INTRODUCTION:

Scientists are interested in nanoparticles (NPs) because they serve as a bridge between bulk materials and molecular or atomic structures¹. NPs vary in diameter from 0.1nm to 100nm, and they can have a number of excellent features, including near-identical strength, active surfaces and distinct laser pulse energies^2,3. Biodegradable self-assembled NPs for the targeted delivery of anticancer medicines and imaging contrast ants are being developed as a result of the action of nano-scale electronics^4,5. Nano-constructs can be used as adaptive, targeted medicine delivery vehicles that are capable of carrying large amounts of carcinogenic agents or restorative properties to threatening cells whilst avoiding interference with solid cells^6,7.

Aluminium (Al) NPs are simple to handle and readily available. Furthermore, these low-cost NPs offer a large surface area and high mechanical strength, along with outstanding chemical stability under high temperature and severe conditions, such as abrasive settings⁸. Electrical conductivity is also low in these materials^9,10. Many biological and pharmaceutical products utilise NPs in their development and quality improvement^11,12,13. The use of metal oxide NPs as antimicrobial agents has several advantages, including increased efficacy against resistant microbial infections, reduced toxicity and heat resistance^14,15. Cancer has surpassed heart diseases as one of the top causes of death worldwide¹⁶. The employed cell type, NP geometry and NP concentration influence the effect of NPs on cancer cells^17,18,19. Several investigations on the effects of nanomaterials on bacteria, the effects of Al₂O₃NPs on pathogenic bacteria and the mechanism of Al₂O₃ NPs’ exact effect on bacteria have been conducted^20,21.

Accordingly, the objective of the current study was to generate Al₂O₃ NPs at various laser pulse energies by using laser ablation liquid technology and to examine their antibacterial, antioxidant and cytotoxic effects.

MATERIALS AND METHODS:

Manufacture of Al₂O₃ NPs:

The laser ablation of an Al metal pellet in deionised water (DIW) was performed to produce Al₂O₃ NPs. As shown in Figure 1. the Al target was placed in a glass vessel. The water level beyond the target was roughly 3 mm, and the vessel contained 1mL of DIW. The NPs were created by a pulsed Nd: YAG laser with the following parameters: ƛ = 1064 nm, F = 1 Hz, and pulse width = 9 ns, at various laser pulse energies (500, 800 and 1000 mJ/pulse). Ablation time was 30 min.

Figure 1: Laser ablation of Al₂O₃ NPs

Characterization:

UV/VIS (Shimadzu UV-1800, Japan)²². XRD analysis (Shimadzu XRD 6000) investigated particle structure²³. Transmission electron microscopy (TEM) (ZEISS LEO 912 AB-100 KV/Germany)²⁴. Fourier transform infrared (FTIR) spectroscopy was performed using an 8000 Series Shimadzu with wave number 500–4000cm⁻¹²⁵. All tests were conducted at the University of Tehran.

Bacterial preparation:

Bacillus cereus and Pseudomonas aeruginosa were obtained from the Department of Biotechnology/University of Technology. Isolates were cultured overnight at (18–24) h. and (37°C) on nutrient broth to prepare cell suspensions that were attuned to (0.5) McFarland standards (5×10⁵ )colony-forming unit.

Antibacterial susceptibility assays:

On a Mueller–Hinton agar plate, the antibacterial activity of Al₂O₃ NPs was tested by the well diffusion method against two species of Gram -ve P. aeruginosa and Gram +ve B. cereus. Al₂O₃ NPs were prepared via laser ablation at different energy level (500, 800 and 1000 mJ). Then, a hole with a diameter of (5) mm was filled with 500, 800 and 1000 mJ of Al₂O₃ NPs. A blank well was created by adding solvent alone (i.e., DIW) to act as negative control. After an incubation period of less than 37°C for (24) h, the growth inhibitory zones were measured. All the tests were performed in triplicate, and the given results were the average of three experiments²⁶. The synergistic/antibacterial activity of Al₂O₃ NPs was investigated when the NPs were coupled with gentamicin and imipenem (antibiotics)²⁷.

