Preparation method and application of hydrogel with inducing and sterilizing functions

Abstract

The invention belongs to the technical field of medical materials, and relates to a preparation method and application of hydrogel with inducing and sterilizing functions, acrylamide, ammonium persulfate, quaternary ammonium salt, N, N-methylene bisacrylamide and methyl aspartic acid are sequentially added into a polydopamine solution, stirring is performed, and finally, the hydrogel with inducing and sterilizing functions is obtained. Adding tetramethylethylenediamine into the solution as an initiator, and standing to obtain the hydrogel with the inducing-sterilizing function. Methyl aspartic acid is adopted as an attractant, quaternary ammonium salt is adopted as a bactericide, bacteria can be efficiently trapped and killed in a platform battlefield far away from a wound, normal cells are not damaged, and wound healing can be promoted; the hydrogel can be used for preparing products such as adhesive bandages and hydrogel dressings, is stable in performance, has excellent biocompatibility and antibacterial activity, and has a wide market application prospect in the fields of chronic wound nursing and infection prevention and control.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, and relates to a hydrogel with dual functions of attracting and killing bacteria, and its preparation method. In particular, it relates to a multifunctional hydrogel prepared in one step using a specific cross-linking agent and functional monomers. This hydrogel can efficiently induce bacterial aggregation under the action of an inducing agent, while simultaneously, the bactericidal agent loaded on its surface effectively kills the aggregated bacteria. Furthermore, this invention also relates to the application method of this hydrogel dressing in the treatment of chronic wounds and infection prevention. Background Technology

[0002] Traditional wound disinfection commonly uses oxidizing bactericides such as iodine, alcohol, and hydrogen peroxide. However, these oxidizing bactericides can easily damage cell membranes, DNA, and mitochondria during the disinfection process, and the residual bacterial corpses and toxins can hinder wound repair and worsen tissue damage. While quaternary ammonium compounds can kill bacteria through physical penetration, free quaternary ammonium compounds indiscriminately damage normal cells, triggering oxidative stress and affecting energy metabolism. Therefore, traditional methods, by treating the wound as the "battlefield" for disinfection, inevitably produce side effects. Thus, a solution is needed that can trap and kill bacteria in a specific "platform battlefield" far from the wound to reduce or avoid these problems.

[0003] In existing research, hydrogels are considered an ideal "platform" for solving the aforementioned traditional disinfection problems because they can create a moist environment for wounds, effectively promote cell migration and proliferation and epithelial tissue regeneration, and their three-dimensional network structure can absorb wound exudate to prevent infection caused by exudate. Furthermore, through structural adjustment, they can also load and fix bactericides. However, hydrogels currently have significant limitations: the range of action of the fixed bactericides is limited, only killing bacteria near the hydrogel. How to effectively guide bacteria from the wound to the interior of the hydrogel and fully contact the fixed bactericide to achieve efficient sterilization has become a key scientific problem that urgently needs to be solved.

[0004] Bacterial chemotaxis is mediated by unique transmembrane receptors. These receptors sense changes in the concentration of chemical substances in the environment and convert them into intracellular signals through the intracellular chemotactic signal transduction network. These signals then control bacterial movement to achieve "seeking advantage and avoiding harm," a survival instinct for most bacteria. Based on this, bacterial chemotaxis behavior can be modulated by selecting inducers, guiding them from wounds to the "battlefield" to avoid the side effects of disinfection on normal cells. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of traditional wound disinfection methods, such as damage to normal cells and impede wound healing caused by oxidizing bactericides. At the same time, it solves the problems of limited range of action of bactericides fixed in existing hydrogel dressings and difficulty for bacteria to fully contact the bactericides, as well as the key technical problems of stable release of inducers in hydrogels and scientific selection of hydrogel monomers. Ultimately, it provides a hydrogel and its preparation method that can efficiently trap and kill bacteria in a "platform battlefield" far away from the wound without damaging normal cells.

[0006] To achieve the above objectives, the present invention relates to a method for preparing hydrogels with attraction-bactericidal functions, the specific process of which is as follows:

[0007] Under ice-water bath conditions, acrylamide, ammonium persulfate, quaternary ammonium salt, N,N-methylenebisacrylamide and methylaspartic acid were added sequentially to an aqueous solution of polydopamine to obtain a mixed system. The mixed system was stirred under magnetic stirring. Finally, tetramethylethylenediamine was added to the above solution as an initiator and allowed to stand to obtain a hydrogel with inducing-bactericidal function.

[0008] The polydopamine solution of the present invention is obtained by dissolving dopamine powder in water, adjusting the pH value to 11, and reacting under magnetic stirring; the mass ratio of dopamine powder to water is 3:10; the stirring time is 15-30 min.

