Preparation method and antibacterial application of metal ion post-modified nano enzyme

Abstract

The invention discloses a preparation method of a metal ion post-modified nano-enzyme, according to the method, Cu < 2 + > or Cu < + > is used for modifying PEI-Mn-CDs, peroxidase-like activity is further enhanced, new enzyme-like activity is endowed, the Cu < + > modified PEI-Mn / Cu < + >-CDs nano-enzyme has a wider pH application range and enzyme catalytic activity, and compared with the PEI-Mn-CDs, the metal ion post-modified PEI-Mn-CDs nano-enzyme has the advantages that the pH application range is wider; the maximum reaction rate Vmax of the enzyme is increased by 29.8 times, and the substrate affinity is higher; mechanism research shows that after Cu < 2 + > or Cu < + > is used as an additional catalytic site and PEI (at) Mn-CDs are modified through a metal amine / carboxyl coordination strategy, the electron transfer efficiency is remarkably improved, the free radical generation amount is remarkably increased, efficient antibiosis is achieved, and the PEI (at) Mn-CDs have excellent antibacterial activity and good cytocompatibility on drug-resistant bacteria and biological membranes of the drug-resistant bacteria; the method has good application potential in resisting drug-resistant bacteria and removing biological membranes.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterial antibacterial technology, and specifically to a preparation method and antibacterial application of a metal ion post-modified nanozyme. Background Art

[0002] Bacterial infections pose a serious threat to human health and healthcare, both in terms of morbidity and treatment costs. Microbial contamination caused by resistant bacteria and their biofilms has been recognized as one of the most serious threats to human health due to antibiotic resistance, and it is necessary to develop innovative strategies to combat multidrug-resistant bacteria and their biofilms. Artificial nanozymes have shown potential in combating resistant bacteria and eliminating biofilms due to their low toxicity, low drug resistance, excellent membrane permeability and biocompatibility. Nanozymes have the ability to produce reactive oxygen species (ROS), thereby causing irreversible damage to bacteria, destroying cell structures and inhibiting bacterial growth, and achieving antibacterial activity. Nanozymes are usually nanomaterials made of carbon-based materials, metal-based nanomaterials, precious metals, polymers, etc., and exhibit various catalytic activities, such as peroxidase-like, oxidase-like and superoxide-like activities.

[0003] Carbon dots (CDs) have the advantages of small size, large specific surface area, low toxicity, and good stability. They are very suitable for binding to substrates and exhibit strong enzyme-like activity. As a nanozyme, carbon dots have received widespread attention in the field of antibacterial and have broad application prospects. They are the most promising nanomaterials to replace natural enzymes. Although carbon dot-based nanozymes have made significant progress in antibacterial applications, the relatively low catalytic activity (compared with natural enzymes) and relatively weak binding ability with bacteria may severely limit the application of carbon dots against drug-resistant bacteria and their biofilms. Modifying or doping nanozymes with metal ions is a reliable and key strategy to obtain highly active antibacterial nanozymes. Metal ions, as active centers, have a significant effect on the enzymatic activity and antibacterial activity of nanozymes. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method for preparing a metal ion post-modified nanozyme. The method firstly uses polyethyleneimine (PEI) and Mn 2+ Carbon dots (PEI@Mn-CDs) were prepared as precursor materials, and then the amine / carboxyl groups on the surface of PEI@Mn-CDs were used to react with Cu 2+ and Cu + Mutual coordination, preparation of PEI@Mn / Cu by post-modification method + -CDs and PEI@Mn / Cu 2+ -CDs nanozymes, Cu 2+ and Cu +The modification can significantly improve the peroxidase-like activity and effectively promote the production of ROS, thereby achieving high efficiency against drug-resistant bacteria and their biofilms.

