ratio of the selenium quantum dots to indocyanine green is (10-15):(1-2).

4. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of the peach gum, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and 3-aminophenylborate salt is (100-120):(47-50):(28-30):(28-30).

5. The preparation method according to claim 1, characterized in that, In step S1, the activation reaction takes 15–30 min, and the reaction time after adding 3-aminophenylborate acid salt is 10–12 h.

6. The preparation method according to claim 1, characterized in that, In step S2, the ratio of fluorescent nanoparticles, gelatin, borate gum, arginine solution, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is (20-25) mg: (42-50) mg: (50-60) mg: (8-10) μL: (8-10) mg: (3-5) mg, and the concentration of the arginine solution is 0.1 g / mL.

7. The preparation method according to claim 1, characterized in that, In step S2, the activation reaction takes 4 to 5 hours.

8. A visual medical hydrogel prepared by the preparation method according to any one of claims 1 to 7.

9. The application of the visual medical hydrogel as described in claim 8 in the preparation of medical materials, wherein the medical materials are used for spinal cord injury repair or for real-time monitoring of the spinal cord injury repair process.

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