Nanostructured γ-Al₂O₃ Synthesis Using an Arc Discharge Method and its Application as an Antibacterial Agent against XDR Bacteria

Abstract

In the last few years, many efforts have been devoted to investigating the antibacterial activity of metal nanoparticles, especially against multidrug-resistant bacteria. Recently extensively drug-resistant (XDR) bacteria have emerged and caused a global threat. The purpose of this manuscript was to synthesize nanostructured γ-Al₂O₃ as an antibacterial agent against some XDRs. The results showed that Al₂O₃ was a mix of rod and spherical shapes in the nano range with diameters of less than 30 nm. The zeta potential was determined to estimate the surface charge for the synthesized γ-Al₂O₃, which was recorded as −34 ± 1.8 mV, indicating good stability. The synthesized nanostructured γ-Al₂O₃ showed a potent antibacterial activity against extensively drug-resistant Acinetobacter baumanii, with an inhibition zone diameter that reached 19 mm and a minimum inhibitory concentration (MIC) value that reached 2 µg/mL. The observed antibacterial activity of the prepared Al₂O₃ nanoparticles confirmed that the main mechanistic actions include bacterial cells apoptosis, ROS increment, cellular membrane disruption, and DNA damage. The cytotoxic effect (CC50) of the prepared γ-Al₂O₃-NPs was 1250 µg/mL in a normal human lung fibroblast cell line (WI-38 cells). It can be concluded that the synthesized γ-Al₂O₃ had an acceptable toxicity, which may pave the way for its use as a potent agent in the fight against XDR bacteria.

1. Introduction

It is critical to investigate the uses of produced nanoparticles in tackling certain severe problems such as the emerging need to synthesize antibacterial substances against some critical illnesses [1]. To address these issues, there is a quest for a new generation of materials that can effectively combat bacterial infections with very low cell toxicity [2]. In the last era, arc discharge machines were used as an ecofriendly physical method to prepare nanometals without using toxic chemicals [3,4]. The arc discharge method was first utilized by Iijima in 1991 to fabricate carbon nanotubes [5]. Methods for the preparation of nanomaterials have been expanded over the decades, showing that the shapes, sizes and phase structures are strongly dependent on the arc discharge parameters, e.g., temperature, current, voltage, type of power supply, pressure, dielectric media, electrode shape, and the gap between the metallic electrodes [6]. Aluminum oxide nanoparticles (AlOxNPs) were obtained in three main forms: spherical, rod-like, and flake-like. Nanomaterials with various architectures, particularly the construction, size, and anisotropy, offer significant promise for bacterial inhibition. However, recent studies have reported the limitation of superhydrophobic surfaces in controlling bacterial infections, such as alumina [7,8]. Researchers focused on nanoparticle research and applications because of the unusual characteristics of Al₂O₃ [9]. However, a strong electric field is required to break the potential barrier (dielectric medium) that separates the electrodes for the arc discharge method to be successful. A strong electric field causes electron emission, producing ions; this is known as the Schottky effect [10]. The most important benefit of applying metal oxide nanoparticles in the biomedical industry (as antimicrobial agents) is their observed effectiveness, even against superbugs (antibiotic-resistant strains). Delaportas et al. [11] and Li et al. [12] successfully synthesized γ-Al₂O₃ using DC electric arc discharge. In the past 10 years, Al₂O₃-NPs have been employed successfully as an antibacterial agent [13]. Even at low dosages, Al₂O₃-NPs have effective antibacterial actions against numerous harmful microorganisms and effectively produce full growth suppression of microorganisms. Al₂O₃-NPs have been identified as potent inhibitors of antibiotic-resistant bacteria [14].

The public health community refers to multidrug-resistant organisms (MDROs) as superbugs with relatively few available treatments. Only one or two antibiotics (with hazardous side effects) may be successful against some MDROs. Boucher et al. [15] described ESKAPE organisms as “Bad Bugs” in 2009, where the E stands for Enterococcus faecium, S stands for Staphylococcus aureus, K stands for Klebsiella pneumoniae, A stands for Acinetobacter baumannii, P stands for Pseudomonas aeruginosa, and E stands for Enterobacter species [15]. Infectious illness rates have risen so dramatically over the past two decades that in many areas of the world public health is already approaching pre-antibiotic levels [16]. The terms “multidrug-resistant” (MDR), “extensively drug-resistant” (XDR), and “pan drug-resistant” (PDR) bacteria have been defined in accordance with standardized international terminology developed by the European Centre for Disease Control (ECDC) and the Centers for Disease Control and Prevention (CDC) [17]. Extensively drug-resistant (XDR) was defined as the inability of a bacterial isolate to be sensitive to more than one agent in any but two or fewer antimicrobial groups [18].

