Preparation method of self-healing and antibacterial dynamic hydrogel and coating application thereof

Abstract

This invention discloses a method for preparing a self-healing, antibacterial dynamic hydrogel and its coating application. The method includes: 1. Reacting konjac glucomannan with sodium periodate to achieve aldehyde functionalization, and mixing it with sodium alginate powder to prepare a precursor polymer solution A; 2. Mixing an acylhydrazide or hydroxylamine small molecule crosslinking agent with an aminoglycoside drug to prepare an amino crosslinking agent solution B, and preparing a calcium salt ionic crosslinking agent solution C; 3. Mixing the three solutions and allowing them to stand to obtain a hydrogel. The application involves coating a hydrogel mixture solution onto a biodegradable metal surface after preparing a polyethyleneimine auxiliary layer and then gelling it. This invention utilizes aldehyde-functionalized oxidized konjac glucomannan and two crosslinking agents to form acylhydrazone, oxime, and imine bond networks, endowing the hydrogel with self-healing, self-responsive, and long-term antibacterial properties. The hydrogel of this invention improves the biocompatibility, antibacterial properties, and stability of biodegradable metal substrates, making it suitable for biodegradable implantation applications.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a method for preparing a self-healing, antibacterial dynamic hydrogel and its coating application. Background Technology

[0002] In implantable biomedical metal materials, unlike traditional medical metals such as titanium alloys, cobalt-chromium alloys, and stainless steel which suffer from stress shielding and require secondary surgery, biodegradable metals such as magnesium-based and zinc-based materials have become a research frontier and hot topic in the field of medical metals due to their excellent biomechanical compatibility and biodegradability. However, biodegradable metals still face challenges such as insufficient biocompatibility and lack of biological functions, which limit their further application in the field of medical implant repair.

[0003] Surface functionalization coatings on biodegradable metals, without affecting their mechanical properties, have become an effective research strategy for regulating degradation behavior, improving biocompatibility, or enhancing biofunctionality, which is of great significance for meeting various clinical applications. References 1 (Singh N, Batra U, Kumar K, Ahuja N, Mahapatro A. Progress in bioactive surface coatings on biodegradable Mg alloys: A critical review towards clinical translation. Bioactive Materials, 19:717-757) and 2 (Shi Y, Xue Z, Li P, Yang S, Zhang D, Zhou S, Guan Z, Li Y, Wang LN. Surface modification on biodegradable zinc alloys. Journal of Materials Research and Technology, 2023, 25:3670-3687) indicate that current research on surface modification of biodegradable metals mainly focuses on magnesium-based metals, with relatively little research on zinc-based metals. Therefore, it is necessary to develop a universal coating strategy to achieve surface modification and functionality of different types of biodegradable metals.

[0004] Hydrogels are a class of soft, wet materials formed by cross-linked polymer networks binding aqueous solutions. They typically exhibit good biocompatibility and drug loading / sustaining release capabilities. Simultaneously, hydrogels possess physicochemical properties similar to the extracellular matrix and an elastic modulus close to that of human soft tissue. As a surface coating for medical metals, they can effectively eliminate mechanical wear and damage to human tissues caused by hard implants. Dynamic covalent gels, formed by reversible C=N bonds (imine bonds, hydrazone bonds, oxime bonds, etc.) cross-linked by aldehyde groups and primary amino, hydrazide, and hydroxylamine groups through Schiff base reactions, typically exhibit self-healing properties and can intelligently release aminoglycoside antibiotics from the unstable imine network under the stimulation of acidic substances produced by bacterial metabolism (Patent CN115785718A). Furthermore, in dynamic coordination network gels formed by carboxyl ligands and calcium ions, excess ligands can chelate magnesium and zinc ions dissolved from degradable metal surfaces, improving the bonding strength between the organic coating and the metal matrix. Combining the aforementioned dynamic covalent network with a metal coordination network to prepare an interpenetrating polymer network is beneficial for fabricating soft, antibacterial, self-healing, and well-adhered dynamic hydrogel coatings to metal substrates. This significantly improves the coating's biocompatibility and extends its service life. Therefore, the preparation of functional dynamic hydrogel coatings on biodegradable metal surfaces plays a crucial role in promoting the implantation and application of biodegradable metals such as magnesium-based and zinc-based materials. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a method for preparing a self-healing, antibacterial dynamic hydrogel, addressing the shortcomings of the prior art. This method utilizes aldehyde-functionalized konjac glucomannan to form a reversible C=N acylhydrazone and oxime crosslinking network with high hydrolytic stability, along with an amino crosslinking agent containing aminoglycosides, to form a pH-sensitive reversible C=N imine network. The combination of these two reversible dynamic covalent networks endows the hydrogel with self-healing and self-responsive dynamic properties. Furthermore, the imine network releases aminoglycosides under the stimulation of the acidic microenvironment of bacteria, achieving highly efficient and long-term antibacterial performance.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing a self-healing, antibacterial dynamic hydrogel, characterized in that the method includes the following steps:

[0007] Step 1: Preparation of hydrogel precursor polymer solution A: Konjac glucomannan was dissolved in deionized water and reacted with sodium periodate in the dark. The reaction was terminated by ethylene glycol. After purification, the product was freeze-dried to obtain aldehyde-functionalized konjac glucomannan. Then, the aldehyde-functionalized konjac glucomannan was mixed with sodium alginate powder and deionized water was added to obtain precursor polymer solution A.

