Injectable self-healing conductive hydrogel with immunoregulation performance

Abstract

The invention relates to the technical field of hydrogel preparation, and particularly discloses injectable self-healing conductive hydrogel with immunoregulation performance, and a preparation method of the hydrogel comprises the following steps: S1, preparing oxidized hyaluronic acid; s2, obtaining a polydopamine nano particle; s3, the polypyrrole nano particles are obtained; s4, preparing two kinds of dispersion liquid and two kinds of premixed solution; s5, adding the two dispersion solutions into oxidized hyaluronic acid, fully stirring to obtain a solution A, adding the two premixed solutions into carboxymethyl chitosan, mixing to obtain a solution B, mixing and fully stirring the solution A and the solution B, and performing Schiff base reaction and self-assembly to form the injectable self-healing conductive hydrogel with the immunoregulation performance. The injectable self-healing conductive hydrogel prepared in the invention has good mechanical properties, can effectively bear stress in application, can effectively control inflammation in the early stage of spinal cord injury, has a good electrical microenvironment, and also has excellent biodegradability.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogel preparation, and in particular relates to an injectable self-healing conductive hydrogel with immune regulation performance. Background Art

[0002] By mimicking the structure of the extracellular matrix, hydrogels not only possess mechanical properties that match those of spinal cord tissue, but can also effectively fill cavities after nerve injury. They also have a unique three-dimensional reticular structure that can carry drugs or cells, release them locally, and regulate secondary injuries. Spinal cord injury repair is a complex and difficult process, and immune regulation is closely related to the nerve repair process. They also have excellent electrical conductivity, can induce cell adhesion and migration, and provide a good microenvironment for neurovascular regeneration. Injectable hydrogels can be combined with minimally invasive interventional therapy techniques to precisely implant hydrogels into deep and anatomically complex areas, thereby achieving precise delivery of bioactive substances to the target site. However, the precise control of injection and solution-gel transition for traditional injectable hydrogels remains challenging. Our research has successfully overcome this technical difficulty, while also combining immune regulation and electrical conductivity, effectively promoting spinal cord nerve repair and improving neurological function.

[0003] The present invention provides a new idea for spinal cord injury repair materials by constructing an injectable self-healing conductive hydrogel with immune regulatory properties. While meeting the immune regulatory properties, it also improves the electrical microenvironment, promotes nerve repair, and improves nerve function.

[0004] The spinal cord nerve repair ability of the conductive hydrogel was evaluated, and the functional and histological repair ability of the scaffold after spinal cord injury was explored through the combined effects of immune regulation and electrical activity.

[0005] In this regard, the present application proposes an injectable self-healing conductive hydrogel with immune regulatory properties to solve the above problems. Summary of the Invention

[0006] The purpose of the present invention is to provide an injectable self-healing conductive hydrogel with immune regulation properties to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] An injectable self-healing conductive hydrogel with immune regulation properties, wherein the preparation method of the hydrogel comprises the following steps:

[0009] S1. Completely dissolve hyaluronic acid in high-temperature sterilized deionized water, and dissolve sodium periodate in high-temperature sterilized deionized water. Mix and stir the two overnight, add ethylene glycol (C2H6O2), and terminate the reaction 2 hours later. Then, place the resulting solution in a dialysis bag and dialyze for three days, changing the water every day. After the dialysis is completed, freeze-dry to oxidize the hyaluronic acid for preparing oxidized hyaluronic acid (OHA).

[0010] S2, dissolving dopamine hydrochloride in a hydrochloride buffer solution of pH 8.5, stirring continuously at 25°C for 12 hours, then ultracentrifuging, washing with deionized water 2-3 times, and freeze-drying to obtain polydopamine nanoparticles;

[0011] S3, dissolving ferric chloride (FeCl3) in sterilized deionized water, adding pyrrole monomer, stirring continuously at 25°C for 12 hours, and then ultracentrifuging, washing with deionized water 2-3 times, and freeze-drying to obtain polypyrrole nanoparticles;

[0012] S4. Dispersing the polydopamine nanoparticles prepared in step S2 and the polypyrrole nanoparticles prepared in step S3 in deionized water to obtain two dispersions, and dissolving the oxidized hyaluronic acid and carboxymethyl chitosan (CMCS) prepared in step S1 in deionized water to obtain two premixed solutions.

[0013] S5. Add the two dispersions obtained in step S4 to oxidized hyaluronic acid and stir thoroughly to obtain liquid A. Add the two premixed solutions obtained in step S4 to carboxymethyl chitosan and mix to obtain liquid B. Mix liquid A and liquid B and stir thoroughly. The aldehyde group of the oxidized hyaluronic acid and the amino group of CMCS undergo a Schiff base reaction to self-assemble to form an injectable self-healing conductive hydrogel with immune regulatory properties.

