Injectable multifunctional magnetic hydrogel and preparation method and application thereof

Abstract

This invention belongs to the field of magnetic materials technology, and relates to an injectable multifunctional magnetic hydrogel, its preparation method, and its applications. The injectable multifunctional magnetic hydrogel comprises the following components: aldehyde-modified Prönnicke F127; carboxymethyl chitosan; ε-polylysine-modified iron(III) oxide; and phosphate buffer. The aldehyde-modified Prönnicke F127 can undergo a Schiff base reaction with amino groups in skin tissue, endowing the hydrogel with excellent tissue adhesion. The ε-polylysine-modified iron(III) oxide not only has the ability to rapidly scavenge reactive oxygen species, but also acts as a small molecule crosslinking agent to accelerate the hydrogeling process, and binds to negatively charged plasma proteins and platelets in the blood through electrostatic interactions. The prepared magnetic hydrogel exhibits rapid gelation, good rheological properties, adhesive properties, hemostatic properties, and excellent biocompatibility, and can synergistically promote wound healing and angiogenesis under external magnetic field stimulation.

Description

Technical Field

[0001] This invention belongs to the field of magnetic materials technology, and relates to an injectable multifunctional magnetic hydrogel, its preparation method and application. Background Technology

[0002] Skin, as the body's first line of defense against external factors, is extremely vulnerable to damage. Severe trauma often results in bleeding and infection, exceeding the skin's own regenerative capacity, necessitating the development of highly efficient, multifunctional dressings. Hydrogels, due to their excellent tissue adhesion, water retention, and biocompatibility, are widely used in wound repair. However, traditional hydrogels primarily function as passive moisturizing dressings, failing to actively reverse the complex pathological microenvironment of refractory wounds, characterized by high oxidative stress, persistent inflammation, and impaired angiogenesis. Therefore, intelligent responsive hydrogels have emerged, with their component structures intelligently designed to sense external stimuli and dynamically regulate the wound microenvironment. Among these, magnetic hydrogels, due to their portable and non-invasive response to external stimuli, and their mediated magnetomechanical stimulation, can apply physical stress to the wound, thereby inducing fibroblast proliferation and angiogenesis, demonstrating significant potential for application in wound repair. Therefore, developing a multifunctional magnetic hydrogel with antibacterial, hemostatic, and antioxidant properties has significant research and application value. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing an injectable multifunctional magnetic hydrogel, its preparation method and application.

[0004] Invention Concept: Pluronic F127 is a temperature-responsive synthetic polymer whose polyoxypropylene ether structural units can achieve temperature-dependent hydrophilic / hydrophobic transitions. Using it as a matrix material can enhance the rheological properties of hydrogels. Furthermore, carboxymethyl chitosan and ε-polylysine, as naturally derived polymers, are rich in highly reactive free amino groups on their molecular chains. Utilizing these natural components to construct a hydrogel network via a dynamic Schiff base reaction not only effectively avoids the toxic side effects of traditional chemical crosslinking agents but also endows the material with excellent injectability, antibacterial activity, and superior hemostatic properties. Ferric oxide nanoparticles, as a commonly used biomedical material, not only possess high biocompatibility and extremely low biotoxicity but also exhibit antioxidant activity and rapid response to external magnetic fields. Therefore, based on the above materials, this invention provides a highly biocompatible, injectable, multifunctional magnetic hydrogel, its preparation method, and its applications. In this invention, aldehyde-modified Pluronic F127 (PF127-CHO), carboxymethyl chitosan (CMCS), and ε-polylysine-modified iron(III) oxide (PIO NPs) are dissolved in phosphate buffer and mixed to prepare a magnetic hydrogel. A reversible Schiff base reaction occurs between PF127-CHO, CMCS, and PIO NPs, forming a magnetic hydrogel with injectability, self-healing properties, good mechanical properties, and biocompatibility.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] This invention discloses an injectable multifunctional magnetic hydrogel, which comprises the following components:

[0007] Component A: Aldehyde-modified Prönnick F127;

[0008] Component B: Carboxymethyl chitosan;

[0009] Component C: ε-polylysine-modified iron(III) oxide;

[0010] Component D: Phosphate buffer.

[0011] In some embodiments, in component A, the aldehyde-modified Pluronic F127 is obtained by introducing an aldehyde group at the molecule terminus of Pluronic F127 using conventional methods.

[0012] In some embodiments, the aldehyde-modified Pluronic F127 is commercially available, or it can be prepared by oxidizing Pluronic F127 via hydroxyl oxidation, or it can be prepared according to the following aldehyde reagent reaction method:

[0013] Pronnic F127 and 4-formylbenzoic acid were esterified in the presence of a condensing agent and a catalyst to prepare aldehyde-modified Pronnic F127.

[0014] In some embodiments, the weight-average molecular weight of Prunic F127 is 12600 Da.

