A fluorescent self-healing injectable hydrogel and a preparation method thereof

Abstract

The application discloses a preparation method of a fluorescent self-healing injectable hydrogel and application thereof. A biocompatible fluorescent small molecule is anchored on a polysaccharide macromolecular chain through an EDC / NHS coupling reaction, and then in-situ cross-linking is carried out through a dynamic imine bond under physiological conditions to prepare a fluorescent self-healing injectable hydrogel. The fluorescent self-healing injectable hydrogel has self-healing, injectability, shape adaptability, biodegradability, cell compatibility, tissue compatibility and outstanding fluorescence effect, and can be used for non-destructive, in-situ and real-time detection, thereby providing a new strategy for synthesizing a polysaccharide-based fluorescent self-healing injectable hydrogel under mild conditions, and simultaneously providing a new idea for establishing a system model of an in-situ real-time monitoring biomedical platform of minimally invasive interventional treatment technology.

Description

Technical Field

[0001] This invention belongs to the field of flexible biomaterials technology and relates to a fluorescent self-healing injectable hydrogel and its preparation method. Background Technology

[0002] Biocompatible self-healing injectable hydrogels hold great promise for applications in the biomedical field. Their key characteristic is their ability to spontaneously recover structure and function after injury, significantly enhancing their performance as intelligent flexible materials. As intelligent multifunctional biomaterials, self-healing injectable hydrogels can be combined with minimally invasive interventional techniques, enabling their widespread application in tissue engineering, drug / cell delivery, and biosensors. The advantages of self-healing injectable hydrogels lie in their ability to gel in situ within a syringe → inject the gel at the target site → allow the gel particles to heal autonomously → reshape themselves at the target site. This departs from the traditional fluid manipulation method of injectable hydrogels, which involves loading the precursor solution into the syringe → injecting the solution at the target site → gelling at the target site. Simultaneously, self-healing injectable hydrogels overcome many shortcomings of traditional injectable hydrogels, such as the loss of drugs and cells due to diffusion before gelation, difficulty in controlling gelation time, and the potential toxicity of chemicals (e.g., monomers, initiators). Despite significant advancements, conventional self-healing injectable hydrogels still lack in-situ real-time monitoring capabilities, limiting non-invasive monitoring and tracking after implantation. To date, non-invasive and tracking capabilities within the body remain a significant challenge for self-healing injectable hydrogels.

[0003] Fluorescence monitoring technology offers advantages such as rapid response, high sensitivity, high contrast, and non-invasiveness, facilitating real-time monitoring of fluorescent hydrogels. Fluorescent hydrogels, with their good biocompatibility, have attracted significant attention in the biomedical field and have specific applications in biomedical platforms, such as bioimaging and biomonitoring. Coupling or doping various fluorescent substances (such as lanthanides, organic dyes, quantum dots, fluorescent proteins, and metal ligands) into the hydrogel matrix can endow the hydrogel with fluorescence properties. However, most fluorescent substances suffer from drawbacks such as biotoxicity, poor water solubility, and complex preparation processes, which greatly limit their practical application in the biomedical field.

[0004] Multifunctional fluorescent self-healing injectable hydrogels hold immense potential for application in biomedical platforms for real-time bioimaging monitoring. While current strategies focus on developing various self-healing injectable hydrogels suitable for the biomedical field, few studies have addressed the application of self-healing injectable hydrogels that simultaneously possess fluorescence, biocompatibility, and biodegradability in biomedical applications. Fluorescent self-healing injectable hydrogels promise to enable real-time in vivo imaging monitoring via non-invasive methods without interfering with treatment efficacy and safety. However, developing simple and effective strategies for immobilizing biocompatible fluorescent substances within dynamic three-dimensional hydrogel networks, and synthesizing fluorescent self-healing injectable hydrogels for biomedical platforms, remains a major challenge for researchers.

[0005] Therefore, it is necessary to combine fluorescent substances with excellent biosafety and easy operation with hydrogels to prepare fluorescent self-healing injectable hydrogels that have both excellent fluorescence properties and the functions of polysaccharide-based dynamic hydrogels (self-healing, injectability, biocompatibility and biodegradability, etc.), thereby realizing non-invasive monitoring and tracking and minimally invasive interventional therapy. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a fluorescent self-healing injectable hydrogel and its preparation method, thereby solving the technical problems of poor biosafety of fluorescent substances, poor fluorescence effect, and lack of in-situ real-time monitoring function in self-healing injectable hydrogels in the prior art.

[0007] This invention is achieved through the following technical solution:

[0008] A method for preparing a fluorescent self-healing injectable hydrogel includes the following steps:

[0009] S1: Dissolve the bioluminescent molecule in a 2-morphine ethanesulfonic acid solution, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and stir to dissolve to obtain a mixed solution; add a 2-morphine ethanesulfonic acid solution of N-carboxyethyl chitosan to the mixed solution, and stir to react to obtain a bioluminescent molecule / N-carboxyethyl chitosan polymer;

[0010] S2: Mix the PBS solution containing the bioluminescent molecule / N-carboxyethyl chitosan polymer with the PBS solution containing sodium alginate to prepare the fluorescent self-healing injectable hydrogel.

[0011] Preferably, the preparation of the bioluminescent molecule specifically involves: mixing citric acid powder and cysteine ​​evenly, heating to react, washing the product, and then recrystallizing to obtain the bioluminescent molecule.

[0012] Preferably, the N-carboxyethyl chitosan is prepared by reacting chitosan with acrylic acid; wherein, in the mixed solution of chitosan and acrylic acid, the concentration of chitosan is 5-10 mM and the concentration of acrylic acid is 20-25 mM.

[0013] Preferably, the degree of deacetylation of the chitosan is 86%–88%, and the molecular weight of the chitosan is 2*10. 5 ~3*10 5 g / mol.

[0014] Preferably, the degree of amino substitution of the N-carboxyethyl chitosan is 48% to 50%.

[0015] Preferably, the oxidized sodium alginate is obtained by oxidizing a mixture of sodium periodate and sodium alginate; wherein, in the mixed solution of sodium periodate and sodium alginate, the concentration of sodium alginate is 5.4*10⁻⁶. -3 ~1.2*10 -2 The concentration of sodium periodate is 1.0 x 10 g / mL. -2 ~1.2*10 -2 g / mL.

[0016] Preferably, the viscosity of the sodium alginate is 350-400 mPa·s.

[0017] Preferably, the degree of oxidation of the oxidized sodium alginate is 48% to 55%.

[0018] Preferably, in step S1, after adding the 2-morphine ethanesulfonic acid solution of N-carboxyethyl chitosan to the mixed solution, the mass fraction of N-carboxyethyl chitosan is 1.5 wt% to 2.5 wt%; in step S2, the mass fraction of sodium alginate in the PBS solution is 0.5 wt% to 1.5 wt%.

