The MBC in two sizes of CuNPs for three bacteria.
Abstract
Copper, a reddish and ubiquitous material in the world, possesses malleable and conductive properties that render copper and its alloys indispensable in vertical integration manufacturing. With advancements in nanotechnology and nanomaterials in recent decades, copper and its related nanoparticles have been engineered. Their applications include engineering, material science, photo−/electro-catalysis, biomedical drug delivery, agriculture, and antipathogen microbicides. Here, we studied the differing toxicity effects of two sizes of copper nanoparticles (CuNPs), recognized for their potent bactericidal properties. Concentration-dependent effects of both 20 and 60 nm CuNPs were significant in Escherichia coli (E. coli), Acinetobacter baumannii (A. baumannii), and Staphylococcus aureus (S. aureus). Sodium dodecyl sulfate, the dispersant of nanoparticles, caused the synergy effects with CuNPs in A. baumannii and S. aureus but not in E. coli. Four modulators were added to CuNP-treated bacteria. By these modulator treatments, programmed cell death was found in E. coli, A. baumannii, and S. aureus. By the BLAST search, caspase-related proteins were commonly identified in gut bacteria and A. baumannii but not in S. aureus. Furthermore, many proteins from E. coli, A. baumannii, and S. aureus were found to harbor the ULK1-catalytic domain. In short, CuNPs can be potent therapeutic agents against bacterial infections.
Keywords
- membrane leakage
- autophagy
- apoptosis
- necroptosis
- cytotoxicity
1. Introduction
Copper, an earth-abundant and inexpensive material, has broad applications in manufacturing and catalysis due to its malleable and conductive properties [1]. Bulk copper is reddish and can be found in its pure form without chemical or physically controlled refining [2]. However, size-dependent color changes from brown to black show the different properties of copper at the nano-scales [3]. Bulk copper and its alloys are applied in material science, engineering, photo-electrical industries, and manufacturing. In contrast, nano-scaled copper is used in agriculture, biomedicine, environmental protection, photo-image, electronics, and superconductors [4]. Fabrication of copper nanoparticles (CuNPs) is not an emerging technology. Medieval red window glasses detected by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) revealed the existence of CuNPs, which might be formed in a series process of heat and redox reactions [5]. In recent decades, various methods have been utilized for the production of copper and copper-related nanoparticles, such as copper nanoclusters and copper oxide nanoparticles. These methods include microemulsion, sol-gel techniques, green synthesis, physical smashing, chemical reduction precipitation, electro−/sono-chemical approaches, and microwave irradiation [6, 7, 8]. These copper and copper-related nanoparticles are categorized as metallic nanoparticles, containing high catalytic activities and easily interacting with organic materials like lipids and proteins [6, 7, 8]. Therefore, CuNPs are considered as potent antimicrobial agents against hazardous pathogens [9].
The increasing prevalence of new and untreatable infections has aroused concerns in recent years. Traditional antibiotics struggle with problems of invalid treatments for multidrug-resistant pathogens, and developing new strategies for these pathogen extirpations becomes an important issue [10, 11]. Gram-negative bacteria, such as
Recently, the mechanisms of bactericide in copper-based nanoparticles have been intensely discussed. Take most metallic nanoparticles, for example. Free ions released from metal, which lead to the production of reactive oxygen species (ROS), are the primary factor for killing bacteria [18]. Copper-based nanoparticles, a type of metallic nanoparticles encompassing CuO/Cu2O, CuS, Cu0, and other core/shell nanoparticles formed from various materials, are known to generate ROS [9, 16]. This is particularly reasonable for them as they exhibit +1 and + 2 oxidation states which enable them to interact with cell membranes through electrostatic attraction [16, 19]. In contrast, CuNPs exist in Cu0 reduction states without charges, which should theoretically make it difficult to generate ROS. Nevertheless, Lia et al. demonstrated that the antimicrobial properties of CuNPs (Cu0) were from ROS [15]. Additionally, Chatterjee et al. indicated that CuNPs released nascent ions via redox reactions when CuNPs were exposed to aqueous conditions, such as cell medium and serum [20]. CuNPs may employ “dry” or “wet” methods for accumulation and dissociation to reduce the proton motive force across cell membranes and increase permeability [9]. Once CuNPs entered cells through damaged cell membranes, ROS-induced oxidative stress led to lipid peroxidation, protein denaturation, and DNA degradation [9, 15, 20]. These series of attacks increased the efficacy of antimicrobial abilities in CuNPs.