[0014] Preferably, in step S1, sodium periodate and hyaluronic acid are mixed in a molar ratio of 1:1 to optimize the degree of hydroformylation and the self-healing rate.

[0015] Preferably, the concentration of dopamine hydrochloride in step S2 is 2 mg / mL, which is used to prepare polydopamine nanoparticles with a particle size of 50-150 nm, and can give the hydrogel excellent immunomodulatory properties.

[0016] Preferably, in step S3, the pyrrole monomer is mixed with ferric chloride (FeCl 3 ) at a weight ratio of 1:1.5 to prepare polypyrrole nanoparticles having an electrical conductivity of not less than 10 S / m.

[0017] Preferably, before mixing the two dispersions and the two premixed solutions in step S4, graphene oxide (GO) may be dispersed in deionized water to further enhance the conductive properties of the hydrogel.

[0018] Preferably, the hydrogel obtained in step S5 undergoes shear thinning under the action of shear force, and after injection, it self-heals through dynamic covalent bonds to restore the initial three-dimensional network structure, thereby achieving minimally invasive injection and efficient remodeling.

[0019] Preferably, the hydrogel obtained in step S5 has a storage modulus of 0.5-2 kPa and a self-healing efficiency of not less than 90%, which is used to match the spinal cord tissue mechanics and maintain the electrical activity environment.

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

[0021] (1) The injectable self-healing conductive hydrogel prepared by the present invention has good mechanical properties, can effectively withstand stress during application, can effectively control inflammation in the early stage of spinal cord injury, and has a good electrical microenvironment, and also has excellent biodegradability. The present invention forms gel in situ in the syringe, and the stress generated during injection induces shear thinning, which facilitates the injection of gel particles into the target area. Subsequently, the extruded gel particles integrate and restore the original hydrogel structure and function after self-healing and remodeling through the interaction of dynamic bonds, effectively improving neurological function after spinal cord injury.

[0022] (2) The hydrogel system of the present invention has multiple functions including injectability, self-healing, electrical conductivity, immune regulation, and three-dimensional network stability. PDA provides ROS regulation and immune regulation functions, effectively alleviating the inflammatory environment; PPy significantly enhances electrical conductivity, facilitating neural signal transmission; GO further enhances mechanical strength and electron transport capacity; the prepared hydrogel is suitable for applications such as spinal cord injury, neural repair, and tissue engineering scaffolds, and has clinical translation potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the process for preparing the injectable self-healing conductive hydrogel with immune regulation performance of the present invention;

[0024] Figure 2 The present invention is a flow chart of the preparation method of the injectable self-healing conductive hydrogel with immune regulation performance. DETAILED DESCRIPTION

[0025] The following will provide a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0026] Example 1:

[0027] See also Figures 1 to 2 As shown, the injectable self-healing conductive hydrogel with immune regulation properties includes:

[0028] (1) Prepare oxidized hyaluronic acid (OHA): completely dissolve hyaluronic acid (HA) in sterilized RO water, then dissolve sodium periodate (NAIO4) in sterilized RO water, mix the two solutions, and stir overnight (protect from light). After overnight reaction, add ethylene glycol (C2H6O2) to terminate the reaction (2 hours), and then place the resulting solution in a dialysis bag and dialyze for three days, changing the water every day. After dialysis, freeze-dry to obtain OHA;

[0029] (2) Prepare polydopamine (PDA) by dissolving dopamine hydrochloride in hydrochloride buffer (10 mmol / L, pH 8.5), stirring continuously, and reacting at 25°C for 12 h. Subsequently, the resulting solution was ultracentrifuged and washed 2-3 times with deionized water. The resulting solution was freeze-dried to obtain PDA nanoparticles. PDA: promotes cell adhesion, reduces inflammation, alleviates oxidative stress, and remodels the extracellular matrix (ECM);

[0030] (3) Preparation of polypyrrole (PPy): After completely dissolving FeCl₃ in sterilized RO water, pyrrole monomer was added and the mixture was allowed to react at 25°C for 12 h with vigorous stirring in the dark. The resulting solution was then ultracentrifuged and washed 2–3 times with deionized water. The mixture was freeze-dried to obtain PPy nanoparticles. PPy: High conductivity and excellent antioxidant properties.

[0031] (4) PDA and PPy control inflammation in the early stages of bone injury through their excellent immune regulatory properties. PPy has good electrical conductivity, which makes the bionic periosteum electrically active, provides an electrical microenvironment for cells, facilitates information exchange between cells, and promotes osteogenic differentiation. HA can absorb and aggregate calcium ions in body fluids and induce osteogenesis. The combination of these three functional components effectively promotes bone regeneration.