[0019] A fluorescent self-healing injectable hydrogel was prepared by the method described above.

[0020] Compared with the prior art, the present invention has the following beneficial technical effects:

[0021] This invention utilizes nitrogen- and sulfur-doped (N, S-CDs) carbon dots, exhibiting significant fluorescence properties. Its main component, sodium 5-oxo-3,5-dihydrothiazol-3,2-pyridine-7-carboxylate (TPCNa), possesses excellent biocompatibility. This invention modifies bioluminescent small molecules onto N-carboxyethyl chitosan macromolecular chains via a coupling reaction, and then in situ crosslinks the bioluminescent molecules / N-carboxyethyl chitosan polymer with oxidized sodium alginate through reversible dynamic imine groups, resulting in the fluorescent self-healing injectable hydrogel of this invention. Simultaneously, in this invention, the bioluminescent molecules are chemically coupled with the hydrogel, resulting in more uniform dispersion of the fluorescent material within the hydrogel and effectively preventing fluorescence quenching. This fluorescent self-healing injectable hydrogel not only possesses the self-healing, injectability, biocompatibility, and biodegradability of polysaccharide-based dynamic hydrogels, but also offers non-invasive real-time monitoring capabilities due to its photoluminescence properties. The fluorescent self-healing injectable hydrogel provided by this invention is easy to prepare, has mild reaction conditions, and excellent biocompatibility, and has great application prospects in the field of bio-imaging for minimally invasive interventional therapy.

[0022] Furthermore, the preparation of the bioluminescent molecule specifically involves: mixing citric acid powder and cysteine ​​evenly, heating and reacting, washing the product, and recrystallizing to obtain the bioluminescent molecule. The obtained bioluminescent molecule has good cell compatibility and tissue compatibility.

[0023] Furthermore, the N-carboxyethyl chitosan is prepared by reacting chitosan with acrylic acid, which can improve the water solubility of chitosan; wherein, in the mixed solution of chitosan and acrylic acid, the concentration of chitosan is 5-10 mM and the concentration of acrylic acid is 20-25 mM, which can ensure that the grafting degree of chitosan meets the application requirements.

[0024] Furthermore, the degree of deacetylation of the chitosan is 86%–88%, and the molecular weight of the chitosan is 2*10. 5 ~3*10 5 A concentration of g / mol can make chitosan have a moderate viscosity and enable it to form a hydrogel well.

[0025] Furthermore, the degree of amino substitution of the N-carboxyethyl chitosan is 48% to 50%, which can improve the water solubility of chitosan while retaining a certain amount of amino groups.

[0026] Furthermore, the oxidized sodium alginate is obtained by oxidizing a mixture of sodium periodate and sodium alginate, which results in the sodium alginate molecule containing a certain amount of aldehyde groups; wherein, in the mixed solution of sodium periodate and sodium alginate, the concentration of sodium alginate is 5.4*10 -3 ~1.2*10 -2 The concentration of sodium periodate is 1.0 x 10 g / mL. -2 ~1.2*10-2 The concentration of g / mL allows sodium periodate to oxidize only some of the hydroxyl groups of sodium alginate.

[0027] Furthermore, the sodium alginate has a viscosity of 350–400 mPa·s, which allows it to form a hydrogel effectively.

[0028] Furthermore, the oxidation degree of the oxidized sodium alginate is 48% to 55%, which allows the aldehyde group on the sodium alginate to form an appropriate amount of imine bond with the amino group on the chitosan.

[0029] Furthermore, in step S1, after adding the 2-morphine ethanesulfonic acid solution of N-carboxyethyl chitosan to the mixed solution, the mass fraction of N-carboxyethyl chitosan is 1.5wt% to 2.5wt%, which allows the chitosan to be well dispersed and dissolved; in step S2, the mass fraction of sodium alginate in the PBS solution is 0.5wt% to 1.5wt%, which makes the viscosity of sodium alginate moderate. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 This is a schematic flowchart of a method for preparing a fluorescent self-healing injectable hydrogel according to the present invention;

[0032] Figure 2 The reaction formulas for synthesizing bioluminescent molecules (i), oxidized sodium alginate (ii), N-carboxyethyl chitosan, and bioluminescent molecule / N-carboxyethyl chitosan polymer (iii) in this invention are shown below.

[0033] Figure 3A The oscillation frequency scan of the TPCNa / CEC-I-OSA hydrogel prepared in Example 1 of this invention;

[0034] Figure 3B Oscillatory strain scan of the TPCNa / CEC-I-OSA hydrogel prepared in Example 1 of this invention;

[0035] Figure 3C The rheological recovery test results are for the TPCNa / CEC-I-OSA hydrogel prepared in Example 1 of this invention.

[0036] Figure 4ATo inject the TPCNa / CEC-I-OSA hydrogel prepared in Example 1 of this invention using different needle sizes (26g, 25g, 22g, 20g);

[0037] Figure 4B A photograph showing the self-healing properties of the head outline of a cartoon bear constructed with TPCNa / CEC-I-OSA hydrogel prepared according to embodiments of the present invention.

[0038] Figure 5A Photoluminescence images and spectral analysis results of TPCNa / CEC-I-OSA hydrogel and TPCNa / CEC solution;

[0039] Figure 5B To analyze the RGB values ​​of solutions and photoluminescent hydrogel photographs using image processing software;

[0040] Figure 5C Fluorescence spectroscopy analysis of TPCNa / CEC-I-OSA hydrogel;

[0041] Figure 6A The image shows the effect of injecting TPCNa / CEC-I-OSA hydrogel into the back of a mouse.

[0042] Figure 6B The biodegradation of fluorescent self-healing injectable hydrogels in mice.

[0043] Figure 7 H&E staining of subcutaneous skin and muscle tissue after injection of TPCA / CEC-OSA hydrogel. Detailed Implementation

[0044] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0045] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0046] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0047] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0048] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0049] like Figure 1 As shown, this invention provides a method for preparing a fluorescent self-healing injectable hydrogel, comprising the following steps:

[0050] S1: Dissolve the bioluminescent molecule in a 2-morphine ethanesulfonic acid solution, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and stir to dissolve to obtain a mixed solution; add a 2-morphine ethanesulfonic acid solution of N-carboxyethyl chitosan to the mixed solution, and stir to react to obtain a bioluminescent molecule / N-carboxyethyl chitosan polymer;

[0051] Among them, such as Figure 2 As shown in (i), the bioluminescent molecule (5-oxo-3,5-dihydro-2H-thiazolo[3,2-a]pyridine-7-carboxylic sodium, 5-oxo-3,5-dihydrothiazol-3,2-pyridine-7-carboxylic acid sodium salt, TPCNa) was synthesized by heating a mixture of citric acid and cysteine ​​powders. The specific synthesis process is as follows: Citric acid powder (0.1 mol, 21.01 g) and cysteine ​​powder (0.1 mol, 12.11 g) were uniformly mixed in a beaker and heated in an oven to 120–180 °C for at least 3–5 h. During the reaction, as the reaction time changed, the white powder melted into a transparent concentrated syrup solution, and the color gradually changed from yellow to dark brown. 100 mL of deionized water was added to the beaker, and the precipitate was collected by filtration and repeated washing. Finally, the product was dissolved in 200 mL of deionized water and recrystallized at 90 °C to obtain TPCNa crystals. The typical yield of TPCNa is approximately 51%.