To the best of our knowledge, very limited information is available about the antibacterial activity of Al₂O₃-NPs against XDR bacteria. Hence, in the present investigation, we aimed to synthesize Al₂O₃ nanoparticles (NPs) through an arc discharge method and conduct in vitro investigations of their antimicrobial activity against extensively drug-resistant strains of Acinetobacter baumannii.

2. Materials and Methods

2.1. Nanoparticle Synthesis

High-purity γ-Al₂O₃ was synthesized using the arc discharge method by using a pure cathodic aluminum electrode with a diameter of 16 mm and a length of 10 cm with an aluminum anode with a diameter of 2 mm and a length of 3 cm. The alternating current was fixed at 15 A, a voltage of 70 was used, the vessel capacity was 3 liters, the rotational speed was 950 rpm, and the experiment was carried out under atmospheric pressure. The pH was adjusted using HCl drops to reach 5, and the electrode gap was about 0.5 mm in deionized water to synthesize the γ-Al₂O₃, as designed [19].

2.2. Filtration and Characterization

Filtration was performed using a refrigerated ultracentrifuge (Hettich MIKRO, Germany), and the nanoparticles separation carried out according to their mass, where the precipitated part contained the solid particles. The relative centrifugal force (RCF) was then calculated using Equation (1) [20]:

$$\mathrm{RCF}=1.118\times {10}^{-5}\times \mathrm{r}\times {\mathrm{N}}^2$$

(1)

where RCF is the relative centrifugal force (cm/s²), r is the rotational radius (cm), and N is the rotating speed (revolutions per minute, rpm). Then, each sample was characterized by a JEOL JEM-2100 HR-TEM (high-resolution transmission electron microscope), XRD (X-ray diffraction), EDX (energy-sispersive X-ray), FTIR (infrared spectroscopy), and a Zetasizer (nano 90 Malvern Panalytical, Malvern, UK).

2.3. In Vitro Studies

2.3.1. Antibacterial Activity

Disc Diffusion Method

The antibacterial activities of the γ-Al₂O₃ nanoparticles were evaluated against different extensively drug-resistant (XDR) bacterial strains of Acinetobacter baumannii, which were identified and provided from the microbiology lab at the Main University Hospital, Alexandria, Egypt. First, 100 μL of freshly prepared 0.5 McFarland bacterial suspensions were swabbed over the surface of Müeller–Hinton agar (MHA) plates. The disc diffusion method was primarily used to assess the antibacterial activity of the prepared γ-Al₂O₃ nanoparticles (20 mg) [21,22].

Minimum Inhibitory Concentration (MIC)

The MIC test was conducted using a serial dilution technique with the prepared γ-Al₂O₃ nanoparticles. First, 20 μL of tween 80, 80 μL of sterile Müeller–Hinton broth (MHB), and 100 μL of γ-Al₂O₃ nanoparticles were mixed and serially diluted in a 96-well microtiter plate. Then, 100 μl of the freshly prepared 0.5 McFarland bacterial suspensions was inoculated in each well. The MIC is the lowest concentration of the tested nanoparticles to inhibit bacterial growth [21,22].

Bacterial Lethality Curve

The optimal time required for γ-Al₂O₃ nanoparticles to inhibit bacterial growth was assessed through bacterial lethality curve plotting. The determined MIC was incubated with 1 mL of 0.5 McFarland bacterial suspension. Then, at different incubation times (0, 2, 4, 6, 8, 12, and 24 h), aliquots from each tube were taken to assess the bacterial growth using the OD at 600 nm [22].

2.3.2. Al₂O₃ Nanoparticle Mechanistic Action

Transmission Electron Microscope (TEM) Study

A transmission electron microscopic (TEM) examination (JEM-100 CX, JOEL, Shanghai, China) was used to assess the ultrastructures of the bacterial cells after treatment with the MIC of the prepared nanoparticles. TEM has a resolution of 3 nm at 30 kV. Ultrathin sections were prepared on grids then stained with 3% uranyl acetate [23].

Reactive Oxygen Species (ROS) and Double-Strand Break Measurements

Reactive oxygen species (ROS) generation was measured using 2,7-dichlorofluorescin diacetate (DCFH-DA) dye. Using this technique, the extracellular ROS were compared between the control and nanoparticle-treated bacterial cells according to Bhuvaneshwari et al. [24]. Moreover, a DNA strand break assay was performed to quantify the genotoxicity of the prepared nanoparticles against the tested bacteria, following the method described by Bhuvaneshwari et al. [24].

