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

Abstract

The application discloses a preparation method of polyvinyl alcohol-based high-strength antibacterial hydrogel and application thereof, and belongs to the field of biomedical materials. In the application, antibacterial peptide NPR is added into a polyvinyl alcohol aqueous solution by one-pot mixing, and a compact antibacterial hydrogel is generated by using the hydrogen bond interaction between the polyvinyl alcohol and the antibacterial peptide NPR and freeze-thaw cycles. The hydrogel serves as high-strength antibacterial hydrogel and has good mechanical strength, which can reach 1.25 MPa. The hydrogel has antibacterial and healing-promoting capacity for wounds, is not prone to breaking, and is convenient to use. The hydrogel can be sealed in plastic packaging and stored at low temperature, and is directly used for wound treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a preparation method of polyvinyl alcohol-based high-strength antibacterial hydrogel with antibacterial peptides and application thereof. BACKGROUND

[0002] Microbial infections account for a large proportion of global deaths. With the discovery of antibiotics, most bacterial infections have been able to be treated, however, the emergence of drug-resistant strains reduces the therapeutic effect of clinically used antibiotics and is associated with blood, urinary tract, respiratory tract and surgical site infections, resulting in serious consequences. However, the development of traditional antibiotics is far slower than the emergence of drug-resistant microorganisms. Therefore, there have been many attempts to find effective alternatives for treating infections in clinics and farms. Antimicrobial peptides (AMPs) have attracted great attention as potential alternatives to traditional antibiotics.

[0003] In addition, PVA hydrogel is a popular polymer material and has great potential in biomedical applications. PVA is a synthetic macromolecular polymer with excellent mechanical properties, biocompatibility, economy and stability. These advantages make it a commonly used hydrogel material in bioengineering and gradually applied to the fields of biological interaction, disease treatment and physiological signal monitoring. However, in biomedical applications, synthetic hydrogels usually face the problem of insufficient mechanical strength and toughness.

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

[0005] In view of the problems existing in the prior art, the present application provides a preparation method of polyvinyl alcohol-based high-strength antibacterial hydrogel and application thereof. The antibacterial peptides are added to the polyvinyl alcohol aqueous solution, and the hydrogen bond interaction between polyvinyl alcohol and antibacterial peptides is utilized, and a dense antibacterial hydrogel is generated by freeze-thaw cycling. The antibacterial hydrogel prepared by the present application has good mechanical strength, which can reach 1.25 MPa. In addition, the hydrogel has antibacterial and healing-promoting ability for wounds and is not easy to break, which is convenient to use. The hydrogel can be sealed in plastic packaging and stored at low temperature, and can be directly used for wound treatment.

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

[0007] The antibacterial peptides are added to the polyvinyl alcohol aqueous solution, mixed uniformly, and then repeatedly frozen and thawed to prepare the polyvinyl alcohol-based high-strength antibacterial hydrogel.

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

[0009] In the system, the adding amount of the polyvinyl alcohol aqueous solution is 1-3 g, and the adding amount of the antibacterial peptide is 100-300 μg.

[0010] The repeated freezing and thawing is a cycle of freezing and thawing of the mixed hydrogel in the previous step, so that the polyvinyl alcohol molecular chain is entangled and hydrogen bonds are formed for multiple times, and the mechanical strength is further enhanced.

[0011] Further, the repeated freezing and thawing is first rapidly frozen at-80℃ for at least 12 h, then thawed at room temperature, and the above steps are repeated after complete thawing. At least 3 cycles of freezing and thawing are performed to obtain excellent mechanical properties.

[0012] Further, the antibacterial peptide is antibacterial peptide NPR (FFRLLFHGRRRRRRRRRRR), and the amino acid sequence is shown as SEQ ID NO: 1.

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

[0014] The antibacterial peptide NPR is added to the polyvinyl alcohol aqueous solution by one-pot mixing method, and the dense antibacterial hydrogel is generated by the hydrogen bond interaction between polyvinyl alcohol and antibacterial peptide NPR and the cycle of freezing and thawing. The hydrogen bond interaction between polyvinyl alcohol and antibacterial peptide NPR enables the close combination of antibacterial peptide NPR and polyvinyl alcohol, and the cycle of freezing and thawing realizes high mechanical strength. After the close combination of the antibacterial peptide NPR and polyvinyl alcohol, the mass fraction of the antibacterial peptide NPR in the hydrogel is 0.002%-0.01%.

