Novel trimetallic (TiO₂–MgO–Au) nanoparticles: Biosynthesis, characterization, antimicrobial, and anticancer activities

2.1 Materials

Banana peels that were collected were supplied from a local store in Giza, Egypt. Most of the chemicals and reagents needed for the experiment were obtained from Sigma Aldrich, including Ti(NO₃)₄·4H₂O (titanium(iv) nitrate tetrahydrate) as a titanium source, magnesium(ii) nitrate hexahydrate (Mg(NO₃)₂·6H₂O), HAuCl₄·3H₂O (hydrogen tetrachloroaurate(iii) hydrate), and sodium hydroxide as a precipitating agent. MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium-bromide), Taxol (paclitaxel), and DMSO (dimethyl sulfoxide) were purchased from Sigma Chemical Co. (Missouri, USA). Fetal bovine serum (FBS), phosphate buffer saline (PBS), trypsin-EDTA, and Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin (Pen/Strep) were procured from Gibco (Gibco, TFS Inc., USA).

2.2 Banana peel extract preparation

Fresh bananas were sourced from local merchants, and their peels were sliced into small pieces and washed three times with distilled water to remove dirt and impurities. The cleaned peel pieces were dried using paper towels and boiled for 20 min at 100°C in a beaker containing 250 mL of double-distilled water and 100 g of peels. The resulting mixture was filtered three times using Whatman No. 1 filter paper, yielding a pale yellow extract, which was then refrigerated at 4°C for further use [32].

2.3 Biosynthesis of trimetallic TiO₂–MgO–Au NPs using banana peel extract

Ten milliliters of 0.01 M solutions of Ti(NO₃)₄·4H₂O, Mg(NO₃)₂·6H₂O, and HAuCl₄·3H₂O were precisely combined and stirred at room temperature for approximately 1 h. After that, the extracted banana peel (30 mL) was added following Kamli et al.’s [33] procedure with a few adjustments. Subsequently, 20 mL of the banana peel extract was added, adjusting the pH to 9.0. The optimal synthesis of trimetallic TiO₂–MgO–Au nanoparticles was achieved by maintaining the reaction conditions at an incubation temperature of 35°C with continuous agitation (250 rpm) in a shaking incubator for approximately 24 h [34]. At the end of the incubation period, the solution changed to a pale brown color, signifying the successful formation of trimetallic TiO₂–MgO–Au nanoparticles. To get rid of peel biomolecules that were weakly attached, the produced trimetallic TiO₂–MgO–Au NPs needed to be washed five times using distilled water. Afterward, the nanoparticles were separated by centrifugation at 15,000 rpm for 5 min, collected, and dried in an oven at 200°C overnight [35].

2.4 Characterization of trimetallic TiO₂–MgO–Au NPs

The color shift of the banana peel extract from pale yellow to pale brown upon mixing with the metal precursors is the first clue that the synthesis of trimetallic TiO₂–MgO–Au NPs has occurred. Then, UV-visible spectroscopy (JENWAY 6305, Staffordshire, UK) was used to determine the absorbance of the faint brown hue at wavelengths between 200 and 800 nm. The synthesized solution (2 mL) was put in a quartz cuvette, and its absorbance was determined at regular intervals at various wavelengths to find the maximum absorbance [36]. Moreover, the chemical functional groups included in the produced trimetallic TiO₂–MgO–Au NPs were determined by FTIR analysis (Cary-660 model, KBr pellet technique, wavenumber range: 400–4,000 cm⁻¹). Moreover, the surface morphology of trimetallic TiO₂–MgO–Au NPs was evaluated using SEM (ZEISS, EVO-MA10, Germany). Using EDX (Bruker, Germany), the elemental composition, purity, and distribution of the constituents in the produced trimetallic TiO₂–MgO–Au NPs were examined. Additionally, we used transmission electron microscopy (TEM) (JEM-2100 Plus, Jeol, Japan) to determine the morphologies, average, and exact sizes of the produced trimetallic TiO₂–MgO–Au NPs. The average particle size distribution and zeta potential analysis of the trimetallic TiO₂–MgO–Au NPs was determined using dynamic light scattering (DLS) (Nano ZS, Malvern, UK) [37]. The crystal size and crystallinity were evaluated by using XRD-6000 (Shimadzu Scientific Instruments, Japan).

