Optimization of probiotic-mediated selenium nanoparticles for superior antibacterial action against methicillin-resistant Staphylococcus aureus

Abstract

The need for new bactericidal agents is becoming increasingly crucial as current antibiotic treatments are becoming less effective against methicillin-resistant Staphylococcus aureus (MRSA). The present study involves the biosynthesis of selenium nanoparticles (SeNPs) using the cell-free supernatant of Limosilactobacillus fermentum (OR553490). To achieve the two objectives of optimizing the biosynthesis of SeNPs and enhancing their antibacterial activity, response surface methodology (RSM) coupled with the Box–Behnken design was employed to vary the components to achieve the desired outcomes. For the maximum production of biosynthesized SeNPs (OD_max), the optimum conditions were found to be a Na₂SeO₃ concentration of 30 mM at pH 7, incubation temperature within the 30–40 °C range for 72 h, and a metabolite-to-precursor ratio within the range of 1:1 to 1:4 (v/v%). Conversely, for maximum antibacterial activity against MRSA (strain ATCC 43300), the optimum conditions are a higher precursor concentration (50 mM) at a 1:1 ratio (30 °C for 48 h). The SeNPs were well characterized; most were coated with a protein layer and had a uniform rod-shaped structure with a mean diameter of 30.94–43.94 nm. The presence of the protein coat was confirmed by the presence and identification of an amine and C–N band in the FT-IR spectrum. The synthesized protein-coated SeNPs exhibited a zeta potential of − 20.93 ± 5 mV, which indicates good stability. Both optimized SeNPs and linezolid were tested against MRSA, with the SeNPs demonstrating greater anti-MRSA activity than linezolid, which served as the clinical benchmark. SeNPs demonstrated identical minimum inhibitory and bactericidal concentrations (MIC/MBC) of 50 µg/ml, effectively doubling the potency of linezolid (MIC/MBC: 100/110 µg/ml). Probiotic-mediated rod-shaped SeNPs may offer an environmentally viable solution for treating drug-resistant bacterial infections.

Background

Antimicrobial resistance (AMR) is one of the abundant risks to public health and global improvement. AMR directly triggered 1.27 million deaths and influenced to 4.95 million deaths as globally reported in 2019 [1]. AMR occurs when microorganisms such as bacteria and fungi cease responding to the antimicrobial drugs [2]. By rendering antimicrobial drugs ineffective and making infections difficult to treat, drug resistance augments the risk of disease transmission, and death rate [3]. Among AMR, bacterial resistance is a major problem that is weakening the efficacy of standard antibiotics against common bacterial diseases [4]. There are several categories of antibiotics that classified according to their structure or antibacterial mechanism. β-lactams (which suppress the synthesis of cell walls), aminoglycosides (which synthesize proteins), macrolides (which synthesize proteins), tetracyclines (which synthesize proteins), glycopeptides (which synthesize cell walls), and daptomycin (which functions as a cell membrane) [5]. Gentamicin, streptomycin, and kanamycin are examples of aminoglycosides, another class of positively charged antibiotics that stick to the outer membrane and encourage accumulation through pores [6]. Aminoglycosides are cationic peptides, such as colistin, that attach to lipid A in lipopolysaccharide to permeabilize the outer membrane and cause cell death [7].

β-Lactams (monobactams, carbapenems, cephalosporins, cephamycins, and penicillins) interact with the 16 S rRNA’s 30S ribosomal subunit, leading to misreading, shortened proteins, and cell death [8]. However, certain bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa, may withstand these antibiotics by inhibiting penicillin-binding proteins that crosslink the peptide chain in the cell wall or by enzymatically inactivating the antibiotic [9]. Nevertheless, S. aureus evolved resistance strategies, such as target modification with reduced affinity for the antibiotic or efflux pumps [10]. In terms of global public health issues, it is among the most well-known and hazardous pathogens. This bacterium is characterized by multidrug resistance and can cause pneumonia, endocarditis, keratitis, osteomyelitis, and sepsis syndrome [11]. Methicillin-resistant S. aureus (MRSA) strains were responsible for most issues, resulting in challenging infections. Thus, several studies have focused on combating this issue.

Recently, a relatively new branch of research called nanobiotechnology examined materials at the nanoscale and altered their characteristics to overcome MRSA resistance [12]. Benefits of nanoparticles (NPs), range in size from 1 to 100 nm, include minimal toxicity, limited microbial resistance, and high stability [13]. Hazardous chemical species are adsorbed on the surface of NPs during their chemical and physical synthesis, which may have an adverse effect on industrial applications [14, 15]. Their employment in clinical settings is restricted by the synthesis process’s use of non-polar solvents and hazardous compounds on the NPs’ surface [16]. Therefore, creating clean, biocompatible, non-toxic, and environmentally acceptable technologies for synthesizing NPs is worthwhile, given that biological techniques are considered secure, economical, environmentally benign, and sustainable.

Selenium NPs (SeNPs) were selected above other NPs due to a number of benefits, such as their vital role in improving human health, cell signaling, antioxidant defense, and other metabolic processes [17]. Additionally, SeNPs have high bioavailability, low toxicity, and biodegradability properties, which make them safe for clinical usage and remarkable in nanomedicine due to their antimicrobial and anticancer qualities [18, 19]. Selenium is also used to treat a number of pathophysiological problems, including diabetes, heart disease, cancer, and neurological disorders, due to its antioxidant and anti-inflammatory characteristics [20].

According to earlier research, the biological techniques to produce SeNPs were endless. Accordingly, employing microorganisms as novel bio-nano factories that use biotransformation to turn ions into metal NPs may attract much interest [21]. The SeNPs can be created chemically, physically, or biologically. Nevertheless, compared to chemical and physical methods, the biological process is safer and more efficient [22]. The biological method is based on green chemistry, which may be carried out by non-living viruses or their naturally occurring secondary metabolites, as well as living organisms including bacteria, fungi, yeasts, algae, and plants [23–25]. One of the key microorganisms in the biosynthesis of NPs, the probiotic lactic acid bacteria (LAB), is proposed as a low-cost bio-nano-factory for the manufacture of NPs that is rapid, safe, simple to culture, and found in dairy products [26]. LAB produces a number of antimicrobial chemicals that may cooperate with NPs, including formic acid, hydrogen peroxide, ethanol, acetone, and bacteriocins [27].