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

[0016] The antibacterial hydrogel has good mechanical strength, and the hydrogel has antibacterial and healing-promoting capacity for wounds, is not easy to break, and is convenient to use. The hydrogel can be sealed in plastic packaging and stored at low temperature, and is directly used for wound treatment.

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

[0018] 1. The application provides a polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel, which comprises polyvinyl alcohol and antibacterial peptide NPR, wherein: the antibacterial peptide NPR is added to the polyvinyl alcohol aqueous solution by one-pot mixing method, and the dense antibacterial hydrogel is generated by the hydrogen bond interaction between polyvinyl alcohol and antibacterial peptide NPR and the cycle of freezing and thawing.

[0019] 2、The polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel has high mechanical strength and is not prone to breakage.

[0020] 3、The polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel has excellent antibacterial performance as a high-strength antibacterial material.

[0021] 4、The polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel has good cell compatibility as a high-strength antibacterial material.

[0022] 5、The polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel is a high-strength antibacterial material, and the preparation method is simple and convenient. BRIEF DESCRIPTION OF DRAWINGS

[0023] The application will be further described below in combination with the drawings and examples.

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

[0025] Figure 2 is a scanning electron microscope image of the polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel of the application.

[0026] Figure 3 is an infrared spectrum diagram of the polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel of the application.

[0027] Figure 4 is a room temperature tensile strength test diagram of the polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel of the application.

[0028] Figure 5 is an antibacterial effect diagram of the polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel of the application.

[0029] Figure 6 is an in vitro L929 cytotoxicity test diagram of the polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel of the application.

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

[0031] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to specific embodiments and with reference to the drawings.

[0032] Polyvinyl alcohol hydrogel is a popular polymer material, which shows great potential in biomedical applications. Polyvinyl alcohol is a synthetic macromolecular polymer with excellent mechanical properties, biocompatibility, economy and stability. However, in biomedical applications, synthetic hydrogels usually face the problem of insufficient mechanical strength and toughness. Antimicrobial peptide NPR has multiple R, which can provide multiple hydrogen bond donors and acceptors, which is conducive to the formation of a more dense network, thereby improving the overall mechanical strength of the hydrogel. And at the same time, antimicrobial peptide NPR also has antibacterial activity, which can inhibit wound infection.

[0033] The present application provides a preparation method of polyvinyl alcohol-based high mechanical strength antibacterial hydrogel, which adopts one-pot mixing method, comprising: taking a certain amount of polyvinyl alcohol aqueous solution, adding antimicrobial peptide NPR to the polyvinyl alcohol aqueous solution, mixing uniformly, and then repeatedly freezing and thawing to obtain polyvinyl alcohol-based high mechanical strength antibacterial hydrogel. The hydrogen bond interaction between polyvinyl alcohol and antimicrobial peptide NPR enables the tight combination of antimicrobial peptide NPR and polyvinyl alcohol, and the combination of freezing and thawing cycle method realizes high mechanical strength; the mass fraction of the antimicrobial peptide NPR and polyvinyl alcohol after tight combination in the hydrogel is 0.002%~0.01%. At the same time, the repeated freezing and thawing method is used for gelation. Both of them can improve the mechanical properties.

[0034] Based on the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel of the above embodiment, the present application provides a preparation method of polyvinyl alcohol-based high mechanical strength antibacterial hydrogel, as shown in Figure 1 The method adopts one-pot mixing method, and specifically comprises the following steps:

[0035] Step 1: taking a certain amount of polyvinyl alcohol aqueous solution, adding antimicrobial peptide NPR to the polyvinyl alcohol aqueous solution, and mixing uniformly;

[0036] Step 2: repeatedly freezing and thawing to obtain polyvinyl alcohol-based high mechanical strength antibacterial hydrogel.

[0037] In the above step 1, the certain amount of polyvinyl alcohol aqueous solution is 1 ~ 3 g of 15% polyvinyl alcohol aqueous solution, and optionally 1 g of 15% polyvinyl alcohol aqueous solution is taken out. The antimicrobial peptide NPR is added to the polyvinyl alcohol aqueous solution, and 100 ~ 300 μg of antimicrobial peptide NPR is added to the polyvinyl alcohol aqueous solution, and optionally 100 μg of antimicrobial peptide NPR is added, and mixed uniformly.