2.5 Antibacterial activity

Green synthesized trimetallic TiO₂–MgO–Au NPs were tested against five different microbial strains: B. subtilis (ATCC 6633), S. aureus (ATCC 6538), P. aeruginosa (ATCC 9027), E. coli (ATCC 25922), and Candida albicans (ATCC 10231). Each microbial strain was evenly spread on sterile Petri plates containing Muller–Hinton agar using the agar diffusion well method, following incubation of pure strains grown in Muller–Hinton broth. Using a clean cork borer, four 7 mm circular wells were created in the plates. To assess antimicrobial efficacy, 0.1 mL of TiO₂ salt, MgO salt, HAuCl₄, and TiO₂–MgO–Au NPs were added to each well. The Petri dishes were then incubated at 37°C overnight, and the inhibition zones were measured [38,39].

2.6 Determination of MIC

Using the broth-based microdilution technique, the minimum inhibitory concentrations (MIC) of TiO₂–MgO–Au NPs were found for B. subtilis (ATCC 6633), S. aureus (ATCC 6538), P. aeruginosa (ATCC 9027), E. coli (ATCC 25922), and C. albicans (ATCC 10231). TiO₂–MgO–Au NPs were already synthesized in various amounts (from 1,000 to 15.62 μg·mL⁻¹). Different amounts of TiO₂–MgO–Au NPs were introduced to sterilized microtiter plate wells after 100 μL of double-strength Mueller–Hinton broth. Microbial cell suspension (20 μL) matching (OD of 0.5 McFarland standard) was added to all wells except the negative control well. Positive control wells were populated with microbial solutions to determine if MH broth would sustain microbial growth. These plates were incubated for 24 h at 37°C. The wells were then filled with 30 µL of HiMedia’s resazurin solution (0.02% wt/v), and then the plate was incubated for a further 6 h to check for microbial growth. In cases when the strains had been propagated properly, the color of the developing control wells changed from blue to red, while the color of the control or negative control wells remained constant in the absence of contamination. Three runs of the examination were conducted [7].

2.7 Assay for biofilm inhibition

Using methicillin-resistant S. aureus (MRSA), clinically relevant isolation with a powerful biofilm-forming agent, the MTP technique was utilized to evaluate the ability of TiO₂–MgO–Au NPs to prevent or diminish the development of bacterial biofilms. We made some changes to the biofilm research to make it better than the previous one [5,40]. MTP-containing TSB media were added to gradient doses of TiO₂–MgO–Au NPs, supplemented with 1% glucose. The organisms investigated were cultivated on MH broth for 24 h at 37°C after being diluted 1:100 in TSB. The growth of the cell intensity (OD620 nm) was monitored during the incubation period, and then the planktonic cells were removed from the plates. The resultant biofilm was fixed for 10 min using 200 μL of 95% methanol as the solvent and washed three times using PBS at pH 7.4. This was done after removing all the components of the well contents so as not to disturb the biofilms that had grown. Once the 200 μL wells were filled with 0.3% w/v crystal violet, they were left at room temperature for around 15 min. Subsequently, the plates were washed using distilled water, and then the wells were filled with a 30% acetic acid reagent for the quantitative evaluation of biofilm formation. The absorbance was measured at OD 540 nm using the STATFAX-USA microplate reader. The results were confirmed by comparing the comparative wells that were treated with the untreated wells [7].

2.8 Anticancer activity

2.9 Statistical analysis

The GraphPad Prism 8.0 (CA, USA) was employed to conduct data assessment and graphical demonstrations. All findings are expressed as mean ± SD, and all investigations were done three times (n = 3). The statistical analysis was performed using a one-way analysis of variance and Tukey’s multiple comparison tests, where P < 0.05 was considered significant.