The current work aimed to bio-reduce Se salt into SeNPs using Limosilactobacillus fermentum OR553490. In addition, different parameters were tested and optimized to obtain efficient and stable NPs and investigate their antibacterial action against MRSA. Unlike previously reported studies for biosynthesis of SeNPs by lactic acid bacteria, Limosilactobacillus fermentum OR553490 combines antimicrobial properties with safety and biocompatibility. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and agar well diffusion tests were used to comprehensively evaluate its antibacterial efficacy of the biosynthesized SeNPs. To the best of our knowledge, this work is the first to look at the optimization parameters to boost and improve the production of NPs and the antibacterial efficacy, particularly against the resistant bacterium MRSA. This opens up new possibilities for future developments in the field. In addition, the current study used response surface methodology (RSM) based on based on the Box–Behnken design (BBD) to systematically investigate and optimize the synergistic effect of key synthesis parameters (temperature, pH, precursor concentration, and cell-to-precursor ratio) in order to guarantee the highest yield and optimal quality of SeNPs for antibacterial applications.

Materials and methods

Materials

Sodium selenite (Na₂SeO₃, MW: 172.94, anhydrous) was purchased from VWR Prolabo Chemicals, Fontenay-sous-Bois, France. Limosilactobacillus fermentum ESA5 was kindly obtained from the Microbiology Laboratory, Faculty of Science, Damietta University, Egypt. This bacterial strain was isolated from an Egyptian yogurt sample at Damietta City, Egypt. Phenotypic and 16 S rRNA gene sequence characterizations were used to confirm its identification, and it was deposited in the database under accession number OR553490. S. aureus ATCC 25,923 and methicillin-resistant S. aureus ATCC 43,300 strains were employed in the investigations of antibacterial activity.

Methods

Preparation of L. fermentum supernatan

On sterile DE Man, Rogosa, Sharpe (MRS, Oxoid, England) agar plates, the strain slant of L. fermentum was streaked, and kept for 48 h at 37 °C. Three colonies were cultivated anaerobically in MRS broth flasks after being aseptically transferred. The infected flasks were incubated at 150 rpm and 37 °C for 48 h. Aseptic centrifugation was used for 20 min at 4000 rpm to collect the culture supernatants after incubation. The supernatants were transferred into fresh flasks after being filtered with a 0.22 μm syringe filter (Millex GV, Millipore).

Biosynthesis of senps

The reaction mixture for SeNPs biosynthesis was prepared throughout mixing the bacterial metabolites with 1 mM Na₂SeO₃ solution (1:1 v/v%). The prepared mixtures were incubated at 37 °C and 150 rpm until the color turned into red. The SeNPs formation was monitored using a double beam UV-Vis spectrophotometer V-630 (JASCO, UK) [23].

Optimizing of senps production

Different parameters were evaluated to optimize the biogenic SeNPs formation. Several Na₂SeO₃ concentrations ranging from 10 to 50 mM were prepared and tested to evaluate the effect of precursor concentration on SeNPs formation after 72 h (pH 7, 37 °C, 150 rpm). Different mixing ratios (1:1–1:16 v/v%) between Na₂SeO₃ and cell-free bacterial metabolites were also tested. The production rates were also evaluated at various temperatures between 10 °C and 60 °C. The pH was changed from 5 to 9 using solutions of 0.1 N HCl or NaOH in order to comprehend the effect of pH on the production of SeNPs. Optimal circumstances for SeNPs formation were ascertained by spectrophotometric investigations.

Response surface methodology

Four independent parameters were used in an RSM technique based on the BBD to optimize the biosynthesis conditions for SeNPs formation [28, 29]. The dependent response variable was the surface Plasmon resonance (SPR) intensity (or OD_max), which serves as a proxy for the yield and quality of the SeNPs. Four independent critical synthesis parameters were selected for optimization including X₁; temperature (A), X₂; pH (B), X₃; concentrations of Na₂SeO₃ (C), and X₄: ratio between Na₂SeO₃ and cell-free bacterial metabolites (D). Each factor was investigated at three levels, designated as − 1 (low), 0 (center), and + 1 (high), based on preliminary single-factor experiments (Table 1). The BBD resulted in a total of 27 experimental runs, including 6 center points, which were randomized to minimize the effects of systematic error.

Table 1.

Variables and levels used for optimization of senps production

Factor	Name	Coded level (− 1)	Coded level (0)	Coded level (+ 1)
X1​	Temperature (°C)	10	30	60
X2​	pH	5	7	9
X3​	Concentration (%)	10	30	50
X4​	Ratio (v/v%)	1:1	1:4	1:16

The experimental data were fitted to a quadratic second-order polynomial equation to model the relationship between the independent factors and the SPR intensity (Eq. 1) :

1

where X_i​ and X_j​ are independent variables, Y is a SPR intensity, β₀ is an intercept term, β_i is linear coefficients, β_ii is quadratic coefficients, and β_ij is interaction coefficients.

The significance of the quadratic model, individual components, and their interactions were assessed at a 95% confidence level (p < 0.05) using analysis of variance (ANOVA). The coefficient of determination (R²) and the lack of fit test were used to evaluate the model’s quality. Minitab Statistical Software (version 22; Minitab, LLC, State College, PA) was used for statistical analysis, including the ANOVA, model fitting, and creation of the RSM plots [30].

Antibacterial activity of SeNPs using agar well diffusion test

Using the agar well diffusion technique, the antibacterial activity of SeNPs during each optimization trial was examined against S. aureus ATCC 25,923 and MRSA ATCC 43,300 [31]. Mueller–Hinton agar (MHA) medium was prepared and added to sterile Petri dishes. Spread plating was used to inoculate 0.5 MacFarland (1–2 × 10⁸ CFU/mL) from each bacterium. A corkborer that had been disinfected was used to make 5 mm holes. The agar plates were kept at 37 °C for 48 h after 100 µL of SeNPs solutions were introduced to the holes. The zones of inhibition (ZOI) were evaluated in millimeters following incubation in order to identify the optimal variables that improved the produced NPs’ antibacterial activity against MRSA.

Minimum inhibition concentration

Mueller–Hinton broth (MHB) medium was used to determine the MIC for SeNPs based on the broth dilution technique [32]. Inoculated MHB test tubes (2 mL) containing 5 × 10⁵ CFU/mL of the investigated bacteria were filled with varying amounts of SeNPs (0–100 µg/mL). For a whole day, tubes were kept at 37 °C and 150 rpm. A UV–Vis Spectrophotometer was used to measure the MIC values of SeNPs at 600 nm. Additionally studied were L. fermentum broth without SeNPs and sodium selenite without bacterial broth. The positive control was linezolid.