[0009] The mass-to-volume ratio of acrylamide to polydopamine solution in this invention is 0.25 g: 1 mL.

[0010] The molar ratio of acrylamide to ammonium persulfate in this invention is (32-41):1.

[0011] In this invention, the amount of N,N-methylenebisacrylamide added is 0.05%-0.5% of the mass of acrylamide, with the optimum being 0.12%.

[0012] In this invention, the quaternary ammonium salt is an ammonium ion ( In this invention, the quaternary ammonium salt is 2-acryloyloxy-ethyl-N,N-dimethyl-6-bromide or other existing quaternary ammonium salts with bactericidal activity; the mass ratio of 2-acryloyloxy-ethyl-N,N-dimethyl-6-bromide to acrylamide is 0.0588:1.

[0013] The mass-to-volume ratio of methylaspartic acid to polydopamine solution in this invention is (13-15) mg:1 mL or 29.4 mg:1 mL; preferably 14.7 mg:1 mL.

[0014] This invention conducted experiments on the content of methylaspartic acid at different concentrations (0-0.2mM), and found that induction occurred at concentrations of 0.1mM and 0.2mM of methylaspartic acid. The concentration selected for the experiment was 0.1mM.

[0015] The amount of tetramethylethylenediamine added in this invention is related to the amount of ammonium persulfate added, and the molar ratio between the two is approximately 13:1.

[0016] The stirring time for the mixing system described in this invention is 5-20 min.

[0017] The present invention also provides a hydrogel with attraction-bactericidal function prepared by the preparation method, wherein the hydrogel has a porous structure, can absorb more than 6 times its own weight in water, and still retains 40% of its original weight after 7 days.

[0018] The hydrogel described in this invention can efficiently induce bacterial aggregation under the action of an inducer (methylaspartic acid). At the same time, the bactericide (quaternary ammonium salt) loaded on its surface plays a role in efficiently killing the aggregated bacteria.

[0019] The present invention also provides the use of the hydrogel in the preparation of products for wound sterilization or disinfection.

[0020] The products include, but are not limited to, adhesive bandages, hydrogel dressings, or other medical dressings.

[0021] The present invention also provides wound dressings or plasters containing the hydrogel.

[0022] The hydrogel prepared in this invention has a porous structure and has been shown to possess both attractant and bactericidal functions. The antibacterial mechanism of this material is as follows: the methylaspartic acid loaded in the hydrogel acts as an attractant, specifically attracting bacterial aggregation, but it does not participate in bacterial metabolism or promote bacterial reproduction. Experimental results show that after incubating the plates at 37°C for 12 h, bacteria significantly aggregated around the pores, indicating that the material has a significant bacterial induction effect. Based on the inherent antibacterial activity of quaternary ammonium salts, it can effectively disrupt the structural integrity of bacterial cell membranes, leading to leakage of cell contents and subsequent cell death, exhibiting excellent antibacterial effects. Experimental results also show that after co-incubating a certain concentration of bacterial suspension with the hydrogel in a 37°C water bath for 3 h, the number of colonies significantly decreased, indicating that the material has good bactericidal effects.

[0023] The hydrogel of this invention can be used to prepare products such as adhesive bandages and hydrogel dressings. It has excellent water absorption, which can maintain a moist environment for wounds; it has an adsorption function to promote wound cleaning; it reduces the interfacial tension between the hydrogel and body fluids, reducing protein adsorption and cell adhesion; it has good elasticity and toughness, and can form a perfect barrier around the wound during the healing process.

[0024] Compared with existing technologies, this invention uses methylaspartic acid as an attractant and quaternary ammonium salt as a bactericide, providing a novel method for preparing hydrogels. This method requires no complex functional modifications, is simple and efficient, and can integrate the dual functions of "attracting and killing" in one step. It can efficiently attract and kill bacteria in a "battlefield" far from the wound, without damaging normal cells and promoting wound healing. The hydrogel of this invention has stable performance, excellent biocompatibility and antibacterial activity, and has broad market application prospects in the fields of chronic wound care and infection control. Attached Figure Description

[0025] Figure 1 The images show a photograph (Figure A), a SEM image (Figure B), a swelling property (Figure C), and a water retention property (Figure D) of the hydrogel prepared according to the present invention.

[0026] Figure 2 The figure shows the experimental results of the induction properties of the hydrogel involved in this invention.

[0027] Figure 3 The images shown are a photograph (Figure A) and a SEM image (Figure B) of the bacterial plate count in the in vitro antibacterial experiment using the hydrogel involved in this invention.