[0052] The preparation process of N-carboxyethyl chitosan and bioluminescent molecules / N-carboxyethyl chitosan polymers is as follows: Figure 2 As shown in (iii).

[0053] The preparation process of N-carboxyethyl chitosan (CEC) involves a Michael addition reaction. First, a mixture of chitosan and acrylic acid is prepared, magnetically stirred, and the pH is adjusted. The mixture is then dialyzed in deionized water, and the solution is freeze-dried to obtain N-carboxyethyl chitosan. The specific steps are as follows: First, chitosan is dissolved in distilled water containing acrylic acid, resulting in a chitosan-acrylic acid mixture. The chitosan is acid-soluble, with a degree of deacetylation of 86%–88% and a molecular weight of 2*10⁻⁶. 5 ~3*10 5 The chitosan and acrylic acid mixture was prepared with a concentration of 5–10 mM chitosan and 20–25 mM acrylic acid, and the mass ratio of chitosan to acrylic acid was controlled at (0.2–0.25):1. The mixture was magnetically stirred at 50–70 °C for 3–4 days at a stirring speed of 90 rpm. The pH was then adjusted to 10–12 by adding 1 M NaOH dropwise. For the next 3 days, the solution was dialyzed in deionized water with a molecular weight cutoff of 8000 Da, and the deionized water was changed twice daily. Finally, CEC was obtained by freeze-drying the solution, with an amino substitution degree of 48%–50%.

[0054] The bioluminescent molecule / N-carboxyethyl chitosan polymer (TPCNa / CEC) was prepared via a typical carbodiimide coupling reaction. Specifically, it was synthesized using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) as an activator and N-hydroxysuccinimide (NHS) as a stabilizer. The specific procedure was as follows: TPCNa (21.9 mg, 0.1 mmol) was dissolved in 10 mL of 2-morphine ethanesulfonic acid solution (MES, pH = 5.5), while EDC·HCl (19.17 mg, 0.1 mmol) and NHS (11.5 mg, 0.1 mmol) were also dissolved in MES solution to obtain a mixed solution. Conversely, CEC (200 mg, 0.324 mmol) was completely dissolved in 50 mL of 2-morphine ethanesulfonic acid (MES, pH = 5.5) buffer solution. The above-mentioned 10 mL TPCNa mixed solution was then mixed with 50 mL CEC solution, and the grafting coupling reaction was carried out under magnetic stirring at room temperature for 24 h. The mixed solution was then transferred to a dialysis bag (MWCO 3500 Da) and dialyzed for 7 days, with the water changed 3 times daily during dialysis. Afterwards, the solution was freeze-dried to obtain luminescent TPCNa / CEC polymer powder. The grafting rate of TPCNa was calculated to be 82% by comparing the characteristic proton peak area of ​​TPCNa with that of CEC using NMR spectroscopy. Simultaneously, the typical yield of the synthesis reaction was calculated to be 69% by weighing.

[0055] S2: The PBS solution of the bioluminescent molecule / N-carboxyethyl chitosan polymer is mixed with the PBS solution of oxidized sodium alginate to prepare the fluorescent self-healing injectable hydrogel.

[0056] The synthesis of oxidized sodium alginate (OSA) is as follows: Figure 2 As shown in (ii), sodium alginate oxide is synthesized by oxidation of sodium periodate. Sodium alginate, sodium periodate and deionized water are prepared into a reaction solution, and after oxidation reaction under light-protected conditions, ethylene glycol is added and stirred to terminate the oxidation reaction. The resulting mixed solution is added to a dialysis bag for dialyzing and then freeze-dried to obtain sodium alginate oxide.

[0057] Specifically, sodium alginate is dissolved in deionized water, with a viscosity of 350–400 mPa·s. Sodium periodate is then added to obtain a mixed solution, in which the concentration of sodium alginate is 5.4 × 10⁻⁶. -3 ~1.2*10 -2 The concentration of sodium periodate is 1.0 x 10 g / mL. -2 ~1.2*10 -2The solution was prepared at a concentration of g / mL and then magnetically stirred at 90 rpm for 5–6 hours at 20–25°C in the dark. Ethylene glycol was then added, and the oxidation reaction was terminated by stirring for another 1–2 hours. The final concentration of ethylene glycol in the reaction system was 1.5–2 vol%. The resulting mixture was dialyzed against deionized water (MWCO 3000 Da) for 3 days, with the deionized water changed twice daily. Finally, freeze-drying was performed to obtain OSA, which had an oxidation degree of 48%–55%.

[0058] The OSA and TPCNa / CEC polymers were then dissolved separately in PBS solution (pH 7.4) to obtain homogeneous solutions. Mixing the two solutions rapidly yielded the TPCNa / CEC-I-OSA hydrogel (approximately 1 minute).

[0059] In step S1, after adding the 2-morphine ethanesulfonic acid solution of N-carboxyethyl chitosan to the mixed solution, the mass fraction of N-carboxyethyl chitosan is 1.5 wt% to 2.5 wt%; in step S2, the mass fraction of sodium alginate in the PBS solution is 0.5 wt% to 1.5 wt%.