Minimum bactericidal concentration

The pour plate method was employed to examine the MBC for SeNPs. Mueller–Hinton agar (MHA) plates were well mixed, seeded with 10 µL aliquots from MIC tubes that did not exhibit any bacterial growth, and allowed to settle. The plates were then kept at 37 °C for a full day. The MBC values were determined by counting the total colony-forming units per milliliter (CFU/mL).

Characterization of optimized SeNPs

UV-Vis spectrophotometer V-630, Fourier transform infrared spectroscopy (FT-IR, FT/IR-4100typeA), transmission electron microscopy (TEM, JEOL JEM-2100, Japan), X-ray diffraction pattern (XRD, X-ray diffractometer, model LabX XRD-6000, Shimadzu, Japan), and zeta potential analysis (Malvern Zetasizer Nano-ZS90, Malvern, UK) were used to examine the optimized SeNPs.

Statistical analysis

The data underwent statistical analysis using SPSS version 18 software. With the exception of bars showing the mean ± standard error (SE), all experiment results were presented as the mean ± standard deviation (SD). There was an ANOVA. A significance criterion of p < 0.05 was established.

Results

Biosynthesis and optimization of SeNPs production

The first indication for the SeNPs formation is the medium’s color changing from pale to red, which was occurred during 84 h. Na₂SeO₃ concentration, temperature, pH, and the ratio of cell-free bacterial metabolites to Na₂SeO₃ were all investigated in order to maximize the bioproduction of SeNPs. The ideal Na₂SeO₃ concentration was first determined by adding several concentrations of Na₂SeO₃ to the supernatant, ranging from 10 to 50 mM (Fig. 1). The formation of SeNPs rises as the concentration of Na₂SeO₃ rises until it reaches 30 mM (the ideal Na₂SeO₃ concentration, which created a colloidal solution that is clearly red in color). After that, it falls and the reaction mixture’s color becomes faintly red. The peak range of 240–290 nm in the UV-Vis spectra verified the production of SeNPs.

Fig. 1.

Optimization of SeNPs biosynthesis using different concentrations (10–50 mM) of Na₂SeO₃

The most ideal conditions for the development of SeNPs were determined at a 1:1 (v/v%) mixing ratio between Na₂SeO₃ and cell-free bacterial metabolites (adsorption peak at 330 nm), respectively (Fig. 2). This mixing ratio greatly enhanced the SeNPs formation, giving a sharp absorption peak and high intensity of the red color. While other mixing ratios, including high amounts from the cell-free bacterial metabolites, produce broad peaks, the intensity of the red color of the reaction mixtures decreased.

Fig. 2.

Optimization of SeNPs biosynthesis using different mixing ratios (1:1–1:16 v/v%) between the Na₂SeO₃ and cell-free bacterial metabolites, respectively

Up to a certain point, increasing the temperature causes an increase in SeNPs production. 30–40 °C (adsorption peaks at 280–295 nm) were the optimum temperatures for NPs formation compared to other temperatures (Fig. 3). At temperatures below 20 °C, SeNPs could not be efficiently biosynthesized. In contrast, as the temperature rose from 50 °C to 60 °C, SeNPs aggregated.

Fig. 3.

Optimization of SeNPs biosynthesis at different temperatures (10–60 °C)

The initial pH of the reaction mixture was measured throughout the synthesis of SeNPs (Fig. 4). The color shift of the Se solutions from light yellow to red would suggest selenite reduction and the production of SeNPs in different sizes and shapes. Consequently, pH would control how different colors are perceived. Increasing the pH value from acidic to neutral and weak alkaline caused an increase in SeNPs production. In contrast, strong alkaline pH 9 inhibited the production rate and led to the NPs perception. The results showed that the most anionic selenite was absorbed at pH 7 (adsorption peak at 285–295 nm). The solution’s red color faded at highly basic and acidic pH levels. In addition, the UV-Vis spectroscopy detected low and red-shifted analytical peaks of NPs compared to the pH 7 peak.

Fig. 4.

Optimization of SeNPs biosynthesis at different pH values (pH5-9)

Optimization of SeNPs production using RSM

The relationship between the SPR intensity (Y) and the four independent variables, where A=temperature, B = pH, C=concentration of Na₂SeO₃, and D=mixing ratio between the Na₂SeO₃ and cell-free bacterial metabolite, is described by the following equation (Eq. 2):

2

This equation allows for the prediction of Y for any factor combination within the experimental design space. By contrasting the magnitude of the factor coefficients, the relative importance of each component in the synthesis process was determined. The concentration of Na₂SeO₃ and pH exhibited the most significant linear effects, with coefficients of − 0.44 and − 0.29, respectively. The negative coefficients show that the SPR intensity decreased by increasing the pH or Na₂SeO₃ concentration. Although the individual linear effects of temperature and mixing ratio were relatively small, a strong positive two-factor interaction (+ 0.43) between the concentration of Na₂SeO₃ and mixing ratio between bacterial metabolites and Na₂SeO₃ suggests that their combined effect increased the SPR intensity. ANOVA thoroughly assessed the model’s quality and statistical significance, as displayed in Table 2. With an F-value of 3.52 and a low p-value of 0.0125 (p < 0.05), the whole model was determined to be highly significant. The non-significant lack of fit p-value of 0.758 (p > 0.10), which indicates that the model properly captures the experimental data and the residual error is just random noise, further supports the model’s strong fitting capacity.

Table 2.

ANOVA and model performance of full quadratic model

Source	Sum of squares	df	Mean square	F-Value	p-Value
Model	8.841	14	0.632	3.52	0.0125	Significant
Linear terms	2.502	4	0.626	3.48	0.038
Quadratic terms	5.861	4	1.465	8.16	0.0016
Interaction terms	0.478	6	0.08	0.44	0.835
Residual	2.155	12	0.18
Lack of fit	1.655	10	0.166	0.66	0.758	Not significant
Pure error	0.5	2	0.25
Cor total	10.996	26

dF = Degree of Freedmen, F = Fisher-ratio

Figure 5A displayed the internally studentized residuals sit along the 45° line, confirming the basic premise of normally distributed errors and guaranteeing the accuracy of the p-values obtained from the ANOVA. Additionally, a random scatter of residuals centered on the zero line across the whole response range is shown in Fig. 5B, verifying constant variance (homoscedasticity) and demonstrating that the model is independent of the projected response size. These diagnostic results, along with the high adequate precision and non-significant lack of fit, clearly demonstrate the validity of the model and its suitability as a reliable tool for interpolating and forecasting ideal synthesis conditions within the designated design space.