2.3.3. Evaluation of the Cytotoxic Effects of the Prepared γ-Al₂O₃

One of the most vulnerable body parts to Acinetobacter baumannii infection is the lung. Hence, a human lung fibroblast (WI-38 cells) normal cell line (ATCC^® number: CCL-75™) was chosen to assess the cytotoxicity of the prepared γ-Al₂O₃. The cytotoxic effects were evaluated using an MTT essay, which is a quantitative method to measure the cells’ viability after incubation with the tested nanoparticles. Eight serially diluted concentrations of the tested nanoparticles were incubated with a precultured cell line (5  ×  10⁴ cell/well) for 24 h. The relation between the living cells and the nanoparticle concentration was plotted using Origin Pro 6.8 software (OriginLab Co., Northampton, MA, USA) to calculate the survival curve and the cytotoxic concentration (CC50) of the WI-38 cells [25,26] according to Equation (2):

$$\mathrm{Cell}\mathrm{Viability}\%=\frac{\mathrm{Treated}\mathrm{cells}\mathrm{OD}}{\mathrm{Untreated}\mathrm{cells}\mathrm{OD}}\times 100$$

(2)

3. Results and Discussion

3.1. Preparation and Characterization of the Nanoyields

After the cathodic plasma process, deionized water was ionized in reactive oxygen, hydrogen, and hydroxide species, which interacted with Al ion clusters to produce γ-Al₂O₃. The prepared nanosample was examined using an HR-TEM study. The results showed that the Al₂O₃ was a mix of rod and spherical shapes in the nano range with diameters of less than 30 nm (Figure 1a). It was important to investigate the corresponding nanoparticle yield [27]. The powder XRD pattern of the sample was used to obtain information about the surface plane (Figure 1b). The pattern assigned to the intensities of the peaks revealed the presence of nano-γ-aluminum oxide, as JCPDS No. 29–0063, with prominent peaks at 2θ values of 37.1°, 45.76°, and 66.79°, corresponding to primitive cubic class values (h, k, and l) of 311, 400, and 440, respectively [28]. The XRD data and HR-TEM results confirmed that the samples were pure and without impurities. Γ-Al₂O₃ were crystalline materials. The EDX analysis proved the purity (100%) of the prepared nanoparticles of γ-Al₂O₃ (Figure 1e). The average crystallite size of the obtained pure Al₂O₃ was calculated to be 3.45 nm using Debye Scherer’s method (

Table 1. Size distribution calculations.

(2Ө) Degree	Intensity (Counts)	(h, k, and l) MillerIndices	FWHM Degree	Grain Size (nm)
35.91	78.21	(311)	8.73	1.00
45.73	102.34	(400)	1.93	4.67
66.79	111.69	(440)	2.13	4.67

).

The crystallinity index was evaluated through the equation expressed as:

$${\mathrm{I}}_{\mathrm{cry}}=\frac{D_{p\left(TEM\right)}}{D_{cry\left(XRD\right)}}$$

(3)

where I_cry is the crystallinity index; D_p is the particle size (obtained from the HRTEM morphological analysis); and D_cry is the particle size (calculated from the Scherrer equation). The crystallinity index displayed a score higher than 1.0, which indicated that the prepared yield was polycrystalline.

The FTIR spectrum of γ-alumina showed a broad band at 3465 cm⁻¹ attributed to the -OH stretching vibrations related to the lattice of water molecules; this may indicate the presence of moisture in the powder. In addition, the available bands at 1637, 1153, and 1072 cm⁻¹ were the consequence of symmetrical bending vibrations of the Al–O–H group. There was a weak band due to Al-O bond vibration at 1332 cm⁻¹ and symmetric bending stretching vibration of Al-O-Al at 701 cm⁻¹ (Figure 1d). The same observations were reported by Wasan et al. [29]. The double regular peaks observed between 2979 cm⁻¹ and 2886 cm⁻¹ indicated the small drops of HCl added to the sample during the preparation process [30].

When electrodes were immersed in dielectric media, an applied strong electric field was sufficient to break down the barrier gap between the aluminum electrodes. As a result, a huge number of charged electrons were eliminated from the electrode-created aluminum ions. The vaporized aluminum ions interfaced with the liquid to condense it as a bottom-up method into its stabilized nanosized form. The zeta potential was determined to estimate the surface charge for γ-Al₂O₃ (−34 ± 1.8 mV), which indicates the good stability of the produced nanoparticles [28]. As shown in Figure 1c, the full width at half-maxima of γ-Al₂O₃ fluctuated nearly to its peak position, which indicated the non-homogenous size distribution of the produced yield. One can conclude that strong applied fields must be provided with enough energy to remove the electron from an electrode and separate aluminum ions in deionized water to properly create the arc plasma between the cathode/anode gap. The difference between the energy required and the work function for the electrons’ removal may be transferred as heat energy in the solution or may play a significant role in changing the produced ions’ crystal order (changing the resultant shapes) [28].

3.2. In Vitro Studies

3.2.1. Antibacterial Activity

The results presented in

Table 2. Antibacterial activity of aluminum oxide nanoparticles.