[0008] Step 2: Preparation of hydrogel precursor crosslinking agent solutions B and C: Mix hydrazide or hydroxylamine small molecule crosslinking agent with aminoglycoside drug, add deionized water to dissolve, and obtain amino crosslinking agent solution B; disperse or dissolve calcium salt in deionized water to obtain ionic crosslinking agent solution C.

[0009] Step 3: Preparation of hydrogel: The precursor polymer solution A obtained in Step 1 is uniformly mixed with the amine crosslinking agent solution B and the ionic crosslinking agent solution C obtained in Step 2 to obtain a hydrogel solution. The mixture is then allowed to stand at room temperature for 5 min to 60 min to allow the hydrogel solution to gel and obtain a healing and antibacterial dynamic hydrogel.

[0010] Typically, in step one of this invention, konjac glucomannan (KGM) is dissolved in deionized water and reacted with sodium periodate (NalO4) in the dark at a water bath at 40°C for 6 hours. After the reaction time is reached, ethylene glycol is added to quench the reaction, and the reaction continues for another 2 hours. After purification, the mixture is freeze-dried to obtain aldehyde-functionalized oxidized konjac glucomannan (OKGM). The reaction mechanism is as follows: sodium periodate selectively oxidizes the hydroxyl groups at the 2 and 3 carbon positions of the sugar ring of konjac glucomannan to obtain an aldehyde substituent, as shown in the following reaction formula:

[0011]

[0012] The aldehyde content in OKGM was determined by hydroxylamine hydrochloride titration. The specific procedure was as follows: 17.500g of hydroxylamine hydrochloride, which had been freeze-dried to constant weight, was dissolved in 150mL of deionized water. Then, 6mL of 0.05% methyl orange solution was added and stirred until homogeneous. The solution was then diluted to 1L with deionized water, and the pH was adjusted to 4.0 with 0.1M NaOH solution. At this point, the solution was orange-yellow, yielding a 0.25M hydroxylamine hydrochloride solution. Subsequently, 0.04g of OKGM was weighed and added to 10mL of the above 0.25M hydroxylamine hydrochloride solution. The solution was stirred in a 40℃ water bath for 6h to obtain a pink mixed solution. This pink mixed solution was titrated with 0.1M NaOH solution, and the pH change of the mixed solution was recorded until the solution changed from orange to yellow. Each sample was tested three times, and the average value was taken.

[0013] The aldehyde content ([-CHO], mmol / g) in OKGM is calculated using the formula [-CHO]=ΔV×cNaOH / mOKGM, where ΔV is the volume of NaOH solution consumed at the titration endpoint (mL), cNaOH is the concentration of NaOH solution (mol / L), and mOKGM is the weight of OKGM weighed (g).

[0014] Typically, the aldehyde content in OKGM ranges from 4 mmol / g to 15 mmol / g.

[0015] The relative molecular weight of aldehyde-functionalized oxidized konjac glucomannan (OKGM) was determined using gel permeation chromatography (GPC). Typically, the average molecular weight of OKGM ranges from 10 kDa to 40 kDa, with a molecular weight distribution index (DPI) of 1.3 to 1.4.

[0016] The method for preparing a self-healing, antibacterial dynamic hydrogel described above is characterized in that the concentration of konjac glucomannan dissolved in deionized water in step one is 0.01 g / mL. Typically, dissolution is performed by magnetic stirring and heating at a temperature of 40℃~60℃ for 4h~10h, resulting in a uniform, stable konjac glucomannan solution free of lumps and insoluble matter.

[0017] The preparation method of the above-mentioned self-healing, antibacterial dynamic hydrogel is characterized in that the mass ratio of sodium periodate to konjac glucomannan in step one is 1:1, and the molar ratio of ethylene glycol to sodium periodate is 2:1.

[0018] The method for preparing a self-healing, antibacterial dynamic hydrogel described above is characterized in that the purification method in step one involves dialysis purification in deionized water. Typically, the reaction product system of konjac glucomannan and periodic acid is placed in a dialysis bag (MWCO: 8000–14000 Da) and dialyzed in deionized water for 7 days.

[0019] The above-mentioned method for preparing a self-healing, antibacterial dynamic hydrogel is characterized in that the total polymer concentration of the precursor polymer solution A in step one is 0.04 g / mL, and the mass ratio of aldehyde-functionalized konjac glucomannan to sodium alginate is 1:1.

[0020] Typically, the viscosity of sodium alginate (SA) powder used in this invention is 100 mPa·s to 1500 mPa·s.

[0021] Typically, in this invention, aldehyde-functionalized konjac glucomannan is mixed with sodium alginate powder, then added to deionized water and heated and stirred to dissolve at a temperature of 40°C to 60°C for 4 to 8 hours to obtain a uniform, stable, and precipitate-free precursor polymer solution A.

[0022] The method for preparing a self-healing, antibacterial dynamic hydrogel described above is characterized in that, in step two, the hydrazide small molecule crosslinking agent is adipic acid dihydrazide (ADH) or succinic acid dihydrazide (SDH), and the hydroxylamine small molecule crosslinking agent is O,O′-1,3-propanediol dihydroxyamine dihydrochloride (PBH); the aminoglycoside drug is tobramycin, amikacin sulfate, gentamicin sulfate, ribosomycin sulfate, neomycin sulfate, or capreomycin sulfate; the concentration of the hydrazide or hydroxylamine small molecule crosslinking agent in the amino crosslinking agent solution B is 0.24 mol / L to 0.84 mol / L, and the concentration of the aminoglycoside drug is 0.02 mol / L to 0.23 mol / L.