Fig. 5.

(A) Normal probability distribution of internally studentized residuals for the optimized SeNPs production. (B) Diagnostic plot of internally studentized residuals against the predicted SPR intensity values

Figure 6 shows a three-dimensional RSM plot between the concentration of Na₂SeO₃ and the mixing ratio between the bacterial metabolites and Na₂SeO₃. The boundary between low Na₂SeO₃ concentration (approaching 10mM) and low ratio (approaching 1:1 v/v%) yields the maximum SPR intensity. The SPR intensity is very sensitive to rising concentration. Regardless of the ratio, the surface rapidly declines into the low-intensity area as concentration rises approaches 50%. This suggests that circumstances with low precursor concentration are optimal for maintaining colloidal stability and appropriate particle. The maximum SPR intensity was precisely predicted by numerical optimization using the fitted quadratic model under the following conditions: temperature of 30 °C, pH 5, 10 mM Na₂SeO₃, and mixing ratio of 1:1 v/v% between bacterial metabolites and Na₂SeO₃. This prediction confirms that high yield and stability of SeNPs are produced under conditions of low precursor concentration and minor acidity, which is consistent with the model’s significant negative linear and quadratic effects for concentration of Na₂SeO₃ and pH value. In comparison to the border condition, the quadratic model demonstrates that a pH 7 offers a more stable process window with less susceptibility to small changes. The use of a neutral pH buffer ensures high, constant SPR intensity while optimizing process resilience. The final suggested ideal settings were determined to be 30 °C, pH 7, 10mM Na₂SeO₃, and mixing ratio of 1:1 v/v% between bacterial metabolites and Na₂SeO₃.

Fig. 6.

Three-dimensional response surface plots. (A) Response surface of optical density (extOD_max) vs. concentration of Na₂SeO₃; X₃​, and pH; X₂ (temperature: 30 °C, ratio: 1:4). (B) Response surface of optical density (extOD_max) vs. concentration of Na₂SeO₃; X₃​, and ratio; X₄ (temperature: 30 °C, pH: 7)

Antibacterial activity of SeNPs during the optimization processes

During the optimization experiments, the anti-staphylococcal action of biosynthesized SeNPs was evaluated against MRSA through the agar well diffusion technique (Figs. 7, 8, 9 and 10; Table 3). Figure 7 shows the effect of varying concentrations of Na₂SeO₃ on the antibacterial action of SeNPs against MRSA. Each well represented SeNPs that were prepared from different concentrations of Na₂SeO₃, and the surrounding clear area is the zone of inhibition (mm). SeNPs had significant antibacterial activity against MRSA as the concentration increased to 60 mM, after which it declined, as shown by the smaller inhibition zones at 70 mM, 80 mM, and 90 mM, and a complete loss of activity at 100 mM.

Fig. 7.

Optimization of SeNPs activity at different concentrations (10–100 mM) of Na₂SeO₃ against MRSA. Bars represent SE. Different letters designate significant differences in access treatment means from repeated-measures ANOVA (P < 0.05)

Fig. 8.

Optimization of SeNPs activity at different mixing ratios (1:1–1:16 v/v%) between the Na₂SeO₃ and cell-free bacterial metabolites against MRSA. Bars represent SE. Different letters designate significant differences in access treatment means from repeated-measures ANOVA (P < 0.05)

Fig. 9.

Optimization of SeNPs activity at different temperatures (10–60 °C) against MRSA. Bars represent SE. Different letters designate significant differences in access treatment means from repeated-measures ANOVA (P < 0.05)

Fig. 10.

Optimization of SeNPs activity at different pH values (pH5-9) against MRSA. Bars represent SE. Different letters designate significant differences in access treatment means from repeated-measures ANOVA (P < 0.05)

Table 3.

Antibacterial activity of SeNPs compared to linezolid against MRSA

Parameter	Range	Zone of inhibition (mm, mean ± SD)	Parameter	Range	Zone of inhibition (mm, mean ± SD)
Concentration of Na2SeO3 (mM)	10	6.0 ± 0.16a	Mixing ratios between the Na2SeO3 and cell-free bacterial metabolites (v/v%)	1:1	13.9 ± 0.16d
	20	6.1 ± 0.14a		1:2	11.5 ± 0.67cd
	30	6.4 ± 0.16ab		1:4	10.6 ± 0.52bc
	40	6.6 ± 0.18ab		1:6	10.2 ± 0.3b
	50	6.8 ± 0.21d		1:8	10.9 ± 0.45c
	60	14.3 ± 0.56e		1:16	8.7 ± 0.32a
	70	12.2 ± 0.48d
	80	10.5 ± 0.32c
	90	5.2 ± 0.06a
Temperature (°C)	10	10.9 ± 0.45bc	pH value	5	6.1 ± 0.06a
	20	11.2 ± 0.41c		6	9.3 ± 0.014b
	30	13.4 ± 0.67d		7	15.9 ± 0.67e
	40	11.8 ± 0.52cd		8	14.5 ± 0.56d
	50	10.5 ± 0.45b		9	11.1 ± 0.52c
	60	9.8 ± 0.42a
Linezolid (60 mM, 30 °C, pH7)					14.7 ± 0.03

Values are presented as mean ± SE of triplicate experiments (n = 3). Different lowercase letters within the same column indicate statistically significant differences between the tested parameters (P < 0.05) according to ANOVA. Values sharing the same letter are not significantly different from each other. Linezolid: Utilized as a positive clinical control under optimized environmental conditions for direct comparison of bactericidal potency

Figure 8 illustrates the antibacterial activity response to different mixing ratios between the cell-free bacterial metabolites and Na₂SeO₃. The range of the mixing ratios was 1:1 (v/v%) to 1:16 (v/v%). When compared to other mixing ratios, the 1:1 (v/v%) mixing ratio between the cell-free bacterial metabolites and Na₂SeO₃ demonstrated the highest and best action of SeNPs against MRSA.

