Abstract

Bacterial infections caused by the misuse of antibiotics have gradually become a growing global problem. Highly toxic reactive oxygen species (ROS) are an ideal means of efficient and rapid sterilization, resulting in the leakage of bacterial contents by destroying bacterial membranes. In this study, we are to develop highly efficient Au@Pd nanospheres with peroxidase activity as antibacterial nanomaterials. The classical TMB chromogenic experiment showed that Au@Pd nanospheres had good peroxidase activity to produce ROS and achieve the antibacterial effect. Bacterial experiments have shown that Au@Pd nanospheres with peroxidase activity have great potential in inhibiting Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus).

1. Introduction

Bacterial infection leads to severe tissue damage and chronic nonhealing wounds, which have become a serious threat to global health. [1-4] ¹ According to Global mortality associated with 33 bacterial pathogens in 2019, bacterial infections were the second leading cause of death after ischemic heart disease, responsible for one in eight deaths worldwide. ⁵ It is emphasized that reducing bacterial infections is an important priority for global public health, especially as the increased risk of drug-resistant bacteria greatly limits the development of antibiotics. ⁶ In addition, the long-term and massive abuse and misuse of antibiotics aggravates the evolution of pathogenic bacteria, resulting in gradually increased pathogenicity and antibacterial properties. ^{7, 8} It was reported that the number of deaths caused by drug-resistant bacteria in the world was at least 700,000 in 2014, and if left unturned, the number of deaths caused by drug-resistant bacteria could surge to 10 million by 2050. [9-12] ⁹ E. coli as the representative of Gram-negative bacteria and S. aureus as the representative of Gram-positive bacteria are the most common and representative bacteria of human infection. Therefore, the development of novel antibacterial agents against this group of bacteria is an urgent task. [13-15] ¹³

Inspired by the enzyme attack bacteria in the body’s immune system to maintain the health of the body, nano-enzymes with the characteristics of low cost, easy to produce, high stability, and easy to store have shown their edge in the field of antibacterial. ¹⁶ As nanomaterials with enzyme-like activity, nanozymes kill bacteria without developing bacterial resistance by catalyzing the production of highly toxic substances (such as ROS) from endogenous substances. [17-20] ¹⁷ At present, a large number of antibacterial nanozymes have been reported, all of which have peroxidase-like (POD) properties. [21-26] ²¹ They can produce highly toxic hydroxyl radicals (•OH) and further cause irreversible oxidative damage to biofilms and bacterial phospholipids, DNA, and proteins through catalysis of hydrogen peroxide (H₂O₂). ^{27, 28} Deng and his colleagues reported multifunctional AuPtNDs capable of catalyzing H₂O₂ to highly toxic ·OH, killing bacteria and promoting wound healing by disrupting the integrity of cells. ²⁹ Wang et al. reported a multifunctional nano platform (APBA-TPE NPs), under the condition of light to produce reactive oxygen species (¹O₂, ·O₂^- and ·OH) kill S. aureus in water samples. ³⁰ Dong et al. have developed a new type of bacteria antibacterial carbon (BAPTCDs), specific binding to specific combination of bacteria, and heating quickly under laser irradiation, to destroy the cell walls of bacteria in order to achieve the purpose of kill bacteria. ³¹

In this study, we report a simple and highly sensitive POD-active nanoenzyme (Au@Pd), which exhibits antibacterial and anticancer properties through the elemental composition of intrinsic POD enzyme activity. Au@Pd Nanospheres have a uniform size of about 20nm and good stability. In addition, Au@Pd nanospheres exhibited peroxidase-like activity and showed significant antibacterial activity against E. coli and S. aureus.

2. Experimental Methods

HAuCl4⋅ 3H₂O, Na₃C₆H₅O₇⋅ 2H₂O, Na₂PdCl₄, L-ascorbic acid (Sigma-Aldrich), 3’,5,5’-tetramethylbenzidine (TMB), glacial acetic acid and anhydrous sodium citrate (Maclean), LB broth and agarose were purchased from Solarbio.