3.1 Biosynthesis of trimetallic TiO₂–MgO–Au NPs using banana peel extract

There are several advantages of using plant extracts for nanoparticle production as opposed to other biological synthesis methods that include microorganisms. Numerous aspects, including the minimal lab needs for nanoparticle synthesis, the appropriateness for commercial production, the avoidance of impurities, the speedier approach, the single-step process, handling safety, and the greater stability of the nanoparticles owing to significant metabolite secretion, all contribute to this conclusion [42]. The primary function of plant secondary metabolites, including phenols, carbohydrates, proteins, flavonoids, steroids, alkaloids, tannins, sugars, and terpenoids, is to reduce and stabilize nanoparticles [43]. Indeed, variability in biochemical composition poses challenges for reproducibility across batches; however, ensuring consistent processing conditions, such as using standardized ripeness levels or controlled extraction methods, is essential to mitigate these issues. In the present study, trimetallic TiO₂–MgO–Au NPs were prepared using an extract from banana peels. The high polyphenolic content in banana peel extract substantially enhances its reducing capacity, facilitating effective nanoparticle synthesis and improving its stability [44]. Banana peel extract contains functional groups such as carboxyl, amine, and hydroxyl groups, which act as reducing agents and stabilize nanoparticles by capping their surface [45]. Nanoparticle stability is influenced by several environmental factors, particularly pH and temperature. The stability of nanoparticles is highly sensitive to the pH of the medium in which they are stored. In highly acidic or alkaline environments, nanoparticles may undergo dissolution, aggregation, or surface modifications that affect their stability. For example, nanoparticles synthesized under more neutral conditions tend to exhibit better long-term stability. Under extreme pH conditions, the surface charge can change, leading to aggregation or destabilization. Studies have shown that nanoparticles with a neutral surface charge are more stable in a wide range of pH levels [46].

Temperature is another crucial factor influencing the long-term stability of nanoparticles. Higher temperatures can accelerate degradation processes such as oxidation, agglomeration, or changes in surface charge. The stability of nanoparticles is generally improved when they are stored at lower temperatures, as this slows down the rate of degradation reactions. However, long-term storage at high temperatures may lead to the transformation or sintering of nanoparticles, potentially resulting in a decrease in their functional properties. For instance, nanoparticles that are stored at room temperature or under refrigeration often exhibit greater stability and retain their biological activity for extended periods [47]. Trimetallic nanostructures, which are subsequently stabilized and capped, are produced as a result of metabolites in the banana peel extract decreasing the metal precursors. A simple and eco-friendly process was developed by Dlugaszewska and Dobrucka [48] to generate Au/Pt/Ag trimetallic nanoparticles using an aqueous Lamii albi flos extract. Furthermore, Meliloti officinalis extract was used to bio-prepare Au–ZnO–Ag trimetallic nanoparticles (∼20 nm) [49]. Aqueous leaf extracts of Froriepia subpinnata and Eryngium campestre were used to biosynthesize Cu/Cr/Ni trimetallic oxide nanoparticles reliably at moderate temperatures [50]. These NPs demonstrated exceptional antibacterial activities against E. coli and S. aureus. Rao and Paria [51], by modifying the mix of phytochemicals in extracts from Aegle marmelos (leaf extract) and Syzygium aromaticum buds, alloy-like Ag–Au–Pd trimetallic NPs (∼8–11 nm) were green-fabricated in 10 min under ambient conditions. In another study, Kaur et al. [52] synthesized TiO₂–Al₂O₃–ZnFe₂O₄ nanocomposites using hydrothermally prepared Hibiscus rosa sinesis flower extract. Furthermore, Aspergillus niger was successfully used in the entire mycosynthesis of trimetallic copper, selenium, and zinc oxide nanoparticles (Tri-CSZ NPs) by Hashem et al. [27].

3.2 Characterization

3.2.1 UV–vis spectroscopy

The first indication of trimetallic TiO₂–MgO–Au NPs production is the plant’s aqueous extract changing from pale yellow to pale brown after being mixed with Ti(NO₃)₄·4H₂O, Mg(NO₃)₂·6H₂O, and HAuCl₄·3H₂O. This change validated the ability of metabolites to reduce metal ions and build nanoscale structures. The new hue’s absorbance was measured between 200 and 800 nm to determine the maximum SPR. There were two maximums of SPR at 270 and 550 nm, which corresponded to (TiO₂, MgO) and Au absorbance, respectively (Figure 1). An absorption band associated with phytobiomolecules may be seen at 280 nm in the UV-visible spectra of banana peel extract. Hassan et al. [53] verified the excitation of green-synthesized TiO₂ NPs using a UV–Vis spectrophotometer at 270 and 290 nm. Furthermore, the specific absorption peak was observed at 267 nm for magnesium oxide nanoparticles synthesized by using Trigonella foenum-graecum leaf extract by Vergheese and Vishal [54]. According to Hassanisaadi et al. [55], the UV-Vis spectra of biosynthesized AuNPs were observed at 545 nm.

Figure 1

UV-Vis spectroscopy of banana peel extract and biosynthesized trimetallic TiO₂–MgO–Au NPs.