[0023] The method for preparing a self-healing, antibacterial dynamic hydrogel described above is characterized in that the calcium salt in step two is calcium hydroxide, calcium carbonate, calcium sulfate, calcium acetate, calcium gluconate, or calcium chloride; and the concentration of the calcium salt in the ionic crosslinking agent solution C is 0.1 mol / L. Typically, the ionic crosslinking agent solution C is a uniform dispersion or a transparent solution.

[0024] Meanwhile, the present invention also discloses an application of a coating of self-healing, antibacterial dynamic hydrogel prepared by the above method, characterized in that: a polyethyleneimine aqueous solution is spin-coated onto a biodegradable metal surface that has undergone alkaline pretreatment, and a polyethyleneimine auxiliary layer is formed after drying; then, the hydrogel mixture obtained in step three is uniformly coated on the polyethyleneimine auxiliary layer; after gelation, a biodegradable metal with a self-healing, antibacterial dynamic hydrogel coating is obtained.

[0025] The above-described coating application is characterized in that the concentration of the polyethyleneimine aqueous solution is 0.1 g / mL.

[0026] The above-mentioned coating application is characterized in that the preparation process of the biodegradable metal after alkaline pretreatment is as follows: magnesium, magnesium alloy or zinc, zinc alloy discs that have been sanded, polished, cleaned and dried are immersed in sodium hydroxide solution for treatment, and then cleaned and dried.

[0027] Typically, the dimensions (diameter × thickness) of the discs are Φ10mm × 3mm; the spin-coating amount of polyethyleneimine aqueous solution (PEI aqueous solution) is 100μL. The discs are immersed in a 5mol / L sodium hydroxide solution at a temperature of 55℃~65℃ for 20min~40min, followed by washing with water and ethanol, drying at 60℃, and then freeze-drying.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] 1. This invention uses aldehyde-functionalized konjac glucomannan and sodium alginate as macromolecular precursors to construct a dynamic hydrogel through crosslinking. The aldehyde-functionalized konjac glucomannan forms a reversible C=N bond acylhydrazone bond and oxime bond crosslinking network with hydrazide or hydroxylamine small molecule crosslinking agents, which has high hydrolytic stability, improving the chemical stability of the gel. This crosslinking network also exhibits self-healing properties. At the same time, the aldehyde-functionalized konjac glucomannan forms a pH-sensitive reversible C=N bond imine bond network with aminoglycoside drug amino crosslinking agents. Under the stimulation of the acidic microenvironment produced by bacteria, it responds by releasing the antibiotic aminoglycoside drug, achieving a highly efficient and long-lasting intelligent antibacterial effect. Since the reversible C=N bond network is a dynamic covalent network, its reversible dynamism can endow the gel with dynamic properties of self-healing and self-response.

[0030] 2. The aldehyde-functionalized oxidized konjac glucomannan used in the preparation of dynamic hydrogels in this invention is obtained by selective oxidation of konjac glucomannan. This process is easy to implement, the preparation method of hydrogels is simple to operate, has good versatility, and other raw materials for preparing dynamic hydrogels are abundant and readily available, with low cost, thus reducing the cost of hydrogel preparation.

[0031] 3. This invention prepares a polyethyleneimine auxiliary layer on the surface of a biodegradable metal to combine a self-healing and antibacterial dynamic hydrogel coating. The hydrogel consists of two interpenetrating dynamic polymer networks: one is a dynamic covalent network formed by aldehyde-modified konjac glucomannan and a small molecule amino crosslinking agent / aminoglycoside drug; the other is a dynamic coordination network crosslinked by sodium alginate and calcium ions. Because the dynamic hydrogel coating has physicochemical properties similar to those of the extracellular matrix, it greatly improves the biocompatibility of the biodegradable metal matrix. At the same time, the dynamic coordination network improves the bonding performance between the dynamic hydrogel coating and the biodegradable metal matrix by chelating magnesium ions, zinc ions, etc. dissolved from the surface of the biodegradable metal. Therefore, the dynamic hydrogel coating improves the biocompatibility, intelligent antibacterial function, and stability and durability of the biodegradable metal matrix, providing a solution to problems such as biotoxicity, bacterial infection, and service life in surgical interventional applications.

[0032] 4. This invention pretreats biodegradable metals with an alkaline solution to form a microporous and microcracked oxide layer on their surface. On the one hand, the hydroxides and oxides in this oxide layer act as a barrier against corrosive media. On the other hand, the micro- and nano-structures in this oxide layer form a mechanical interlocking structure with the polyethyleneimine auxiliary layer, enhancing the bonding ability between the biodegradable metal and the polyethyleneimine auxiliary layer. Furthermore, the amine groups in the polyethyleneimine auxiliary layer form imine bonds with the aldehyde-functionalized oxykonjac glucomannan in the dynamic covalent network of the hydrogel, further improving the bonding ability between the dynamic hydrogel coating and the metal substrate.

[0033] 5. This invention regulates the gelation rate and microstructure of dynamic hydrogels by controlling the type and concentration of amine crosslinking agents in the amine crosslinking agent solution and ionic crosslinking agent solution, thereby ensuring the self-crosslinking rate of the hydrogel precursor solution on the biodegradable metal surface and improving the preparation efficiency and success rate of dynamic hydrogel coatings on biodegradable metal surfaces.

[0034] 6. The oxidized konjac glucomannan and sodium alginate used in this invention to prepare dynamic hydrogel coatings are both biocompatible and low-cost biopolymers that have been widely developed for use in wound repair, drug delivery, tissue engineering and other fields. This is conducive to the promotion and application of such dynamic hydrogel coating materials in the field of biodegradable implants.