The UV-vis spectrum was measured by a UV-Vis spectrophotometer (Thermo Scientific™ GENESYS™ 150). Transmission electron microscopy (TEM) images were obtained using a Jeol transmission electron microscope at 100 kV, hydrodynamic particle sizes were obtained using a nanoparticle particle size and Zeta potential analyzer Zetasizer (Brook Haven), and bacterial counts and area calculations were performed using ImageJ software.

Au@Pd nanospheres were synthesized according to the reported literature. ³² Add 1ml of 25mM HAuCl₄⋅3H₂O solution to a round-bottom flask containing 99ml ultrapure water (ddH₂O), heat and stir, add 3ml of Na₃C₆H₅O₇⋅2H₂O solution, keep boiling for 30min, and cool to room temperature to obtain Au nanospheres with a diameter of 15nm. Subsequently, 2.4ml ultrapure water, 299ul Na₂PdCl₄ solution, and newly synthesized 15nm Au nanospheres were heated and stirred; after mixing, 300ul L-ascorbic acid solution was added and continued stirring for 30min to obtain 20nm Au@Pd nanospheres of the target product.

The morphology, absorption spectrum, particle size, and other data of Au@Pd were characterized by transmission electron microscope, UV-vis spectrometer, Zetasizer (OMN) nanoparticle particle size and Zeta potential analyzer, and SONY camera.

A mixture containing 0.2M NaOAc/HOAc buffer (pH 2, 4, 6, 8, and 10), H₂O₂ solution (0.5mM, 1.0mM, 1.5mM, and 2.0mM), and TMB solution (2mM, 4mM, 6mM, 8mM, and 10mM) was added to the cuvette with a path (l) of 10mM. The absorption peak at 652nm of Au@Pd nanospheres in different pH, TMB solution, and H₂O₂ solution was detected by UV-vis spectrometer and fully functional microplate detector (Synergy Neo2) to evaluate the optimal peroxidase activity of Au@Pd nanospheres.

The MIC of Au@Pd nanospheres incubated with E. coli was determined by adding different concentrations of Au@Pd nanospheres (0, 1, 2, 4, 8, 16 μg/mL) to the same amount of bacterial suspension (106 CFU/mL dispersed in 25 g/L LB agar solution). Then, the suspension was incubated overnight at 37°C. Bacterial concentration was determined at 560 nm on a microplate reader. The bacterial suspension was diluted 106-fold and incubated on AGAR plates for 12 h, and the number of colonies was counted. AGAR plates were prepared by curing a PBS solution containing 0.04 g/mL LB AGAR medium. The MIC of Au@Pd nanospheres incubated with S. aureus was the same.

3. Results and Conclusions

According to the existing references, Au nanospheres were first prepared, and then Au@Pd nanospheres were formed by coating the surface of the Au nanospheres with a Pd shell. ³³ The morphology of the above nanoparticles was examined by transmission electron microscopy (TEM), as shown in Figure 1A, the Au nanospheres showed a uniform spherical shape with a diameter of about 15nm, and when coated with a Pd shell, Au@Pd nanospheres with a diameter of about 20nm were observed (Figure 1B and 1C). The characteristic absorption of the UV-visible spectra of Au nanospheres and Au@Pd nanospheres at 520nm further indicated the successful preparation of Au nanospheres and the successful coating of Pd shells (Figure 1D).

The peroxidase-like activity of Au@Pd nanospheres was assessed by canonical colorimetry with 3,3 ', 5,5 '-tetramethylbenzidine (TMB). ³⁴ As shown in Figures 2A and 2B, the oxidation of TMB (colorless) to oxTMB (blue) by H₂O₂ in the presence of Au@Pd nanospheres showed a distinct color change and a distinct UV-Vis absorption peak at 652 nm. However, in the treatment group without Au@Pd nanospheres, the solution was lighter in color. In addition, negligible color changes were observed in the control group with only H₂O₂ and Au@Pd nanospheres, indicating that the mixed solution of H₂O₂ and Au@Pd did not cause any oxidation reaction. The above results indicate that Au@Pd nanospheres have peroxidase-like catalytic activity. ³⁵