[0005] The preparation method of nanozymes based on metal ion post-modification and their antibacterial applications of the present invention are as follows: (1) Synthesis of polyethyleneimine-manganese carbon dots (PEI@Mn-CDs) Dissolve 0.1-0.3g polyethyleneimine, 0.1-0.3g citric acid, 0.4-0.5g manganese acetate and 0.4-0.5g o-phenylenediamine in 10-30mL deionized water; after ultrasonic treatment for 20-40min, transfer the solution to a microwave digestion tank and heat the reaction at 170-190℃ for 1-3h; after cooling the solution to room temperature, filter it with a 0.22μm filter membrane to obtain a PEI@Mn-CDs solution; The molecular weight of the polyethyleneimine is 300-10000; (2) Polyethyleneimine-manganese-cuprous nanozyme (PEI@Mn / Cu + Preparation of -CDs Take 3-8 mL of the polyethyleneimine-manganese carbon dot solution obtained in step (1) and 3-8 mL of the cuprous chloride solution, stir for 10-30 min, let stand overnight, centrifuge, filter the liquid after centrifugation with a 0.22 μm filter membrane to obtain a polyethyleneimine-manganese-cuprous nanozyme solution, wash the solid with deionized water 2-3 times after centrifugation, and obtain a polyethyleneimine-manganese-cuprous nanozyme solid; The centrifugation is carried out at 4000-10000 r / min for 10-15 min; the concentration of the cuprous chloride solution is 10-30 mg / mL; (3) Polyethyleneimine-manganese-copper nanozyme (PEI@Mn / Cu 2+ Preparation of -CDs Take 3-8 mL of the polyethyleneimine-manganese carbon dot solution obtained in step (1) and 3-8 mL of the copper chloride solution, stir for 10-30 min, let stand overnight, and then filter with a 0.22 μm filter membrane to obtain PEI@Mn / Cu 2+ -CDs.

[0006] The concentration of the cupric chloride solution is 10-30 mg / mL.

[0007] Another object of the present invention is to provide the use of the metal ion post-modified nanozyme prepared by the above method in the preparation of antibacterial preparations.

[0008] The advantages of the present invention are: 1. The present invention adopts Cu 2+ and Cu +To modify PEI@Mn-CDs, PEI@Mn / Cu 2+ -CDs and PEI@Mn / Cu + -CDs can significantly enhance the catalytic activity of peroxidase-like enzymes, and the addition of Cu + It can produce other multi-enzyme activities, including oxidase-like activity, laccase-like activity and catalase-like activity, which can more effectively promote the production of ROS and make Cu 2+ and Cu + The modified PEI@Mn / CDs can achieve better antibacterial and anti-biofilm effects; 2. PEI@Mn / Cu of the present invention + -CDs nanozymes have a wider pH range (pH 3-8), which is more conducive to practical antibacterial applications. Compared with PEI@Mn-CDs, the PEI@Mn / Cu + -The maximum catalytic rate of CDs ( V max ) increased by 29.8 times, and had a stronger affinity for its substrate; 3. The nanozyme of the present invention has good antibacterial effect on Escherichia coli, Staphylococcus aureus, drug-resistant Escherichia coli and drug-resistant Staphylococcus aureus. + -CDs antibacterial efficiency reaches 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a transmission electron microscopy image of polyethyleneimine-manganese-cuprous nanozyme; Figure 2 This is the UV-visible absorption spectrum of the nanozyme peroxidase activity detection; Figure 3 This is the UV-visible absorption spectrum of the nanozyme laccase activity detection; Figure 4 This is the UV-visible absorption spectrum of the nanozyme oxidase activity detection; Figure 5 This is the UV-visible absorption spectrum of the nanozyme catalase activity detection; Figure 6 The UV-visible absorption spectra of the effects of different pH on the activity of peroxidase-like enzymes; Figure 7 UV-visible absorption spectrum for reactive oxygen species (ROS) determination; Figure 8 This is the result of the nanozyme cytotoxicity test; Fig. 9 Figure 2 shows the antibacterial effects of different nanozymes; Fig.10Figure 1 shows the effect of crystal violet on biofilm removal (a) and the image of live (green) bacteria remaining in the biofilm (b). DETAILED DESCRIPTION