[0015] In some embodiments, in the aldehyde reaction method: the condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0016] In some embodiments, in the aldehyde reaction method: the catalyst is 4-dimethylaminopyridine.

[0017] In some embodiments, in the aldehyde reaction method, the molar ratio of the hydroxyl group in the Pluronic F127 to the 4-formylbenzoic acid is 1.0:(10.0~30.0), preferably 1.0:10.0.

[0018] In some embodiments, in the aldehyde reaction method, the molar ratio of the hydroxyl group in the Pluronic F127 to the condensing agent and catalyst is 1.0:(1.0~2.0):(10.0~20.0), preferably 1.0:1.0:10.0.

[0019] In some embodiments, in the aldehyde reagent reaction method: the solvent used in the esterification reaction is any one or a combination of several of dichloromethane, dimethyl sulfoxide, and dimethylformamide; wherein, all solvents used are anhydrous solvents; optionally, the solvent used in the esterification reaction is anhydrous dichloromethane.

[0020] In some embodiments, in the aldehyde reagent reaction method: the amount of solvent used in the esterification reaction is not particularly required, as long as the raw materials are dispersed and / or dissolved evenly and the viscosity of the reaction system is appropriate.

[0021] In some embodiments, in the aldehyde reagent reaction method: the esterification reaction is carried out under the protection of an inert gas, wherein the inert gas is preferably nitrogen.

[0022] In some embodiments, in the aldehyde reagent reaction method: the esterification reaction is carried out at room temperature; the esterification reaction time is 10 h to 30 h, preferably 24 h.

[0023] In some embodiments, in component B, preferably, the degree of deacetylation of the carboxymethyl chitosan is ≥96%.

[0024] In some embodiments, in component C: the ε-polylysine-modified iron oxide is prepared according to the following method:

[0025] Ferric chloride, ferrous chloride, and concentrated hydrochloric acid were mixed to obtain an iron salt solution. The pH of the iron salt solution was adjusted to 10-14 using an alkaline solution, followed by a salt-alkali neutralization reaction. After the reaction was completed, post-treatment was performed to obtain magnetite magnetic nanoparticles. Subsequently, the magnetite magnetic nanoparticles and ε-polylysine were dispersed in a first solvent and subjected to an electrostatic adsorption reaction. After the reaction was completed, post-treatment was performed to obtain ε-polylysine-modified magnetite magnetic nanoparticles.

[0026] In some embodiments, the molar ratio of ferric chloride to ferrous chloride is (1.0~3.0):1.0; the alkaline solution is 25wt%~28wt% ammonia or 1.5~4.5 mol / L sodium hydroxide aqueous solution; the salt-alkali neutralization reaction is carried out under inert gas protection; the reaction temperature of the salt-alkali neutralization reaction is 70℃~90℃; the reaction time of the salt-alkali neutralization reaction is 1 h~3 h; the first solvent is deionized water; the mass ratio of the magnetite magnetic nanoparticles to ε-polylysine is 1.0:(1.0~5.0); the amount of the first solvent used is 0.5 mL~2.0 mL based on 1.0 mg of ε-polylysine; the electrostatic adsorption reaction is carried out at room temperature; the reaction time of the electrostatic adsorption reaction is 12 h~36 h.

[0027] In some embodiments, the molar ratio of ferric chloride to ferrous chloride is 2.0:1.0; the alkaline solution is 25wt%~28wt% ammonia or 2.8 mol / L sodium hydroxide aqueous solution; the salt-alkali neutralization reaction is carried out under inert gas protection; the salt-alkali neutralization reaction is carried out at a reaction temperature of 80℃; the salt-alkali neutralization reaction is carried out for 2 h; the first solvent is deionized water; the mass ratio of the magnetite magnetic nanoparticles to ε-polylysine is 1.0:1.0; the amount of the first solvent used is 1.0 mL based on 1.0 mg of ε-polylysine; the electrostatic adsorption reaction is carried out at room temperature; the electrostatic adsorption reaction is carried out for 24 h.

[0028] In some embodiments, preferably, the weight-average molecular weight of the ε-polylysine is 3500 Da.

[0029] In some embodiments, the amount of concentrated hydrochloric acid used is not particularly limited, as long as it disperses and / or dissolves the raw materials in the reaction system and the viscosity of the reaction system is appropriate.

[0030] In some embodiments, the concentrated hydrochloric acid serves to provide an acidic environment to prevent ferrous ions from being oxidized to ferric ions. The amount used is unlimited, as long as the raw material is dissolved and the viscosity is appropriate.

[0031] In some embodiments, the iron salt solution is prepared under inert gas protection, preferably under nitrogen protection.

[0032] In some embodiments, the salt-base neutralization reaction is carried out under inert gas protection, preferably under nitrogen protection.

[0033] In some embodiments, the specific post-processing operation after the salt-alkali neutralization reaction is as follows: the product is dried in a vacuum drying oven to obtain ferric oxide magnetic nanoparticles.

