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Universidade Federal de Santa Maria
Ci. e Nat., Santa Maria, v. 47, e84264, 2025
DOI: 10.5902/2179460X84264
ISSN 2179-460X
Submitted: 27/06/2023 • Approved: 01/08/2024 • Published: 29/04/2025
Chemistry
Synthesis and antimicrobial activity of iron oxide/silver nanocomposites against Pseudomonas aeruginosa biofilms
Síntese e atividade antimicrobiana de nanocompósitos de óxido de ferro/prata contra biofilmes de Pseudomonas aeruginosa
Aline Fernandes BarcelosI
Alliny das Graças AmaralI
Lílian Carla CarneiroII
Plínio Lázaro Faleiro NavesI
Luciana Rebelo GuilhermeI
I Universidade Estadual de Goiás, Goiás, GO, Brasil
II Universidade Federal de Goiás, Goiás, GO, Brasil
RESUMO
As Pseudomonas aeruginosa são reconhecidas pela patogenicidade, resistência aos antimicrobianos e habilidade de crescer em biofilmes o que as tornam ainda mais preocupantes na área da saúde. Neste contexto, a nanotecnologia permite o desenvolvimento de novos materiais contra micro-organismos multirresistentes. No presente trabalho foram preparadas dispersões coloidais de nanopartículas de óxido de ferro (IONPs), prata (AgNPs) e de compósito de óxido de ferro/prata (IO/Ag-NCs) que foram testados quanto à toxicidade e à atividade contra biofilmes de isolados clínicos de P. aeruginosa. Os nanocompósitos heterodímeros apresentaram morfologia esférica, potencial zeta indicando relativa estabilidade coloidal apesar da sua polidispersão. As nanopartículas não apresentaram toxicidade para Artemia salina nas concentrações testadas e inibiram a formação de biofilmes de alguns dos isolados clínicos de P. aeruginosa. As NPs resultaram na inibição da formação de biofilme de alguns dos isolados clínicos de P. aeruginosa. As AgNPs inibiram a formação de biofilme em 3 isolados, enquanto as IONPs a reduziram em 4 e as IO/Ag-NCs inibiram em 3 isolados de P. aeruginosa. Nossos resultados indicam que novas pesquisas devem ser direcionadas com estratégias que considerem o aumento da concentração de prata e a utilização de IO/Ag-NCs como nanocarreadores para o controle da formação de biofilmes microbianos.
Keywords: Composto híbrido; Heterodímero; Artemia salina
ABSTRACT
Pseudomonas aeruginosa are known for their pathogenicity, antimicrobial resistance, and ability to grow in biofilms, making them even more problematic in health care. In this context, nanotechnology allows the development of new materials against multiresistant microorganisms. The present study prepared colloidal dispersions of iron oxide (IONPs) and silver (AgNPs) nanoparticles and iron oxide/silver composite (IO/Ag-NC), testing them for toxicity and activity against biofilms of P. aeruginosa clinical isolates. Heterodimer nanocomposites showed spherical morphology and zeta potential, indicating relative colloidal stability despite their polydispersity. The nanoparticles did not present toxicity to Artemia salina at the tested concentrations and inhibited the biofilm formation of some P. aeruginosa clinical isolates. Nanoparticles (NPs) inhibited the biofilm formation of some P. aeruginosa clinical isolates: AgNPs inhibited biofilm formation in three isolates, IONPs reduced it in four, and IO/Ag-NCs inhibited it in three P. aeruginosa isolates. Further research should focus on strategies that consider increasing silver concentration and using IO/Ag-NCs as nanocarriers for controlling microbial biofilm formation.
Palavras-chave: Colloidal dispersion; Heterodimer; Artemia salina
Individual nanoparticles (NPs) have specific characteristics and applications, such as iron oxide nanoparticles (IONPs) and silver nanoparticles (AgNPs), which individually have antimicrobial properties (Chen et al., 2013; Ismail et al., 2015). The synthesis of nanocomposites containing IONPs and AgNPs (IO/Ag-NCs) combines the characteristics of each nanostructure to improve the applications and limitations of an individual component, including their use in microbial contaminant treatments (Sharma & Jeevanandam, 2013).
In the case of IONPs, they have been tested against the formation of biofilms by microorganisms such as Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa, and the results of inhibiting the growth of these biofilms have been promising. (Bruckmann et al., 2022). On the other hand, AgNPs can significantly reduce the mass of pathogenic bacteria that are resistant to antibiotics, such as Burkholderia pseudomallei, S. aureus (ATCC 43300, ATCC 25923 e 29213) e P. aeruginosa (ATCC 15442 e ATCC 27853), E. coli (ATCC 35218) and Salmonella Typhi (ATCC 14028) (Diniz et al., 2020; Hadi et al., 2024; Maheshwari, 2024; Tun et al., 2024).