[0060] This invention proposes a method for preparing a fluorescent self-healing injectable hydrogel. Using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride as an activator and N-hydroxysuccinimide as a stabilizer, a bioluminescent molecule (sodium 5-oxo-3,5-dihydrothiazol-3,2-pyridine-7-carboxylate) is coupled to N-carboxyethyl chitosan via a coupling reaction, generating a bioluminescent molecule / N-carboxyethyl chitosan polymer. This anchors a biocompatible fluorescent small molecule to a polysaccharide macromolecular chain. Then, under physiological conditions, in-situ cross-linking occurs via dynamic imine bonds, specifically, the bioluminescent molecule / N-carboxyethyl chitosan polymer is in-situ cross-linked with oxidized sodium alginate, resulting in the fluorescent self-healing injectable hydrogel of this invention. In this fluorescent self-healing injectable hydrogel, the bioluminescent molecules exhibit excellent biocompatibility. These molecules bind to the hydrogel via chemical bonding, effectively enhancing the binding effect between the fluorescent substance and the hydrogel. Furthermore, the fluorescent substance is bonded to N-carboxyethyl chitosan before hydrogel synthesis, and then to sodium alginate oxide, thus being added to the hydrogel in situ, resulting in more uniform dispersion of the fluorescent substance within the hydrogel. The use of N-carboxyethyl chitosan in this reaction effectively improves solubility. During the reaction, the amino groups on the N-carboxyethyl chitosan macromolecules react with the aldehyde groups on the sodium alginate oxide macromolecules through a Schiff base reaction, cross-linking through imine bonds to obtain the hydrogel. Moreover, the uniformly dispersed fluorescent substance in this method effectively avoids fluorescence quenching caused by aggregation, effectively enhancing the fluorescence performance of the fluorescent substance. Therefore, this method yields a self-healing injectable hydrogel with excellent fluorescence performance, effectively expanding the application prospects of self-healing injectable hydrogels for real-time monitoring during non-invasive and in-situ treatment processes. The hydrogel prepared by this invention possesses self-healing, injectability, shape adaptability, biodegradability, biocompatibility, and fluorescence properties. The preparation method includes: preparing N-carboxyethyl chitosan (CEC) via Michael addition reaction, and obtaining N-carboxyethyl chitosan (CEC) powder by freeze-drying; synthesizing oxidized sodium alginate (OSA) by completely dissolving sodium alginate in deionized water, then adding sodium periodate and stirring, dialyzing, and obtaining oxidized sodium alginate powder by freeze-drying; and synthesizing a biocompatible luminescent small molecule (TPCNa) by heating a mixture of citric acid and cysteine ​​powder. Then, N-carboxyethyl chitosan (CEC) was modified with a luminescent small molecule (TPCNa) to obtain the TPCNa / CEC luminescent polymer. The TPCNa / CEC luminescent polymer was prepared via a typical carbodiimide coupling reaction, synthesized under conditions where 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) was used as the activator and N-hydroxysuccinimide (NHS) as the stabilizer. Finally, sodium alginate oxide and the TPCNa / CEC polymer were dissolved separately in PBS solution to obtain homogeneous solutions.The TPCNa / CEC-I-OSA hydrogel can be rapidly obtained by mixing the two solutions. This invention discloses a fluorescent self-healing injectable hydrogel and its preparation method. A fluorescent self-healing injectable hydrogel is prepared by anchoring biocompatible fluorescent small molecules to polysaccharide macromolecular chains via an EDC / NHS coupling reaction, followed by in-situ cross-linking through dynamic imine bonds under physiological conditions. The fluorescent self-healing injectable hydrogel possesses comprehensive properties such as self-healing, injectability, shape adaptability, biodegradability, cell compatibility, and tissue compatibility, and can be used for non-destructive, in-situ, and real-time monitoring.

[0061] The bioluminescent small molecule TPCNa in the fluorescent self-healing injectable hydrogel of this invention exhibits excellent biocompatibility. The fluorescence characteristics of TPCNa originate from the co-doping of nitrogen and sulfur (N, S-CDs) on carbon dots, resulting in significant fluorescence. Furthermore, the bioluminescent small molecule TPCNa is modified onto the polymer chain of the CEC biomolecule via a coupling reaction, resulting in stable bonding and good fluorescence dispersion. In this invention, the amino group (-NH2) linked to TPCNa / CEC and the aldehyde group (-CHO) suspended on OSA form a reversible crosslink through a Schiff base reaction, yielding a hydrogel with excellent mechanical properties, injectability, self-healing properties, and biocompatibility.

[0062] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0063] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0064] Example 1

[0065] Step 1: Prepare N-carboxyethyl chitosan (CEC) via Michael addition reaction.

[0066] The specific steps are as follows: First, chitosan is dissolved in distilled water containing acrylic acid to obtain a chitosan-acrylic acid mixture. The concentration of chitosan in the mixture is 6.2 mM, and the concentration of acrylic acid is 21.3 mM. The mixture is then magnetically stirred at 50°C for 3 days at a speed of 90 rpm. The pH of the solution is then adjusted to 11 by adding 1 M NaOH dropwise. For the next 3 days, the solution is dialyzed in deionized water (MWCO 8000 Da), with the deionized water changed twice daily. Finally, the solution is freeze-dried to obtain CEC, which has an amino substitution degree of 48%.

[0067] Step 2: Synthesize sodium oxidized alginate (OSA) by oxidation with sodium periodate.

[0068] The specific steps are as follows: Dissolve 1.0 g of sodium alginate in 100 mL of deionized water. The viscosity of the sodium alginate is greater than 350 mPa·s. Add 1.08 g of sodium periodate. Stir magnetically for 5 hours at 90 rpm under light-protected conditions at 25°C. Then add 1.5 mL of ethylene glycol and stir for another 1 hour to terminate the oxidation reaction. Dialyze the resulting mixture in deionized water (MWCO 3000 Da) for 3 days, changing the deionized water twice daily. Finally, freeze-dry to obtain OSA, with an oxidation degree of 50%.

[0069] Step 3: Synthesis of biocompatible luminescent small molecule (TPCNa)

[0070] TPCNa was synthesized by heating a mixture of citric acid and cysteine ​​powders. The synthesis process is as follows: Citric acid powder (0.1 mol, 21.01 g) and cysteine ​​powder (0.1 mol, 12.11 g) were uniformly mixed in a beaker and heated in an oven at 150°C for 3 hours. During the reaction, as the reaction time changed, the white powder melted into a transparent, concentrated syrup solution, and the color gradually changed from yellow to dark brown. 100 mL of deionized water was added to the beaker, and the precipitate was collected by filtration and repeated washing. Finally, the product was dissolved in 200 mL of deionized water and recrystallized at 90°C to obtain TPCNa crystals. The typical yield of TPCNa is approximately 51%.

[0071] Step 4: N-Carboxyethyl chitosan (CEC) is used to modify the luminescent small molecule (TPCNa) and a TPCNa / CEC-I-OSA hydrogel is prepared.