[0034] In some embodiments, the post-treatment after the electrostatic adsorption reaction is as follows: the product is dried in a vacuum drying oven to obtain ε-polylysine-modified iron oxide magnetic nanoparticles.

[0035] In some embodiments, in component D, the phosphate buffer solution has a pH of 7.2-7.6 and is 0.01 M.

[0036] In some embodiments, the injectable multifunctional magnetic hydrogel obtained by mixing components A, B, C, and D contains: component A, aldehyde-modified Prönnicke F127, at a concentration of 50 mg / mL to 150 mg / mL; component B, carboxymethyl chitosan, at a concentration of 20 mg / mL to 60 mg / mL; and component C, ε-polylysine-modified iron(III) oxide, at a concentration of 0.5 mg / mL to 3 mg / mL.

[0037] In some embodiments, the injectable multifunctional magnetic hydrogel obtained by mixing components A, B, C, and D contains: component A, aldehyde-modified Prönnicke F127, at a concentration of 80 mg / mL to 120 mg / mL; component B, carboxymethyl chitosan, at a concentration of 30 mg / mL to 50 mg / mL; and component C, ε-polylysine-modified iron(III) oxide, at a concentration of 1 mg / mL to 3 mg / mL.

[0038] In some embodiments, the injectable multifunctional magnetic hydrogel obtained by mixing components A, B, C, and D contains: component A, aldehyde-modified Prönnicke F127, at a concentration of 100 mg / mL; component B, carboxymethyl chitosan, at a concentration of 40 mg / mL; and component C, ε-polylysine-modified iron(III) oxide, at a concentration of 2 mg / mL.

[0039] The aforementioned injectable multifunctional magnetic hydrogel is also within the scope of protection of this invention.

[0040] Furthermore, the present invention provides a method for preparing an injectable multifunctional magnetic hydrogel, wherein the above-mentioned components A, B, C and D are mixed to obtain the injectable multifunctional magnetic hydrogel.

[0041] In some embodiments, the injectable multifunctional magnetic hydrogel is prepared on demand; after mixing the components, the standing time does not exceed 100 seconds.

[0042] In some embodiments, the injectable multifunctional magnetic hydrogel contains: component A, aldehyde-modified Prönnicke F127, with a final concentration of 50 mg / mL to 150 mg / mL; component B, carboxymethyl chitosan, with a final concentration of 20 mg / mL to 60 mg / mL; and component C, ε-polylysine-modified iron(III) oxide, with a final concentration of 0.5 mg / mL to 3 mg / mL.

[0043] In some embodiments, the injectable multifunctional magnetic hydrogel contains: component A, aldehyde-modified Prönnicke F127, with a final concentration of 80 mg / mL to 120 mg / mL; component B, carboxymethyl chitosan, with a final concentration of 30 mg / mL to 50 mg / mL; and component C, ε-polylysine-modified iron(III) oxide, with a final concentration of 1 mg / mL to 3 mg / mL.

[0044] In some embodiments, the injectable multifunctional magnetic hydrogel contains: component A, aldehyde-modified Prönnicke F127, with a final concentration of 100 mg / mL; component B, carboxymethyl chitosan, with a final concentration of 40 mg / mL; and component C, ε-polylysine-modified iron(III) oxide, with a final concentration of 2 mg / mL.

[0045] Furthermore, the present invention provides a method for preparing a multifunctional magnetic hydrogel, wherein the above-mentioned components A, B, C and D are mixed to obtain an injectable multifunctional magnetic hydrogel; after the injectable multifunctional magnetic hydrogel is left to stand, it becomes a multifunctional magnetic hydrogel.

[0046] In some embodiments, the injectable multifunctional magnetic hydrogel contains: component A, aldehyde-modified Prönnicke F127, with a final concentration of 50 mg / mL to 150 mg / mL; component B, carboxymethyl chitosan, with a final concentration of 20 mg / mL to 60 mg / mL; and component C, ε-polylysine-modified iron(III) oxide, with a final concentration of 0.5 mg / mL to 3 mg / mL.

[0047] In some embodiments, the injectable multifunctional magnetic hydrogel contains: component A, aldehyde-modified Prönnicke F127, with a final concentration of 80 mg / mL to 120 mg / mL; component B, carboxymethyl chitosan, with a final concentration of 30 mg / mL to 50 mg / mL; and component C, ε-polylysine-modified iron(III) oxide, with a final concentration of 1 mg / mL to 3 mg / mL.

[0048] In some embodiments, the injectable multifunctional magnetic hydrogel contains: component A, aldehyde-modified Prönnicke F127, with a final concentration of 100 mg / mL; component B, carboxymethyl chitosan, with a final concentration of 40 mg / mL; and component C, ε-polylysine-modified iron(III) oxide, with a final concentration of 2 mg / mL.

[0049] In some embodiments, the settling time is 120 s to 600 s, optionally 300 s.