Bacterial resistance to antimicrobials is a natural phenomenon that may occur due to inappropriately using antibiotics without prescriptions and discontinuing drug use before the predetermined period (Loureiro et al., 2016). In this context, developing antimicrobial biological agents has become necessary due to the decline of clinically effective antibiotics, especially for refractory biofilm-related infections. Biofilm provides a practical defense against antimicrobial agents, facilitating bacterial spread throughout the patient’s body and the development of antimicrobial resistance, such as those from P. aeruginosa infections (Ribeiro et al., 2018; Zhang et al., 2020). P. aeruginosa is a Gram-negative bacterium responsible for hospital infections due to its high adaptability, exhibiting multidrug resistance phenotypes that limit the efficacy of most available antimicrobials. In this context, new methods are required to reduce the morbidity and mortality of patients infected by these microorganisms (Armijo et al., 2020; Boucher et al., 2013; Zamperini et al., 2017).
The lethality test with Artemia salina shows a good relationship with other laboratory assays. A. salina is a small halophilic invertebrate from the Crustacea class and Brachiopoda subclass living in saltwater. Its life cycle comprises cyst, nauplii, and metanauplii stages. A. salina is highly valued for detecting toxicity through hatched nauplii (lethality assay). The lethality test with A. salina is fast, convenient, and inexpensive, thus extensively used in research and applied toxicology (Ntungwe N et al., 2020).
Besides their antimicrobial activity, the toxicity of novel nanoparticles must be assessed, as composite material toxicity may vary from individual components. So, the present study synthesized AgNP, IONP, and IO/Ag-NC colloids and verified nanocomposite toxicity with the A. salina lethality assay and antimicrobial activity against biofilms of P. aeruginosa clinical isolates.
2.1 Materials
The nanocomposites of IONPs, AgNPs, and IO/Ag-NCs were synthesized using the chemical reagents, FeCl2.4H2O (JT Baker, USA), FeCl3.6H2O (Neon, Brazil), NH4OH (Neon, Brazil), citric acid (Dinâmica, Brazil), AgNO3 (Sigma-Aldrich, USA), sodium citrate (Sigma-Aldrich, India), and NaBH4 (Sigma-Aldrich, USA).
2.2 Synthesis
IONPs were synthesized with the coprecipitation method, according to the methodology by Khalafalla and Reimers (1980). In a beaker, 6 g of FeCl2.4H2O and 12 g of FeCl3.6H2O were dissolved in 50 mL of deionized water; then, 25 mL of 30% NH4OH was added by mechanical stirring at 600 rpm. The beaker with the resulting precipitate was separated by magnetic decantation using a permanent neodymium magnet, which helped wash the solid ten times with deionized water (Khalafalla & Reimers, 1980).
IONPs were functionalized by heating a beaker containing the nanoparticles at 60°C with mechanical stirring at 250 rpm. Then, a 0.87 mol L-1 citric acid solution was added in drops until reaching a pH of 2.5 to 3.5. The dispersion was washed ten times with deionized water aided by a permanent neodymium magnet. Next, 150 mL of water was added, and NH4OH was included in drops to reach a pH of 7. A colloid obtained by nanoparticle functionalization was filtered on a 250-nm membrane filter and sterilized in an autoclave.
AgNPs were synthesized by adding 3 mL of a 0.016 mol L-1 AgNO3 solution and water up to 200 mL. Then 5 mL of a 0.0054 mol L-1 sodium citrate solution was added, starting magnetic stirring and stopping the timer. At 15 minutes, 8 mL were added at a time to the NaBH4 40 mmol L-1 solution stored under refrigeration. At 20 minutes, magnetic stirring stopped, and the reaction mixture remained uncapped in the dark for two hours (Kereselidze et al., 2012). Finally, the resulting colloid was sterilized in an autoclave.
IO/Ag-NC synthesis occurred in a beaker, which received 400 µL of IONPs and 20 mL of deionized water, and 100 mL of an AgNO3 0.01 mol L-1 solution was dripped in the tip sonicator. Subsequently, 400 µL of 1% (w/v) sodium borohydride was added, and the mixture was ultrasonicated for 20 minutes at 40% power. The solution was heated under vigorous mechanical stirring at 80°C for two hours. The IO/Ag-NCs were washed thrice with deionized water using magnetic decantation (adapted from LIU et al., 2008). Finally, the IO/Ag-NCs were dispersed in water, sterilized in an autoclave, and stored.