[0028] Figure 4 The image shows a product of the hydrogel involved in this invention, where A is a hydrogel dressing and B is a band-aid. Detailed Implementation

[0029] The invention will be further described below with reference to specific examples and accompanying drawings.

[0030] Example 1:

[0031] This embodiment relates to a method for preparing a hydrogel with attraction-bactericidal function, the specific steps of which are as follows:

[0032] S1. At room temperature, dissolve 3 mg of dopamine powder in 10 mL of deionized water, add 70 μL of NaOH aqueous solution to adjust the pH to 11, and magnetically stir for 20 min to obtain a polydopamine (PDA) solution.

[0033] S2. Under ice-water bath conditions, 2.5 g acrylamide (AM), 250 mg ammonium persulfate, 0.14 g 2-acryloyloxy-ethyl-N,N-dimethyl-6-ammonium bromide (AEDMHA), and 3 mg N,N-methylenebisacrylamide were added sequentially to the PDA solution to obtain a PDA-AEDMHA-PAM solution. Then, 0.147 g of N-methyl-D-aspartic acid (NMDA) was added to the above solution to obtain a mixed system. The mixed system was stirred under magnetic stirring for 10 min, and 10 μL of tetramethylethylenediamine was added as an initiator. After standing for 3-5 h, a hydrogel with attraction-bactericidal multifunctionality (PDA-AEDMHA-NMDA-PAM) was obtained.

[0034] The prepared PDA-AEDMHA-NMDA-PAM hydrogel was characterized by SEM, and the results are as follows: Figure 1 As shown in B. From Figure 1 As can be seen from B, the hydrogel has a porous structure.

[0035] The swelling properties of the PDA-AEDMHA-NMDA-PAM hydrogel were tested, and the experimental procedure is as follows:

[0036] (1) Prepare hydrogel samples of the same size and freeze-dry them.

[0037] (2) Set up three parallel samples, weigh the original weight of the samples and record it. Put the samples into PBS buffer for swelling rate experiment, and then place them on a shaker at 37 ℃. Weigh the samples at 0 min, 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 150 min and 180 min respectively.

[0038] (3) Calculate the swelling ratio of the hydrogel using the formula SR=(Ws-Wd) / Wd×100% and plot the swelling ratio-time change graph. Where SR: swelling ratio, Wd: initial freeze-dried hydrogel mass, Ws: mass of the swollen hydrogel.

[0039] Experimental results are as follows Figure 1 As shown in Figure C, the PDA-AEDMHA-NMDA-PAM hydrogel exhibits excellent water absorption properties. It can absorb up to 6 times its own weight in water and expands rapidly within 1 hour, with an expansion rate exceeding 630%. It reaches expansion equilibrium within 3 hours, highlighting its rapid and high-capacity liquid absorption capacity.

[0040] The water retention properties of the PDA-AEDMHA-NMDA-PAM hydrogel were tested, and the experimental procedure is as follows:

[0041] (1) Prepare the hydrogel into a film of uniform thickness, weigh and record the weight, and lay it flat in a desiccator.

[0042] (2) Prepare a 90wt% potassium hydroxide aqueous solution, place it in a desiccator containing the hydrogel sample and keep the humidity in the container at about 79%, and measure the humidity with a hygrometer.

[0043] (3) Take out the film hydrogel every 24 hours to weigh it for 7 days, and finally calculate the water retention rate of the hydrogel and plot the water retention rate-time change graph.

[0044] The calculation formula is: Q = (MI / MO) × 100%

[0045] Where Q is the water retention rate, MI is the mass of the film hydrogel taken out and weighed each day, and MO is the initial mass of the film hydrogel.

[0046] from Figure 1 As can be seen from D, the PDA-AEDMHA-NMDA-PAM hydrogel can still retain about 40% of its original weight after 7 days, demonstrating excellent long-term water retention performance.

[0047] Example 2:

[0048] This embodiment relates to a method for preparing a hydrogel with attraction-bactericidal function, the specific steps of which are as follows:

[0049] S1. At room temperature, dissolve 3 mg of dopamine powder in 10 mL of deionized water, add 70 μL of NaOH aqueous solution to adjust the pH to 11, and magnetically stir for 15 min to obtain a polydopamine (PDA) solution.