According to many studies conducted by Alshehri and Malik, the biosynthesis of trimetallic Cu–Co–Ni NPs utilizing an extract of Origanum vulgar L. shows absorption peaks in its UV–visible spectra at 270 and 320 nm [34]. The green-produced NPs from the combined leaf extracts of F. subpinnata and E. campestre had the highest absorbance at about 220 nm, according to Vaseghi et al. [50]. It is crucial to keep in mind that the interaction of numerous chemicals in the reaction mixture may prevent separate peaks from forming for each of the individual metals that make up the trimetallic structure. As shown by Kannaiyan et al. [56], the UV–Vis absorbance of the C. sativum extract was compared with the collected tri-metallic oxide NPs. The three distinctive peaks of tri-metallic oxide NPs, which exhibited wavelengths of 261 nm, 426 nm, and 564 nm, correspond to the tri-metallic oxide Ni/Cr/Cu NPs, as previously reported. Furthermore, Vaseghi et al. [50] observed that the nanocomposite’s absorption spectra showed the appearance of the largest absorption peak at 265 nm, which suggested the existence of a distinct surface plasmon resonance.

3.2.2 FT-IR spectroscopy

FT-IR spectroscopy was used to identify the trimetallic TiO₂–MgO–Au NPs that were biosynthesized. The FT-IR spectra of the synthetic composite are in the 400–4,000 cm⁻¹ spectral region. Ten distinct peaks at wavenumbers 3,200, 2,668, 2,340, 2,076, 1,830, 1,643, 1,405, 1,095, 570, and 439 cm⁻¹ were found in the FT-IR spectra of trimetallic TiO₂–MgO–Au NPs (Figure 2). Several groups found in banana peel extract belonged to a variety of substances, including proteins, amino acids, amines, and polysaccharides. The peaks at 3,200, 2,668, 2,340, 2,076, 1,830, and 1,095 cm⁻¹ are caused by the stretching bonds of O–H, C–H, C═O, C═C, and C–O, respectively, that are found in bioorganic molecules such as phenolics, amino acids, and carboxylic acid compounds [57]. Conversely, the nanocomposite’s spectrum displayed all of these vibrations at a greater intensity because of the weak van der Waals contacts between the metallic and biological NPs [58]. According to Botteon et al. [59], metal–metal interactions inside the nanocomposite are responsible for the new, strong bands in the 400–600 cm⁻¹ range. Both the new bands at 1,643 and 1,405 cm⁻¹, which are often associated with the stretching and bending vibrations of the carboxylate anion [60], most likely resulted from the oxidation of –C–OH in phytochemicals during the reduction of metal ions [61]. The results are consistent with previous studies [34,50,57] on the manufacture of trimetallic nanoparticles using plant aqueous extract.

Figure 2

FTIR analysis of biosynthesized trimetallic TiO₂–MgO–Au NPs using banana peel extract.

3.3 Morphological and elemental analysis

TEM and SEM were used to examine the surface morphology and particle size of the biosynthesized TiO₂–MgO–Au NPs (Figure 3). In this case, an extract from banana peels was used to create trimetallic TiO₂–MgO–Au NPs; while many plant extracts share similar properties, the composition of banana peel extract, especially its polysaccharides and high antioxidant levels, offers distinct benefits in creating smaller, more uniform nanoparticles with robust stability [62]. TEM was used to examine their distribution, size, and shape. The produced trimetallic TiO₂–MgO–Au NPs, in this instance, had spherical forms and ranged in size from 50 to 70 nm, with an average size of 55 nm (Figure 3a). Therefore, smaller nanoparticles are more reactive in biological environments [63]. Similarly, Hussein et al. [57] claim that TEM images display the spherical form of nanoparticles with a particle size of 50–90 nm. Vaseghi et al. [50] revealed that the TEM images of Cu/Cr/Ni trimetallic oxide NPs biosynthesized by aqueous leaf extracts of E. campestre and F. subpinnata show a variety of sizes and shapes.

Figure 3

(a) TEM image, (b) SEM image, and (c) EDX analysis of biosynthesized trimetallic TiO₂–MgO–Au NPs by using banana peel extract.