2.3 Characterization
Atomic absorption spectroscopy (AAS) identified metal concentrations. For AAS analysis, 100 μL of each nanocomposite sample (IONPs, AgNPs, and IO/Ag-NCs) was opened with concentrated nitric acid. Then, 20 mL of deionized water was added, and the solutions were heated for 20 minutes. Subsequently, the samples were transferred to 100-mL volumetric flasks. The Perkin Elmer AAnalyst 400 atomic absorption spectrophotometer reads the atomic absorptions. IONP samples had 3,560 mg L-1 of iron ions, and AgNPs had 100 mg L-1 of silver ions. The IO/Ag-NC sample showed iron and silver ion concentrations of 72 and 280 mg L-1, respectively.
Absorption spectroscopy was analyzed in the ultraviolet and visible (UV-vis) region in a Perkin Elmer Lambda 35 UV-Visible Spectrometer equipment. The technique determined optical properties and calculated the samples’ band gap (Eg) using the Tauc plot in OriginLab. Eg was determined by extrapolation from the absorption edge provided by the following relationship: αhν = α0 (hν - Eg)n. In this equation, hν is the energy of incident photons, Eg is the optical gap value corresponding to the transitions indicated by n, and α٠ is constant depending on the transition probability (BASAK et al., 2021). The best linear fit was for n = 2.
Vibrational absorption spectroscopy in the infrared region was analyzed in a Perkin-Elmer Spectrum Frontier FT-IR/NIR (Perkin-Elmer Corp., Norwalk, CT). The samples were prepared on KBr tablets.
The zeta potential and dynamic light scattering (DLS) were examined in a Malvern polystyrene U-shaped cell of the zeta potential in the Malvern ZetaSizer apparatus, Nano-ZS90 model. The dispersion index (DI) from the DLS analysis was evaluated according to (ISO, 2017).
The samples were analyzed with Transmission Electron Microscopy (TEM) and Selected Area Electron Diffraction (SAED) using the Jeol apparatus, JEM-2100 model, equipped with an energy-dispersive X-ray detector (EDS). The samples’ mean diameters and DIs were determined by analyzing the micrographs using ImageJ software.
2.4 Artemia salina toxicity test
The method by Molina-Salinas and Said-Fernández (2006), with minor modifications, determined the toxicity of compounds using the A. salina lethality assay. The artificial seawater medium (ASM) was prepared by dissolving sea salt (36 g L-1) and yeast extract (6 g L-1) in distilled water, and the solution was sterilized by autoclaving, measuring a pH of 8.5 (Molina-Salinas & Said-Fernández, 2006).
Then, 0.3 g of A. salina cysts were incubated in 500 mL of ASM at 25°C for 36 hours under a light shelter with constant oxygenation. After hatching, ten nauplii were attracted with light and transferred in 100 μL of fresh ASM to wells of a microplate previously prepared with 100 μL of nanoparticle dispersions, composing the concentrations of 20.5, 10.25, 5.125, 2.562, and 1.281 μg mL-1. The microplates were incubated at 25°C for 24 hours, and the number of dead and live nauplii per well was counted after this period.
2.5 Biofilm formation inhibition assay of P. aeruginosa
Thirteen (13) P. aeruginosa were tested: 11 clinical isolates and two American Type Culture Collection (ATCC) strains of P. aeruginosa (ATCC 27853 and ATCC 9027) from the laboratory collection.
The bacterial inocula were prepared from colonies grown for 24 hours on cetrimide agar in a sterile physiological solution (0.9% NaCl) with turbidity corresponding to 0.5 on the McFarland scale. Then, 100 µL of bacterial suspensions were added to 9.9 mL of TSB (trypticase soy broth), and 50 µL aliquots of the broth were transferred to 96-well polystyrene microplate compartments containing the nanocompound samples, an initial inocula of approximately 7.5x104 bacteria, and final concentrations of 0.258 µg mL-1 (IONPs), 1 µg mL-1 (AgNPs), and 0.258/1 µg mL-1 (IO/Ag-NCs) per well.
The microplates were incubated at 35.5ºC for 24 hours and then subjected to visual inspection, growth broth removal, and well washing twice with 150 µL of sterile physiological solution (SPS) in an Aquari® (MA ٦١٥, Brazil) automatic microplate washer to remove non-adhered cells. Next, the wells received ١٥٠ µL of ١٪ crystal violet and were washed thrice with ٢٠٠ µL of distilled water after incubating the dye for ١٠ minutes, and microplates were dried in an incubator at ٣٥.٥ºC for ٢٠ minutes. Finally, each well received ١٥٠ µL of absolute ethanol to stain the adhered bacteria, and the microplates were incubated at room temperature for ten minutes to read the optical densities of the wells at ٤٩٢ nm in the BioTech Epoch™ microplate reader (Naves et al., 2008).
