Preparation method and application of polyvinyl alcohol-based high-strength antibacterial hydrogel

Abstract

The invention discloses a preparation method and application of polyvinyl alcohol-based high-strength antibacterial hydrogel, and belongs to the field of biomedical materials. A one-pot mixing method is adopted, the antibacterial peptide NPR is added into a polyvinyl alcohol aqueous solution, and compact antibacterial hydrogel is generated through hydrogen bond interaction of polyvinyl alcohol and the antibacterial peptide NPR and freeze-thaw cycle. As a high-strength antibacterial hydrogel, the hydrogel has better mechanical strength, and the mechanical strength can reach 1.25 MPa. The hydrogel has antibacterial and healing promoting capabilities on wounds, is not easy to break and is convenient to use. The hydrogel can be sealed in a plastic package, is stored at low temperature, and is directly used for wound treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a preparation method and application of a polyvinyl alcohol-based high-strength antibacterial hydrogel modified with antibacterial peptides. Background Art

[0002] Microbial infections account for a significant proportion of global deaths. While the discovery of antibiotics has made it possible to treat most bacterial infections, the emergence of drug-resistant strains has reduced the effectiveness of clinically used antibiotics and has been linked to bloodstream, urinary tract, respiratory tract, and surgical site infections, resulting in serious consequences. However, the development of traditional antibiotics has been far slower than the emergence of drug-resistant microorganisms. Consequently, there have been numerous recent attempts to identify effective alternatives for treating infections in clinics and on the farm. Antimicrobial peptides (AMPs) have attracted significant attention as potential alternatives to traditional antibiotics.

[0003] In addition, PVA hydrogel is a popular polymer material showing great potential in biomedical applications. PVA is a synthetic macromolecular polymer with excellent mechanical properties, biocompatibility, affordability, and stability. These advantages make it a commonly used hydrogel material in bioengineering, with applications in areas such as bio-interaction, disease treatment, and physiological signal monitoring. However, in biomedical applications, synthetic hydrogels often suffer from insufficient mechanical strength and toughness.

[0004] In summary, there is an urgent need to develop a polyvinyl alcohol-based antibacterial hydrogel with high mechanical strength that can adapt to daily life, be easy to process and transport, and be commercialized. Summary of the Invention

[0005] To address the problems of the prior art, the present invention provides a method for preparing a polyvinyl alcohol-based high-strength antimicrobial hydrogel and its application. The present invention adds antimicrobial peptides to a polyvinyl alcohol aqueous solution, utilizes hydrogen bonding interactions between polyvinyl alcohol and antimicrobial peptides, and employs freeze-thaw cycles to generate a dense antimicrobial hydrogel. The antimicrobial hydrogel prepared by the present invention has good mechanical strength, reaching 1.25 MPa. Furthermore, the hydrogel of the present invention exhibits antimicrobial and wound-healing properties, is resistant to breakage, and is easy to use. The hydrogel of the present invention can be sealed in plastic packaging, stored at low temperatures, and used directly for wound treatment.

[0006] The method for preparing the polyvinyl alcohol-based high-strength antibacterial hydrogel of the present invention comprises the following steps:

[0007] Antimicrobial peptides were added to a polyvinyl alcohol aqueous solution, mixed evenly, and then subjected to repeated freezing and thawing to prepare a polyvinyl alcohol-based high-strength antimicrobial hydrogel.

[0008] The mass concentration of the polyvinyl alcohol aqueous solution is 15%.

[0009] In the system, the addition amount of polyvinyl alcohol aqueous solution is 1~3g, and the addition amount of antimicrobial peptide is 100~300 μg.

[0010] The repeated freezing and thawing is to freeze and thaw the hydrogel mixed in the previous step in a cycle, so that the polyvinyl alcohol molecular chains are entangled multiple times and form hydrogen bonds, thereby making it denser and enhancing the mechanical strength.