Our previous study on CuNPs corroborates these findings regarding ROS-induced results [15]. We specifically investigated CuNP toxicities in
2. Materials and methods
2.1 Nanoparticle preparation
Two sizes of CuNPs, 25 nm (marked as 20 nm) and 60–80 nm (marked as 60 nm), were purchased from Sigma-Aldrich Company (Sigma-Aldrich, St. Louis, MO, USA). As a dispersant, 1.0 mM of sodium dodecyl sulfate (SDS) (Sigma-Aldrich) was applied. The aggregation of CuNPs was prevented by capping with SDS and ultrasonic bath at 40°C for 30 min. The aqueous CuNPs were freshly prepared and used immediately. To ensure the sizes and the physicochemical properties of CuNPs, different equipment, such as transmission electron microscope (TEM; H-7500; Hitachi, Tokyo, Japan), liquid particle attractor (FlowVIEW, Hsinchu, Taiwan), Flow AOI (FlowVIEW), and Zetasizer Nano ZS (MalvernInstruments, Worcestershire, UK), were used in this study [15].
2.2 Bacterial cell culture
2.3 Viability determination
To understand the antimicrobial activities of 20 and 60 nm CuNPs, three bacteria were treated with PBS (the negative control), 0 (mock), 1, 5, 10, 50, and 100 μg/mL of 20 or 60 nm CuNPs, respectively, at 37°C for 24 h. The difference between the group of negative control and mock is that mock contains dispersant while negative control does not. Bacteria were treated with 70% alcohol for 24 h as the positive control. For viability measurement, PrestoBlue® Cell Viability Reagent (Invitrogen, Carlsbad, CA, USA) was added to each treated bacterium. After 2 h of incubation at 37°C and 200 rpm, the fluorescence at 590 nm was measured by a Varioskan LUX multimode microplate reader (ThermoFisher Scientific, MA, USA). The value of fluorescence represented the survival of cells.
To detect the minimum bactericidal concentration (MBC), three bacteria were treated with either 20 nm or 60 nm of CuNPs at the concentrations of 0, 1, 5, 10, 50, and 100 μg/mL for 24 h. After overnight treatment, each group of bacteria was transferred to the LB agar plates and incubated for another overnight. The standard colony counting method was used for colony number calculation, and MBC was determined by the colony number.
2.4 Cell death mechanism measurement
To comprehend the bactericidal mechanism of 20 and 60 nm CuNPs, we chose four modulators of programmed cell death to apply in CuNP-treated bacteria. Z-VAD-FMK (marked as Z-VAD; Sigma-Aldrich, Saint Louis, MO, USA) is an apoptosis inhibitor that binds to caspase-family proteins [21]. SBI-0206965 (marked as SBI; BioVision, Milpitas, CA, USA) and wortmannin (marked as Wort; Abcam, MA, USA) are autophagy inhibitors, which work on serine/threonine kinase ULK1 and the class III phosphatidylinositol 3-kinase (PI3K), respectively [22, 23]. Necrosulfonamide (NSA; Sigma-Aldrich, Saint Louis, MO, USA) blocking mixed lineage kinase domain-like pseudokinase (MLKL) serves as the necroptosis suppressor [24]. Before the CuNP treatments, bacteria were pretreated with 100 nM Z-VAD for 30 min, 5 μM SBI for 2 h, 100 nM Wort for 30 min, or 0.5 μM NSA for 1 h. The pretreated bacteria were then incubated with 20 nm or 60 nm of CuNPs at 0, 1, 5, 10, 50, and 100 μg/mL for 24 h, containing (or not) the same modulator at the same concentration in the pre-treatment stage. The cell viabilities of bacteria rescued by modulators were detected with PrestoBlue® Cell Viability Reagent. The protocol of the PrestoBlue assay was the same as previously described.
2.5 Statistical analysis
Data are expressed as mean ± standard deviation (SD). Mean values and SDs were calculated from at least three independent experiments carried out in triplicates for each treatment group. Statistical comparisons were performed by one-way ANOVA and the Student’s t-test, using statistical significance at
![](/media/chapter/a043Y000010Jz6iQAC/a093Y00001h6BHrQAM/media/F1.png)
Figure 1.