Tested Strains	γ-Al2O3 NPs
	IZ (mm)	MIC (mg/mL)
A. baumannii 1	15	8
A. baumannii 2	10	16
A. baumannii 3	19	2
A. baumannii 4	15	8
A. baumannii 5	6	64
A. baumannii 6	13	16
A. baumannii 7	6	64
A. baumannii 8	10	16
A. baumannii 9	7	64
A. baumannii 10	17	8

IZ: inhibition zone diameter, MIC: minimum inhibitory concentration.

proved the promising antibacterial activity of the prepared nanoparticles. The most susceptible strain was A. baumannii strain 3, with an inhibition zone diameter that reached 19 mm and an MIC value that reached 2 mg/mL. A. baumannii strain 3 was chosen for further investigations. The data in Figure 2 showed a significant reduction in the bacterial growth after a 12 h incubation with aluminum oxide nanoparticles. This result provided an additional advantage for aluminum oxide nanoparticles to completely eradicate the bacterial growth in a short time. The antibacterial activity of Al₂O₃ nanoparticles was previously tested against several pathogens, namely Staphylococcus aureus, Streptococcus mutans, Escherichia coli, and Proteus vulgaris. The results presented the promising antibacterial activity of the prepared nanoparticles due to the nanoparticles’ large surface area [31]. Jwad et al. [32] tested the antibacterial activity of aluminum oxide nanoparticles (10-60 nm diameter size), and it was represented by inhibition zone diameters that reached 25.55 mm against S. aureus and 20.56 and 19.33 mm against E. coli and P. aeruginosa, respectively.

3.2.2. γ-Al₂O₃ Nanoparticle Mechanistic Action

Several techniques were used to determine the γ-Al₂O₃ nanoparticle mechanism of action against the tested bacteria. A TEM study of the bacteria-treated cells revealed the complete distortion of the bacterial cells and leakage of the intracellular components (Figure 3a). Furthermore, oxidative stress was investigated. The bacterial cell response to interactions with γ-Al₂O₃ nanoparticles usually resulted in a reactive oxygen species (ROS) increment. It was observed that by increasing the nanoparticle concentration from 0.25 to 1.25 mg/mL the ROS content increased by almost 32% (Figure 3b). The Al³⁺ ions released by Al₂O₃ NPs produced ROS, which in turn caused membrane damage and a cell viability reduction. Internalization and γ-Al₂O₃ nanoparticle attachment can potentially harm cell organelles, such as the nucleus (genomic DNA and plasmids) [33].

The genotoxic effect of the prepared nanoparticles was investigated by assessing the rate at which a double-stranded DNA (dsDNA) transited to single-stranded DNA (ssDNA), which was directly proportional to the number of breaks in the phosphodiester backbone. A. baumannii cells treated with Al₂O₃ nanoparticles (0.25, 0.5, and 1 mg/L) exhibited significant (p < 0.05) DNA damage, with F values of 19.3 ± 0.4, 52.7 ± 0.2, and 75.1 ± 0.6%, respectively. Bhuvaneshwari et al. [24] compared the genotoxic effect of bulk and nano-Al₂O₃ and reported that Al₂O₃ nanoparticles showed more potent effects on Gram-negative bacteria than Gram-positive bacteria. DNA damage can cause cell apoptosis or necrosis by triggering signal transduction pathways, leading to cell death [24].

3.3. Cytotoxic Effects of the Prepared Nanoparticles

In a trial to study the in vitro cytotoxic effects of the prepared γ-Al₂O₃-NPs, the cell proliferation using a normal human lung fibroblast cell line (WI-38 cells) was tested. A lung fibroblast cell line was chosen due to the possible biomedical application of the prepared γ-Al₂O₃-NPs as a potent treatment against Acinetobacter baumannii infection. It was found that the CC50 of γ-Al₂O₃-NPs was 1250 µg/mL (Figure 4).

4. Conclusions

γ-Al₂O₃-NPs were successfully synthesized on a nano scale using an arc discharge method. Clearly, the structure phase of γ-Al₂O₃ and its size influenced the bacterial inhibition activity. In summary, γ-Al₂O₃ had a diameter less than 30 nm and a zeta potential of −34 ± 1.8 mV. In vitro studies were applied to assess the antibacterial activity of the prepared nanoparticles, focusing on their mechanistic action. γ-Al₂O₃ nanoparticles inhibited bacterial growth by disrupting the bacterial cell membrane and inducing ROS. The CC50 of the prepared γ-Al₂O₃-NPs was 1250 µg/mL in a normal human lung fibroblast cell line (WI-38 cells). According to the high antibacterial activity, the prepared nanoparticles can be considered as potent saviors in the combat against extensive resistant nosocomial strains.