[0050] S2. Under ice-water bath conditions, 2.5 g acrylamide (AM), 228 mg ammonium persulfate, 0.14 g 2-acryloyloxy-ethyl-N,N-dimethyl-6-ammonium bromide (AEDMHA), and 1.25 mg N,N-methylenebisacrylamide were added sequentially to the PDA solution to obtain a PDA-AEDMHA-PAM solution. Then, 0.147 g of N-methyl-D-aspartic acid (NMDA) was added to the above solution to obtain a mixed system. The mixed system was stirred under magnetic stirring for 5 min, and 10 μL of tetramethylethylenediamine was added as an initiator. After standing for 3-5 h, a hydrogel with attraction-bactericidal multifunctionality (PDA-AEDMHA-NMDA-PAM) was obtained.

[0051] Example 3:

[0052] This embodiment relates to a method for preparing a hydrogel with attraction-bactericidal function, the specific steps of which are as follows:

[0053] S1. At room temperature, dissolve 3 mg of dopamine powder in 10 mL of deionized water, add 70 μL of NaOH aqueous solution to adjust the pH to 11, and magnetically stir for 30 min to obtain a polydopamine (PDA) solution.

[0054] S2. Under ice-water bath conditions, 2.5 g acrylamide (AM), 195 mg ammonium persulfate, 0.14 g 2-acryloyloxy-ethyl-N,N-dimethyl-6-ammonium bromide (AEDMHA), and 12.5 mg N,N-methylenebisacrylamide were added sequentially to the PDA solution to obtain a PDA-AEDMHA-PAM solution. Then, 0.147 g of N-methyl-D-aspartic acid (NMDA) was added to the above solution to obtain a mixed system. The mixed system was stirred under magnetic stirring for 20 min, and 10 μL of tetramethylethylenediamine was added as an initiator. After standing for 3-5 h, a hydrogel with attraction-bactericidal multifunctionality (PDA-AEDMHA-NMDA-PAM) was obtained.

[0055] Example 4:

[0056] This embodiment applies the hydrogel prepared in Example 1 to bacterial induction. Holes were punched in an agar plate, with a central well surrounded by five smaller wells. The central well had a diameter of 14 mm, and the smaller wells had a diameter of 7 mm. Hydrogels (0.5 mm × 0.5 mm × 0.7 mm in volume) containing 0.147 g (0.1 mM), 0.2058 g (0.14 mM), 0.2205 g (0.15 mM), and 0.294 g (0.2 mM) of methylaspartic acid, respectively, were added to wells 1-5. 100 μL of bacterial suspension was added to the central well, and the agar plates were incubated overnight in a 30°C oven. Colonies on the plates were observed after 12 h. Each independent experiment was performed in triplicate. The results are as follows: Figure 2 As shown, bacterial aggregation occurred at concentrations of 0.1 mM and 0.2 mM.

[0057] This embodiment applies the hydrogel (0.1 mM methyl aspartic acid concentration) prepared in Example 1 to sterilization. The experiment was divided into a blank control group (without hydrogel material) and a PDA-AEDMHA-NMDA-PAM hydrogel group, with three samples in each group. *Escherichia coli* and *Staphylococcus aureus* were revived and passaged twice to maintain bacterial viability, and bacterial suspensions were prepared separately. The bacterial suspensions at a certain concentration were incubated with the hydrogel in a 37°C water bath for 3 h. After incubation, 100 μL of the bacterial suspension was inoculated onto the surface of a solid culture medium and cultured in a 37°C constant temperature aerobic incubator for 12-24 h. The colonies on the solid culture dishes were counted, and the antibacterial rate of each group was calculated.

[0058] Antibacterial rate (%) = (Number of colonies in blank control group - Number of colonies in experimental group) / Number of colonies in blank control group × 100%. Results are as follows: Figure 3 As shown in Figure A, after the bacterial suspension was treated with hydrogel, the number of Escherichia coli and Staphylococcus aureus colonies was significantly reduced. The hydrogel showed an antibacterial rate of 99.99% against Escherichia coli and 99.45% against Staphylococcus aureus, indicating that the material has a good bactericidal effect.

[0059] The hydrogel prepared in Example 1 was applied to bacterial scanning electron microscopy (SEM) imaging. *Escherichia coli* and *Staphylococcus aureus* were revived and passaged twice to maintain bacterial viability, and bacterial suspensions were prepared separately. The bacterial suspensions of a certain concentration were incubated with the hydrogel for 4.5 h. The bacterial samples were centrifuged at 3000 r / min for 10 min, and the bacterial precipitate was collected. 1 mL of sterile water was added, and the sample was centrifuged at 3000 r / min for 5 min. Subsequently, the precipitate was fixed with 2.5% glutaraldehyde solution at 4°C for 12–20 h, centrifuged at 3000 r / min for 3 min to discard the glutaraldehyde, and then rinsed with 1 mL of sterile water and centrifuged at 3000 r / min for 3 min. Elution was then performed sequentially with 30%, 50%, 70%, 90%, and 100% (v / v) ethanol aqueous solutions, followed by centrifugation at 3000 r / min for 3 min, and the precipitate was collected. Finally, anhydrous ethanol was added and mixed thoroughly. A small amount was dropped onto a silicon wafer for SEM characterization. Each sample group was repeated three times. Results are as follows: Figure 3 As shown in Figure B, compared with Escherichia coli and Staphylococcus aureus before hydrogel treatment, Escherichia coli and Staphylococcus aureus after hydrogel treatment showed shrinkage and biofilm damage, indicating that the antibacterial mechanism of quaternary ammonium salt is to destroy the biofilm on the bacterial surface.