The process steps in this study.
3. Results
3.1 Bactericidal activities in CuNPs
In our previous study, we disclosed the effects of different sizes and concentrations of CuNPs in
![](/media/chapter/a043Y000010Jz6iQAC/a093Y00001h6BHrQAM/media/F2.png)
Figure 2.
The bactericidal abilities of CuNPs in three pathogens.
To determine the minimum bactericidal concentration (MBC) in CuNP treatments, three bacteria were incubated with either 20 or 60 nm CuNPs, and a standard colony counting assay was performed (Figure 2C–E). In
MBC (μg/mL) | ||
---|---|---|
20 nm CuNPs | 60 nm CuNPs | |
10 | 50 | |
50 | 100 | |
10 | 10 |
Table 1.
3.2 Bactericidal mechanism studies in CuNPs
To understand the bactericidal mechanisms triggered by CuNPs, four modulators, SBI, Z-VAD, NSA, and Wort, were chosen for subsequent experiments. These modulators are frequently employed in research on mammalian cell death mechanisms. SBI binds to Unc-51-like kinase (ULK) and interrupts the initiation of autophagy [22], while Z-VAD binds to caspase-like family proteins and inhibits apoptotic programmed cell death [21]. NSA interacts with MLKL to prevent necroptic cell death [24], and Wort increases survival rates by depleting autophagosome formation [23]. In our experiments,
![](/media/chapter/a043Y000010Jz6iQAC/a093Y00001h6BHrQAM/media/F3.png)
Figure 3.
Effects of CuNPs and programmed cell death modulators on bacterial cell viability.
In
In contrast,
Interestingly, the mechanisms of bactericidal effects from the dispersant of nanoparticles, SDS, were distinct in
3.3 NCBI protein database alignment of programmed cell death modulators
To verify the effects of modulators on programmed cell death in
![](/media/chapter/a043Y000010Jz6iQAC/a093Y00001h6BHrQAM/media/F4.png)
Figure 4.
Sequence alignment of the possible Z-VAD- and SBI-responsive domains in
SBI is a potent inhibitor of the kinase ULK1 [25]. Through our analysis, many proteins from
Despite searching for the bacterial homologs of the NSA activation domain, 4HB domain of MLKL (NP_689862.1), and PI3K-like catalytic domains for Wort in the NCBI protein database using BLAST, no hits were found in
4. Discussion
In this study, we demonstrated that both 20 and 60 nm CuNPs were effective bactericides in
Metallic and metallic oxide nanoparticles have gathered interest in recent years due to their biomedical applications. They are capable of generating ROS and attacking pathogens. Our previous study confirmed this phenomenon [15], demonstrating that both 20 and 60 nm CuNPs destroyed the cell membranes of
Programmed cell death triggered by different stressors in
There are many similarities between prokaryotes and eukaryotes, yet certain properties have long been accepted as essential eukaryotic characteristics that distinguish them from prokaryotes. For instance, endocytosis was traditionally considered a primary survival behavior in eukaryotes. However, discoveries such as cyanobacteria’s ability to engage in energy-dependent endocytosis challenged this notion [41]. Subsequent research revealed that diverse gram-positive bacteria like
5. Conclusion
Metallic and metallic oxide nanoparticles, including CuNPs, have been shown to reduce viabilities and colony formation abilities in
Acknowledgments
We are grateful for the support from the Core Facility Center, Tzu Chi University, Taiwan. This work was supported by the Grant No. MOST 106-2813-C-320-005-B (to L.-I. T. and B. R. L.) from the National Science and Technology Council, Taiwan.
Appendices and nomenclature
silver nanoparticles | |
apoptotic-like death | |
copper-transporting adenosine triphosphatases | |
copper nanoparticles | |
copper oxide nanoparticles | |
alcohol | |
U.S. Food and Drug Administration | |
Luria–Bertani | |
minimum bactericidal concentration | |
mixed lineage kinase domain-like pseudokinase | |
necrosulfonamide | |
optical densities | |
phosphatidylinositol 3-kinase | |
reactive oxygen species | |
SBI-0206965 | |
standard deviation | |
sodium dodecyl sulfate | |
scanning electron microscopy | |
transmission electron microscopy | |
Unc-51-like kinase | |
wortmannin | |
zinc oxide nanoparticles | |
Z-VAD-FMK |
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