[0010] The present invention is further explained by examples below, but these examples do not limit the scope of protection of the present invention. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified; Example 1: Preparation and antibacterial effect of metal ion post-modified nanozymes 1. Synthesis of polyethyleneimine-manganese carbon dots (PEI@Mn-CDs) 0.2 g polyethyleneimine (molecular weight 2000), 0.2 g citric acid, 0.458 g manganese acetate and 0.4 g o-phenylenediamine were dissolved in 20 mL deionized water. After ultrasonic treatment for 30 min, the solution was transferred to a microwave digestion instrument and heated at 180 °C for 2 h. The solution was cooled to room temperature and finally filtered through a 0.22 μm filter membrane to obtain a PEI@Mn-CDs solution. 2. Polyethyleneimine-manganese-cuprous (PEI@Mn / Cu + Preparation of nanozymes Take 5 mL of PEI@Mn-CDs solution and 5 mL of cuprous chloride (18 mg / mL) solution, stir for 20 min, let stand overnight, centrifuge at 8000 rpm for 10 min, and filter the liquid with a 0.22 μm filter membrane to obtain PEI@Mn / Cu + -CDs solution, and after centrifugation, the solid was washed twice with deionized water to obtain PEI@Mn / Cu + -CDs solid; PEI@Mn / Cu by transmission electron microscopy + -CDs solid was characterized and analyzed. The results are as follows Figure 1 As shown, PEI@Mn / Cu + -CDs have a flocculent aggregate morphology structure; 3. Polyethyleneimine-manganese-copper nanozyme (PEI@Mn / Cu 2+ Preparation of -CDs Take 5 mL of PEI@Mn-CDs solution and 5 mL of copper chloride (18 mg / mL) solution, stir for 20 min, let stand overnight, and filter with a 0.22 μm filter membrane to obtain PEI@Mn / Cu 2+ -CDs solution; 4. Determination of peroxidase-like activity of nanozymes 20 μL of PEI@Mn-CDs solution and PEI@Mn / Cu + -CDs solution, PEI@Mn / Cu +-CDs solid (dispersed with deionized water to obtain a 4 mg / mL dispersion), PEI@Mn / Cu 2+ -CDs solution was mixed with 100 µL TMB solution (5 mM) and 100 µL HO 2 O 2 The solution (50mM) was mixed, 300µL NaAc-Hac (pH=4) buffer solution was added, and then the volume was adjusted to 4mL with deionized water. After thorough mixing, it was incubated at room temperature for 15min, and the absorbance at 654nm was measured using a UV-visible spectrophotometer; each sample was measured 3 times and the average value was taken. The results are shown in Figure 2 As shown, it can be seen that the order of peroxidase activity is: PEI@Mn / Cu + -CDs (solid) > PEI@Mn / Cu + -CDs (liquid) > PEI@Mn / Cu 2+ -CDs>PEI@Mn-CDs.

[0011] The studies were conducted to investigate TMB and H 2 O 2 As substrate, the maximum reaction rate of the nanozyme was further analyzed by the Michaelis-Menten equation. V max and Michaelis constant K m The results are shown in Table 1. It can be seen that when TMB is used as the substrate, PEI@Mn / Cu + -CDs (Solid) V max Increased by 29.8 times, K m The lower the value, the stronger its ability to bind to the substrate; Table 1 Enzyme reaction kinetics data of nanozymes

[0012] 5. Determination of other types of enzyme activity of nanozymes ① Take 4-AP+2,4-DP as substrate and add PEI@Mn-CDs solution, PEI@Mn / Cu + -CDs solution, PEI@Mn / Cu + -CDs solid (concentration 4 mg / mL dispersion), PEI@Mn / Cu 2+ -CDs solution, the laccase-like activity of the nanozyme was determined. After the substrate and enzyme bind, the product will be red and have a characteristic absorption peak at 510 nm. The results are as follows Figure 3 As shown, PEI@Mn / Cu +-CDs (solid) has the highest absorption peak and the strongest laccase activity. PEI@Mn-CDs and PEI@Mn / Cu 2+ -CDs do not generate red products and therefore have no laccase-like activity; ② Only TMB is used as substrate. Other determination steps are the same as 4. The results are as follows Figure 4 As shown, PEI@Mn / Cu + -CDs (solid) can also significantly catalyze the oxidation of TMB to blue ox-TMB, indicating that it also has oxidase-like activity; ③ Through H 2 O 2 With Ti 4+ The reaction generates complex H 2 O 2 -Ti 4+ , verifying that PEI@Mn-CDs, PEI@Mn / Cu + -CDs (solid or liquid) and PEI@Mn / Cu 2+ -CDs has a catalase-like activity. The complex has a characteristic absorption peak at 376nm. Adding nanozymes with catalase activity will reduce the characteristic absorption peak, and then study the catalase activity of nanozymes. The results are as follows Figure 5 , it can be seen that PEI@Mn / Cu 2+ -CDs, PEI@Mn / Cu + -CDs (solid or liquid) all showed catalase-like activity, and the order of catalytic activity was PEI@Mn / Cu + -CDs (solid) > PEI@Mn / Cu 2+ -CDs>PEI@Mn / Cu + -CDs (liquid) > PEI@Mn-CDs.