[0050] The application of the above-mentioned injectable multifunctional magnetic hydrogel, or the injectable multifunctional magnetic hydrogel prepared by the above-mentioned preparation method, in the preparation of magnetic biomaterials is also within the scope of protection of this invention.

[0051] In some embodiments, and optionally further, the use of the above-described injectable multifunctional magnetic hydrogel, or the injectable multifunctional magnetic hydrogel prepared by the above-described preparation method, or the multifunctional magnetic hydrogel prepared by the above-described preparation method in the preparation of magnetic biomaterials that assist in wound hemostasis, and / or assist in wound antibacterial activity, and / or promote wound angiogenesis, and / or promote wound repair is also within the scope of protection of this invention.

[0052] Beneficial effects:

[0053] Compared with the prior art, the present invention has the following advantages:

[0054] (1) The magnetic hydrogel prepared by the present invention forms rapidly, and the gelation time can be shortened again after the introduction of ε-polylysine-modified iron oxide nanoparticles. At the same time, it has good mechanical properties and cell compatibility.

[0055] (2) The magnetic hydrogel prepared by the present invention introduces ε-polylysine-modified iron oxide. The addition of ε-polylysine-modified iron oxide increases the cross-linking sites inside the hydrogel, improves the mechanical properties of the hydrogel, and at the same time endows the hydrogel with magnetic responsiveness. In addition, it can also exert enzyme-like activity to scavenge reactive oxygen species and improve the survival ability of cells in a high reactive oxygen environment.

[0056] (3) In this invention, Pluronic F127 and carboxymethyl chitosan are selected as hydrogel raw materials. The two raw materials can undergo cross-linking through simple modification, and the preparation is simple. The raw materials have been commercialized. Therefore, their selection and the establishment, promotion and application of such gelation methods in tissue engineering and regenerative medicine are of great value.

[0057] (4) The magnetic hydrogel of the present invention can respond to an external magnetic field and promote wound closure and angiogenesis.

[0058] (5) In this invention, aldehyde-modified Pluronic F127 can undergo Schiff base reaction with amino groups in skin tissue structure, giving the hydrogel excellent tissue adhesion ability; ε-polylysine-modified iron oxide not only has the ability to quickly scavenge reactive oxygen species, but also can act as a small molecule crosslinking agent to accelerate the hydrogel gelation process, and bind to negatively charged plasma proteins and platelets in the blood through electrostatic interaction; the prepared magnetic hydrogel has rapid gelation, good rheological properties, adhesion properties, hemostatic properties and excellent biocompatibility, and can synergistically promote wound healing and angiogenesis under external magnetic field stimulation. Attached Figure Description

[0059] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0060] Figure 1 The structural characterization diagrams of aldehyde-modified Prönnicke F127 and ε-polylysine-modified iron oxide prepared in Example 1 are shown in Figure a. Figure a is the NMR spectrum of aldehyde-modified Prönnicke F127, Figure b is the XRD curve of ε-polylysine-modified iron oxide, and Figure c is the TEM image of ε-polylysine-modified iron oxide magnetic nanoparticles.

[0061] Figure 2 Figure 1 shows the gelation time curves of different hydrogel materials, the modulus-time curves of hydrogel materials before and after self-healing, and the results of the injectability test. Figure 2 shows the gelation time curves of different hydrogel materials, Figure 3 shows the modulus-time curves of hydrogel materials before and after self-healing, and Figure 4 shows the results of the injectability test.

[0062] Figure 3 Figure 1 shows the results of the hydrogel functional tests; Figure 2 shows the macroscopic results of hydrogel adhesion on different tissues, Figure 3 shows the macroscopic results of the hydrogel hemostasis test in vivo, and Figure 4 shows the results of the hydrogel antibacterial test in vitro.

[0063] Figure 4The images show the staining results of hydrogels promoting wound closure and angiogenesis in repair tissues; image a is a macroscopic photograph of wound closure, and image b is a staining result of angiogenesis in repair tissues. Detailed Implementation

[0064] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0065] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0066] The Pronnick F127 used in the examples: manufacturer Sigma-Aldrich, model P2443, weight-average molecular weight 12600 Da.

[0067] The ε-polylysine used in the examples was manufactured by Aladdin, model P192512, with a weight-average molecular weight of 3500 Da.

[0068] The carboxymethyl chitosan used in the examples was manufactured by McLean, model C902396, with a degree of deacetylation ≥96%.

[0069] Example 1: Synthesis and characterization of aldehyde-modified Pluronic F127 and ε-polylysine-modified iron(III) oxide

[0070] (1) Synthesis of aldehyde-modified Prönnick F127

[0071] Weigh out 12.6 g of Pluronic F127 (containing approximately 2.0 mmol of hydroxyl groups), 3.0 g of 4-formylbenzoic acid (20.0 mmol), 0.31 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 2.0 mmol), and 2.44 g of 4-dimethylaminopyridine (DMAP, 20.0 mmol), dissolve them in 100 mL of anhydrous dichloromethane, and then add the solution to a round-bottom flask. Stir thoroughly until homogeneous. Purge with nitrogen for 15 min beforehand and carry out the esterification reaction at room temperature under a nitrogen atmosphere for 24 h. After the reaction is complete, remove some of the dichloromethane by rotary evaporation, and then separate the precipitate by passing diethyl ether through an ice bath. The white flocculent polymer ligand was obtained by filtration through a Buchner funnel and then dried overnight in a vacuum drying oven at 40°C to obtain aldehyde-modified Prönnick F127, denoted as PF127-CHO, which was then stored in a desiccator for later use.