[0035] 7. This invention applies a self-healing, antibacterial dynamic hydrogel to the surface of biodegradable metals, which not only enhances the biocompatibility of biodegradable metals, but also achieves long-lasting intelligent antibacterial effect, preventing stress damage to soft tissues and surgical infection caused by hard implants. It can serve as an important reference for the application of biofunctional coatings in the field of biodegradable surgical interventions and can be further promoted and learned from.

[0036] 8. The method of preparing biodegradable metals with self-healing and antibacterial dynamic hydrogel coatings on the surface according to the present invention has high versatility and good repeatability, and has good prospects for industrial application.

[0037] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the self-healing, antibacterial dynamic hydrogel of the present invention and its application in a biodegradable metal coating.

[0039] Figure 2 The Fourier transform infrared spectra of SA, KGM, OKGM, G4-0 and NetworkNo.1 and NetworkNo.2 in Embodiment 4 and G4-2 in Embodiment 8 are shown.

[0040] Figure 3 This is a physical image of the dynamic hydrogel prepared in Example 4 of the present invention to verify its self-healing properties.

[0041] Figure 4 SEM images of the dynamic hydrogels prepared in Examples 3-6 of this invention.

[0042] Figure 5a The survival rate of L929 cells after 24 hours of treatment with the dynamic hydrogels prepared in Examples 4, 7 and 8 of this invention.

[0043] Figure 5bThe survival rate of L929 cells after 72 hours of treatment with the dynamic hydrogels prepared in Examples 4, 7 and 8 of this invention.

[0044] Figure 5c This is a live-cell fluorescence staining image of L929 cells after 72 hours of treatment with the dynamic hydrogels prepared in Examples 4, 7 and 8 of this invention.

[0045] Figure 6 This is a comparison of bacterial colonies obtained by plate counting of the dynamic hydrogels prepared in Examples 8 and 9 of this invention.

[0046] Figure 7a This is a SEM image of the surface of the magnesium alloy WE43 disc after alkaline pretreatment in Example 24 of the present invention.

[0047] Figure 7b This is a SEM image of the surface of the magnesium alloy WE43 disc in Embodiment 24 of the present invention, in which a polyethyleneimine auxiliary layer is formed.

[0048] Figure 7c This is a SEM image of the surface of the magnesium alloy WE43 disc with G4-2 dynamic hydrogel coating in Embodiment 24 of the present invention.

[0049] Figure 8a This is a SEM image of the zinc disc surface after alkaline pretreatment in Example 25 of the present invention.

[0050] Figure 8b This is a SEM image of the zinc disc surface in Example 25 of the present invention where a polyethyleneimine auxiliary layer is formed.

[0051] Figure 8c This is a SEM image of the zinc disc surface with a G4-2 dynamic hydrogel coating in Embodiment 25 of the present invention. Detailed Implementation

[0052] Figure 1 This is a schematic diagram of the self-healing, antibacterial dynamic hydrogel of the present invention and its application in a biodegradable metal coating. Figure 1 It can be seen that OKGM in precursor polymer solution A forms crosslinking network 1 (Network No. 1, where rhombuses represent hydrazone or oxime crosslinking and stars represent imine bond networks) with hydrazide or hydroxylamine small molecule crosslinking agent through Schiff base reaction. SA in precursor polymer solution A forms crosslinking network 2 (Network No. 2) with ionic crosslinking agent through coordination bond crosslinking, which interpenetrates with network 1. This is coated on a biodegradable metal surface that has been pretreated with alkaline solution and coated with polyethyleneimine (PEI) aqueous solution to form a polyethyleneimine auxiliary layer, resulting in a biodegradable metal with a self-healing, antibacterial dynamic hydrogel coating.

[0053] The preparation method of the self-healing and antibacterial dynamic hydrogel of the present invention is described in detail through Examples 1 to 9.

[0054] Example 1

[0055] This embodiment includes the following steps:

[0056] Step 1: Preparation of hydrogel precursor polymer solution A: Dissolve 5g of konjac glucomannan in 500mL of deionized water and stir in a water bath at 40℃~60℃ for 8h. Add 40mL of 0.125g / mL NaIO4 solution dropwise under dark conditions, completing the addition in about half an hour. Continue the reaction under dark conditions for 6h. Add 2.9g of ethylene glycol and stir for 2h to terminate the reaction. Purify the reaction product system by dialyzing in deionized water for 7 days. Obtain the aldehyde group by freeze-drying. Functionalized konjac glucomannan OKGM was tested and found to have an aldehyde content of no more than 7.8 mmol / g, an average molecular weight of 16.8 kDa, and a molecular weight distribution DPI of 1.39. Then, 0.08 g of aldehyde-functionalized konjac glucomannan was mixed with 0.08 g of sodium alginate powder with a viscosity of 200 mPa.s ± 20 mPa.s, and 4 mL of deionized water was added. The mixture was stirred in a water bath at 40℃~60℃ for 6 h to obtain precursor polymer solution A.

[0057] Step 2: Preparation of hydrogel precursor crosslinking agent solutions B and C: Adipic acid dihydrazide (ADH) was mixed with amikacin sulfate (Ami) and dissolved in deionized water to obtain 0.24 mol / L amine crosslinking agent solution B. Calcium gluconate was dissolved in deionized water to obtain 0.1 mol / L ionic crosslinking agent solution C.