[0060] Example 5:

[0061] This embodiment applies the hydrogel prepared in Example 1 to product preparation.

[0062] Band-Aid Preparation: After the hydrogel has reacted, it is removed from the mold and trimmed into a 25mm × 18mm cuboid using a cutting device, ensuring smooth, burr-free edges. Then, a multi-layer structure is assembled: using medical-grade light yellow elastic fabric as the base material, a highly absorbent fiber pad is fixed using a hot-melt adhesive spraying process; the hydrogel layer (located in the central area) is precisely adhered to the surface of the absorbent pad, followed by a breathable protective composite pad (medical non-woven fabric) and an anti-adhesion covering film. Slight pressure (0.1-0.3 MPa) is applied using a rolling process to ensure tight adhesion between layers and no air bubbles. The assembled band-aid is then sterilized with ultraviolet light and subsequently vacuum-sealed using a medical aluminum-plastic composite film, with the sealing temperature controlled at 160-180℃ to ensure sealing strength. After packaging, the product is stored in a cool, dry environment to maintain its sterile state.

[0063] Application Preparation: After the hydrogel has reacted, it is removed from the mold and trimmed into a 3.4 cm × 4 cm cuboid, ensuring smooth, burr-free edges. Multi-layer assembly is then performed: using medical tape (hypoallergenic acrylic material) as the base material, a highly absorbent fiber pad (approximately 1.5 mm thick) is fixed using a hot melt adhesive spraying process; the hydrogel layer (located in the central area) is precisely adhered to the surface of the absorbent pad, and a release paper is placed over the hydrogel surface. Slight pressure (0.1-0.3 MPa) is applied using a rolling process to ensure tight adhesion between layers and no air bubbles. The assembled dressing is then sterilized with UV light and subsequently vacuum-sealed using a medical aluminum-plastic composite film, with the sealing temperature controlled at 160-180℃ to ensure sealing strength. After packaging, the product is stored in a cool, dry environment to maintain its sterile state.

Claims

1. A method for preparing a hydrogel with attraction-bactericidal function, characterized in that, The specific steps are as follows: Acrylamide, ammonium persulfate, quaternary ammonium salt, N,N-methylenebisacrylamide and methyl aspartic acid were added sequentially to a polydopamine solution and stirred. Finally, tetramethylethylenediamine was added to the above solution as an initiator and allowed to stand to obtain a hydrogel with inducing-bactericidal function.

2. The method for preparing the hydrogel with attraction-bactericidal function according to claim 1, characterized in that, The polydopamine solution is obtained by dissolving dopamine powder in water, adjusting the pH value to 11, and reacting under magnetic stirring.

3. The method for preparing the hydrogel with attraction-bactericidal function according to claim 1, characterized in that, The molar ratio of acrylamide to ammonium persulfate is (32-41):1; the amount of N,N-methylenebisacrylamide added is 0.05%-0.5% of the mass of acrylamide.

4. The method for preparing the hydrogel with attraction-bactericidal function according to claim 1, characterized in that, The quaternary ammonium salt is 2-acryloyloxy-ethyl-N,N-dimethyl-6-ammonium bromide.

5. The method for preparing a hydrogel with attraction-bactericidal function according to claim 1, characterized in that, The mass-to-volume ratio of the methylaspartic acid to the polydopamine solution is (13-15) mg: 1 mL.

6. The hydrogel with attraction-bactericidal function prepared by the preparation method according to any one of claims 1-5.

7. The use of the hydrogel with attraction-bactericidal function as described in claim 6 in the preparation of products for wound sterilization or disinfection.

8. The application of the hydrogel with attraction-bactericidal function according to claim 7 in the preparation of products for wound sterilization or disinfection, characterized in that, The products include, but are not limited to, adhesive bandages and hydrogel dressings.

9. A bandage or dressing containing the hydrogel of claim 6.

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