[0072] (2) Synthesis of ε-polylysine-modified iron(III) oxide

[0073] Nitrogen gas was introduced for 15 min beforehand. Then, ferric chloride hexahydrate (0.1378 g, 0.5 mmol) and ferrous chloride tetrahydrate (0.0501 g, 0.25 mmol) were weighed and dissolved in 1.0 mL of concentrated hydrochloric acid to obtain an iron salt solution.

[0074] A ferric salt solution was added to a three-necked flask, followed by 15 mL of ammonia water (25wt%–28wt%). The pH of the reaction mixture was adjusted to 10–14. A salt-base neutralization reaction was then carried out at 80°C under a nitrogen atmosphere for 2 h. After the reaction, the mixture was dried in a vacuum drying oven to obtain black granular magnetite magnetic nanoparticles, denoted as IO NPs.

[0075] 0.2 g of IO NPs and 0.2 g of ε-polylysine were weighed and dispersed in 200 mL of deionized water. Electrostatic adsorption was carried out by mechanical stirring at room temperature for 24 h. After the reaction was completed, the nanoparticles were dried in a vacuum drying oven to obtain black granular ε-polylysine-modified magnetite nanoparticles, denoted as PIO NPs.

[0076] (3) Characterization of materials

[0077] The molecular structure of the aldehyde-modified Pluronic F127 (PF127-CHO) prepared in this embodiment was characterized by nuclear magnetic resonance (NMR). The results are as follows: Figure 1 As shown in Figure a, it can be seen that a proton peak from the aldehyde structure of 4-formylbenzoic acid appeared at 10.1 ppm, and proton peaks from the benzene ring structure of 4-formylbenzoic acid appeared at 8.2 ppm and 7.9 ppm. These results indicate the successful preparation of PF127-CHO.

[0078] The crystal structures of the magnetite nanoparticles (IO NPs) and ε-polylysine-modified magnetite nanoparticles (PIO NPs) prepared in this embodiment were characterized by XRD. The results are as follows: Figure 1 As shown in Figure b, it can be seen from the figure that the prepared IO NPs and PIO NPs can correspond to the main crystal planes of the standard magnetite crystal (Fe3O419-0629) and have an anticubic spinel structure.

[0079] The size of the ε-polylysine-modified iron oxide magnetic nanoparticles (PIO NPs) prepared in this embodiment was characterized by TEM. The results are as follows: Figure 1 As shown in Figure c, the prepared PIO NPs have a clearly visible crystal structure with a particle size between 10 and 20 nm.

[0080] Example 2: Preparation of non-magnetic and magnetic hydrogels and testing of their self-healing and injectability.

[0081] The aldehyde-modified Pluronic F127 (PF127-CHO) and ε-polylysine-modified iron oxide magnetic nanoparticles (PIO NPs) prepared in Example 1 were applied in this example.

[0082] (1) Preparation of nonmagnetic hydrogels

[0083] PF127-CHO was dissolved in phosphate buffer (pH=7.2-7.6, 0.01 M) at a concentration of 200 mg / mL to obtain an aldehyde-modified Prönnick F127 dispersion.

[0084] Carboxymethyl chitosan (CMCS) was dissolved in phosphate buffer (pH=7.2-7.6, 0.01 M) at a concentration of 80 mg / mL to obtain a carboxymethyl chitosan dispersion.

[0085] Then, the above-mentioned aldehyde-modified Pluronic F127 dispersion was mixed with an equal volume of carboxymethyl chitosan dispersion to obtain a non-magnetic hydrogel precursor solution material. After uniform mixing, it was allowed to stand for 300 s to form a non-magnetic hydrogel, denoted as CP.

[0086] (2) Preparation of multifunctional magnetic hydrogels

[0087] PF127-CHO was dissolved in phosphate buffer (pH=7.2-7.6, 0.01 M) at a concentration of 200 mg / mL to obtain an aldehyde-modified Prönnick F127 dispersion.

[0088] Carboxymethyl chitosan (CMCS) and PIO NPs were dissolved in phosphate buffer (pH=7.2-7.6, 0.01 M) at concentrations of 80 mg / mL and 4 mg / mL, respectively, to obtain a mixed dispersion of carboxymethyl chitosan and ε-polylysine-modified iron oxide.