[0072] The TPCNa / CEC luminescent polymer was prepared via a typical carbodiimide coupling reaction, synthesized using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) as an activator and N-hydroxysuccinimide (NHS) as a stabilizer. Specifically, TPCNa (21.9 mg, 0.1 mmol) was dissolved in 10 mL of 2-morphine ethanesulfonic acid solution (MES, pH = 5.5), while EDC·HCl (19.17 mg, 0.1 mmol) and NHS (11.5 mg, 0.1 mmol) were also dissolved in MES solution to obtain a mixed solution. Conversely, CEC (200 mg, 0.324 mmol) was completely dissolved in 50 mL of 2-morphine ethanesulfonic acid (MES, pH = 5.5) buffer solution. The above-mentioned 10 mL TPCNa mixed solution and 50 mL CEC solution were then mixed and subjected to a full grafting coupling reaction under magnetic stirring at room temperature for 24 h. The mixture was then transferred to a dialysis bag (MWCO 3500 Da) and dialyzed for 7 days, with the water changed 3 times daily during dialysis. Afterwards, the solution was freeze-dried to obtain luminescent TPCNa / CEC polymer powder. The grafting rate of TPCNa was calculated to be 82% by comparing the characteristic proton peak area of ​​TPCNa with that of CEC using NMR spectroscopy. The typical yield of this synthesis reaction was calculated to be 69% by weighing.

[0073] OSA and TPCNa / CEC polymers were dissolved separately in PBS solution (phosphate buffer, pH 7.4) to obtain homogeneous solutions. Mixing the two solutions rapidly yielded a TPCNa / CEC-I-OSA hydrogel (approximately 1 min).

[0074] Example 2

[0075] This embodiment provides a method for preparing a fluorescent self-healing injectable hydrogel. The preparation method described in this invention refers to Example 1. The degree of oxidation of the prepared sodium alginate is 50%, the degree of amino substitution of the prepared N-carboxyethyl chitosan is 48%, the grafting rate of TPCNa is 80%, and the typical yield of this synthesis reaction is 67% by weighing.

[0076] Example 3

[0077] This embodiment provides a method for preparing a fluorescent self-healing injectable hydrogel. The preparation method described in this invention refers to Example 1. The degree of oxidation of the prepared sodium alginate is 55%, and the degree of amino substitution of the prepared N-carboxyethyl chitosan is 50%. The grafting rate of TPCNa is 81%, and the typical yield of this synthesis reaction is calculated to be 68% by weighing.

[0078] Example 4

[0079] A method for preparing a fluorescent self-healing injectable hydrogel includes the following steps:

[0080] (1) Preparation of bioluminescent molecule TPCNa: 21.01 g of citric acid powder and 12.11 g of cysteine ​​were uniformly mixed in a beaker and heated to 120 °C in an oven for 3 h. After the reaction was completed, 100 mL of deionized water was added to the beaker, and the product was filtered, washed, and the precipitate was collected. Finally, the product was dissolved in 200 mL of deionized water and recrystallized at 90 °C to obtain TPCNa crystals.

[0081] (2) Preparation of N-carboxyethyl chitosan (CEC): Chitosan was dissolved in distilled water containing acrylic acid to obtain a chitosan-acrylic acid mixture. The chitosan was acid-soluble, with a degree of deacetylation of 86% and a molecular weight of 2*10. 5 The chitosan and acrylic acid mixture was prepared at a concentration of 5 mM chitosan and 20 mM acrylic acid. The mixture was magnetically stirred at 50°C for 4 days at 90 rpm. The pH was then adjusted to 10 by adding 1 M NaOH. For the next 3 days, the solution was dialyzed in deionized water (with a molecular weight cutoff of 8000 Da), with the deionized water changed twice daily. Finally, CEC was obtained by freeze-drying the solution, and the amino group of the CEC was determined.

[0082] (3) Preparation of bioluminescent molecule / N-carboxyethyl chitosan polymer (TPCNa / CEC): TPCNa (21.9 mg, 0.1 mmol) was dissolved in 10 mL of 2-morphine ethanesulfonic acid solution (MES, pH = 5.5), while EDC·HCl (19.17 mg, 0.1 mmol) and NHS (11.5 mg, 0.1 mmol) were also dissolved in MES solution to obtain a mixed solution. On the other hand, CEC (200 mg, 0.324 mmol) was completely dissolved in 50 mL of 2-morphine ethanesulfonic acid (MES, pH = 5.5) buffer solution. Then, the above 10 mL TPCNa mixed solution and 50 mL CEC solution were mixed, and the grafting coupling reaction was carried out for 24 h at room temperature with magnetic stirring. Then, the above mixed solution was transferred to a dialysis bag (MWCO 3500Da) for dialysis for 7 days, with the water changed 3 times a day during dialysis. After that, it was freeze-dried to obtain luminescent TPCNa / CEC polymer powder.

[0083] (4) Preparation of oxidized sodium alginate (OSA): Sodium alginate was dissolved in deionized water. The viscosity of sodium alginate was 350 mPa·s. Sodium periodate was then added to obtain a mixed solution. The concentration of sodium alginate in the mixed solution was 5.4 × 10⁻⁶. -3 The concentration of sodium periodate is 1.0 x 10 g / mL. -2 The concentration of ethylene glycol was increased to g / mL, and then the mixture was magnetically stirred at 90 rpm for 6 hours at 20°C in the dark. Ethylene glycol was then added, and the oxidation reaction was terminated by stirring for another 2 hours. The final concentration of ethylene glycol in the reaction system was 1.5 vol%. The resulting mixture was dialyzed against deionized water (MWCO 3000 Da) for 3 days, with the deionized water changed twice daily. Finally, freeze-drying yielded OSA with an oxidation degree of 48%.

[0084] (5) Then, OSA and TPCNa / CEC polymers were dissolved in PBS solution (pH 7.4) to obtain homogeneous solutions. After mixing the two solutions, TPCNa / CEC-I-OSA hydrogel was prepared.

[0085] In step S1, after adding the 2-morphine ethanesulfonic acid solution of N-carboxyethyl chitosan to the mixed solution, the mass fraction of N-carboxyethyl chitosan is 1.5 wt%; in step S2, the mass fraction of sodium alginate in the PBS solution is 0.5 wt%.

[0086] Example 5

[0087] A method for preparing a fluorescent self-healing injectable hydrogel includes the following steps:

[0088] (1) Preparation of bioluminescent molecule TPCNa: 21.01 g of citric acid powder and 12.11 g of cysteine ​​were uniformly mixed in a beaker and heated to 180 °C in an oven for 5 h. After the reaction was completed, 100 mL of deionized water was added to the beaker, and the product was filtered, washed, and the precipitate was collected. Finally, the product was dissolved in 200 mL of deionized water and recrystallized at 90 °C to obtain TPCNa crystals.