[0058] Step 3: Preparation of hydrogel: Mix 250 μL of precursor polymer solution A obtained in Step 1 with 25 μL of amine crosslinking agent solution B obtained in Step 2 and 25 μL of ionic crosslinking agent solution C obtained in Step 2 to obtain a hydrogel solution. Let it stand at room temperature for 5 min to allow the hydrogel solution to gel, and obtain a self-healing, antibacterial dynamic hydrogel, named G1-0.

[0059] In this embodiment, the hydrazide small molecule crosslinking agent can be replaced with succinic dihydrazide SDH, or with the hydroxylamine small molecule crosslinking agent O,O′-1,3-propanediol dihydroxyamine dihydrochloride PBH. The aminoglycoside drug can also be replaced with tobramycin, amikacin sulfate, gentamicin sulfate, ribosomycin sulfate, neomycin sulfate, or capreomycin sulfate. The calcium salt can also be replaced with calcium hydroxide, calcium carbonate, calcium sulfate, calcium acetate, or calcium chloride.

[0060] Example 2

[0061] The difference between this embodiment and Example 1 is that the concentration of ADH in the amine crosslinking agent solution B prepared in step two is 0.32 mol / L, and the hydrogel mixture solution in step three is allowed to stand at room temperature for 10 minutes to form a gel. The obtained self-healing, antibacterial dynamic hydrogel is named G2-0.

[0062] Example 3

[0063] The difference between this embodiment and Example 1 is that the concentration of ADH in the amine crosslinking agent solution B prepared in step two is 0.48 mol / L, and the hydrogel mixture solution in step three is allowed to stand at room temperature for 12 minutes to form a gel. The obtained self-healing, antibacterial dynamic hydrogel is named G3-0.

[0064] Example 4

[0065] The difference between this embodiment and Example 1 is that the concentration of ADH in the amine crosslinking agent solution B prepared in step two is 0.6 mol / L, and the hydrogel mixture solution in step three is allowed to stand at room temperature for 60 min to form a gel. The obtained self-healing, antibacterial dynamic hydrogel is named G4-0.

[0066] Example 5

[0067] The difference between this embodiment and Example 1 is that the concentration of ADH in the amine crosslinking agent solution B prepared in step two is 0.72 mol / L, and the hydrogel mixture solution in step three is allowed to stand at room temperature for 30 minutes to form a gel. The obtained self-healing, antibacterial dynamic hydrogel is named G5-0.

[0068] Example 6

[0069] The difference between this embodiment and Example 1 is that the concentration of ADH in the amine crosslinking agent solution B prepared in step two is 0.84 mol / L, and the hydrogel mixture solution in step three is allowed to stand at room temperature for 10 minutes to form a gel. The obtained self-healing, antibacterial dynamic hydrogel is named G6-0.

[0070] Example 7

[0071] The difference between this embodiment and Example 1 is that the concentrations of ADH and amikacin sulfate (Ami) in the amine crosslinking agent solution B prepared in step two are 0.6 mol / L and 0.02 mol / L, respectively, and the hydrogel mixture solution in step three is allowed to stand at room temperature for 60 min to form a gel. The obtained self-healing, antibacterial dynamic hydrogel is named G4-1.

[0072] Example 8

[0073] The difference between this embodiment and Example 1 is that the concentrations of ADH and amikacin sulfate (Ami) in the amine crosslinking agent solution B prepared in step two are 0.6 mol / L and 0.23 mol / L, respectively, and the hydrogel mixture solution in step three is allowed to stand at room temperature for 30 min to form a gel. The obtained self-healing, antibacterial dynamic hydrogel is named G4-2.

[0074] Example 9

[0075] The difference between this embodiment and Example 1 is that the concentrations of ADH and amikacin sulfate (Ami) in the amine crosslinking agent solution B prepared in step two are 0.84 mol / L and 0.23 mol / L, respectively, and the hydrogel mixture solution in step three is allowed to stand at room temperature for 10 min to form a gel. The obtained self-healing, antibacterial dynamic hydrogel is named G6-2.

[0076] (I) Fourier Transform Infrared Spectroscopy (FTIR) Characterization

[0077] The SA, KGM, OKGM, dynamic hydrogel G4-0 of Example 4, Network No. 1, Network No. 2, and dynamic hydrogel G4-2 of Example 8 were characterized by FTIR. The specific process was as follows: all samples were freeze-dried before testing; then, the dried KBr and the sample to be tested were mixed evenly at a mass ratio of 100:1 and ground into powder. After pressing into tablets, Fourier transform infrared spectroscopy was used for testing, with a scanning range of 400 cm⁻¹. -1 ~4000cm -1 The result is as follows Figure 2 As shown.

[0078] from Figure 2 It can be seen that after selective oxidation with NaIO4, the FTIR curve of OKGM is at 1730 cm⁻¹. -1 A stretching vibration peak of the aldehyde group, which is not present in KGM, appeared at 880 cm⁻¹; at the same time, a new absorption peak of the hemiacetal generated by the condensation of the aldehyde group and hydroxyl group in OKGM also appeared at 880 cm⁻¹. -1 The presence of these peaks demonstrates the successful synthesis of aldehyde-functionalized OKGM. The disappearance of the OKGM aldehyde characteristic peaks in Network No. 2, G4-0, and G4-2 confirms that the aldehyde group reacts with the amine crosslinking agent to successfully construct a dynamic covalent network of acylhydrazone / imine bonds.