The SeNPs maintains a high level of activity across a broad range of temperatures, from 10 to 60 °C (Fig. 9). The zones of inhibition are all relatively large and consistent, suggesting that the SeNPs is thermally stable and its antimicrobial properties are not significantly degraded by heat within this range. Up to a certain point, raising the temperature increases the anti-staphylococcal activity. The ideal temperature for production was 30 °C.

Figure 10 shows how the pH affects the SeNPs’s antimicrobial activity. The SeNPs was tested at pH levels ranging from 5 to 9. The SeNPs is most effective at a pH of 7, which corresponds to a neutral medium. The increase in pH value from acidic pH to neutral values enhanced the SeNPs activity, while the alkaline values had lower antibacterial action.

MIC and MBC

Excessive Se intake leads to a side effect condition called selenosis which may be a combined with hair and nail changes, gastrointestinal issues such as nausea, vomiting, and diarrhea, or neurological effects such as peripheral neuropathy [33]. The symptoms can range from mild to severe, depending on the dosage and duration of exposure [34]. To mitigate these risks and ensure safe application, the current work investigate the MIC studies according the CLSI guidelines to determine the most effective low dose from SeNPs against the tested bacteria. For adults, the recommended daily allowance (RDA) is usually 60 µg. For optimum health, this is the bare minimum [35]. However, 400 µg per day is the adult tolerable upper intake level (UL). This is the highest dosage that is thought to be safe for long-term use without a doctor’s supervision [36].

At 2 and 50 µg/mL SeNPs (MIC values), the biocidal effect against S. aureus ATCC 25,923 and MRSA growth, respectively, was noticeably greater than at lower concentrations (Fig. 11). MRSA revealed a complete resistance against the cell-free bacterial metabolites of L. fermentum ESA5. Compared to the SeNPs, linezolid inhibited S. aureus ATCC 25,923 and MRSA at 4 and 100 µg/mL, respectively. In contrast to Na₂SeO₃ (MIC 6 µg/mL and MBC 7 µg/mL), the bacterial metabolites of L. fermentum ESA5 shown antibacterial activity against S. aureus ATCC 25,923 at MIC 14 µg/mL and MBC 15 µg/mL. Figure 11 displays the MBC results, indicating that the MIC values of the SeNPs are consistent with the MBC values. The MBC values of SeNPs against S. aureus ATCC 25,923 and MRSA were 2 and 50 µg/mL, respectively, while those of the conventional antibiotic were 5 and 110 µg/mL. Crucially, the biogenic SeNPs exhibited an exceptional MIC of 2 µg/mL against S. aureus ATCC 25,923, marking them as significantly more potent (up to 100-fold) than most previously reported chemically or myco-synthesized SeNPs (Table 4). Furthermore, the activity against MRSA (MIC = 50 µg/mL) is superior to the 80 µg/mL MIC reported for similar materials. The highly significant finding that the MIC is equivalent to the MBC for both tested strains confirms that the L. fermentum-stabilized SeNPs exert a strong microbicidal effect, which is highly desirable for topical antimicrobial formulations targeting resistant pathogens.

Fig. 11.

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of SeNPs against S. aureus ATCC 25,923 and MRSA ATCC 43,300 compared to linezolid, cell-free bacterial metabolites of L. fermentum ESA5, and Na₂SeO₃. Bars represent SE. Different letters are indicated to the significant variations in access treatment means from repeated-measures ANOVA (P < 0.05)

Table 4.

Comparative MIC/MBC of SeNPs with previously published against S. aureus and MRSA

Pathogen	SeNPs type/stabilizer	Synthesis method	MIC (µg/mL)	MBC (µg/mL)	References
S. aureus ATCC 25,923	Current biogenic SeNPs (L. fermentum metabolites)	Biological (bacterial)	2	2	Current study
S. aureus (ATCC 6538)	Myco-synthesized (Penicillium chrysogenum MZ945518)	Biological (Fungal)	312.5	N/A	El-Sayed et al. [37].
S. aureus (ATCC 25923)	Chemically synthesized (Bovine serum albumin)	Chemical reduction	≈ 7.8 (Growth Inhibition)	N/A	Webster & Tran [38]
S. aureus	Phyto-synthesized (Cassia javanica)	Biological (Plant)	500	N/A	Soliman et al. [39].
B. subtilis			500
S. aureus ATCC 25,213	Chemically synthesized (NaBH4)	Chemical reduction	8	N/A	Huang et al. [40].
K. pneumonia R12K2637			24
E. coli E76227			32
P. aeruginosa BJ915			24
P. aeruginosa NCIM 5031	Biogenic synthesis (Shewanella sp.)	Biological (Bacterial)	160	80	Annamalai et al. [41].
Listeria monocytogenes ATCC 15,313	Chemically synthesized (Ascorbic acid)	Chemical reduction	60	30	Alagawany et al. [42].
Salmonella enterica MTCC 1253			100	50
K. pneumoniae ATCC 11,296	Biogenic synthesis (P. mirabilis PQ350419)	Biological (Bacterial)	110	110	Elshikiby et al. [43].
P. mirabilis PQ350419			150	150
Bacillus cereus BIPC04	Biogenic synthesis (B. cereus BIPC04)	Biological (Bacterial)	75 (mM)*	75 (mM)*	Pouri et al. [44].
S. aureus (Clinical Strains)	Biogenic synthesis (L. acidophilus)	Biological (Bacterial)	1–10	N/A	Alam et al. [18].
MRSA (ATCC 43300)	Current biogenic SeNPs (L. fermentum metabolites)	Biological (Bacterial)	50	50	Current study
MRSA (ATCC 700699)	Chemically synthesized (Ascorbic acid)	Chemical reduction	80	160	Han et al. [45].
MRSA (Clinical Strains)	Phyto-synthesized (P. guajava)	Biological (Plant)	50 − 800	N/A	Ibrahim et al [46].

N/A (not applicable); indicates that the MBC was not determined in the cited study, which focused primarily on growth inhibition (MIC). ^*While the majority of antimicrobial data is standardized to µg/mL for clinical relevance, results reported in mM (e.g., Pouri et al. [44]). are kept in their original format to maintain the accuracy of the source data without introducing rounding errors

Characterization of the optimized SeNPs

FT-IR measurements for SeNPs revealed two main regions: the functional group and the fingerprint region (Fig. 12). The peak in the spectra at 3343 cm⁻¹ indicates the solvent, O–H stretching vibration. Asymmetric amines (2919, 2219 cm⁻¹) appeared at SeNPs spectrum. The assignment of aliphatic and aromatic C-N at 1632, 1588, 1412, 132, and 1260 cm⁻¹, respectively, verified the existence of proteins as capping and stabilizing agents throughout biosynthesis. The SeNPs pattern showed peaks at 2919, 2219, 1632, 1588, 1390, 1412, 1320, and 1250 cm⁻¹, which showed how the amine groups in L. fermentum proteins interacted with Se. The stretching vibration of the carbonyl group (C = O) within the amide is detected at 1632 cm⁻¹ and 1588 cm⁻¹. The attendance of SeNPs as a Se–O was verified by the metal–oxygen vibrations that showed at 1053 cm⁻¹ [26].