[0013] 6. Effect of different pH on the activity of nanozyme peroxidase 20 µL PEI@Mn / Cu + -CDs solid (concentration 4 mg / mL dispersion) and TMB solution (5 mM), H 2 O 2 100 µL of each solution (50 mM) was mixed, and then 300 µL of buffer solutions with pH values ​​of 2, 3, 4, 5, 6, 7, 8, and 9 were added, and the volume was adjusted to 4 mL with deionized water. After thorough mixing, the mixture was incubated at room temperature for 15 min, and the absorbance was measured at 654 nm using a UV-visible spectrophotometer; each sample was measured 3 times and the average value was taken. The results are shown in Figure 6 As shown, it can be seen that PEI@Mn / Cu + -CDs solids maintain peroxidase-like activity over a wide pH range of 3-8.

[0014] 7. Determination of reactive oxygen species (ROS) production Methylene blue (MB) dye was used as a colorimetric probe for analysis. MB has a strong absorption peak at 675nm, but after interacting with ROS, the absorbance decreases. The results are as follows Figure 7 As shown, it can be seen that at 675nm, PEI@Mn / Cu + -CDs (solid) + MB + H 2 O 2 The peak value is the lowest, indicating that the most •OH is generated.

[0015] 8. Nanozyme cytotoxicity test MTT method was used to evaluate the PEI@Mn / Cu + -CDs (solid) and HeLa cells, HCT116 cells. The cells were placed in a sterile 96-well plate and incubated with different concentrations (0, 10, 30, 50 and 70 μg / mL) of PEI@Mn / Cu + -CDs were incubated at 37°C for 24 h; 50 µL MTT solution was added to the 96-well plate for 2 h, and the absorbance of each well at 490 nm was measured to evaluate cell viability. The results are shown in Figure 8 As shown, it can be seen that PEI@Mn / Cu + -CDs (solid) have good cell compatibility.

[0016] 9. Nanozyme plate antibacterial experiment Escherichia coli ( E. coli , ATCC 25922), Staphylococcus aureus ( S. aureus , ATCC 25923), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC BAA-1720) and ampicillin-resistant Escherichia coli (AREC) were used as experimental strains; for each strain, 3-5 single colonies were inoculated into fresh tryptone soy broth (TSB) and cultured at 37°C for 16-18h until the stable phase; 40 μL of bacterial solution was diluted 100 times with fresh TSB and cultured at 37°C until the mid-log phase (OD 600 =0.5-0.7); after obtaining the bacterial cells, centrifuge and wash once with sterile PBS, and adjust the concentration to 1.5×10 6 CFU / mL; the nanozyme dispersion was diluted 2-fold in series with sterile PBS buffer, and each nanozyme dilution (100 μL) was added to each dilution well of a 96-well microplate. 50 μL of the adjusted bacterial suspension was inoculated into each dilution well of the pre-set microplate to reach 5×10 5CFU / mL (150μL); the microplate was then incubated at 37°C for 3h; it was subsequently diluted 10-fold with sterile PBS buffer, and the diluted bacterial solution (20μL) was spread on a TSB agar plate and cultured overnight at 37°C to form colonies visible to the naked eye; the inoculum size was indicated by a control sample containing similarly treated bacteria but without nanozymes; each test was performed in parallel 3 times, and the test results were the average of two independent tests; the minimum inhibitory concentration (MIC) value was defined as the lowest concentration of antibiotics or nanozymes that inhibited 90% bacterial growth.

[0017] PEI@Mn / Cu + -The minimum inhibitory concentration (MIC) of CDs (immobilized) nanozyme was 4µg / mL; Fig. 9 The results of coating showed that the nanozyme exhibited excellent antibacterial properties; PEI@Mn / Cu + -CDs and PEI@Mn / Cu 2+ -CDs have an antibacterial efficiency of more than 91.7% against Staphylococcus aureus and drug-resistant Staphylococcus aureus; PEI@Mn / Cu 2+ The antibacterial efficiency of Cu-CDs against E. coli and drug-resistant E. coli was 74.9% and 41.9%, respectively. 2+ Replaced by Cu + After that, the activity of nanozymes was significantly improved, and the antibacterial efficiency reached 92.8% and 75.8% respectively; at the same time, PEI@Mn / Cu + -CDs (solid) further significantly enhanced the antibacterial effect of the four bacteria, and the inhibition rate could reach 100%.