[0089] Then, the above-mentioned aldehyde-modified Pronic F127 dispersion was mixed with an equal volume of a mixed dispersion of carboxymethyl chitosan and ε-polylysine-modified iron oxide to obtain a multifunctional magnetic hydrogel precursor solution material. After uniform mixing, it was allowed to stand for 300 s to form a multifunctional magnetic hydrogel, denoted as CP / PIO.

[0090] (3) Preparation of magnetic hydrogels

[0091] PF127-CHO was dissolved in phosphate buffer (pH=7.2-7.6, 0.01 M) at a concentration of 200 mg / mL to obtain an aldehyde-modified Prönnick F127 dispersion.

[0092] Carboxymethyl chitosan (CMCS) and IO NPs were dissolved in phosphate buffer (pH=7.2-7.6, 0.01 M) at concentrations of 80 mg / mL and 4 mg / mL, respectively, to obtain a mixed dispersion of carboxymethyl chitosan and iron oxide.

[0093] Then, the above aldehyde-modified Pluronic F127 dispersion was mixed with an equal volume of a mixed dispersion of carboxymethyl chitosan and iron oxide to obtain a magnetic hydrogel precursor solution material. After uniform mixing, it was allowed to stand for 300 s to form a magnetic hydrogel, denoted as CP / IO.

[0094] (4) Tests on gelation time, self-healing properties and injectability of materials.

[0095] (4-1) Hydrogel gelation time was tested using a DHR-1 rheometer. 0.2 mL of hydrogel precursor solution material (the non-magnetic hydrogel precursor solution material (CP hydrogel precursor solution material) prepared in step (1) of this embodiment, and the multifunctional magnetic hydrogel precursor solution material (CP / PIO hydrogel precursor solution material) prepared in step (2)) was placed on a parallel plate, and the liquid gelation time was tested in the time range of 0-500 s at 25°C using a 20 mm upper clamp.

[0096] See results Figure 2 Figure a shows that, compared to CP hydrogel, the gelation time of CP / PIO hydrogel is reduced from 279 s to 116 s due to the introduction of PIO NPs.

[0097] (4-2) The self-healing performance of the hydrogel was tested using a DHR-1 rheometer. 0.2 mL of the hydrogel precursor solution (the multifunctional magnetic hydrogel precursor solution (CP / PIO hydrogel precursor solution) prepared in step (2) of this embodiment) was added to a 15 mm diameter silicone mold. After the hydrogel was allowed to stand and fully gel, it was cut open and the cross-section was brought into full contact for 5 min. The hydrogel was placed on a parallel plate and tested using a 20 mm upper clamp. The modulus-time curve was obtained by testing at 25°C and a frequency of 1 Hz over a time range of 0-300 s.

[0098] Figure 2Figure b shows the modulus-time curves of the CP / PIO hydrogel material before and after self-healing. Figures A and B distinguish portions from two separate CP / PIO hydrogels. The figure shows that the completely separate hydrogels undergo significant cross-linking after a period of contact, forming a fused structure. The modulus-time curves indicate that the hydrogel's stability is not affected by the structural damage and re-cross-linking, and its modulus shows no significant difference before and after self-healing.

[0099] (4-3) Test the injectability of the hydrogel using a syringe. Mix 0.2 mL of the hydrogel precursor solution material (the multifunctional magnetic hydrogel precursor solution material (CP / PIO hydrogel precursor solution material) prepared in step (2) of this embodiment) evenly and place it in a 1 mL syringe. Let it stand for 5 min, then inject the hydrogel into deionized water and draw a pattern on a plate, while taking pictures for observation.

[0100] Figure 2 Figure c shows the macroscopic results of the injectable experiment of CP / PIO hydrogel. It can be observed that the CP / PIO hydrogel precursor solution material forms obvious gel filaments under the push of the syringe, and can complete the secondary binding of the filaments to form a specific pattern.

[0101] Example 3: Hydrogel adhesion performance test, hemostatic performance test, and antibacterial performance test

[0102] (1) The non-magnetic hydrogel precursor solution material, CP non-magnetic hydrogel, magnetic hydrogel precursor solution material, CP / IO magnetic hydrogel, multifunctional magnetic hydrogel precursor solution material, and CP / PIO multifunctional magnetic hydrogel prepared in Example 2 are applied to this example.

[0103] (2) Hydrogel adhesion performance test

[0104] The mice used were male ICR mice, 4-6 weeks old, with an average weight of 25g, purchased from Nanjing Qinglongshan Animal Breeding Farm.

[0105] Mouse skin and internal organ tissues (heart, liver, spleen, lung, and kidney) were collected, and CP / PIO multifunctional hydrogel precursor solution (multifunctional magnetic hydrogel precursor solution) was applied or injected in situ onto the surface of each organ. After the hydrogel had completely gelled, it was peeled off with tweezers, and the adhesion effect was observed.