[0032] (5) OHA was added to the solution containing PDA and PPy and stirred thoroughly to obtain liquid A. Carboxymethyl chitosan (CMCS) was added to the solution containing PDA and PPy and stirred thoroughly to obtain liquid B. Liquid A and liquid B were then mixed and stirred thoroughly to obtain an injectable self-healing conductive hydrogel through a Schiff base reaction (without the need for an initiator or high temperature).

[0033] (6) Graphene oxide (GO) has similar electroactivity to HA.

[0034] From the above, it can be seen that the injectable self-healing conductive hydrogel prepared by the present invention has good mechanical properties, can effectively withstand stress during application, can effectively control inflammation in the early stage of spinal cord injury, has a good electrical microenvironment, and also has excellent biodegradability.

[0035] The present invention forms a gel in situ within the syringe. The stress generated during injection induces shear thinning, facilitating injection of the gel particles into the target area. Subsequently, the extruded gel particles self-heal and reshape through dynamic bond interactions, integrating and restoring the original hydrogel structure and function.

[0036] Effectively improve neurological function after spinal cord injury.

[0037] Example 2:

[0038] Preparation and performance evaluation of hydrogels containing PDA / PPy nanoparticles:

[0039] 1. Preparation steps:

[0040] Preparation of oxidized hyaluronic acid (OHA)

[0041] Take 100 mg of sodium hyaluronate (HA) and dissolve it in 10 mL of deionized water.

[0042] Prepare 10 mL of 10 mg / mL sodium periodate (NaIO4) solution and slowly add it dropwise to the HA solution.

[0043] The reaction was carried out under magnetic stirring in the dark for 12 h, and the small molecular impurities were removed by dialysis using a dialysis bag (MWCO 3500 Da) and then freeze-dried to obtain OHA powder.

[0044] Preparation of PDA nanoparticles

[0045] 100 mg of dopamine hydrochloride was dissolved in 100 mL of Tris-HCl buffer (pH=8.5, 10 mM).

[0046] After reacting at 25°C under magnetic stirring for 12 h, the product was collected by centrifugation (12000 rpm, 15 min) and the supernatant was removed.

[0047] The mixture was washed three times and freeze-dried to obtain PDA nanoparticles (particle size 90±10 nm).

[0048] Preparation of PPy nanoparticles

[0049] Take 200 μL of pyrrole monomer and dissolve it in 50 mL of deionized water, and add 1.5 times the molar amount of FeCl3 solution (20 mL, 1.5 mM).

[0050] After vigorous stirring at room temperature for 12 h, the mixture was centrifuged and washed to obtain a black precipitate of PPy, which was freeze-dried for later use (particle size 110±15 nm, conductivity about 11.2 S / m).

[0051] Hydrogel assembly

[0052] 5 mL each of OHA (10 mg / mL) and carboxymethyl chitosan (CMCS) (10 mg / mL) were dissolved and mixed with 1 mL of PDA dispersion (2 mg / mL) and 1 mL of PPy dispersion (2 mg / mL).

[0053] After mixing, the mixture is sheared and injected to form a gelling system, which is cured at room temperature for 5 minutes and self-assembled to form a three-dimensional cross-linked network hydrogel.

[0054] Example 3:

[0055] The steps are the same as in Example 1, but before assembling the hydrogel, graphene oxide (GO) dispersion is added to enhance the conductivity and mechanical properties:

[0056] GO nanosheet dispersion (1 mg / mL), 1 mL was added to the above four solutions.

[0057] Gelling system (total 10 mL): OHA (10 mg / mL), CMCS (10 mg / mL), PDA (2 mg / mL), PPy (2 mg / mL), and GO (1 mg / mL) were mixed and quickly injected to form a gel.

[0058] The comparative example was compared with the traditional hydrogel without the introduction of conductive components and PDA.

[0059] Comparative sample 1: OHA-CMCS hydrogel without PDA and PPy (basic self-healing hydrogel);

[0060] Comparative sample 2: only PDA nanoparticles without PPy and GO;

[0061] Comparative sample 3: containing only PPy nanoparticles without PDA and GO;

[0062] The test items and methods are shown in Table 1 below:

[0063] Table 1

[0064]

[0065] The technical data comparison is shown in Table 2 below:

[0066] Table 2

[0067]

[0068]

[0069] Data acquisition:

[0070] The G' modulus test was performed using an ARES-G2 rheometer at a frequency of 1 Hz and a shear strain of 1% at room temperature.

[0071] The conductivity was tested using a standard four-probe instrument (RTS-8). The hydrogel was dehydrated and then pressed into sheets for thickness correction.