[0079] To confirm SA and Ca 2+ A metal coordination network was successfully formed. The FTIR of SA and Network No. 1 were tested separately. The curve comparison results show that the carboxyl group (COO) of SA... - The asymmetric stretching vibration peak of ) in Network No. 1 is from 1610 cm⁻¹-1 The blue shift was to 1630cm -1 The absorption peak intensity decreased at this location, indicating that the association between carboxyl groups dissociated and Ca... 2+ With COO - Coordination occurred.

[0080] In summary, the FTIR test results demonstrate the successful cross-linking of the dynamic covalent network and dynamic coordination network of the gel.

[0081] (II) Self-healing properties of dynamic hydrogels

[0082] A dynamic hydrogel disc (5 mm high, 10 mm in diameter) was cut in half using a utility knife blade, and one half was stained with methyl orange. The two separate hemispherical gel fragments were then brought into contact with each other under a small amount of external force and left at room temperature for a certain period of time to observe the boundary changes at the gel joint. Subsequently, one half of the gel was adhered to a metal sheet and placed vertically to observe the self-healing properties of the dynamic hydrogel.

[0083] Upon testing, it was found that after the dynamic hydrogel hemispherical fragments prepared in Examples 1 to 6 of this invention were spliced ​​together, the boundary at the splicing point became difficult to observe within 2 to 10 minutes, indicating that the two hemispherical gel fragments underwent material exchange and recombination at the cut.

[0084] Figure 3 This is a physical image of the dynamic hydrogel prepared in Example 4 of this invention to verify its self-healing properties. Figure 3 It can be seen that under the action of gravity, the semi-circular gel fragments at the bottom of the dynamic hydrogel did not separate after being left to stand for 3 to 5 minutes, proving that the dynamic hydrogel has achieved self-healing. This is because the self-healing property provided by the reversible cross-linking of two interpenetrating dynamic polymer networks in the dynamic hydrogel is beneficial to extending the service life of the biodegradable metal coating.

[0085] (III) Observation by Scanning Electron Microscopy (SEM)

[0086] After sputtering gold onto the surface of the freeze-dried dynamic hydrogel sample, the surface morphology was observed using SEM.

[0087] Figure 4 The images shown are SEM images of the dynamic hydrogels prepared in Examples 3-6 of this invention, where (a) is G3-0 prepared in Example 3, (b) is G4-0 prepared in Example 4, (c) is G5-0 prepared in Example 5, and (d) is G6-0 prepared in Example 6. Figure 4As can be seen, all hydrogels in the figure exhibit the interconnected network structure characteristic of this type of material. As a coating for biodegradable metals, it facilitates cell migration and nutrient transport, providing a favorable extracellular matrix environment for promoting tissue repair and regeneration of metal implants. Simultaneously, from G3-0, G4-0, G5-0 to G6-0, with the increase of amine crosslinking agent content, the network structure of the dynamic hydrogel becomes more uniform. This may be due to more dynamic acylhydrazone networks interspersed in the dynamic coordination network, effectively alleviating the hydrophobic aggregation of the dynamic covalent network. Furthermore, since aminoglycoside drugs with antibacterial functions can also serve as amine crosslinking agents for generating imine bonds, selecting the G4-0 system (G4-2 in Example 8 of this invention), with a total amine crosslinking agent concentration similar to G6-0 after increasing amikacin sulfate, as the antibacterial gel system is beneficial for obtaining a more uniform gel network and facilitating the release of amikacin, thus achieving the antibacterial function of the coating.

[0088] (iv) Biocompatibility of dynamic hydrogel coatings

[0089] The biocompatibility of the dynamic hydrogels of Examples 4 (G4-0), 7 (G4-1), and 8 (G4-2) of this invention was evaluated using mouse fibroblasts (L929 cells). The specific procedure was as follows: Dynamic hydrogel samples sterilized by UV for 30 min were added to MEM medium at a concentration of 0.01 g / mL and soaked at 37°C for 24 h. 100% extract was collected, and then serially diluted with MEM medium to obtain 50% and 25% extracts. L929 cells were seeded in 96-well plates, and the medium was replaced with 100%, 50%, and 25% gel extracts. Cells without dynamic hydrogel extract served as a control group. After 24 h and 72 h of culture, medium containing 10% CCK-8 reagent was added. After 1 h, the absorbance at 450 nm was measured using a microplate reader to calculate cell viability. The cytotoxicity test results are as follows: Figures 5a-5c As shown.

[0090] from Figures 5a-5c It can be seen that from G4-0, G4-1 to G4-2, the toxicity of the dynamic hydrogel to cells increases with the increase of Ami content. Figure 5b Cell viability after 72 hours showed that the cytotoxicity of the dynamic hydrogel increased significantly with increasing Ami content. L929 cells cultured in the control group and 100% gel extract for 72 hours were observed after fluorescent staining using a live / dead cell staining kit. Live cell staining results are shown below. Figure 5c ,and Figure 5bThe results from CCK-8 assays were largely consistent. These results further confirm that the loading of aminoglycoside antibiotics significantly affects the biocompatibility of the gel. However, the G4-0 hydrogel without Ami showed no cytotoxicity to L929 cells at both 24h and 72h, and the G4-1 hydrogel containing a small amount of Ami also showed no cytotoxicity to L929 cells at 24h, indicating that dynamic hydrogels possess good biocompatibility and can promote the biocompatibility of degradable metal substrates as functional coatings.