[0089] (2) Preparation of N-carboxyethyl chitosan (CEC): Chitosan was dissolved in distilled water containing acrylic acid to obtain a chitosan-acrylic acid mixture. The chitosan was acid-soluble, with a degree of deacetylation of 88% and a molecular weight of 3*10. 5The chitosan and acrylic acid mixture contained 10 mM chitosan and 25 mM acrylic acid. The mixture was magnetically stirred at 70°C for 3 days at 90 rpm. The pH was then adjusted to 12 by adding 1 M NaOH. For the next 3 days, the solution was dialyzed in deionized water with a molecular weight cutoff of 8000 Da, and the deionized water was changed twice daily. Finally, CEC with a degree of amino substitution of 50% was obtained by freeze-drying the solution.

[0090] (3) Preparation of bioluminescent molecule / N-carboxyethyl chitosan polymer (TPCNa / CEC): 21.9 mg of TPCNa was dissolved in 10 mL of 2-morphine ethanesulfonic acid solution (MES, pH = 5.5), while 19.17 mg of EDC·HCl and 11.5 mg of NHS were also dissolved in the MES solution to obtain a mixed solution. Meanwhile, 200 mg of CEC was completely dissolved in 50 mL of 2-morphine ethanesulfonic acid (MES, pH = 5.5) buffer solution. The above 10 mL TPCNa mixed solution and 50 mL CEC solution were then mixed and subjected to magnetic stirring at room temperature for 24 h to allow for a complete graft coupling reaction. The mixed solution was then transferred to a dialysis bag (MWCO 3500 Da) and dialyzed for 7 days, with the water changed 3 times daily during dialysis. Afterwards, the solution was freeze-dried to obtain the bioluminescent TPCNa / CEC polymer powder.

[0091] (4) Preparation of oxidized sodium alginate (OSA): Sodium alginate was dissolved in deionized water to obtain a viscosity of 400 mPa·s. Sodium periodate was then added to obtain a mixed solution with a concentration of 1.2 × 10⁻⁶ mPa·s. -2 The concentration of sodium periodate is 1.2 * 10 g / mL. -2 The solution was concentrated at g / mL, and then magnetically stirred at 90 rpm for 5 hours at 25°C in the dark. Ethylene glycol was then added, and the oxidation reaction was terminated by stirring for another 2 hours. The final concentration of ethylene glycol in the reaction system was 2 vol%. The resulting mixture was dialyzed against deionized water (MWCO 3000 Da) for 3 days, with the deionized water changed twice daily. Finally, freeze-drying yielded OSA with an oxidation degree of 55%.

[0092] (5) Then, OSA and TPCNa / CEC polymers were dissolved in PBS solution (pH 7.4) to obtain homogeneous solutions. After mixing the two solutions, TPCNa / CEC-I-OSA hydrogel was prepared.

[0093] In step S1, after adding the 2-morphine ethanesulfonic acid solution of N-carboxyethyl chitosan to the mixed solution, the mass fraction of N-carboxyethyl chitosan is 2.5 wt%; in step S2, the mass fraction of sodium alginate in the PBS solution is 1.5 wt%.

[0094] Example 6

[0095] A method for preparing a fluorescent self-healing injectable hydrogel includes the following steps:

[0096] (1) Preparation of bioluminescent molecule TPCNa: 21.01 g of citric acid powder and 12.11 g of cysteine ​​were uniformly mixed in a beaker and heated to 150 °C in an oven for 4 h. After the reaction was completed, 100 mL of deionized water was added to the beaker, and the product was filtered, washed, and the precipitate was collected. Finally, the product was dissolved in 200 mL of deionized water and recrystallized at 90 °C to obtain TPCNa crystals.

[0097] (2) Preparation of N-carboxyethyl chitosan (CEC): Chitosan was dissolved in distilled water containing acrylic acid to obtain a chitosan-acrylic acid mixture. The chitosan was acid-soluble, with a degree of deacetylation of 87% and a molecular weight of 2.5 × 10⁻⁶. 5 The chitosan and acrylic acid mixture contained 7 mM chitosan and 22 mM acrylic acid. The mixture was magnetically stirred at 60°C for 3 days at 90 rpm. The pH was then adjusted to 11 by adding 1 M NaOH. For the next 3 days, the solution was dialyzed in deionized water with a molecular weight cutoff of 8000 Da, and the deionized water was changed twice daily. Finally, CEC with a degree of amino substitution of 49% was obtained by freeze-drying.

[0098] (3) Preparation of bioluminescent molecule / N-carboxyethyl chitosan polymer (TPCNa / CEC): 21.9 mg of TPCNa was dissolved in 10 mL of 2-morphine ethanesulfonic acid solution (MES, pH = 5.5), while 19.17 mg of EDC·HCl and 11.5 mg of NHS were also dissolved in the MES solution to obtain a mixed solution. Meanwhile, 200 mg of CEC was completely dissolved in 50 mL of 2-morphine ethanesulfonic acid (MES, pH = 5.5) buffer solution. The above 10 mL TPCNa mixed solution and 50 mL CEC solution were then mixed and subjected to magnetic stirring at room temperature for 24 h to allow for a complete graft coupling reaction. The mixed solution was then transferred to a dialysis bag (MWCO 3500 Da) and dialyzed for 7 days, with the water changed 3 times daily during dialysis. Afterwards, the solution was freeze-dried to obtain the bioluminescent TPCNa / CEC polymer powder.

[0099] (4) Preparation of oxidized sodium alginate (OSA): Sodium alginate was dissolved in deionized water. The viscosity of sodium alginate was greater than 350 mPa·s. Then, sodium periodate was added to obtain a mixed solution. The concentration of sodium alginate in the mixed solution was 1.0 × 10⁻⁶. -2 The concentration of sodium periodate is 1.1 x 10 g / mL. -2 The concentration of ethylene glycol was increased to g / mL, and then the mixture was magnetically stirred at 90 rpm for 5.5 h at 22 °C in the dark. Ethylene glycol was then added, and the oxidation reaction was terminated by stirring for another 1 h. The final concentration of ethylene glycol in the reaction system was 1.7 vol%. The resulting mixture was dialyzed against deionized water (MWCO 3000 Da) for 3 days, with the deionized water changed twice daily. Finally, freeze-drying yielded OSA with an oxidation degree of 50%.

[0100] (5) Then, OSA and TPCNa / CEC polymers were dissolved in PBS solution (pH 7.4) to obtain homogeneous solutions. After mixing the two solutions, TPCNa / CEC-I-OSA hydrogel was prepared.

[0101] In step S1, after adding the 2-morphine ethanesulfonic acid solution of N-carboxyethyl chitosan to the mixed solution, the mass fraction of N-carboxyethyl chitosan is 2.0 wt%; in step S2, the mass fraction of sodium alginate in the PBS solution is 1.3 wt%.