Amikacin and imipenem (antibiotics) minimum inhibitory concentration (MIC) analysis:

Imipenem and amikacin were determined via microdilution assay²⁸.

Evaluation of bacterial cell viability:

The vitality of bacterial cells was determined using an acridine orange/ethidium bromide (AO/EB) staining method for B. cereus and P. aeruginosa after treating them with the prepared Al₂O₃ NPs. The cell viability was performed in accordance with the manufacturer’s protocol. The antibacterial efficacy of Al₂O₃ NPs against the examined bacterial strains was measured using a fluorescent microscope. Firstly, (50) μl of the treated samples (bacterial suspension) and untreated one was combined with (50) μl of AO/EB (prepared from 10 µg/ml of AO/EB stock solution) and then left for (2) min. After staining, a thin layer of the mixture was transferred onto a glass slide and examined under an immunofluorescent microscope (ZeissAxiovert S100 microscope), EB-stained dead cells fluoresced red, whilst AO-stained live cells fluoresced green²⁹.

Antioxidant action of Al₂O₃ NPs:

The antioxidant activity of Al₂O₃NPs was determined by 1,1-diphenyl-2-picryl-hydrazyl (DPPH)³⁰. Al₂O₃NPs were prepared at different laser energies (500, 800 and 1000 mJ). Then, 0.5ml of DPPH completed with 490ml of methanol solution was added to 10μl of Al₂O₃NPs of different final 1000ml and allowed to react at (25°C). After (30) min, the reduction of absorbance at (517)nm was calculated using Formula (1)³¹.

SA %= OD^A ─ OD^B/OD^A × 100 …………………… (1)

SA% = Scavenging activity %, OD = Optical density,

A = Control, B = Test.

Determination of anticancer activity of Al₂O₃ NPs via methyl thiazolyl tetrazolium (MTT) assay:

The anticancer effect of Al₂O₃NPs at different laser energies (500, 800 and 1000 mJ) was estimated on MCF-7 (breast cancer) cell line from the Iraq Biotech Cell Bank Unit. Cancer cells were converted in fetal bovine serum (10%) and 1% glutamine after being treated with NPs for (48) h at (37)°C with 5% carbon dioxide. The negative control was cancer cell without NP treatment. Then, (1%) dimethyl sulphoxide and MTT dye were added for (4) h at (5) μg/ml and maintained at (37)°C. After incubation, isopropanol was added^32,33, and then colour density was measured at (570) nm by using an enzyme-linked immunosorbent assay reader. Cytotoxicity was calculated using Formula 2³⁴:

Inhibition rate = A−B/A×100 ……………….……… (2)

Where A, and B are the OD of the control and the test, respectively.

Statistical analysis:

Graph Pad Prism 6³⁵ was used to conduct statistical analysis on the acquired data. The consequences were presented as the mean and standard deviation of three independent measurements³⁶.

RESULTS AND DISCUSSION:

Figure 2 shows the colors of Al₂O₃ NPs colloidal. The laser powers of 500, 800 and 1000 mJ determines the degree of colour of the colloidal solution. An increase in the number of laser leads to an increase in the production of NPs. The change in colour of Al₂O₃ NPs is due to the slow oxidation by oxygen dissolved in the liquid, with a high refractive index³⁷. The colour NPs are arranged depending on the size and shape of the NPs and the dielectric constant of the surrounding DIW³⁸.