Fig. 12.

FT-IR of the biosynthesized SeNPs

XRD analysis examined the crystalline structure of the optimized SeNPs generated by L. fermentum (Fig. 13). At angles 23.750°, 30.079°, 41.640°, 43.918°, 45.746°, 52.068°, 56.118°, 61.705°, and 65.429°, the results showed clear, distinctive peaks of SeNPs. Accordingly, these angles match the crystallographic planes of (100), (101), (110), (102), (111), (201), (212), (202), and (210). The hexagonal structure of SeNPs is responsible for the detected diffraction peaks’ lattice constants, a = 4.357 A and c = 4.945 A, which match with the standard JCPDS data No. 06-0362. The final product was pure since there were no diffraction peaks from contaminants. SeNPs’ crystallite size was assessed using Scherrer’s equation: D = Kλ/(β cos θ); θ is the angle of diffraction, λ is the x-ray wavelength, β is the full width at half maximum, K is the constant = 0.94, and D is the particle size. It has been determined that the crystallite size of SeNPs is ≈ 43.38 nm.

Fig. 13.

XRD of the biosynthesized SeNPs

The TEM micrograph of SeNPs confirmed the efficient biogenesis of rod-shaped SeNPs with average diameters ranging from 30.94 to 43.94 nm (Fig. 14A). SeNPs possessed a negative charge (-20.93 ± 5 mV) based on the Zeta potential data (Fig. 14B).

Fig. 14.

TEM; (A), and Zeta potential; (B), investigations of the biosynthesized SeNPs. Bar scale = 100 nm. Zeta potential distribution of SeNPs is measured as − 20.93 ± 5 mV

Discussion

MRSA is a harmful bacterium that is resistant to β-lactam medications. It is challenging to treat the infections brought on by this strain because of its resistance [47]. S. aureus causes might cause several human illnesses such as urinary tract infections, meningitis, lung infections, gastroenteritis, bacteremia, osteomyelitis, and other infections [48]. The goal of this research is to identify a different, environmentally friendly method of treating infections, particularly those caused by MRSA that are resistant to the available commercial medications. The cell-free supernatant of L. fermentum OR553490 was employed in this investigation as a simple, low-cost, and secure one-step technique for the extracellular production of SeNPs. The red coloration that L. fermentum’s cell-free supernatant produces indicates that the bacterium’s metabolites may cause a bio-reduction of colorless Se into non-toxic red metallic SeNPs [26]. Using P. aeruginosa culture supernatant, Dwivedi et al. [49]. hypothesized that NADH and NADH-dependent reductases act as redox agents to reduce selenite metal ions to Se nanospheres. Reductases can also act as capping agents to ensure the formation of thermodynamically stable nanostructures [50]. It has been observed that L. fermentum produces NADH and NADH-dependent reductase enzymes [51]. Thus, it is anticipated that a multi-component redox system comprising NADH and probably NADH-dependent reductases in the cell-free supernatant of L. fermentum will catalyze the synthesis of SeNPs.

In metals, the surface SPR is excited to produce the color look. This phenomenon was verified by measuring the reaction mixture’s maximal SPR peak using UV-visible spectrum analysis. UV-visible absorbance spectroscopy is a particularly helpful technique for examining metal NPs since the locations and forms of the peaks are affected by particle size. The optical characteristics of SeNPs were confirmed by the SPR peak of SeNPs produced in the reaction combination of L. fermentum cell-free supernatant and Na₂SeO₃ solution, which was found at 262–272 nm and is in line with earlier research [26]. Testing many factors, including Na₂SeO₃ concentrations, the ratio of cell-free bacterial metabolites to Na₂SeO₃, temperature, and pH, allowed for the optimization of SeNP production. It was demonstrated that a 1:1 (v/v%) combination of cell-free bacterial metabolites and 30 mM Na₂SeO₃ at 30 °C were the ideal conditions for SeNPs’ action on S. aureus. According to El-Dein et al. [52]., a mixing ratio of 1:3 v/v% improved the production of NPs using bacterial metabolites. The low levels of enzyme substrate may cause the minimal production of SeNPs at lower concentrations of Na₂SeO₃ solutions (10, 20 mM). Still, the toxicity of the higher concentration of Na₂SeO₃ solution (100 mM) prevented the development of SeNPs [26, 50]. Abdelmoneim et al. [53]. found that 40 °C was optimal for the consistent synthesis of NPs. High temperatures lead to the breakdown or inactivation of biomolecules involved in the biosynthesis processes which may decrease the rate of NPs formation. Moreover, a redshift in the absorption peak indicated that higher temperatures and precursor concentrations were mainly responsible for the formation of bigger NPs [54].

While the RSM model suggested a potentially higher SPR intensity and NPs yield at an acidic pH 5, the current study strategically selected pH 7 for the optimized protocol. In accordance to the laboratory observations, it was found that although reduction occurred rapidly at pH 5, the resulting SeNPs exhibited a higher susceptibility to irreversible aggregation and precipitation within the 72-hour synthesis window. This is likely due to the partial denaturation or reduced solubility of the L. fermentum capping proteins near their isoelectric points in acidic conditions [55, 56]. By maintaining a neutral pH, it was ensured that the proteinaceous capping layer remained structurally robust, providing superior electrostatic and steric stabilization [57, 58]. This choice was critical for preserving the uniform rod-shaped morphology and ensuring that the particles remained in a stable colloidal suspension for pharmaceutical testing [59]. Furthermore, from a practical standpoint, the synthesis at pH 7.0 proved more reproducible and easier to scale, as it avoided the rapid shifts in metabolite activity often observed in more acidic environments [60].