[0011] Furthermore, the repeated freeze-thaw process involves first rapidly freezing the sample at -80°C for at least 12 hours, then thawing the sample at room temperature, and then repeating the above steps after the sample is completely thawed. At least three freeze-thaw cycles are performed to obtain excellent mechanical properties.

[0012] Furthermore, the antimicrobial peptide is antimicrobial peptide NPR (FFRLLFHGRRRRRRRRRRR), and its amino acid sequence is shown in SEQ ID NO: 1.

[0013] The nucleotide sequence encoding the antimicrobial peptide is shown in SEQ ID NO: 2.

[0014] The present invention uses a one-pot mixing method to add antimicrobial peptide NPR to a polyvinyl alcohol (PVA) aqueous solution. The method utilizes hydrogen bonding interactions between the PVA and NPR, along with freeze-thaw cycles, to generate a dense antimicrobial hydrogel. The hydrogen bonding between the PVA and NPR allows for a tight bond between the PVA and NPR. Combined with freeze-thaw cycles, this method achieves high mechanical strength. Once tightly bound to the PVA, the mass fraction of the PVA NPR in the hydrogel ranges from 0.002% to 0.01%.

[0015] The invention discloses an application of the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel in the preparation of medical antibacterial dressings.

[0016] The antibacterial hydrogel of the present invention has good mechanical strength, and at the same time, the hydrogel has antibacterial and wound healing capabilities, is not easy to break, and is easy to use. The hydrogel can be sealed in a plastic package, stored at low temperature, and directly used for wound treatment.

[0017] The beneficial effects of the present invention are embodied in:

[0018] 1. The present invention provides a polyvinyl alcohol-based antibacterial hydrogel with high mechanical strength, comprising polyvinyl alcohol and an antimicrobial peptide NPR, wherein: the antimicrobial peptide NPR is added to a polyvinyl alcohol aqueous solution by a one-pot mixing method, and a dense antimicrobial hydrogel is generated by utilizing the hydrogen bond interaction between polyvinyl alcohol and the antimicrobial peptide NPR and freeze-thaw cycles.

[0019] 2. The polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel provided by the present invention has high mechanical strength and is not prone to breakage. Its mechanical tensile strength can reach 1.25 MPa.

[0020] 3. The polyvinyl alcohol-based, high-mechanical-strength antimicrobial hydrogel provided by the present invention, as a high-strength antimicrobial material, exhibits excellent antimicrobial properties. When tested against Escherichia coli and Staphylococcus aureus, the hydrogel exhibited significant antimicrobial activity.

[0021] 4. The polyvinyl alcohol-based high mechanical strength antibacterial hydrogel provided by the present invention, as a high-strength antibacterial material, has good cell compatibility.

[0022] 5. The polyvinyl alcohol-based high mechanical strength antibacterial hydrogel provided by the present invention is a high-strength antibacterial material with a simple and convenient preparation method. After preparation, it can be used for skin wound repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The present invention will be further described below with reference to the accompanying drawings and examples.

[0024] Figure 1 The present invention is a flow chart of the preparation method of the polyvinyl alcohol-based antibacterial hydrogel with high mechanical strength.

[0025] Figure 2 The scanning electron micrographs of the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel of the present invention are shown in Figure A, wherein Figure A is pure PVA and Figure B is PVA-NPR.

[0026] Figure 3 This is an infrared spectrum of the polyvinyl alcohol-based antibacterial hydrogel with high mechanical strength of the present invention.

[0027] Figure 4 This is a test chart of the tensile strength at break of the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel of the present invention at room temperature.

[0028] Figure 5 This is a diagram showing the antibacterial effect of the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel of the present invention.

[0029] Figure 6 This is a graph showing the in vitro L929 cytotoxicity test of the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel extract of the present invention.