Figure 2 shows that the UV/Vis spectroscopy within the wavelength range of 300–1000 nm the measurement of the absorption of Al₂O₃ NPs via the laser ablation method, when laser energy is increased, the size of NPs increased and the peaks of their absorption spectra shift towards shorter wavelengths, and vice versa³⁹. Figure 3 presents the XRD patterns of the Al target and the particles generated at laser energies of 500, 800 and 1000 mJ. The strength of XRD peaks increases as laser energy is increased. The average particle size of Al₂O₃NPs is (48, 26 and 21) nm, this phenomenon can lead to the conclusion that particle concentrations in DIW produced at laser energy of 1000 mJ are higher than those obtained at laser energies of 500 mJ and 800 mJ. These findings are consistent with those previously published in^40,41,42,43.

Fig.4. shows the average particle size of Al₂O₃ NPs as a size distribution. The particle sizes ranging from 20nm to 90nm at 500 mJ, 15nm to 60nm at 800 mJ and 16nm to 32nm at 1000 mJ. The laser raises the temperature of the target, causing the material to melt, and eventually, the ablated amount is ejected off the surface of the target. Furthermore, the micrographs reveal that small nanomaterials with narrow size distributions are generated at low energy, but the width of the NP size distribution clearly expands at high energy. Low energy encourages evaporation, resulting in the production of nanomaterials with a homogeneous size distribution from evaporated atoms. These findings are in line with those previously published in⁴⁴.

Figure 5 shows the FTIR spectra of Al₂O₃ NPs from the 500–4000 cm⁻¹ generated by laser of various (500, 800 and 1000 mJ) in DIW. The absorption peaks at 3356.14, 3417.26 and 3424.23 cm⁻¹ are associated with water (O–H) stretching vibration mode and water bending. The absorption band at around 1643.35, 1619.69 and 1630.52 cm⁻¹ is a well-known H-O-H angle bending vibration band of weakly bound molecular water. When bond bending in the FTIR spectra of the samples is stretched, a significant difference is only observed in terms of the peak shifting of 480.5, 582.50 and 620.71 cm⁻¹. These bands are blue shifted in the Al₂O₃ FTIR spectrum, possibly due to the size confinement effect. The Group bands (2926.98cm^-1) may be attributed to C-H stretching compound class alkane.

Figure 2: UV/Vis spectra of Al₂O₃ NPs at different laser energies;

Figure 3: XRD patterns of Al₂O₃NPs synthesised at different laser energies

Figure 4: TEM of Al₂O₃ NPs synthesised at different laser energies

Figure 5: FTIR spectrum of Al₂O₃ NPs at different laser energies

Antibacterial assay:

The antibacterial activity of Al₂O₃ NPs in vitro at different laser energies (500, 800 and 1000 mJ) revealed the evolution inhibitory effects of NPs against B. cereus and P. aeruginosa. As illustrated in Figures 6 and 7, Gram (+ve) bacteria are more susceptible to Al₂O₃ NPs than Gram (-ve) bacteria. Meanwhile, the antibiotic activity of Al₂O₃ NPs when mixed with gentamicin and imipenem was studied via the well diffusion method at a concentration of 32μg/ml (antibiotic).

The observed synergistic effect of Al₂O₃ NPs–imipenem against B. cereus is depicted in Fig.6, with a marked inhibition area of 16.42±0.03, 17.32±2.13 and 18.41±0.73 mm at different laser energies (500, 800 and 1000 mJ, respectively). Meanwhile, imipenem alone obtained an inhibition area of 12.01±0.12 mm. With regard to the synergistic effect of the Al₂O₃ NPs–gentamicin complex against P. aeruginosa, the area of inhibition (15.12±1.02, 15.21±1.01 and 16.11±0.32mm) was recorded at different laser energies (500, 800 and 1000 mJ, respectively). Meanwhile, gentamicin obtained inhibition areas of 12.20±0.12mm, as shown in Fig.7.