[0038] In the above step 2, the repeated freezing and thawing is a plurality of freezing and thawing for a sufficient length of time, for example, can take -80℃ rapid freezing for at least 12 h, and then thawing at room temperature. After complete thawing, repeat the above steps for at least 3 cycles to obtain excellent mechanical properties. Thus, polyvinyl alcohol is combined with the antibacterial peptide NPR to form a network.

[0039] Further, the embodiment of the present application also provides a polyvinyl alcohol-based high mechanical strength antibacterial hydrogel in medical applications. The polyvinyl alcohol-based high mechanical strength antibacterial hydrogel provided by the embodiment of the present application is stored in a plastic package at low temperature and used directly for hand wound repair without thawing after taking out, as a daily wound.

[0040] The polyvinyl alcohol-based high mechanical strength antibacterial hydrogel, the preparation method and the application thereof in medical treatment provided by the present application are described in detail below in combination with specific embodiments.

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

[0042] In this embodiment, the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel is prepared. 1 g of 15% polyvinyl alcohol solution is mixed with 100 μg of antibacterial peptide NPR, and then rapidly frozen at -80℃ for at least 12 h, and then thawed at room temperature. After complete thawing, repeat the above steps 3 times to obtain the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel.

[0043] The vertical surface of the above polyvinyl alcohol-based high mechanical strength antibacterial hydrogel (PVA-NPR) is detected by a scanning electron microscope. As shown in Figure 2 , the vertical section of the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel according to the embodiment of the present application presents a layered structure. The layered ladder structure of pure PVA is obvious, and there is a hole structure (A); in the PVA-NPR group, the layered structure boundary disappears, and the section structure is more dense (B). The statistical results of the vertical section spacing of the hydrogel sheet show that the vertical section spacing j in the PVA group is larger, and the average value is 5.15±0.34 μm; the average value of the vertical section spacing in the PVA-NPR group is 1.65±0.11 μm, which is one third of the pure polyvinyl alcohol group. This may be due to the sequence (FFRLLFHGRRRRRRRRRRR) of the antibacterial peptide NPR itself, which has a large number of R residues, and they have rich amino groups, which can be used as a potential hydrogen bond crosslinking agent to enhance the interchain interaction by strong hydrogen bond. The freeze-dried sample is analyzed by an infrared spectrometer. As shown in Figure 3 , there is an increase in the absorption intensity near 1650 cm -1 , which is caused by the stretching vibration of the guanidine group C=N in the R group in the added antibacterial peptide. Another point worth noting is that there is an absorption peak at 3265 cm-1 ~3270 cm -1 The peak area increased slightly, accompanied by a slight red shift. This is because the added antimicrobial peptide provided additional NH, which formed hydrogen bonds and increased the crystallinity of the entire hydrogel system.

[0044] To characterize the mechanical properties of PVA-NPR, this study used a fracture tensile test to evaluate the mechanical strength of the hydrogel. Its fracture tensile curve is shown below. Figure 4 As shown, the mechanical strength of PVA-NPR is significantly improved compared to the pure PVA group. The tensile strength at break of polyPVA-NPR is roughly proportional to the content of antimicrobial peptide NPR in the hydrogel. When the amount of antimicrobial peptide NPR added is 100 μg / mL, the tensile strength at break is increased by 8 times compared to pure PVA. Compared to other antimicrobial peptides, taking WT (FFRLLFHGVHHVGKIKPRA) as an example, PVA-NPR shows significant differences in mechanical properties compared to hydrogels with the same formulation but with other antimicrobial peptides added. The tensile strength at break is increased by 7.3 times compared to hydrogels with added WT. This may be due to the additional hydrogen bond crosslinking formed 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 this invention were evaluated using *Escherichia coli* and *Staphylococcus aureus* as pathogenic models. 0.1 g of the hydrogel, irradiated with UV light for 30 min, was mixed with 10... 8 Antimicrobial performance was assessed by co-culturing a bacterial suspension of CFU / mL for 24 hours and then plating it onto a plate. Figure 5 As shown, the polyvinyl alcohol-based high mechanical strength antibacterial hydrogel according to the embodiments of the present invention has an inhibition rate of 60% against Escherichia coli and an inhibition rate of up to 92% against Staphylococcus aureus. This demonstrates that the PVA-NPR of the embodiments of the present invention has good prospects for antibacterial applications.