[0018] 10. Study on the anti-biofilm effect of nanozymes ① Take 10 μL of E. coli, AREC, Staphylococcus aureus and MRSA (1×10 8 CFU mL -1 ) suspension and 990 μL of liquid culture medium were added to a 24-well plate and cultured at 37°C for 12 h; the culture medium in each well was slowly aspirated and then rinsed 2 to 3 times with PBS buffer to obtain the biofilm; PBS buffer, H 2 O 2 PEI@Mn-CDs+H 2 O 2 PEI@Mn / Cu + -CDs (solid) + H 2 O 2 PEI@Mn / Cu + -CDs (liquid) + H 2 O 2, respectively, mixed with bacterial biofilms and further cultured at 37.0°C for 48 h until the bacteria formed mature biofilms; after washing with methanol to remove planktonic bacteria and materials, they were fixed with 200 µL 4% paraformaldehyde for 2 h; The crystal violet staining method was used to evaluate the removal effect of nanozymes on biofilms: First, 200 µL of 0.1% crystal violet solution was added to each well. After natural air drying, the biofilm was incubated for 20 min; 100 µL of 33% acetic acid solution was added to each well to dissolve the crystal violet, and the absorbance was measured at 595 nm using an ELISA reader.

[0019] Results Fig.10 a, REI@MnCu-CDs (solid) has a removal efficiency of 98.21% and 98.3% for AREC and MRSA, respectively, while PEI@Mn / Cu + -CDs (solid) can effectively remove biofilm; ② Sterile cell slides with AREC or MRSA biofilms were constructed on 24-well plates; then, E. coli, AREC, Staphylococcus aureus, and MRSA biofilms were treated as follows: control (untreated), PEI@Mn-CDs+H 2 O 2 、PEI@Mn / Cu + -CDs (solid) + H 2 O 2 、PEI@Mn / Cu + -CDs (liquid) + H 2 O 2 , and incubated at 37°C for 3 h; to observe live bacteria, the biofilm on the slide was stained with 50 μg / mL SYTO-9 in the dark, and then the slide containing the biofilm was gently rinsed three times with PBS buffer (pH = 7.4), and fluorescence imaging was performed using a fluorescence microscope: the excitation wavelength of the green fluorescence image was 510-550 nm.

[0020] The results are as follows Fig.10 As shown in b, in PEI@Mn / Cu + -CDs (solid or liquid) group, there was only a weak green fluorescence signal in the four different biofilms, indicating that the number of surviving bacteria was very small; according to the fluorescence intensity, quantitative analysis showed that PEI@MnCut +-CDs (solid) showed a removal rate of 91.3%, 85.8%, 86.0% and 88.4% against E. coli, AREC, S. aureus and MRSA biofilms, respectively, while under the same conditions, the removal rates of PEI@Mn-CDs against E. coli, AREC, S. aureus and MRSA biofilms were 27.8%, 55.5%, 50.1% and 7.8%, respectively.

[0021] The above results show that PEI@Mn / Cu + -CDs (solid) can effectively destroy the biofilms of E. coli, AREC, S. aureus and MRSA.

Claims

1. A method for preparing a metal ion post-modified nanozyme, characterized in that: Here are the steps: (1) Dissolve 0.1-0.3 g polyethyleneimine, 0.1-0.3 g citric acid, 0.4-0.5 g manganese acetate, and 0.4-0.5 g o-phenylenediamine in deionized water, treat with ultrasound for 20-40 min, heat in a microwave at 170-190 °C for 1-3 h, cool and filter to obtain a polyethyleneimine-manganese carbon dot solution; (2) taking 3-8 mL of polyethyleneimine-manganese carbon dot solution and 3-8 mL of cuprous chloride solution, stirring and mixing, standing overnight, centrifuging, filtering the liquid with a 0.22 μm filter membrane to obtain a polyethyleneimine-manganese-cuprous nanozyme solution, and washing the solid with deionized water 2-3 times after centrifugation to obtain a polyethyleneimine-manganese-cuprous nanozyme solid; Alternatively, 3-8 mL of polyethyleneimine-manganese carbon dot solution and 3-8 mL of copper chloride solution are taken, stirred and mixed, and then allowed to stand overnight, and then filtered with a 0.22 μm filter membrane to obtain polyethyleneimine-manganese-copper nanozyme liquid.

2. The method for preparing the metal ion post-modified nanozyme according to claim 1, characterized in that: The concentration of the cuprous chloride solution or cupric chloride solution is 10-30 mg / mL.

3. The method for preparing the metal ion post-modified nanozyme according to claim 1, characterized in that: The molecular weight of polyethyleneimine is 300-10000.

4. The method for preparing the metal ion post-modified nanozyme according to claim 1, characterized in that: Centrifugation is carried out at 4000-10000 r / min for 10-15 min.

5. Use of the metal ion post-modified nanozyme obtained by the preparation method of the metal ion post-modified nanozyme according to any one of claims 1 to 4 in the preparation of antibacterial preparations.

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