The nanocomposite’s morphology was examined using SEM. Figure 3b displays the recorded SEM images. Because of the high surface energy-imposed strong interparticle contact, the trimetallic composite structure displayed an oval and irregular crystalline porous structure, which is characteristic of metallic nanocomposites. When smaller nanostructures merge to form larger ones, the result is a mixed nanostructure. This is evident in the SEM images and indicates the formation of a nanocomposite [57]. Additional research by Alshehri and Malik [34] demonstrates through SEM micrographs of biosynthesized Cu–Co–Ni trimetallic nanoparticles that structural porosity is caused by the agglomeration of nanoflakes.

Verifying the presence and distribution of the required metals inside the trimetallic structure can be facilitated by EDX analysis, which offers information on the elemental composition of a nanostructure. Knowing the elemental composition of NPs is essential to creating materials with appropriate qualities and their use for certain applications. Using EDX analysis, the trimetallic TiO₂–MgO–Au NPs’ elementary mapping was determined. According to the acquired data, the as-formed sample mostly consists of Ti, Mg, O, and Au ions, as shown by their respective weight percentages (26.3%, 9.4%, 40.4%, and 7.1%) and atomic percentages (22.3%, 5.2%, 53.9%, and 5.2%) (Figure 3c). The trimetallic synthesis that was previously studied using chemical techniques and plant or microbial extracts was consistent with our findings [34,50]. Plant extract capping agents that coat nanoparticles for stability may be the cause for the C and K peaks [64]. Alshehri and Malik [34], using EDX analysis, determined the weight percentages of 17.34% Co, 10.37% Ni, and 34.01% Cu, resulting in a 2:1:3 ratio for the Cu–Co–Ni trimetallic nanoparticles made with the extract from Origanum vulgare L. in a different study. It appears that surface biomolecules act as capping agents when weak C and O signals are present, and in the elemental mapping, Ni is shown in white, Co in green, and Cu in red.

3.4 XRD, DLS, and zeta potential analysis

The trimetallic TiO₂–MgO–Au NPs were examined using XRD. The trimetallic TiO₂–MgO–Au NPs XRD spectra, as displayed, revealed ten powerful reflection peaks at 2θ of 25.4°, 36.8°, 38.0°, 42,7°, 48.1°, 54.5°, 62.2°, 64.5°, 74.5°, and 77.6°, respectively. These represent the Bragg diffraction of the (101), (111), (200), (211), (220), and (311) planes, as shown in Figure 4a. Upon analyzing the produced nanoparticles using XRD, they found unique diffraction peaks at four different angles: 25.4°, 38.0°, 48.1°, and 54.5°. These angles correspond to the (101), (111), (200), and (211) planes, respectively, and are in line with a typical TiO₂-NP phase pattern (JCPDS No. 21-1272) [65]. MgO-NPs exhibited characteristic peaks at 2θ values of 36.8°, 42,7°, 62.2°, and 74.5°, corresponding to the (111), (200), (220), and (311) planes (JCPDS: 9000493) [66]. Five distinct diffraction peaks were discovered for the hexagonal structure of Au-NP, and they were indexed with the planes (111), (220), and (311) at 38.0°, 64.5°, and 77.6°, respectively (JCPDS: 00-407-84) [67]. These peaks lined up with certain crystallographic planes, such as (220), (211), (211), (111), (200), and (311). Notably, the crystalline structure of the trimetallic TiO₂–MgO–Au NPs was verified by these peaks. Using Scherrer’s equation and the XRD pattern, the average crystallite size of the trimetallic TiO₂–MgO–Au NPs was found to be 60 nm. The discrepancy between DLS and TEM’s recorded size values of vesicles is expected. This is because the sample preparation for TEM measurement involves dehydration and staining, which can affect the size. Moreover, DLS provides an average size for particles moving in dispersion and is highly sensitive to particle agglomeration or clustering. Even minor aggregation can significantly increase the measured hydrodynamic diameter, as DLS calculates an average size weighted by the intensity of scattered light, but TEM captures a specific field. A similar discrepancy was noticed in previous work and was discussed similarly [68]. The trimetallic nanocomposite (Ru/Ag/Pd) XRD patterns were shown by Hussein et al. [57], and the (111), (200), and (220) planes of Pd-NP were matched with the high-intensity peaks at 2θ = 40.11, 47.75, and 68.31, which match JCPDS 87-0641. At 2θ values of 38.45°, 44.85°, 67.55°, and 77.5°, Ag-NPs showed peaks that corresponded to the (111), (200), (220), and (311) planes, respectively (JCPDS: 04-0783). Ru-NPs revealed peaks for the (100), (002), (101), (102) planes (69.42°, 43.82°, 46.12°, 58.32°, and 69.42°) (JCPDS: 06–0663). Pure crystalline trimetallic nanoparticles were present since no impurity peaks were seen. By using Scherrer’s equation, the crystal size was determined to be 15.67 nm. The same result was obtained by Alshehri and Malik [34] for biogenic Cu–Co–Ni trimetallic nanoparticles.