The cell walls of Gram-positive bacteria have a thin layer of peptidoglycan on top of teichoic acid and numerous pores that allow foreign particles to pass through them, causing cell membrane damage and even cell death. By contrast, Gram-ve bacteria have lipoproteins, lipopolysaccharides and phospholipids, which form a penetration barrier that only allows large molecules to pass through, explaining why B. cereus is more sensitive than P. aeruginosa. Furthermore, Gram+ve bacteria have a larger negative charge on their cell walls than Gram-negative bacteria, attracting NPs⁴⁵. For bacterial resistance to the outside world, cell walls and membranes are crucial defence barriers. The natural shape of a bacterium depends on its cell wall in particular. The adsorption pathways for NPs, Gram+ve bacteria and Gram-ve bacteria are generated by the cell membrane components⁴⁶. The combination of NPs with antibiotics is believed to be responsible for the observed synergistic activity against microorganisms⁴⁷.

Although Al₂O₃ NPs have been widely explored for their antimicrobial properties, however their mechanism of action remains unknown. This activity is attributed to a number of variables, the most notable of which is the small size and increased surface area of NPs, which allow for interactions with bacterial cells due to increased membrane permeability and bacterial cell death. Cell disintegration and alterations in cell membrane permeability can be caused by Al₂O₃ NPs. In addition, Al₂O₃NPs adhere onto the surface of the cell membrane, infiltrating bacteria and affecting cell function.

Figure 6: Antibacterial action of Al₂O₃NPs synthesised via laser ablation at different laser energies (500, 800 and 1000 mJ) against B. cereus. A+G = Al₂O₃ NPs + gentamicin. A = Al₂O₃NPs only, G = gentamicin only and C=Control (*, **P≤0.05, ***P≤0.01).

Figure 7: Antibacterial activity of Al₂O₃ NPs synthesised via laser ablation at different laser energies (500, 800 and 1000 mJ) against and P. aeruginosa. A+G = Al₂O₃NPs + gentamicin. A = Al₂O₃ NPs only, G = gentamicin only and C=control (*, **P≤0.05, ***P≤0.01).

The interactions of Al₂O₃ NPs with amino acids and enzymes include bonding with amino acids (particularly the –SH group) and the formation of reactive oxygen species (ROS). Cells are largely composed of thin bases, such as phosphorus and sulphur, and DNA contains these important components; NPs can interact with these thin bases and destruction DNA, causing cell decease^48,49. The decrease in antibacterial action may have been caused by the calcination of Al₂O₃ NPs, which increased particle size. Al₂O₃ NPs demonstrated an unusual decrease in antibacterial activity due to their higher specific surface area.

Prepared NPs induce death of bacterial strains:

Fluorescence microscopy was assessed using the antibacterial activity of the produced NPs on P. aeruginosa and B. cereus bacterial strains. The colour of live cells will be green, whilst the colour of dead cells will be red. For both types of bacterial strains, all untreated bacterial cells fluoresced green in Figure 8 (A and B), indicating that they are alive. In comparison with the untreated cells, the number of red cells increased when bacteria were treated with Al₂O₃NPs, as shown in Figure 8 (C and D). Individual treatment with Al₂O₃NPs caused DNA damage and kill bacterial cells. Consequently, Al₂O₃NPs exerted the greatest effect on both types of bacteria. NPs exerted greater effect on B. cereus than on P. aeruginosa due to differences in cell membrane structure.

Figure 8: Fluorescence microscopic images of (A) B. cereus and (B) P. aeruginosa untreated bacterial strains. (C and D) Bacterial strains treated with Al₂O₃ NPs (50×)

Figure 9: Antioxidant activity of Al₂O₃NPs synthesised via laser ablation at different laser energies (1000, 800 and 500 mJ), via DPPH assay

Antioxidant activity:

The DPPH radical scavenging (%) action of Al₂O₃NPs is shown in Figure 9. The antioxidant activity of Al₂O₃NPs at 500 mJ is 59.667±0.11%, that at 800 mJ is 70.56±0.17% and that at 1000 mJ is 86.65±0.12%. The result demonstrated that Al₂O₃ NPs can give more hydrogen atoms and remove more unstable electrons from DPPH at 1000 mJ than at 500 mJ. A substrate that can provide hydrogen atoms can be combined with DPPH. The hue transition from violet to yellow can result in an increasing reduced form⁴⁷.