[0030] Figure 7 This is a diagram of the wound area treated by the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel of the present invention. DETAILED DESCRIPTION

[0031] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0032] Polyvinyl alcohol (PVA) hydrogel is a popular polymer material that shows great potential in biomedical applications. Polyvinyl alcohol (PVA) is a synthetic macromolecular polymer with advantages such as excellent mechanical properties, biocompatibility, economy, and stability. However, in biomedical applications, synthetic hydrogels often face problems with insufficient mechanical strength and toughness. Antimicrobial peptides (NPRs) have poly-Rs, which can provide multiple hydrogen bond donors and acceptors, facilitating the formation of a denser network, thereby improving the overall mechanical strength of the hydrogel. At the same time, antimicrobial peptides (NPRs) also have antibacterial activity and can inhibit wound infection.

[0033] The present invention provides a method for preparing a polyvinyl alcohol-based antimicrobial hydrogel with high mechanical strength. The method utilizes a one-pot mixing process, comprising: taking a certain amount of polyvinyl alcohol aqueous solution, adding an antimicrobial peptide (NPR) to the aqueous solution, mixing uniformly, and then repeatedly freezing and thawing to produce the polyvinyl alcohol-based antimicrobial hydrogel with high mechanical strength. Hydrogen bonding interactions between the polyvinyl alcohol and the antimicrobial peptide (NPR) allow the NPR to bind tightly to the polyvinyl alcohol. Combined with freeze-thaw cycles, high mechanical strength is achieved. After the NPR binds tightly to the polyvinyl alcohol, its mass fraction in the hydrogel ranges from 0.002% to 0.01%. Repeated freeze-thaw cycles are also employed for gelation. Both methods enhance mechanical properties.

[0034] Based on the polyvinyl alcohol-based antibacterial hydrogel with high mechanical strength in the above embodiment, the present invention provides a method for preparing the polyvinyl alcohol-based antibacterial hydrogel with high mechanical strength, such as Figure 1 As shown, the method adopts a one-pot mixing method, which specifically includes the following steps:

[0035] Step 1: Take a certain amount of polyvinyl alcohol aqueous solution, add antimicrobial peptide NPR into the polyvinyl alcohol aqueous solution, and mix well;

[0036] Step 2: After repeated freezing and thawing, polyvinyl alcohol-based antibacterial hydrogel with high mechanical strength was prepared.

[0037] In step 1 above, the step of taking a certain amount of polyvinyl alcohol aqueous solution is 1 to 3 g of a 15% polyvinyl alcohol aqueous solution, or alternatively, 1 g of a 15% polyvinyl alcohol aqueous solution. The step of adding the antimicrobial peptide NPR to the polyvinyl alcohol aqueous solution is 100 to 300 μg of the antimicrobial peptide NPR, or alternatively, 100 μg of the antimicrobial peptide NPR, and uniformly mixing.

[0038] In step 2 above, the repeated freeze-thaw cycles involve multiple freeze-thaw cycles of sufficient duration, such as rapid freezing at -80°C for at least 12 hours followed by thawing at room temperature. After complete thawing, the above steps are repeated for at least three cycles to achieve excellent mechanical properties. This allows the polyvinyl alcohol and antimicrobial peptide NPR to form a network.

[0039] Furthermore, embodiments of the present invention provide a polyvinyl alcohol-based antibacterial hydrogel with high mechanical strength for medical use. The polyvinyl alcohol-based antibacterial hydrogel provided in embodiments of the present invention can be used as a wound dressing, sealed in plastic packaging and stored at low temperatures. After removal, it can be used directly for hand wound repair without thawing, as well as for daily abrasions.

[0040] The following describes in detail the polyvinyl alcohol-based antibacterial hydrogel with high mechanical strength, its preparation method and its application in medical treatment provided by the present invention in conjunction with specific examples.

[0041] Example 1: Preparation and performance testing of polyvinyl alcohol-based high mechanical strength antibacterial hydrogel

[0042] In this example, a polyvinyl alcohol-based antimicrobial hydrogel with high mechanical strength was prepared by first mixing 1 g of a 15% polyvinyl alcohol solution with 100 μg of the antimicrobial peptide NPR. The mixture was then rapidly frozen at -80°C for at least 12 hours and then thawed at room temperature. After complete thawing, the above steps were repeated three times to obtain a polyvinyl alcohol-based antimicrobial hydrogel with high mechanical strength.