[0102] The performance of the TPCNa / CEC-I-OSA hydrogel obtained in Example 1 of this invention was tested using the following methods:

[0103] Test 1: Rheological testing of TPCNa / CEC-I-OSA hydrogel

[0104] The rheological properties of the TPCNa / CEC-I-OSA hydrogel obtained in Example 1 were tested, specifically, the mechanical properties of the TPCNa / CEC-I-OSA hydrogel were quantitatively evaluated by frequency scanning. The test results are as follows: Figure 3A As shown, the fluorescent hydrogel exhibits an almost constant storage modulus (G') while the strain remains at γ = 1.0%, ranging from 0.1 to 100 rad·s. -1 The entire dynamic frequency scan within the range revealed that the storage modulus was significantly higher than the corresponding loss modulus (G”). These data indicate that the hydrogel exhibits a typical gel state, and the TPCNa / CEC-I-OSA hydrogel showed almost no frequency dependence within the test range, suggesting that there are enough dynamically reversible imine bonds in the polymer network to form a stable hydrogel.

[0105] The linear viscoelasticity of the hydrogel was further investigated through strain oscillation rheology experiments, and the results are as follows: Figure 3B As shown. By Figure 3B It was found that under low strain (1% to 80%), the hydrogel exhibited linear viscoelastic behavior, while the storage modulus and loss modulus remained unchanged and independent of the strain amplitude. γ = 80% was the critical strain point for both storage modulus and loss modulus. Due to the partial reversible dissociation of imine bonds at relatively high strain, the TPCNa / CEC-I-OSA hydrogel entered a gel-sol state. This critical point indicates that when γ < 80%, the hydrogel exhibits elastic gel behavior of an associated or cross-linked polymer network; when γ > 80%, with further increases in strain, a sudden decrease in storage modulus and a rapid increase in loss modulus were observed, indicating that the dynamic hydrogel underwent a gel-sol transition, changing from a solid state with gel properties to a fluid state. The results show that by adjusting the strain-induced shear thinning of the hydrogel, its rheological behavior and injectability can be tuned.

[0106] The reversibility of the gel-sol transition of the hydrogel was further verified by rheological recovery performance testing, revealing the dynamic self-healing behavior of the fluorescent hydrogel. The results are as follows: Figure 3C As shown. By Figure 3C It was observed that the hydrogel underwent a gel-sol transition under low shear strain (1%, 200 s) and high shear strain (800%, 200 s) cycles. When the shear strain increased from 1% to 800%, the reversible imine bonds in the hydrogel dissociated under shear thinning, and the storage modulus decreased by approximately two orders of magnitude. When the shear strain decreased from 800% to 1%, the imine bonds in the hydrogel reformed, and the storage modulus almost completely recovered to the initial level of the gel state.

[0107] The experimental results above demonstrate that the gel-sol transition of the fluorescent hydrogel is reversible, and it can self-heal to its original state without affecting its mechanical properties. The cross-linking and dissociation processes of the abundant dynamic imine bonds in the polysaccharide chains are the reason why the fluorescent hydrogel exhibits rapid alternating stress relaxation.

[0108] Test 2: Self-healing performance test of TPCNa / CEC-I-OSA hydrogel

[0109] The hydrogel could be injected into a syringe using needles of different sizes (20g, 22g, 25g, 26g), demonstrating the excellent injectability of the fluorescent hydrogel. The results are as follows: Figure 4A As shown.

[0110] Figure 4B The image above shows the results of irradiating the nearly colorless, fluorescent, self-healing injectable hydrogel obtained in this invention with ultraviolet light. The results show that after irradiation, the hydrogel turned a bright blue. Furthermore, after 10 minutes, when the cartoon panda head formed by the hydrogel was lifted, the hydrogel exhibited strong mechanical properties. Additionally, Figure 4B The image below shows the process of staining the hydrogel. One part is stained with methylene blue to represent the cartoon panda's face, while the other part is stained pink with eosin to represent the panda's ears. After the pink ear hydrogel comes into contact with the blue face hydrogel, the pink and blue parts will self-heal without any external intervention, resulting in a complete cartoon panda pattern. Figure 4B iii), and can withstand large deformation and tension after 10 minutes ( Figure 4B (iv) Through the reshaping and integration of a cartoon panda's head and a bear's head, the macroscopic function of hydrogel, including injectability and self-healing, was demonstrated. To further prove that the cartoon bear could maintain its integrity after self-healing, a simple lifting test was conducted. Using tweezers, the two ears that had healed with the face were lifted. Obvious deformation of the healed boundary was observed, but no splitting was observed, indicating that the two ears had completely fused with the face into a single unit. Furthermore, the cracks tended to smooth out and almost disappeared after 30 minutes, and the boundary between the ears and the head became blurred. Figure 4B (iv) The results showed that the merged hydrogel exhibited good self-healing properties and remained stable overall, able to withstand slight stretching and deformation caused by facial weight. This was mainly attributed to the "flow phase" healing process, including the rapid flow of polymer chains around the damaged area and the spontaneous dissociation-binding reaction of dynamic imine bonds between amino and aldehyde groups on the TPCNa / CEC and OSA biomacromolecules.

[0111] Test 3: Fluorescence performance test of TPCNa / CEC-I-OSA hydrogel

[0112] The photoluminescence properties were evaluated by in-situ gelation in quartz cuvettes. Figure 5A The results showed that the transparent TPCNa / CEC-I-OSA hydrogel emitted a very bright violet-blue fluorescence under ultraviolet light (λ = 365 nm), which was very similar to that of the TPCNa / CEC PBS solution. To evaluate the difference in fluorescence intensity in color, RGB colors were analyzed using image processing software and compared with those of the TPCNa / CEC and TPCNa / CEC-I-OSA PBS solutions. The results are as follows: Figure 5B As shown. By Figure 5B It can be seen that the two samples exhibit similar color morphology, with the B value being higher than the R and G values, indicating that the photoluminescent hydrogel exhibits a stronger purplish-blue hue. Furthermore, the fluorescence color is uniformly distributed within the hydrogel, a phenomenon further confirmed by RGB value analysis. TPCNa is stably linked to the CEC polymer chain, effectively blocking the aggregation of fluorophores in the hydrogel matrix. After gelation, the distribution of TPCNa within the hydrogel matrix is ​​uniform and stable.