[0072] RAW264.7 macrophages were used for the expression of immune factors. The supernatant was collected after 24 h of stimulation and IL-6 and IL-10 were measured by ELISA.

[0073] The compression test was performed using an INSTRON tension-compression machine with a compression of 30% and the stress value was recorded.

[0074] From the above, it can be seen that the hydrogel system has multiple functions including injectability, self-healing, conductivity, immune regulation, and three-dimensional network stability.

[0075] PDA provides ROS regulation and immune regulation functions, effectively alleviating the inflammatory environment;

[0076] PPy significantly enhances electrical conductivity, facilitating nerve signal transmission;

[0077] GO further improves mechanical strength and electron transport capabilities;

[0078] The prepared hydrogel is suitable for application scenarios such as spinal cord injury, nerve repair, and tissue engineering scaffolds, and has clinical transformation potential.

[0079] In the description of this specification, the reference terms "one embodiment", "some embodiments", "examples", "specific examples" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0080] In the drawings of the embodiments disclosed in the present invention, only the structures related to the embodiments disclosed in the present invention are involved. Other structures can refer to the general design. In the absence of conflict, the same embodiment and different embodiments of the present invention can be combined with each other.

[0081] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An injectable self-healing conductive hydrogel with immune regulation properties, characterized in that: The preparation method of the hydrogel comprises the following steps: S1. Completely dissolving hyaluronic acid in high-temperature sterilized deionized water and sodium periodate in high-temperature sterilized deionized water, mixing and stirring the two overnight, adding ethylene glycol, and terminating the reaction 2 hours later. Then, placing the resulting solution in a dialysis bag and dialyzing for three days with daily water changes. After the dialysis is completed, freeze-drying is performed to oxidize the hyaluronic acid for preparing oxidized hyaluronic acid (OHA); S2, dissolving dopamine hydrochloride in a hydrochloride buffer solution of pH 8.5, stirring continuously at 25°C for 12 hours, then ultracentrifuging, washing with deionized water 2-3 times, and freeze-drying to obtain polydopamine nanoparticles; S3, dissolving ferric chloride (FeCl3) in sterilized deionized water, adding pyrrole monomer, stirring continuously at 25°C for 12 hours, and then ultracentrifuging, washing with deionized water 2-3 times, and freeze-drying to obtain polypyrrole nanoparticles; S4, dispersing the polydopamine nanoparticles prepared in step S2 and the polypyrrole nanoparticles prepared in step S3 in deionized water to obtain two dispersions, and dissolving the oxidized hyaluronic acid and carboxymethyl chitosan prepared in step S1 in deionized water to obtain two premixed solutions; S5. Add the two dispersions obtained in step S4 to oxidized hyaluronic acid and stir thoroughly to obtain liquid A. Add the two premixed solutions obtained in step S4 to carboxymethyl chitosan and mix to obtain liquid B. Mix liquid A and liquid B and stir thoroughly. Self-assemble through a Schiff base reaction to form an injectable self-healing conductive hydrogel with immune regulatory properties.

2. The injectable self-healing conductive hydrogel with immune regulation properties according to claim 1, characterized in that: In step S1, sodium periodate and hyaluronic acid are mixed in a molar ratio of 1:1 to optimize the degree of hydroformylation and the self-healing rate.

3. The injectable self-healing conductive hydrogel with immune regulation properties according to claim 1, characterized in that: In step S2, the concentration of dopamine hydrochloride is 2 mg / mL, which is used to prepare polydopamine nanoparticles with a particle size of 50-150 nm.

4. The injectable self-healing conductive hydrogel with immune regulation properties according to claim 1, characterized in that: In step S3, the pyrrole monomer and ferric chloride (FeCl 3 ) are mixed in a weight ratio of 1:1.5 to prepare polypyrrole nanoparticles having an electrical conductivity of not less than 10 S / m.

5. The injectable self-healing conductive hydrogel with immune regulation performance according to claim 1, characterized in that: Before mixing the two dispersions and the two premixed solutions in step S4, the graphene oxide is first dispersed in deionized water to further enhance the conductive properties of the hydrogel.

6. The injectable self-healing conductive hydrogel with immune regulation properties according to claim 1, characterized in that: The hydrogel obtained in step S5 undergoes shear thinning under the action of shear force, and after injection, it self-heals through dynamic covalent bonds to restore the initial three-dimensional network structure, thereby achieving minimally invasive injection and efficient remodeling.

7. The injectable self-healing conductive hydrogel with immune regulation properties according to claim 1, characterized in that: The hydrogel obtained in step S5 has a storage modulus of 0.5-2 kPa and a self-healing efficiency of not less than 90%, which is used to match the spinal cord tissue mechanics and maintain the electrical activity environment.

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