Figure 4

XRD (a) and DLS (b) of the biosynthesized trimetallic TiO₂–MgO–Au NPs.

The size distribution of the particles in a suspension or solution may be measured using the DLS method. It offers details on how different particle sizes are distributed within a sample; this information is commonly shown as a histogram or intensity-weighted size distribution curve. Particle populations that are polydisperse (varying in size) or monodisperse (uniform in size) can be identified by the analysis. DLS examination of the trimetallic TiO₂–MgO–Au NPs revealed an average particle size distribution of about 70 nm (Figure 4b). They came to the conclusion that the greater size might have been caused by trace quantities of larger particles formed by contamination or agglomeration, which could add uncertainty to particle size measurements [69]. Similarly, the particle size was roughly 190 nm for the Cu–Fe–Ag NPs that were subjected to DLS analysis by Roy et al. [70]. Monodisperse models are more consistent with the polydispersity index (PDI) values of less than 0.05. Conversely, values higher than 0.7 are expected to indicate the dispersion of polydispersity particles [71]. In this study, the PDI value was 0.472 for the approved PDI levels. The present data show that the trimetallic TiO₂–MgO–Au NPs biosynthesized have a moderately polydisperse size distribution.

The zeta potential is a key parameter in assessing the colloidal stability of nanoparticle suspensions, influenced by factors such as surface chemistry, particle roughness, and adsorbed biomolecules [101]. A high absolute zeta potential value (greater than ±30 mV) typically indicates strong electrostatic repulsion, preventing aggregation and ensuring stability [102]. Conversely, values approaching 0 mV suggest reduced repulsive forces, leading to particle agglomeration. Some studies suggest that particles with zeta potential values exceeding ±20 mV can still exhibit moderate stability, depending on other stabilizing interactions [103,104].

In this study, the biosynthesized trimetallic TiO₂–MgO–Au NPs exhibited a bimodal zeta potential distribution, with peaks at approximately −20 and +25 mV, indicating a heterogeneous surface charge, as shown in Figure 5. This variation may result from differences in the particle size, surface composition, or functional groups adsorbed from the biological extract used in the synthesis [102]. In this study, the observed zeta potential values indicate a moderate level of colloidal stability, which aligns with the PDI value (0.472), confirming the polydisperse nature of the synthesized nanoparticles. El-Sawaf et al. [105] reported a zeta potential of 21.5 mV for the trimetallic CuO/Ag/ZnO nanocomposite synthesized using Ziziphus spina-christi plant extract.

Figure 5

Zeta potential analysis of the biosynthesized trimetallic TiO₂–MgO–Au NPs.

3.5 Antimicrobial activity of trimetallic TiO₂–MgO–Au NPs

In the present investigation, the agar well-diffusion technique was used to evaluate the antimicrobial performance of biologically generated TiO₂–MgO–Au NPs against a variety of five microbial pathogens, including Gram-positive (S. aureus ATCC 6538, B. subtilis ATCC 6633), Gram-negative (E. coli ATCC 25922, P. aeruginosa ATCC 9027), and C. albicans ATCC 10231. TiO₂–MgO–Au NPs strongly inhibited C. albicans, S. aureus, B. subtilis, E. coli, and P. aeruginosa by 33.8 ± 0.25 mm, 36.6 ± 0.75 mm, 24.7 ± 0.55 mm, 23.16 ± 0.4 mm, and 25.9 ± 0.32 mm, respectively, when compared to each microbe’s area of inhibition (Figure 6).

Figure 6

Antimicrobial activity of trimetallic TiO₂–MgO–Au NPs using the agar well technique.