[0113] The optimal excitation (Eex) and emission (Eem) wavelengths for the fluorescent hydrogel were 360 ​​nm and 434 nm, respectively. The numerical results are very close to those of the TPCNa / CEC PBS solution (Eex = 360 nm, Eem = 433 nm), but slightly different from TPCNa (Eex = 339 nm, Eem = 418 nm). This may be because the chemical reaction between TPCNa and CEC has a relatively small impact on its photoluminescence properties. The excitation-related emission spectrum of the TPCNa / CEC-I-OSA hydrogel is shown below. Figure 5C As shown. By Figure 5C It was observed that when the excitation wavelength increased from 300 nm to 360 nm, the emission wavelength of the photoluminescent hydrogel remained almost unchanged, while the corresponding photoluminescence intensity increased. When the excitation wavelength was further increased to 375 nm and 390 nm, the corresponding emission wavelengths decreased sharply. Simultaneously, when the excitation wavelength was 390 nm, the maximum emission wavelength shifted to approximately 450 nm. These results indicate that the multifunctional hydrogel exhibits fluorescence properties similar to TPCNa because the biocompatible organic fluorophore is stably grafted onto the CEC polymer chain through chemical modification.

[0114] Test 4: Testing the in vivo injectability and biodegradability of TPCNa / CEC-I-OSA hydrogel

[0115] Using rats as a model, the hydrogel can be successfully implanted into their bodies via injection, and its shape can be clearly observed on the dorsal skin of the mice. Figure 6A As shown. Using Balb / c mice as a model, the real-time monitoring of biodegradable hydrogels was analyzed. The experimental procedure is as follows: Figure 6BAs shown, the degradation of TPCNa / CEC-I-OSA fluorescent hydrogel in vivo was analyzed by subcutaneous injection into the dorsal side of mice. The hydrogel was smoothly injected into the mouse back, and fluorescence was observed under ultraviolet light. Subcutaneous bioimaging confirmed that the hydrogel possessed sufficiently strong fluorescence intensity. After 42 days, the fluorescence intensity decreased as the hydrogel size decreased, indicating that the subcutaneously injected hydrogel gradually degraded over time. Figure 6B Furthermore, it takes a relatively long time for the hydrogel to completely degrade. The results indicate that fluorescence intensity and in vivo bioimaging can serve as indicators of hydrogel biodegradation.

[0116] Test 5: Biocompatibility test of TPCNa / CEC-I-OSA hydrogel

[0117] like Figure 7 As shown, live-dead staining revealed that the released cells maintained high cell viability after 72 hours of growth on the culture dish surface, indicating that the fluorescent hydrogel has excellent cell compatibility. The hydrogel was injected into the back of mice to study its effects on skin and muscle tissue, further evaluating the hydrogel's tissue compatibility. Figure 7 As shown in Figure a. Glutaraldehyde-crosslinked chitosan hydrogel served as the negative control group, while CaCl2-crosslinked sodium alginate hydrogel, which has good biocompatibility, served as the positive control group. H&E staining results for skin tissue are shown below. Figure 7 As shown in b, by Figure 7 As shown in b, TPCNA / CEC-l-OSA fluorescent hydrogel did not induce any inflammatory response in mice one day after injection; on the second day, inflammatory cells infiltrated the tissue containing chitosan hydrogel; and on the third day, a large number of inflammatory cells appeared around the tissue. In contrast, the tissue injected with TPCNA / CEC-l-OSA fluorescent hydrogel contained only a small number of inflammatory cells, similar to the results of the sodium alginate hydrogel test. This indicates that TPCNA / CEC-l-OSA fluorescent hydrogel has good tissue compatibility and does not induce a significant inflammatory response.

[0118] The results of H&E staining of muscle tissue are as follows Figure 7 As shown in c. From Figure 7 As shown in Figure c, no inflammatory response occurred in any of the muscle tissues injected with the hydrogel on day 1 post-injection; on day 2, a significant inflammatory response appeared around the tissues injected with chitosan hydrogel; and after day 3, the number of inflammatory cells around the tissues injected with chitosan hydrogel increased significantly. In contrast, no significant inflammatory cell infiltration was observed around the tissues injected with the other two hydrogels. This indicates that the TPCNa / CEC-l-OSA fluorescent hydrogel has good biocompatibility in muscle tissue.

[0119] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing a fluorescent self-healing injectable hydrogel, characterized in that, Includes the following steps: S1: Dissolve the bioluminescent molecule in a 2-morphine ethanesulfonic acid solution, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and stir to dissolve to obtain a mixed solution; add a 2-morphine ethanesulfonic acid solution of N-carboxyethyl chitosan to the mixed solution, and stir to react to obtain a bioluminescent molecule / N-carboxyethyl chitosan polymer; S2: Mix the PBS solution containing the bioluminescent molecule / N-carboxyethyl chitosan polymer with the PBS solution containing sodium alginate to prepare the fluorescent self-healing injectable hydrogel. The preparation of the bioluminescent molecule specifically involves: mixing citric acid powder and cysteine ​​evenly, heating to react, washing the product, and recrystallizing to obtain the bioluminescent molecule.

2. The method for preparing a fluorescent self-healing injectable hydrogel according to claim 1, characterized in that, The N-carboxyethyl chitosan is prepared by reacting chitosan with acrylic acid; wherein, in the mixed solution of chitosan and acrylic acid, the concentration of chitosan is 5~10 mM and the concentration of acrylic acid is 20~25 mM.

3. The method for preparing a fluorescent self-healing injectable hydrogel according to claim 2, characterized in that, The degree of deacetylation of the chitosan is 86%~88%, and the molecular weight of the chitosan is 2*10. 5 ~3*10 5 g / mol.

4. The method for preparing a fluorescent self-healing injectable hydrogel according to claim 1, characterized in that, The degree of amino substitution of the N-carboxyethyl chitosan is 48%~50%.

5. The method for preparing a fluorescent self-healing injectable hydrogel according to claim 1, characterized in that, The oxidized sodium alginate is prepared by oxidizing a mixture of sodium periodate and sodium alginate; wherein, in the mixed solution of sodium periodate and sodium alginate, the concentration of sodium alginate is 5.4 × 10⁻⁶. -3 ~1.2*10 -2 The concentration of sodium periodate is 1.0 x 10 g / mL. -2 ~1.2*10 -2 g / mL.

6. A method for preparing a fluorescent self-healing injectable hydrogel according to claim 5, characterized in that, The viscosity of the sodium alginate is 350~400 mPa·s.

7. The method for preparing a fluorescent self-healing injectable hydrogel according to claim 1, characterized in that, The oxidation degree of the oxidized sodium alginate is 48%~55%.

8. A method for preparing a fluorescent self-healing injectable hydrogel according to claim 1, characterized in that, In step S1, after adding the 2-morphine ethanesulfonic acid solution of N-carboxyethyl chitosan to the mixed solution, the mass fraction of N-carboxyethyl chitosan is 1.5 wt%~2.5 wt%; in step S2, the mass fraction of sodium alginate in the PBS solution is 0.5 wt%~1.5 wt%.

9. A fluorescent self-healing injectable hydrogel, characterized in that, It is prepared by the method described in any one of claims 1 to 8.

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