Visual medical hydrogel as well as preparation method and application thereof

Abstract

The embodiment of the invention provides visual medical hydrogel as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials and biomedical engineering. The preparation method comprises the following steps: adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide into a peach gum solution, carrying out an activation reaction, then adding 3-aminophenylboronic acid hydrochloride, carrying out a reaction, and after the reaction is completed, carrying out freeze drying on a product to obtain boronic acid peach gum; the preparation method comprises the following steps: dissolving fluorescent nanoparticles in water, adding gelatin, boric acid peach gum and an arginine solution, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and carrying out an activation reaction to obtain the visual medical hydrogel. The visual medical hydrogel has excellent fluorescence, anti-inflammation and self-repairing functions, and has good spinal cord injury repairing performance and injury repairing real-time monitoring performance when applied to spinal cord injury repairing biological tissue materials.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials and biomedical engineering, and relates to a visualized medical hydrogel and a preparation method and application thereof. Background Art

[0002] Spinal cord injury (SCI) is devastating. In SCI, strong traumatic forces impact the spinal cord, resulting in permanent loss of neurological function below the level of injury. Spinal cord injury is one of the most important and fundamental problems faced by the entire medical community. In the past 20 years, these problems have attracted great attention and have been systematically and deeply studied as an independent subject. Endogenous neural repair remains a challenge due to poor microenvironmental conditions and limited intrinsic regenerative capacity of nerve cells. At present, the clinical treatment of SCI is mainly surgical decompression, but due to the complexity of the microenvironment at the injury site, it is often difficult to achieve effective therapeutic effects. Nowadays, various biomaterial-based tissue engineering methods have been used to treat SCI, including nanoparticles, decellularized extracellular matrix (dECM) and hydrogels. Among them, hydrogels have attracted much attention because of their ability to provide a hydrated biological environment and encapsulate drugs that are beneficial to tissue regeneration. However, existing hydrogel materials rely on the drugs they load to promote neural repair, but cannot monitor the repair of 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 the present invention is to at least partially solve the above technical problems, and to provide a visualized medical hydrogel and its preparation method and application. The visualized medical hydrogel prepared by the present invention has excellent in vivo fluorescence imaging performance, adhesion performance, anti-inflammatory and self-repairing performance, and has good biocompatibility and injectability, and can be used 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. adding peach gum to PBS buffer, adjusting the pH to 4-6, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to carry out activation reaction, and then adding 3-aminophenylborate hydrochloride to react. After the reaction is completed, the product is freeze-dried to obtain borated peach gum;

[0007] S2. Dissolve the fluorescent nanoparticles in water, add gelatin, the borated peach gum and arginine solution, stir evenly, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to activate the reaction to obtain a visualized medical hydrogel.

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

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

[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 reactor, heated at 160°C-180°C for 6-10 hours, 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 peach gum, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and 3-aminophenylborate hydrochloride is (100-120):(47-50):(28-30):(28-30).

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

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

[0015] In some embodiments of the present invention, in step S2, the amount ratio of the fluorescent nanoparticles, gelatin, the borated peach gum, the 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, in step S3, the activation reaction time is 4 to 5 hours.

[0017] The second aspect of the present invention provides a visualized medical hydrogel prepared by the above-mentioned preparation method.

[0018] The third aspect of the present invention provides a use of the above-mentioned visualized medical hydrogel in medical materials, wherein the medical material is used for repairing spinal cord injuries or for real-time monitoring of the spinal cord injury repair process.

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

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

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

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

[0023] (4) The hydrogel can achieve visual repair of spinal cord injury. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0025] Figure 1 is a self-repair effect test process diagram of test case 1;

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

[0027] Figure 3 This is a graph of the in vivo imaging test results of selenium quantum dots in Test Example 3;

[0028] Figure 4 Schematic diagram of visualized medical hydrogel adhesion prepared in Example 1; A is a schematic diagram of the spinal cord without the use of hydrogel; B is a schematic diagram of the spinal cord after the use of hydrogel. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further specifically described below by examples and in conjunction with the accompanying drawings. In the 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 of the present invention.

[0030] Example 1

[0031] 1) Dissolve 5.26 mg of sodium selenite pentahydrate, 38.42 mg of citric acid, and 139.36 mg of arginine in 40 mL of distilled water, adjust the pH to 11, add the mixed solution to a reactor, 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). The selenium quantum dots have a maximum emission peak at a maximum excitation of 590 nm and 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), react for 10 to 12 hours, 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 PBS buffer without calcium and magnesium ions. Use 0.004 mol / mL hydrochloric acid to adjust the pH value to 5. 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 hydrochloride and react for 12 h. Dialyze for 3 days and freeze-dry to obtain borated 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 borated peach gum to the Se@ICG solution.

[0035] 5) After dissolution, add 10 μL of 0.1 g / mL arginine solution, stir evenly, add 10 mg EDC·HCl and 5 mg NHS, and let stand for 4 to 5 hours to obtain borated peach gum gelatin selenium quantum dot indocyanine green hydrogel, that is, visualized 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 distilled water, adjust the pH to 11, add the mixed solution into a reactor, 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).

[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), react for 10 to 12 hours, 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 PBS buffer without calcium and magnesium ions. Use 0.004 mol / mL hydrochloric acid to adjust the pH value to 5. 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 hydrochloride and react for 12 h. Dialyze for 3 days and freeze-dry to obtain borated 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 borated peach gum to the Se@ICG solution.

[0041] 5) After dissolution, add 10 μL of 0.1 g / mL arginine solution, stir evenly, add 10.02 mg EDC·HCl and 4.86 mg NHS, and let stand for 4 to 5 hours to obtain borated peach gum gelatin selenium quantum dot indocyanine green hydrogel, i.e., visualized 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 distilled water, adjust the pH to 11, add the mixed solution into a reactor, 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).