The inhibitory impact of various concentrations of TiO₂–MgO–Au NPs (15.62–1,000 µg·mL⁻¹) was examined to determine the MICs of the NPs against the indicated microbial pathogens. The lowest MIC for S. aureus was 125 µg·mL⁻¹, whereas the MIC for P. aeruginosa and C. albicans was 250 µg·mL⁻¹, and that for B. subtilis and E. coli was 500 µg·mL⁻¹ (Figure 7). The obtained result was consistent with studies concerning the antibacterial efficacy of trimetallic nanoalloys [72,73]. Multiple studies indicated that the efficacy of nanoparticles versus diverse pathogenic bacteria was ranked (from highest to lowest) as follows: tri, bi, and monometallic. The trimetallic Au/Pt/Ag exhibited superior antibacterial efficacy against E. faecalis, S. aureus, E. coli, and C. albicans in comparison to monometallic variants [48]. One possible explanation for this action is the additive nature of trimetallic compounds as opposed to bi- or monometallic ones [74].

Figure 7

MIC of trimetallic TiO₂–MgO–Au NPs toward the tested microbial strains.

The FDA has also approved MgO NPs for use as harmless substances [75]; they received research attention for their potential use in biological fields. Magnesium oxide nanoparticles (MgO NPs) are non-toxic and easily obtainable and have antibacterial characteristics against both Gram-positive and Gram-negative bacteria, fungi, and viruses, along with traits that limit biofilm formation [76,77]. MgO NPs have shown their ability to inhibit S. aureus, P. aeruginosa, along with E. coli [75,78]. They prevented K. pneumoniae and S. aureus from forming biofilms [77]. TiO₂ NPs were demonstrated to have antimicrobial capabilities against a variety of bacteria, including E. coli, S. aureus, P. aeruginosa, and P. expansum [79]. Enhanced antimicrobial effectiveness of AuNPs towards Gram-negative bacteria has been found; this may be because of thinner cell walls and more sustained electrostatic contacts [80]. It has been noted that the type of microbe and strain, as well as the size, functionalization, and quantity of AuNPs, directly affect the antibacterial action [81]. Here, NPs serve as antimicrobial substances that can be employed to administer traditional antimicrobials or to fight resistance to antimicrobial treatments directly. The potential of NPs to enter and damage microbial cell membranes through membrane-damaging hardness, reduce cellular permeability, or generate antimicrobial properties (e.g., the generation of ROS, interactions between proteins and nucleic acids, deactivation of enzymes, excessive expression regarding efflux pumps and expulsion of metal ions), and prevent the development of biofilms [82–84]. The trimetallic (TiO₂–MgO–Au) NPs can inhibit pathogenic microbes via impact on the cell wall, cell membrane, and protein synthesis that leads to suppressing the activity of the microbes as mentioned in previous studies [28,85,86].

3.6 Anti-biofilm activity of trimetallic TiO₂–MgO–Au NPs

The antibiofilm efficacy of TiO₂–Mg–ZnO) NPs against S. aureus MRSA in the current investigation showed significant results. Thus, when used at quantities below the MIC amount, TiO₂–MgO–Au NPs displayed the greatest efficacy against the development of biofilms caused by MRSA: 250, 125, 62.5, 31.25, and 15.62 μg·mL⁻¹ decreased the generation of biofilm by 86.8%, 77.1%, 63.3%, 53.4%, 37.6%, and 25.1%, respectively. (Figure 8). According to their respective quantitative and qualitative assessments, TiO₂–MgO–Au NPs prevented the very initial stages of MRSA biofilm creation. The biofilm inhibition results using crystal violet were in agreement with those of Khan et al. [87].

Figure 8

Antibiofilm activity of trimetallic TiO₂–MgO–Au NPs.

It was found that P. aeruginosa biofilms were inhibited by intracellularly produced gold nanoparticles employing Laccaria fraternal. The nanoparticles’ gold content was about 15%, and they had a 93% reduction in biofilms [88]. AuNPs showed notable biofilm inhibition against P. aeruginosa and E. coli with sub-MICs, according to Anwar et al. [89]. At concentrations that ranged from 0.25 to 0.5× MICs, the nanocomposite, including AuNPs along with reduced graphene oxide (Au-RGO), showed comparable results, dependent on concentration, removal of established mature biofilms of MRSA and P. aeruginosa [90]. Moreover, the MIC of TiO₂ NPs demonstrated strong antibiofilm action towards P. aeruginosa by successfully obstructing the planktonic cells’ adhesion to the substratum [91]. According to Achudhan et al., G-TiO₂ NPs were evaluated against fungus (C. albicans) as well as bacteria (Citrobacter freundii and S. mutans) to determine their antimicrobial and antibiofilm properties. At 100 μg·mL⁻¹, each of the fungus and bacterial biofilms were significantly suppressed [92]. Mg-NPs inhibited the biofilms of S. pyogenes, S. epidermis, and P. aeruginosa by 87.15%, 76.35%, and 49.14%, respectively, at an average concentration of 0.98 µg·mL⁻¹. The bacteria in concern had the same minimal biofilm inhibitory concentrations (MBICs) of 1.95 µg·mL⁻¹ for the first two strains and 7.81 µg·mL⁻¹ for P. aeruginosa [93]. The effectiveness of TMNC in inhibiting biofilm generation by S. aureus as well as E. coli was assessed by employing crystal violet staining, as noted in prior research, at different inhibition percentages. Elevated TMNC doses inhibited biofilm creation by S. aureus and E. coli by 85% and 83%, respectively [94]. The biofilm inhibition proportion of the nanocomposite for both bacterial strains increased with a higher TMNC ratio. This is due to the antibiofilm capability of TMNCs. The quantity of TMNC enhances biofilm inhibition and also increases for both bacterial strains. This followed a prior publication by Garza-Cervantes et al. about the progressive reduction in biofilm development as the amount of composite nanoparticles increased [95].