[0106] The results are as follows Figure 3As shown in Figure a, Figure a is a macroscopic diagram of the adhesion effect of CP / PIO hydrogel on various tissues. It can be seen from the figure that CP / PIO hydrogel can stably bind to the tissue and firmly adhere to its surface by reacting the aldehyde group in the PF127-CHO molecular structure with the amino group in the skin tissue through a Schiff base reaction.

[0107] (3) Hydrogel hemostatic performance test

[0108] The rats used were male SD rats, 3-4 weeks old, with an average weight of 200 g, purchased from Nanjing Qinglongshan Animal Breeding Farm.

[0109] A rat liver puncture injury model was used to evaluate the in vivo hemostatic activity of the hydrogel, with gelatin sponge (purchased from Nanjing Jinling Pharmaceutical Factory, Jinling Pharmaceutical Co., Ltd., batch number H32024096) used as a control group. Male SD rats were anesthetized with isoflurane, the abdominal cavity was opened, and the liver was exposed. Filter paper was placed beneath the liver. The liver surface was punctured using a 1 mL syringe, and immediately 300 μL of CP / PIO multifunctional hydrogel precursor solution (multifunctional magnetic hydrogel precursor solution) was injected into the wound. After bleeding stopped, the hemostatic effect was observed by photography. The control group (Control) did not use any material for hemostasis.

[0110] The results are as follows Figure 3 As shown in Figure b, which presents the macroscopic experimental results of the in vivo hemostasis test, it can be seen from the figure that compared with the gelatin sponge group, the CP / PIO multifunctional magnetic hydrogel can significantly reduce the area of ​​blood adsorbed on the filter paper.

[0111] (4) Antibacterial performance test of hydrogel

[0112] 50 μL of bacterial suspension (Escherichia coli ATCC 8739 or Staphylococcus aureus ATCC29213, respectively, both at a concentration of 2 × 10⁻⁶) was prepared. 5The bacterial suspension (CFU / mL) was mixed with 950 μL of phosphate buffer solution (pH=7.2-7.6, 0.01 M), 3 μL of Luria-LB liquid medium (Shanghai Yuanye, batch number R20125), and 200 μL of hydrogel precursor solution material (non-magnetic hydrogel precursor solution material, i.e., CP group; magnetic hydrogel precursor solution material, i.e., CP / IO group; multifunctional magnetic hydrogel precursor solution material, i.e., CP / PIO group), and placed in 48-well plates and incubated at 37℃ for 24 hours. After incubation, the bacterial suspension was diluted 1000 times and 20 μL was evenly spread on agar plates. The agar plates were incubated at 37℃ for 24 h, and the bacterial colony formation was observed by photography to evaluate the antibacterial effect. The blank control group (Control) did not receive any hydrogel precursor solution material.

[0113] The results are as follows Figure 3 As shown in Figure c, which represents the results of the in vitro antibacterial test, it can be seen from the figure that: compared with the blank control group, the CP hydrogel played a certain role in inhibiting bacterial growth, which is mainly attributed to the amino groups present in the CMCS molecular chain; after the introduction of PIO NPs, the inhibitory ability of the CP / PIO hydrogel on bacteria was further enhanced, which can be attributed to the additional amino groups brought by the ε-polylysine component.

[0114] Example 4: Evaluation of the in vivo effects of hydrogel on promoting wound healing and angiogenesis

[0115] (1) The non-magnetic hydrogel precursor solution material, CP non-magnetic hydrogel, multifunctional magnetic hydrogel precursor solution material and CP / PIO multifunctional magnetic hydrogel prepared in Example 2 were applied to this example.

[0116] (2) The rats used were male SD rats, 3-4 weeks old, with an average weight of 200 g, purchased from Nanjing Qinglongshan Animal Breeding Farm.

[0117] SD rats were randomly divided into a blank control group (Control group), a CP group, a CP / PIO group, and a CP / PIO (SMF+) group under static magnetic field (grouping was based on the treatment method used). A circular wound with a diameter of 1.5 cm was created on the back of the rats using a skin sampler, and then a hydrogel precursor solution was applied to the wound site in situ. The wounds were photographed on days 0, 3, 7, and 14 to record the condition. The wound closure rate was statistically analyzed using ImageJ. Regenerated tissue was collected and CD31 stained to assess angiogenesis within the tissue.

[0118] Among them, the blank control group did not use hydrogel materials in the wound; the CP group used non-magnetic hydrogel precursor solution materials; the CP / PIO group used multifunctional magnetic hydrogel precursor solution materials; the CP / PIO (SMF+) group under static magnetic field action used multifunctional magnetic hydrogel precursor solution materials, with an external magnetic field applied, the magnet material being neodymium iron boron magnets, and the field strength being 89mT.

[0119] Macroscopic images of the wound, such as Figure 4 As shown in Figure a, it can be seen that the wound area of ​​all treatment groups gradually decreased throughout the healing process. On day 3, the wound closure of the CP / PIO group and the CP / PIO(SMF+) group was significantly better than that of the other two groups, indicating that the multifunctional magnetic hydrogel promoted wound healing in the initial stage. By day 7 and day 14, the wound color of the CP / PIO group and the CP / PIO(SMF+) group was lighter, similar to normal tissue, indicating that the synergistic effect of magnetic stimulation and PIO NPs promoted wound healing.