[0091] (V) Antibacterial properties of dynamic hydrogel coatings

[0092] The antibacterial properties of the dynamic hydrogels loaded with Ami in Examples 8 and 9 of this invention were investigated using the plate count method with Staphylococcus aureus and Escherichia coli. The specific procedure was as follows: dynamic hydrogel samples were prepared in 24-well plates, and 10... 6 A bacterial solution of CFU / mL was incubated at 37°C for 2 hours. The bacterial suspension was then inoculated onto an agar plate and incubated at 37°C for 18 hours as the experimental group. The bacterial colony count was recorded, and the inhibition rate (%) was calculated as (CFU of the anode control group - CFU of the experimental group) / CFU of the anode control group. The anode control group consisted of bacterial liquid culture medium that did not come into contact with the gel. Results are as follows: Figure 6 As shown.

[0093] from Figure 6 The results showed that the G4-2 and G6-2 gels exhibited inhibition rates of 91% and 96% against Staphylococcus aureus, respectively, indicating that the dynamic hydrogel loaded with aminoglycoside drugs possesses excellent in vitro antibacterial properties and can effectively prevent bacterial infection of degradable metal implants as a coating. Both G4-2 and G6-2 gels contained the same concentration of aminoglycosides (Ami), but the antibacterial ability of G4-2 was slightly lower than that of G6-2. This is because the concentration of ADH amino crosslinking agent in G4-2 was lower than that in G6-2, resulting in more Ami participating in the construction of the imine bond network in G4-2. Therefore, within the same time frame, less Ami was released from G4-2 gel than from G4-6. This demonstrates that by controlling the crosslinking agent composition of the dynamic covalent network in the dynamic hydrogel, the release amount of aminoglycoside antibacterial agents can be controlled, thereby prolonging the antibacterial ability of the gel coating.

[0094] The application of the biodegradable metal coating of the self-healing, antibacterial dynamic hydrogel of the present invention is described in detail through Examples 10-27.

[0095] Example 10

[0096] This embodiment includes the following steps:

[0097] Step 1: After sanding, polishing, cleaning, and drying, magnesium alloy WE43 discs with dimensions (diameter × thickness) of Φ10mm × 3mm are immersed in a 5mol / L sodium hydroxide solution for 40 minutes at a temperature of 55℃~65℃. Then, they are washed with water and ethanol and dried to obtain magnesium alloy WE43 discs that have undergone alkaline pretreatment.

[0098] Step 2: Spin-coat 100 μL of a 0.1 g / mL polyethyleneimine aqueous solution onto the surface of a pre-treated alkali magnesium alloy WE43 disc, place it in a 60°C oven to dry and freeze-dry to form a polyethyleneimine auxiliary layer, and then uniformly coat the polyethyleneimine auxiliary layer with the hydrogel mixture solution obtained in Step 3 of Example 1 of this invention. After gelation, a biodegradable magnesium alloy WE43 with a self-healing, antibacterial dynamic hydrogel coating is obtained.

[0099] Example 11

[0100] This embodiment includes the following steps:

[0101] Step 1: After sanding, polishing, cleaning, and drying, zinc discs with dimensions (diameter × thickness) of Φ10mm × 3mm are immersed in a 5mol / L sodium hydroxide solution for 20 minutes at a temperature of 55℃~65℃. Then, they are washed with water and ethanol and dried to obtain zinc discs that have undergone alkaline pretreatment.

[0102] 100 μL of a 0.1 g / mL polyethyleneimine aqueous solution was spin-coated onto the surface of a zinc disc that had undergone alkaline pretreatment. The disc was then dried in an oven at 60°C and freeze-dried to form a polyethyleneimine auxiliary layer. The hydrogel mixture obtained in step three of Example 1 of this invention was then uniformly coated onto the polyethyleneimine auxiliary layer. After gelation, a biodegradable zinc with a self-healing, antibacterial dynamic hydrogel coating was obtained.

[0103] Example 12

[0104] The difference between this embodiment and Embodiment 10 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 2 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0105] Example 13

[0106] The difference between this embodiment and Embodiment 11 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 2 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0107] Example 14

[0108] The difference between this embodiment and Embodiment 10 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 3 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0109] Example 15

[0110] The difference between this embodiment and Embodiment 11 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 3 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0111] Example 16

[0112] The difference between this embodiment and Embodiment 10 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 4 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0113] Example 17

[0114] The difference between this embodiment and Embodiment 11 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 4 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0115] Example 18

[0116] The difference between this embodiment and Embodiment 10 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 5 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0117] Example 19

[0118] The difference between this embodiment and Embodiment 11 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 5 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0119] Example 20

[0120] The difference between this embodiment and Embodiment 10 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 6 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0121] Example 21

[0122] The difference between this embodiment and Embodiment 11 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 6 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0123] Example 22

[0124] The difference between this embodiment and Embodiment 10 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 7 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0125] Example 23

[0126] The difference between this embodiment and Embodiment 11 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 7 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0127] Example 24

[0128] The difference between this embodiment and Embodiment 10 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 8 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0129] Example 25

[0130] The difference between this embodiment and Embodiment 11 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 8 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0131] Scanning electron microscopy (SEM) was performed on the magnesium alloy WE43 discs and zinc discs from each processing stage in Examples 24-25 of this invention. The results are as follows: Figures 7a-7c and Figures 8a-8c As shown.