3.7 Cytotoxic effect of TiO₂–MgO–Au NPs

The cytotoxic activities of TiO₂–MgO–Au NPs were assessed on HepG2 and MCF-7. The MCF-7 cells exhibited the most significant cytotoxic impact, as evidenced by their minimal IC₅₀ values (Figure 9). TiO₂–MgO–Au NPs resulted in a minimal IC₅₀ value of 11.09 ± 1.02 µg·mL⁻¹, while the IC₅₀ for taxol was 8.96 ± 0.98 µg·mL⁻¹. The MCF-7 cells are more susceptible to the cytotoxic effects of TiO₂–MgO–Au NPs due to their increased expression of receptors and cell surface markers in breast cancer cells, which increase susceptibility. Conversely, HepG2 cells exhibit a greater antioxidant capacity than MCF-7 cells. This antioxidant defense can potentially reduce the cytotoxic activity of TiO₂–MgO–Au nanoparticles toward HepG2 cells, thereby mitigating their cytotoxic effects [96,97].

Figure 9

In vitro cytotoxic effects of TiO₂–MgO–Au towards MCF-7 and HepG2 cell lines. The data are displayed as the mean ± SD obtained from three separate and independent trials. *Statistically significant within the Taxol group at p < 0.001.

3.8 Effect of TiO₂–MgO–Au NPs on caspase-8 activity

TiO₂–MgO–Au NPs influence the apoptotic marker caspase-8 (Table 1). Exposure of MCF-7 cells to TiO₂–MgO–Au NPs significantly increased caspase-8 activity (1.095 ± 0.04 ng·mL⁻¹) in comparison to the control (0.341 ± 0.02 ng·mL⁻¹). Furthermore, when exposed to TiO₂–MgO–Au NPs, caspase-8 activity was significantly higher compared with Taxol treatments (0.986 ± 0.03 ng·mL⁻¹). Apoptosis triggers the activation of DNA fragmentation enzymes by activating caspase-8 [98]. These compounds induced apoptosis in MCF-7 cells by activating caspase-8.

3.9 Effect of TiO₂–MgO–Au NPs on VEGFR-2 activity

Using the inhibition concentration–response curve, the 50% inhibition concentration value (IC₅₀) was found. The positive control used in this experiment was sorafenib. The comparison of IC₅₀ values for VEGFR-2 inhibition between TiO₂–MgO–Au and the established inhibitors like sorafenib reveals significant differences in their potential anti-angiogenic activities. The close IC₅₀ values of TiO₂–MgO–Au to sorafenib suggest that these differences may not be substantial. An IC₅₀ value of 0.305 ± 0.018 µg·mL⁻¹ for the TiO₂–MgO–Au NPs indicated a strong inhibitory effect (Table 2).

Breast cancer cells exhibit elevated VEGF expression in comparison to normal tissues. The anti-angiogenic effect is suggested by the decreased expression of the VEGF receptor (VEGFR-2). VEGF is a powerful angiogenic agent that stimulates the formation of blood vessels, angiogenesis, and the development of tumors in breast cancer. However, a reduction in VEGFR-2 expression hampers the capacity of VEGF to promote angiogenesis. The simultaneous decrease in VEGFR expression might result in a decline in the formation of new blood vessels, which can be advantageous in cancer therapy, where excessive blood vessel formation can promote tumor development and spread [99,100].