[0044] 2) Add 19.68 mg of purified selenium quantum dots to 50 mL of distilled water, add 1.97 mg of indocyanine green (ICG), react for 10 to 12 hours, wash with water, centrifuge three times, and freeze-dry to obtain Se@ICG.

[0045] 3) Weigh 1024 mg of purified peach gum and dissolve it in 100 mL of PBS buffer without calcium and magnesium ions. Use 0.004 mol / mL hydrochloric acid to adjust the pH value to 5. 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 hydrochloride and react for 12 h. Dialyze for 3 days and freeze-dry to obtain borated 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 borated peach gum to the Se@ICG solution.

[0047] 5) After dissolution, add 10 μL of 0.1 g / mL arginine solution, stir evenly, add 9.98 mg EDC·HCl and 5.06 mg NHS, and let stand for 4 to 5 hours to obtain borated peach gum gelatin selenium quantum dot indocyanine green hydrogel, i.e., visualized medical hydrogel.

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

[0049] 1) Weigh 1000 mg of purified peach gum and dissolve it in 100 mL of PBS buffer without calcium and magnesium ions. Use 0.004 mol / mL hydrochloric acid to adjust the pH value to 5. 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 hydrochloride and react for 12 h. Dialyze for 3 days and freeze-dry to obtain borated 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 borated peach gum.

[0051] 3) After dissolution, 10 μL of 0.1 g / mL arginine solution was added, and after stirring evenly, 10 mg of EDC·HCl and 5 mg of NHS were added, and the mixture was allowed to stand for 4 to 5 hours 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 1 As shown. The specific process is: take 0.5mL of the visualized medical hydrogel (A) prepared in Example 1, and cut it evenly into four small pieces (B). Gently put the four small pieces of visualized adhesive self-repairing hydrogel close together, wait for 5 minutes, and the biological glue cut into four pieces forms a whole after a period of time (C), and it will not break after being lifted up by clamping one end with tweezers (D). It can be seen that the visualized adhesive self-repairing hydrogel provided by the present invention is cut into four pieces of biological glue, which forms a whole after a period of time and does not break when stretched, proving that the biological glue has good self-repairing properties.

[0054] The hydrogel without selenium fluorescent particles prepared in Comparative Example 1 was evenly cut into four small pieces and gently pressed together. After waiting for 5 minutes, it was found that it did not form a whole. This shows that the glue without adding selenium fluorescent particles does not have self-healing properties, which 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 visualized medical hydrogel or Se@ICG solution (20 mg / mL) prepared in Example 1 was injected into the back of the rat. Live imaging was performed on the rat 24 hours after the injection to observe whether there was a fluorescent signal on the back of the rat. The results are as follows: Figure 2 shown.

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

[0059] Test Example 3

[0060] 1x10 cells were seeded in a 24-well plate 5 After adherent culture, 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 see if there was any fluorescence signal. The cell imaging results are shown in the figure below. Figure 3 As shown in the left figure, the results show that selenium quantum dot solution can cause cells to produce red fluorescence signals.

[0061] 1 mL of selenium quantum dot solution (20 mg / mL) was injected subcutaneously on the back of rats. Live imaging was performed immediately after the injection to observe whether there was a fluorescent signal on the back. The results of animal live imaging are as follows: Figure 3 As shown in the figure on the right, the results show that the selenium quantum dot solution cannot be imaged under the skin of the rat's back. This proves that although selenium quantum dots have fluorescent properties, they cannot achieve in vivo imaging.

[0062] Figure 4 1 is a diagram showing the adhesion effect of the visualized medical hydrogel prepared in Example 1. Figure 4 It can be seen that the disconnected spinal cords can be adhered together after applying the bioglue, proving that the bioglue has good adhesion.

[0063] Although some embodiments of the present general inventive concept have been shown and described, it will be appreciated by those skilled in the art 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 comprises the following steps: S1. adding peach gum to PBS buffer, adjusting the pH to 4-6, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to carry out activation reaction, and then adding 3-aminophenylborate hydrochloride to react. After the reaction is completed, the product is freeze-dried to obtain borated peach gum; S2. Dissolve the fluorescent nanoparticles in water, add gelatin, the borated peach gum and arginine solution, stir evenly, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide to activate the reaction to obtain the visualized medical hydrogel.

2. The preparation method according to claim 1, characterized in that The fluorescent nanoparticles have one or more of carboxyl, amino and aldehyde groups on their surfaces.

3. The preparation method according to claim 1, characterized in that: The fluorescent nanoparticles are fluorescent nanoparticles Se@ICG, and the fluorescent nanoparticles are prepared by the following method: Adding selenium quantum dots into water and indocyanine green, after the reaction is complete, freeze-drying the product to obtain the fluorescent nanoparticles; The selenium quantum dots have carboxyl groups and amino groups on their surfaces, and have a maximum emission peak at a maximum excitation of 590 nanometers, with an emission wavelength of 650 to 750 nanometers.

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

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

6. The preparation method according to claim 1, characterized in that: In step S1, the activation reaction time is 15 to 30 minutes, and the reaction time after adding 3-aminophenylborate hydrochloride is 10 to 12 hours.

7. The preparation method according to claim 1, characterized in that: In step S2, the usage ratio of the fluorescent nanoparticles, gelatin, the borated peach gum, the 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.

8. The preparation method according to claim 1, characterized in that: In step S3, the activation reaction time is 4 to 5 hours.

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

10. Use of the visualized medical hydrogel according to claim 9 in medical materials, wherein the medical materials are used for repairing spinal cord injuries or for real-time monitoring of the repair process of spinal cord injuries.

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