[0043] The vertical surface of the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel (PVA-NPR) was examined using a scanning electron microscope. Figure 2 As shown, the vertical sections of the polyvinyl alcohol-based high mechanical strength antibacterial hydrogels according to the embodiments of the present invention all exhibit a layered structure. Among them, the pure PVA layered ladder structure is obvious, and there is a pore structure (A); in the PVA-NPR group, the layered structure boundary disappears, and the cross-sectional structure is more dense (B). Statistics of the interlamellar spacing in the longitudinal section of the hydrogel sheet show that the longitudinal section spacing j is larger in the PVA group, with an average value of 5.15±0.34 μm; the average longitudinal section spacing in the PVA-NPR group is 1.65±0.11 μm, which is one-third of that in the pure polyvinyl alcohol group. This may be because the sequence of the antimicrobial peptide NPR itself (FFRLLFHGRRRRRRRRRRR) has more R residues, which are rich in amino groups and can act as potential hydrogen bond crosslinkers to enhance interchain interactions through strong hydrogen bonds. The freeze-dried samples were analyzed using an infrared spectrometer. As shown Figure 3 As shown, at 1650 cm -1 There is an increase in absorption intensity near 3265 cm, which should be caused by the stretching vibration of the guanidine C=N in the R group contained in the added antimicrobial peptide. Another noteworthy absorption intensity is at 3265 cm-1 ~3270 cm -1 The peak area partially increases, accompanied by a slight red shift, which is due to the fact that the added antimicrobial peptide provides additional NH, forming hydrogen bonds while increasing the crystallinity of the entire hydrogel system.

[0044] In order to characterize the mechanical properties of PVA-NPR, this study used the tensile test method to evaluate the mechanical strength of the hydrogel. Figure 4 As shown in the figure, the mechanical strength of PVA-NPR is significantly improved compared to the pure PVA group. The tensile strength at break of poly PVA-NPR is roughly proportional to the content of antimicrobial peptide NPR in the hydrogel. When the antimicrobial peptide NPR addition amount is 100 μg / mL, the tensile strength at break is increased by 8 times compared to pure PVA. Compared with other antimicrobial peptides, taking WT as an example here (FFRLLFHGVHHVGKIKPRA), PVA-NPR has significant differences in mechanical properties compared to hydrogels with the same scheme but with the addition of other antimicrobial peptides. The tensile strength at break is increased by 7.3 times compared to the hydrogel with WT. This may be due to the formation of additional hydrogen bonds between the polyamino structure on the antimicrobial peptide and the hydroxyl group on the polyvinyl alcohol.

[0045] The antibacterial properties of the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel of the present invention were evaluated using Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as pathogenic bacteria models. 0.1 g of the hydrogel was irradiated with UV light for 30 min and then mixed with 10 8 CFU / mL of bacterial suspension were co-cultured for 24 hours and then plated to evaluate the antibacterial properties. Figure 5 As shown, the polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel according to the present invention has an inhibition rate of 60% against Escherichia coli and a 92% inhibition rate against Staphylococcus aureus, indicating that the PVA-NPR according to the present invention has good antibacterial application prospects.

[0046] The biocompatibility of the materials was evaluated according to the in vitro cytotoxicity standard in GB / T 16886. 0.1 g of each PVA and PVA-NPR hydrogel was weighed and incubated in RPMI 1640 medium at 37°C for 24 hours on a shaker to prepare hydrogel extracts. At least three samples were prepared for each group. Mouse fibroblast L929 cells were seeded in RPMI 1640 complete medium supplemented with 1% double-antibody antibody and 10% fetal bovine serum and cultured at 37°C in a 5% CO2 incubator. L929 cells were seeded at a density of 3000 cells per well in 96-well plates and cultured statically. After cell attachment, the medium was removed and replaced with the PVA and PVA-NPR hydrogel extracts. The biocompatibility of the PVA and PVA-NPR hydrogels was assessed using the CCK8 assay after 1, 3, and 5 days of co-culture with the cells. The specific description of the CCK8 assay method is as follows: After the cells are incubated with the hydrogel extract for the target number of days, the culture medium is replaced with RPMI 1640 complete medium containing 10% CCK8. After incubation at 37°C in a 5% CO2 incubator for 1 hour, 100 μL of supernatant is aspirated from each well into a new 96-well plate and the absorbance is measured at 450 nm. Figure 6 It can be seen that the polyvinyl alcohol-based antibacterial hydrogel with high mechanical strength in this example has good biocompatibility.