The multivariate optimization technique was validated by the RSM-optimized SeNPs, which not only showed maximum yield but also the highest antibacterial activity against MRSA. The optimal conditions effectively determined as 30 °C, pH 7, 10–30 mM Na₂SeO₃, and a mixing ratio of no more than 1:4 v/v% between cell-free supernatant of L. fermentum and Na₂SeO₃ solution. There is an ideal threshold, beyond which too much precursor material inhibits the probiotic’s reductase enzymes and reduces yield, as seen by the quadratic dependency on Se concentration.

The antibacterial activity against MRSA was recorded during the optimization investigations for SeNPs production. The findings indicated that the physicochemical properties of SeNPs may enhance the antibacterial action and the interactions between the biogenic NPs and the bacterial cells. These findings are consistent with the well-established shape- and size-dependent antimicrobial mechanisms of SeNPs. Combined, these findings support SeNPs’ possible antibacterial effectiveness and point to their prospective uses in the pharmacological, environmental, and therapeutic domains. More research is needed to improve the selectivity and optimize treatment settings to assist in the development of targeted SeNPs-based antimicrobial formulations. A dose-dependent response was suggested by the fact that SeNPs generated with the greatest concentration of Na₂SeO₃ often showed higher antibacterial activity than those synthesized with ≤ 50 mM. The synthesis of SeNPs was unexpectedly prevented at 100 mM, which was the single exception. Additionally, the increased antibacterial potential of SeNPs in conjunction with L. plantarum S14 against S. aureus, B. subtilis, E. coli, and P. aeruginosa was reported by Yanez-Lemus et al. [54]. However, other NPs, like zinc oxide NPs, which were biosynthesized using the probiotic-bacteria L. fermentum, demonstrated more potent antibacterial activity against Serratia marcescens, Vibrio harveyi, and V. parahaemolyticus, with inhibition zones ranging from 3 to 15 mm at concentrations of 30 to 60 mM, while it diminished at higher concentrations [61]. While AgNPs were biosynthesized using L. fermentum HwOs-2, and at a concentration of 50 mM, they demonstrated strong antibacterial activity against MRSA and Salmonella typhi, with zones of inhibition extending from 20.3 mm to 27.6 mm and 20.3 mm, respectively [62].

Compared to lower temperatures, the higher inhibition zone against MRSA demonstrated that the synthesis of SeNPs at 30 °C was the most effective. The antibacterial efficacy was lower at 20 °C and 50 °C, as evidenced by lower inhibition zone values. The higher antibacterial activity of NPs at 30 and 40 °C was probably caused by the improved solubility and increased availability of Se ions. In a similar panner, the biosynthesis of SeNPs using B. licheniformis F1 at 37 °C improved the biosynthesis processes compared to lower and higher temperatures [63]. SeNPs were synthesized at 30 °C and 37 °C, with the highest synthesis occurred at 37 °C, according to Wadhwani et al. [64]. and Lortie et al. [65]. They were also reported the unable biosynthesis of SeNPs at higher temperatures. The influence of pH on NPs synthesis and antibacterial activity was also studied. At a constant temperature of 30 °C, reactions were carried out at pH 5–6 (acidic circumstances), pH 7 (the secretome’s normal pH), and pH 8–9 (basic conditions). Inhibition zones measured 6–9 mm at pH 5 and 6 and 11–14.5 mm at pH 8 and 9. A less effective antibacterial action is suggested by the measured inhibition zone of 15.9 mm at pH 7, which is consistently smaller than the values at pH 5–6 and 8–9. According to our hypothesis, Se ions may react with OH⁻ ions at this pH level to generate precipitates of Se hydroxide, which may then oxidize into Se oxide, decreasing the amount of Se ions available for the creation of NPs. Furthermore, a crucial factor for the uses of SeNPs is their stability when synthesized at different pH levels. According to Wang et al. [63]., SeNPs were more stable at pH 7–9 after two days than at pH 1–5, despite some precipitation and instability. When the pH is between 7 and 9, there is very little difference in the particle size range of SeNPs with zeta potential values below − 30 mV, demonstrating remarkable stability of SeNPs. Based on these results, SeNPs for subsequent characterizations were synthesized by incubating the cell-free supernatant at 30 °C, pH 7, with 60 mM and a mixing ratio of 1:1 (v/v%) for 84 h. This finding raises the possibility that other elements could affect antibacterial activity, such as particular interactions between the NPs and microbial cells. Crucially, the antibacterial activity of the SeNPs in this investigation is either equal to or better than that seen in the literature.

The stability of NPs is associated with common issues. NPs’ aggregation and agglomeration are among the main problems that limit their utilization in various applications. A discernible function of capping agents is to stop NPs from aggregating [50]. The incidence of proteins surrounding the NPs as capping agents was verified by the FT-IR analysis. Protein as capping agents improves the stability of NPs by preventing their accumulation and aggregation through cysteine and amine residues [26]. It was discovered that SeNPs-L. fermentum has a negative charge of -20.93 ± 5 mV, which promotes the stability of the NPs grains by creating a repulsive force. Laslo et al. [66]. documented that L. casei produced SeNPs with a maximum Zeta potential of − 23 mV. It’s crucial to keep in mind that zeta potential surface charge of NPs contributes significantly to their antibacterial action through electrostatic adhesion contact with microbial cell membranes [67]. The disadvantage of a green nanofactory that used a range of bacteria, such as Lactobacillus sp., Bifidobacter sp., and L. lactis, to create SeNPs was that it produced big NPs with sizes ranging from 100 nm to 550 nm [26].

Several studies showed that the biosynthesized SeNPs have strong antibacterial properties. SeNPs have potent antibacterial characteristics with inhibitory zones ranging from 12 to 15 mm [68]. Determining the MIC values of SeNPs is crucial as antibacterial action is frequently dose-dependent. The biosynthesized SeNPs using L. fermentum showed a stronger biocidal impact against MRSA than the traditional anti-MRSA linezolid, inhibiting the bacterium at 110 µg/mL with MIC and MBC values of 50 µg/mL. On the other hand, Soliman et al. [68]. found that the MIC of SeNPs against B. subtilis and S. aureus was 500 µg/mL. Alam et al. [18]. synthesized SeNPs using L. acidophilus and recorded their antimicrobial action against K. pneumonia, E. coil, S. aureus, and P. aeruginosa with MIC values of 1–10 µg/mL.