In addition, the effects of different TMB concentrations, H₂O₂ concentrations, pH, and temperature on the peroxidase activity of Au@Pd were further evaluated by the control variable method. As shown in Figure 2C, when pH=2, the color of Au@Pd NPs, TMB, and H₂O₂ reaction solution turns green. This is because, under physiological conditions, the peroxidase (Au@Pd NPs) catalyzes the amino group of TMB to lose an electron and become a cationic radical, which exists in the form of a dimeric charge-transfer complex in the system. This dimer has a maximum absorption at 370nm and 652nm and appears blue. At low pH, the complex will lose an electron again and form a stable conjugate monomer structure with a maximum absorption wavelength at 450nm and a yellow color. Blue and yellow overlap to give a green color visible to the naked eye. When pH=4, the reaction solution of Au@Pd NPs, TMB, and H₂O₂ was dark blue. When the pH is basic, the reaction solution of Au@Pd NPs, TMB, and H₂O₂ is light blue and becomes lighter with the increase of alkalinity. ¹⁵ The above results indicate the optimal enzymatic activity of Au@Pd NPs at pH=4. At the same time, the absorption at 652nm was gradually enhanced with the increase of TMB concentration. The absorption at 652nm was gradually enhanced with increasing H₂O₂ concentration. At the same TMB concentration, the absorption peak at 652nm was gradually enhanced with the extension of time. At the same time, the enzyme activity increased gradually with the increase of temperature. At the same temperature, the enzyme activity increased gradually with the extension of time. Unlike native enzymes, which are easily inactivated at high temperatures, the enzymatic activity of Au@Pd NPs gradually increased with increasing temperature. However, Au@Pd nanospheres are difficult to achieve above 40°C for antibacterial application in vivo. Therefore, 37°C, the normal temperature of the organism, was selected as the reaction temperature, considering the limitation of the environment in which the test strip was used. In conclusion, the highest catalytic efficiency and enzyme activity of Au@Pd were observed at pH 4 with 10 mM TMB and 10 mM H₂O₂.

To evaluate the antibacterial properties of Au@Pd nanospheres, we determined the minimum inhibitory concentration of Au@Pd nanospheres against E. coli or S. aureus. ³⁶ When the concentration of Au@Pd nanospheres was 16ug/ml, the inhibition rate of E. coli was 93.12%, and the inhibition rate of S. aureus was 93.12%. Therefore, a liquid medium mixed with different concentrations of Au@Pd nanospheres with E. coli or S. aureus was diluted and coated onto a solid LB medium for culture to assess bacterial clearance. Figure 3 shows the bacterial solid plate after different material treatment groups. The optical density of the liquid medium at 560nm is positively correlated with the growth of the bacteria. Therefore, OD560 reflects bacterial reproduction. Bacteria treated with Au@Pd nanospheres at concentrations of 0.25, 0.5, 1, 2, 4, 8, and 16ug/ml had lower OD560 values compared with PBS, suggesting that Au@Pd nanospheres inhibited the growth of bacteria. In particular, when the concentration of Au@Pd nanospheres was 16ug/ml, only a very small number of E. coli or S. aureus colonies were formed, indicating that Au@Pd nanospheres had a high antibacterial rate and good bactericidal ability, which effectively reduced the use of antibiotics.

4. Conclusion

In this study, we synthesized Au@Pd nanospheres to explore the peroxidase properties of Au@Pd nanospheres for antibacterial tests. It was found that Au@Pd is a brown nanosphere with a 20-nm diameter of gold core and palladium shell structure with peroxidase properties. By producing toxic ROS, it causes damage to the bacterial membrane and induces leakage of intracellular substances, to achieve the purpose of bacterial eradication. Systematic antibacterial tests showed that Au@Pd nanospheres had high bactericidal rates against E. coli and S. aureus.

Author Contributions

Conceptualization, Leilei Yu and Peifeng Li; methodology, Leilei Yu; validation, Leilei Yu; formal analysis, Leilei Yu; investigation, Leilei Yu and Peifeng Li; resources, Peifeng Li; writ-ing—original draft preparation, Leilei Yu; writing—review and editing, Peifeng Li. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Integrated Project of Major Research Plan of National Natural Sci-ence Foundation of China, grant number 92249303.

References