[0132] from Figure 7a and Figure 8a It can be seen that after alkaline pretreatment, the surfaces of the biodegradable magnesium alloy WE43 discs and zinc discs acquired micro-nano structures with micropores and microcracks, which is beneficial for the formation of a mechanically interlocking surface bond between the polyethyleneimine auxiliary layer and the metal surface, thereby improving the bonding ability between the metal substrate and the dynamic hydrogel coating; from Figure 7b and Figure 8b It can be seen that after treatment with polyethyleneimine, both the magnesium alloy WE43 discs and the zinc discs are uniformly coated with a polyethyleneimine film, forming a PEI film. Even when cracks appear in the dried PEI film, no micro- or nano-structures that can degrade the metal surface are observed, indicating that the polyethyleneimine auxiliary layer has successfully and completely covered the metal substrate surface. Figure 7c and Figure 8c It can be seen that after coating with the dynamic hydrogel coating, the surface morphology of the magnesium alloy WE43 discs and zinc discs exhibits a micro-network porous structure of hydrogel. The surface gel network structure of the magnesium alloy WE43 discs differs from that of the zinc disc substrate; the gel network structure on the magnesium alloy surface is more dense. This may be because, compared to the zinc substrate, the magnesium substrate is more prone to degradation of dissolved Mg. 2+ I participated in the establishment of the gel dynamic ligand network.

[0133] Example 26

[0134] The difference between this embodiment and Embodiment 10 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 9 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0135] Example 27

[0136] The difference between this embodiment and Embodiment 11 is that in step two, the hydrogel mixture solution obtained in step three of Embodiment 9 of the present invention is uniformly coated on the polyethyleneimine auxiliary layer.

[0137] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing a self-healing, antibacterial dynamic hydrogel, characterized in that, The method includes the following steps: Step 1: Preparation of hydrogel precursor polymer solution A: Konjac glucomannan was dissolved in deionized water and reacted with sodium periodate in the dark. The reaction was terminated by ethylene glycol. After purification, the product was freeze-dried to obtain aldehyde-functionalized konjac glucomannan. Then, the aldehyde-functionalized konjac glucomannan was mixed with sodium alginate powder and deionized water was added to obtain precursor polymer solution A. Step 2: Preparation of hydrogel precursor crosslinking agent solutions B and C: A small molecule crosslinking agent, either acylhydrazide or hydroxylamine, is mixed with an aminoglycoside drug and dissolved in deionized water to obtain amino crosslinking agent solution B. Calcium salt is dispersed or dissolved in deionized water to obtain ionic crosslinking agent solution C. The acylhydrazide small molecule crosslinking agent is adipic acid dihydrazide (ADH) or succinic acid dihydrazide (SDH), and the hydroxylamine small molecule crosslinking agent is O,O′-1,3-propanediol dihydroxylamine dihydrochloride (PBH). Step 3: Preparation of hydrogel: The precursor polymer solution A obtained in Step 1 is uniformly mixed with the amine crosslinking agent solution B and the ionic crosslinking agent solution C obtained in Step 2 to obtain a hydrogel solution. The mixture is then allowed to stand at room temperature for 5 min to 60 min to allow the hydrogel solution to gel and obtain a self-healing, antibacterial dynamic hydrogel.

2. The method for preparing a self-healing, antibacterial dynamic hydrogel according to claim 1, characterized in that, The concentration of konjac glucomannan dissolved in deionized water in step one is 0.01 g / mL.

3. The method for preparing a self-healing, antibacterial dynamic hydrogel according to claim 1, characterized in that, In step one, the mass ratio of sodium periodate to konjac glucomannan is 1:1, and the molar ratio of ethylene glycol to sodium periodate is 2:

1.

4. The method for preparing a self-healing, antibacterial dynamic hydrogel according to claim 1, characterized in that, The purification method described in step one is dialysis purification in deionized water.

5. The method for preparing a self-healing, antibacterial dynamic hydrogel according to claim 1, characterized in that, In step one, the total polymer concentration of the precursor polymer solution A is 0.04 g / mL, and the mass ratio of aldehyde-functionalized konjac glucomannan to sodium alginate is 1:

1.

6. The method for preparing a self-healing, antibacterial dynamic hydrogel according to claim 1, characterized in that, The aminoglycoside drug mentioned in step two is tobramycin, amikacin sulfate, gentamicin sulfate, ribosomycin sulfate, neomycin sulfate, or capreomycin sulfate; the concentration of the amide crosslinking agent solution B is 0.24 mol / L to 0.84 mol / L, and the concentration of the aminoglycoside drug is 0.02 mol / L to 0.23 mol / L.

7. The method for preparing a self-healing, antibacterial dynamic hydrogel according to claim 1, characterized in that, The calcium salt mentioned in step two is calcium hydroxide, calcium carbonate, calcium sulfate, calcium acetate, calcium gluconate, or calcium chloride; the concentration of the calcium salt in the ion crosslinking agent solution C is 0.1 mol / L.

8. An application of a coating of a self-healing, antibacterial dynamic hydrogel prepared by the method according to any one of claims 1 to 7, characterized in that, A polyethyleneimine aqueous solution is spin-coated onto the surface of a biodegradable metal that has undergone alkaline pretreatment. After drying, a polyethyleneimine auxiliary layer is formed. Then, the hydrogel solution obtained in step three is uniformly coated onto the polyethyleneimine auxiliary layer. After gelation, a biodegradable metal with a self-healing, antibacterial dynamic hydrogel coating is obtained.

9. The coating application according to claim 8, characterized in that, The concentration of the polyethyleneimine aqueous solution is 0.1 g / mL.

10. The coating application according to claim 8, characterized in that, The preparation process of the biodegradable metal after alkaline pretreatment is as follows: magnesium, magnesium alloy or zinc, zinc alloy discs that have been sanded, polished, cleaned and dried are immersed in sodium hydroxide solution for treatment, and then cleaned and dried.

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