Highly reactive oxygen species, such as OH⁻, H₂O₂⁻, and O₂²⁻ could be produced by SeNPs. The generation of H₂O₂ from the surface of SeNPs was one of the main chemical species responsible for the antibacterial activity [69–71]. Electron-hole pairs (e⁻h⁺) can form in Se with defects because they can be triggered by both visible and ultraviolet light. The holes in SeNPs split H₂O molecules into H⁺ and OH⁻. Radical dots O^− 2, are produced when oxygen molecules dissolve in water. Hydrogen peroxide anions (HO₂-) are created when these radicals combine with H⁺ to form the HO₂ radical, which then interacts with electrons. When these anions mix with hydrogen ions, H₂O₂ molecules are produced. Bacteria can be killed by the generated H₂O₂ because it can pass through their cell membranes [72]. Superoxide and hydroxyl radicals, on the other hand, cannot cross the cell membrane and remain in touch with the bacteria’s surface due to their negative charge. However, the cell can be exposed to H₂O₂ [73].

The recognized safety criteria for Se were taken into consideration when designing the microbiological investigation. Although elemental selenium is necessary, too much of it can cause selenosis, a side-effect syndrome that manifests as peripheral neuropathy, gastrointestinal distress, and changes in the hair and nails [33]. Since reducing these dangers is crucial, the current study concentrated on figuring out the MIC in accordance with CLSI criteria in order to identify the lowest effective dosage. SeNPs must be shown to be effective at concentrations far within acceptable limits, as the Recommended Daily Allowance (RDA) is 60 µg/day and the Tolerable Upper Intake Level (UL) is 400 µg/day for adults [35, 36]. The outcomes validate the great potency of the SeNPs that were produced. The biocidal effect was achieved with remarkably low MIC values of 2 µg/mL against S. aureus ATCC 25,923 and 50 µg/mL against the challenging MRSA strain. These concentrations provide a significant margin of safety when compared to the UL. Most significantly, the SeNPs worked better than the conventional antibiotic linezolid, which required higher dosages to cause inhibition (MIC of 4 µg/mL against S. aureus and 100 µg/mL against MRSA). The extremely significant fact that the MBC values of the SeNPs (2 and 50 µg/mL) were equivalent to their respective MIC values against both strains indicate a robust, dose-dependent bactericidal effect rather than a purely static inhibition. The higher effectiveness of the biogenic SeNPs in comparison to the precursor, Na₂SeO₃ (MIC 6 µg/mL), and the L. fermentum ESA5 metabolites (MIC 14 µg/mL) highlights the enhanced antibacterial mechanism offered by the nanosized delivery system. This suggests that SeNPs offer a means of delivering a strong antibacterial payload while lowering the risk of systemic toxicity, particularly against resistant bacteria like MRSA. Therefore, SeNPs may be considered a viable substitute for conventional antibiotics in the treatment of MRSA-related infections.

Conclusion

The current study provided an effectively and sustainably production method for the biosynthesis of SeNPs using the crude metabolite of Lactobacillus fermentum (OR553490). This method produces SeNPs with competitive size (30.94 and 43.94 nm), shape (uniform rod-shaped), stable (proteins as a capping and stabilizing agent), and antibacterial properties especially against MRSA. The biosynthesis of SeNPs was optimized by investigating different parameters including concentration of Se precursor (Na₂SeO₃), mixing ratio between the cell-free bacterial metabolites, pH 7 value of the reaction mixture, and temperature. Also, response surface methodology was used for optimizing SeNPs formation. The optimal conditions were recorded as 30 mM Na₂SeO₃, mixing ratio of 1:1 to 1:4 v/v% between Na₂SeO₃ and cell-free bacterial metabolites of L. fermentum, pH 7, and 30–40 °C. The finding implies that the tested variables could influence the antibacterial activity of SeNPs against MRSA. Future in vitro and in vivo studies should be conducted to investigate the cytotoxicity and antibacterial mode of action for the optimized SeNPs.

Abbreviations

AMR

Antimicrobial resistance

ANOVA

One-way analysis of variance

BBD

Box–Behnken design

CFU

Colony-forming unit

FT-IR

Fourier transform-infrared spectroscopy

LAB

Lactic acid bacteria

MBC

Minimum bactericidal concentration

MHA

Mueller–Hinton agar

MHB

Mueller–Hinton broth

MIC

Minimum inhibition concentration

MRSA

Methicillin-resistant Staphylococcus aureus

RSM

Response surface methodology

SE

Standard error

SeNPs

Selenium nanoparticles

SPR

Surface plasmon resonance

TEM

Transmission electron microscopy

UV-Vis

Ultraviolet-visible

XRD

The X-ray diffraction

Author contributions

Contribution the idea and the design of the study: Mohamed El‑Zahed, Mahmoud Khalifa; Methodology: Mohamed El‑Zahed, Hajar El-Sheikh, Mahmoud Khalifa; Data Curation and Formal Analysis: Mohamed El‑Zahed, Hajar El-Sheikh, Mahmoud Khalifa; Investigation and Resources: Mohamed El‑Zahed, Hajar El-Sheikh, Mahmoud Khalifa; Original draft preparation: Mohamed El‑Zahed, Hajar El-Sheikh, Mahmoud Khalifa; Writing, Reviewing and Editing: Mohamed El‑Zahed, Hajar El-Sheikh, Mahmoud Khalifa; Supervision: Mohamed El‑Zahed, Mahmoud Khalifa. All authors read and approved the final manuscript.

Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). Not applicable.

Data availability

The partial sequence of the 16 S ribosomal RNA gene of *Limosilactobacillus fermentum* strain ESA5 obtained in the current study was deposited in the NCBI GenBank database under accession number: OR553490 and is available at the following URL: (https://www.ncbi.nlm.nih.gov/nuccore/OR553490). The datasets generated during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethical approval

Not applicable. This study does not involve human participants, data, or animals. The bacterial strains used are standard laboratory and clinical isolates.

Consent to participate

Not applicable.

Consent to publish

All authors have read the final version of the manuscript and give their formal consent for the publication of this research in the journal Discover Nano.

Clinical trial number

Not applicable.

AI disclosure statement

During the preparation of this work, the authors used Gemini for the purpose of linguistic refinement and grammatical checking to improve readability. Following the use of this tool, the authors performed a comprehensive review and manually edited the content to ensure scientific accuracy. The authors take full responsibility for the integrity of the experimental data, the response surface methodology (RSM) calculations, the interpretation of the results, and the final content of the publication. The experimental findings and statistical analyses reported herein were generated entirely by the authors without the assistance of artificial intelligence.

Competing interests

The authors declare no competing interests.