The free radicals present are unstable, they induce cellular damage by creating ROS, which interacts with other molecules in biological reactions and causes cellular damage. The qualities of absorbing, neutralizing, or quenching singlet and triplet oxygen²² are critical variables that are responsible for antioxidant action. The presence of several bio-reductive groups on the surface of Al₂O₃ NPs is credited with the best antioxidant activity⁵⁰. Compared with conventional ascorbic acid, the radical scavenging property of Al₂O₃ NPs is relatively similar, as shown in Figure 10, with the highest percentage of inhibition observed at 1000 mJ. The hue of the solution changed from deep violet to pale yellow in the presence of Al₂O₃ NPs dissolved in DPPH during the experiment, indicating that the scavenging of free radicals was complete⁵⁰.

Anticancer activity of NPs:

The cytotoxic effect of Al₂O₃ NPs at different laser energies (1000, 800 and 500 mJ) against MCF-7 cancer cells was studied and calculated using Formula (2). The capacity of Al₂O₃NPs to inhibit the proliferation of cancer cell lines was used to investigate their anticancer activity. Al₂O₃ NPs exhibited a highly substantial cytotoxic action against MCF-7 cells in accordance with the findings. Figures 10, and 11 Illustrates the cytotoxic effect of Al₂O₃NPs on MCF-7 cells. Cytotoxicity at 500 mJ is 62.33±2.333%, and that at 800 mJ is 73.00±2.082%. Meanwhile, cytotoxicity at1000 mJ is more effective than that of individual Al₂O₃ NPs and more efficient for penetrating the membrane of the cell line, increasing the percentage of kills, i.e. 85.00±1.732%.

Figure 6 shows the morphological changes of the cells untreated by NPs (control). The anticancer activity of NPs at different laser energies is strongly dependent on the nature of NPs. The shape of Al₂O₃ NPs is spherical, indicating that the kill percentage for Al₂O₃ NPs at 1000 mJ is more effective than that at 500 mJ. These findings are consistent with those of earlier research that suggested that Al₂O₃ NPs have anticancer properties^39,41. Control cells have their natural morphological shape, and they adhere to the tissue culture, as shown in the photographs of untreated cells. After 72 h of treatment, cancer cells exhibit morphological abnormalities, lose interaction with surrounding cells and decrease in number, this is more efficient for penetrating the membrane of the cell line. Therefore, the percentage of kill increased, i.e. 85.00±1.732%, indicating that Al₂O₃ NPs play an important role in increasing the kill percentage.

Figure 10: Cell viability of Al₂O₃ NPs in MCF-7 cells. (A) 500; (B) 800; and (C) 1000 mJ;

Figure 11: (A) Control, i.e. no-treated MCF-7 cells and (B) MCF-7 cells after treatment with Al₂O₃ NPs

CONCLUSION:

We successfully proved that Al₂O₃nanoparticles may be produced from the Al disc using varied energies of laser ablation. It was discovered that the particle size of Al₂O₃ ablated at higher laser energy was lower than that achieved at lower laser energy. Al₂O₃ nanoparticles that were synthesized have a spherical form through XRD and TEM images. Antibacterial activities of Al₂O₃NPs were shown to be more effective against Gram-positive bacteria than Gram-negative bacteria. Al2O3 nanoparticles have cytotoxic effects on the breast cancer cell line MCF-7 as well as antioxidant capabilities.

ACKNOWLEDGEMENTS:

The authors wish to direct his appreciation to the chairman and members of the Department of Laser and Optoelectronics Engineering at the University of Technology in Iraq (www.uotechnology.edu.iq) for their support with this project.

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Received on 30.06.2022 Modified on 12.08.2022

Research J. Pharm. and Tech 2023; 16(3):1267-1273.

DOI: 10.52711/0974-360X.2023.00209