[0046] The biocompatibility of the materials was evaluated according to the standard of in vitro cytotoxicity in GB / T 16886. 0.1 g of PVA and PVA-NPR hydrogel was weighed in RPMI 1640 medium, and the hydrogel extract was prepared by shaking at 37°C for 24 hours. Each group of samples was not less than 3. Mouse fibroblast L929 cells were inoculated into RPMI 1640 complete medium containing 1% double antibody and 10% fetal bovine serum, and cultured in a 37°C 5% CO2 incubator. L929 cells were inoculated in a 96-well plate at a density of 3000 cells per well, and incubated. After the cells adhered, the medium was removed and replaced with PVA and PVA-NPR hydrogel extract. After co-culturing with cells for 1, 3, and 5 days, the biocompatibility of PVA and PVA-NPR hydrogel was evaluated by CCK8 method. The CCK8 determination method is described as follows: after the hydrogel extract and the cells were incubated for the target number of days, the medium was replaced with RPMI 1640 complete medium containing 10% CCK8, and incubated in a 37°C 5% CO2 incubator for 1 hour. Then 100 μL of supernatant was taken from each well and placed in a new 96-well plate, and the absorbance was measured at 450 nm. The cell survival rate was calculated according to the following formula: Figure 6 It can be seen that the polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel of the present example has good biocompatibility.

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

[0048] The polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel (PVA-NPR) of the present example has multiple processing properties. The polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel (PVA-NPR) of the present example was used as a wound dressing to detect its performance in promoting wound healing. Sprague-Dawley (SD) rats were used to perform rat bacterial infection full-thickness wound injury repair experiments to verify the in vivo antibacterial activity of PVA-NPR hydrogel. The experimental scheme was approved by the ethics committee (approval number: YSY-DWLL-2021031) and met the accreditation standards of the Association for Assessment and Accreditation of Laboratory Animal Care. Isoflurane inhalation anesthesia was used in this study. After the rats were anesthetized, the skin was prepared with a 10 mm biopsy punch to create a full-thickness skin defect on the back of the rat. Then 100 μL of S. aureus bacterial suspension (PBS, OD 600 =0.5) was added to simulate wound contamination. After 5 minutes, the wounds were treated with PBS, PVA, and PVA-NPR hydrogel, respectively, and the PBS-treated group served as a blank control.

[0049] To evaluate the specific performance of the polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel (PVA-NPR) of the present example as a wound dressing, the area of the mouse wound was counted at 1, 4, and 7 days. 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, which indicates that the polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel of the embodiment of the present application has the performance of promoting wound healing as a wound dressing.

[0050] The above-described specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the present application, and it should be understood that the above-described is only a specific embodiment of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing a polyvinyl alcohol-based high-strength antibacterial hydrogel, characterized by The method comprises the following steps: An antibacterial peptide NPR is added into a polyvinyl alcohol aqueous solution, mixed uniformly, and then repeatedly frozen and thawed to prepare a polyvinyl alcohol-based high-strength antibacterial hydrogel; The amino acid sequence of the antibacterial peptide NPR is shown as SEQ ID NO:1; The mass concentration of the polyvinyl alcohol aqueous solution is 15%, the addition amount of the polyvinyl alcohol aqueous solution is 1-3 g, and the addition amount of the antibacterial peptide is 100-300 μg; The repeated freezing and thawing is first rapidly frozen at-80℃ for at least 12 h, then thawed at room temperature, and the above steps are repeated after complete thawing, and the freezing-thawing cycle is performed for at least 3 times.

2. The preparation method according to claim 1, characterized in that: The nucleotide sequence encoding the antibacterial peptide is shown as SEQ ID NO:

2.

3. The preparation method according to claim 1, characterized in that: The number of freezing-thawing cycles is 3-6 times.

4. Application of the polyvinyl alcohol-based high-mechanical-strength antibacterial hydrogel prepared by the preparation method in any one of claims 1-3 in preparation of a medical antibacterial dressing, wherein the antibacterial dressing has antibacterial activity against Escherichia coli and Staphylococcus aureus.

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