[0120] The results of CD31 immunohistochemical staining of endothelial cells in the healing tissue are shown in the figure below. Figure 4 Figure b shows the angiogenesis within the tissue. It can be seen that in the CP / PIO hydrogel group, the tubular structures formed by CD31 on day 3 were clearer and denser. This indicates that the magnetic hydrogel promoted rapid capillary formation in the early stages and provided sufficient nutrients for tissue repair. This can be attributed to the antioxidant effect of PIO NPs, which created a microenvironment conducive to angiogenesis within the tissue.

[0121] Furthermore, under the influence of an external magnetic field, the angiogenesis of the CP / PIO (SMF+) group was significantly better than that of the CP / PIO group, indicating that the magnetic stimulation generated by the magnetic hydrogel in response to the magnetic field promotes tissue angiogenesis.

[0122] This invention provides an injectable multifunctional magnetic hydrogel, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. An injectable multifunctional magnetic hydrogel, characterized in that, It includes the following components: Component A: Aldehyde-modified Prönnick F127; Component B: Carboxymethyl chitosan; Component C: ε-polylysine-modified iron(III) oxide; Component D: Phosphate buffer.

2. The injectable multifunctional magnetic hydrogel of claim 1, wherein, In component A, the aldehyde-modified Pluronic F127 is obtained by introducing an aldehyde group at the molecule terminus of Pluronic F127 using conventional methods.

3. The injectable multifunctional magnetic hydrogel of claim 1, wherein, In component C: the ε-polylysine-modified iron oxide was prepared according to the following method: Ferric chloride and ferrous chloride are mixed with concentrated hydrochloric acid to obtain an iron salt solution; After adjusting the pH of the iron salt solution to 10-14 with an alkaline solution, a salt-alkali neutralization reaction was carried out. After the reaction was completed, post-processing was performed to obtain ferric oxide magnetic nanoparticles. Subsequently, the magnetite nanoparticles and ε-polylysine were dispersed in the first solvent and subjected to electrostatic adsorption reaction. After the reaction was completed, post-treatment was performed to obtain ε-polylysine-modified magnetite nanoparticles.

4. The injectable multifunctional magnetic hydrogel of claim 3, wherein, The molar ratio of ferric chloride to ferrous chloride is (1.0~3.0):1.0; the alkaline solution is 25wt%~28wt% ammonia or 1.5~4.5 mol / L sodium hydroxide aqueous solution; the salt-alkali neutralization reaction is carried out under inert gas protection; the reaction temperature of the salt-alkali neutralization reaction is 70℃~90℃; the reaction time of the salt-alkali neutralization reaction is 1 h~3 h; the first solvent is deionized water; the mass ratio of the magnetite magnetic nanoparticles to ε-polylysine is 1.0:(1.0~5.0); the amount of the first solvent used is 0.5 mL~2.0 mL based on 1.0 mg ε-polylysine; the electrostatic adsorption reaction is carried out at room temperature; the reaction time of the electrostatic adsorption reaction is 12 h~36 h.

5. The injectable multifunctional magnetic hydrogel of claim 1, wherein, In component D, the phosphate buffer solution has a pH of 7.2-7.6 and a strength of 0.01 M.

6. The injectable multifunctional magnetic hydrogel of claim 1, wherein, In the injectable multifunctional magnetic hydrogel obtained by mixing components A, B, C, and D: the concentration of aldehyde-modified Pronnic F127 in component A is 50 mg / mL to 150 mg / mL; the concentration of carboxymethyl chitosan in component B is 20 mg / mL to 60 mg / mL; and the concentration of ε-polylysine-modified iron(III) oxide in component C is 0.5 mg / mL to 3 mg / mL.

7. A method for preparing an injectable multifunctional magnetic hydrogel, characterized in that, Mixing components A, B, C, and D as described in any one of claims 1 to 6 yields an injectable multifunctional magnetic hydrogel.

8. A method for preparing a multifunctional magnetic hydrogel, characterized in that, The injectable multifunctional magnetic hydrogel prepared according to the preparation method of claim 7 is then left to stand to obtain a multifunctional magnetic hydrogel.

9. The preparation method according to claim 8, characterized in that, The settling time is 120 s to 600 s.

10. The application of the injectable multifunctional magnetic hydrogel according to any one of claims 1 to 6, or the injectable multifunctional magnetic hydrogel prepared by the preparation method according to claim 7, or the multifunctional magnetic hydrogel prepared by the preparation method according to claim 8, in the preparation of magnetic biomaterials, and optionally, in the preparation of magnetic biomaterials that assist in wound hemostasis and / or assist in wound antibacterial activity and / or promote wound angiogenesis and / or promote wound repair.

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