[0047] Example 2: Polyvinyl alcohol-based high mechanical strength antibacterial hydrogel for wound dressing

[0048] The polyvinyl alcohol-based high mechanical strength antibacterial hydrogel (PVA-NPR) of the embodiment of the present invention has multiple processabilities. The polyvinyl alcohol-based high mechanical strength antibacterial hydrogel (PVA-NPR) of the embodiment of the present invention was used as a wound dressing to detect its performance in promoting wound healing. Sprague-Dawley (SD) rats were used to conduct a full-thickness wound damage repair experiment on rats infected with bacteria to verify the in vivo antibacterial activity of the PVA-NPR hydrogel. The experimental protocol was approved by the Ethics Committee (approval number: YSY-DWLL-2021031) and complies with the certification standards of the Association for Assessment and Accreditation of Laboratory Animal Care. In this study, isoflurane inhalation anesthesia was used. After the rats were anesthetized, the skin was prepared and a full-thickness skin defect was created on the back of the rat using a 10 mm biopsy instrument. Subsequently, 100 μL of S. aureus bacterial suspension (PBS, OD 600 = 0.5) was used to simulate wound contamination. After waiting for 5 minutes, the wounds were treated with PBS, PVA, and PVA-NPR hydrogel, respectively. The PBS-treated group served as a blank control.

[0049] In order to evaluate the specific performance of the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel (PVA-NPR) of the present invention as a wound dressing, the area of ​​the wounds of mice at 1, 4 and 7 days was statistically analyzed. The statistical results are as follows: Figure 7As shown, the wound dressing can play a significant role in promoting wound healing in the first 4 days, indicating that the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel of the embodiment of the present invention has the performance of promoting wound healing when used as a wound dressing.

[0050] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a polyvinyl alcohol-based high-strength antibacterial hydrogel, characterized in that The steps include: The antimicrobial peptide NPR was added to the polyvinyl alcohol aqueous solution, mixed evenly, and then subjected to repeated freezing and thawing to obtain a polyvinyl alcohol-based high-strength antimicrobial hydrogel.

2. The preparation method according to claim 1, characterized in that: The amino acid sequence of the antimicrobial peptide NPR is shown in SEQ ID NO:

1.

3. The preparation method according to claim 2, characterized in that: The nucleotide sequence encoding the antimicrobial peptide is shown in SEQ ID NO:

2.

4. The preparation method according to claim 1, characterized in that: The mass concentration of the polyvinyl alcohol aqueous solution is 15%.

5. The preparation method according to claim 4, characterized in that: In the system, the addition amount of polyvinyl alcohol aqueous solution is 1~3g, and the addition amount of antimicrobial peptide is 100~300 μg.

6. The preparation method according to claim 1, characterized in that: The repeated freezing and thawing is to subject the hydrogel mixed in the previous step to a freezing-thawing cycle, so that the polyvinyl alcohol molecular chains are entangled multiple times and form hydrogen bonds, thereby making it denser and enhancing the mechanical strength.

7. The preparation method according to claim 6, characterized in that: The repeated freezing and thawing first involves rapid freezing at -80°C for at least 12 h, followed by thawing at room temperature, and repeating the above steps after complete thawing, for at least 3 freezing-thawing cycles.

8. The preparation method according to claim 7, characterized in that: The number of freeze-thaw cycles was 3-6 times.

9. Use of the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel prepared by the preparation method according to any one of claims 1 to 8 in the preparation of medical antibacterial dressings.

Images