The bacteria were classified for biofilm formation into four categories according to the ODCW (optical density of the control well) and ODAB (optical density of adhered bacteria) ratio, with the following criteria: If ODAB ≤ ODCW, bacteria are considered non-biofilm formers; if ODAB > ODCW ≤ 2x ODCW, bacteria are weak biofilm formers; if ODAB > 2x ODCW ≤ 4X ODCW, bacteria are moderate biofilm formers; and if ODAB > 4x ODCW, bacteria are strong biofilm formers (Stepanović et al., 2000).
The impact of IONPs, AgNPs, and IO/Ag-NCs on P. aeruginosa biofilm formation was evaluated by comparing the results of biofilm formation tests without the nanocomposites (untreated control).
2.6 Statistical analyses
All tests occurred in independent triplicates, and the results were organized by calculating mean and standard deviation values. Nanocomposite toxicity was defined by calculating the median lethal concentration (LC50) with the number of dead and alive nauplii per well in the A. salina lethality assay by Probit analysis with R software. The readings were compared with the untreated viability control to analyze biofilm formation results in the presence of IONPs, AgNPs, and IO/Ag-NCs.
3.1 Synthesis and characterization
Figure 1a presents the electronic absorption spectra in the ultraviolet and visible (UV-vis) region of the prepared colloidal dispersions (IONPs, AgNPs, and IO/Ag-NCs). The IONP spectrum shows a broadband bandwidth between 200 and 350 nm, indicating a very energetic band and charge transfer transition (LITTER; BLESA’, 1992). The image also presents an intense plasmonic band centered at 395 nm for AgNPs, confirming AgNP formation and suggesting a spherical and regular shape (Shervani et al., 2008).
The IO/Ag-NC spectrum suggests that the synthesized nanocomposite comprises silver and iron oxide nanoparticles. This spectrum shows two bands: one centered at 277 nm and another quite intense band centered at 431 nm. The former can be attributed to IONPs and suggests charge transfer transition (Kumar et al., 2014; Litter & Blesa, 1992), and the latter corresponds to a plasmonic band typical of silver nanoparticles with irregular shapes (Shervani et al., 2008)
Figures 1b, 1c, and 1d present Tauc plot extrapolations to determine the optical gap value of the samples. IONP (Figure 1b) and AgNP (Figure 1c) samples obtained optical gaps of 3.37 eV and 2.75 eV, respectively. These results were consistent with the literature, which identified values of 2.51 eV for AgNPs (Aziz et al., 2018) and 3.5 eV and 3.23 eV for IONPs (Basak et al., 2021). Figure 1d shows two optical gaps for IO/Ag-NCs (2.3 and 3.9 eV), with displaced values of individual AgNPs and IONPs, respectively. These values suggest that the gap energy in the nanocomposite decreases for AgNPs, shifting the plasmonic band to longer wavelengths (Caro et al., 2016; Sallam et al., 2018). The results indicate that the samples are semiconducting compounds and may serve as catalysts.
Figure 1 – a) Electronic absorption spectra in the UV-vis region; b) Tauc Plot of IONPs; c) Tauc Plot of AgNPs; and d) Tauc Plot of IO/Ag-NCs
|
a |
b |
|
c |
d |
Source: authors (2023)
Figure 2 shows the FTIR analysis spectra for NP samples in the 4,000 to 400 cm-1 region. IONP, AgNP, and IO/Ag-NC samples have bands in the region of 3,450 cm-1. Bands in the region of 3,400 cm-1 refer to O-H bond stretching due to moisture. The samples show bands in the region of 1,630 cm-1, and the absorption in the range of 1,715 ± 100 cm-1 is usually due to the C-O bond (carbonyl group) in molecules. All samples also presented bands in the region of 1,400 cm-1, and the absorption in this region refers to the CH2 folding in citric acid and sodium citrate used to synthesize NPs (Pavia et al., 2010).
Figure 2 - FTIR spectra of nanocomposites
Source: authors (2023)
The band (Figure 2) at approximately 1,270 cm-1 corresponds to the C-O bond stretching in citric acid and sodium citrate used to synthesize NPs. The bands between 1,000 and 400 cm-1 of the AgNP spectrum are characteristic of C-H bond deformation and out-of-plane bending of C-H and CH2 (Pavia et al., 2010).
The high-intensity bands in the region of 600 cm-1 in the IONP and IO/Ag-NC spectrum are typical of Fe-O stretching in inverse spinel iron oxide. Two bands around 630 and 590 cm-1 represent the γ-Fe2O3 (maghemite) Fe-O bond (Karimzadeh et al., 2016; Wang et al., 2014). Thus, the bands in the 620 and 570 cm-1 FTIR spectrum suggest that the IONPs in this study are maghemite.
Table 1 shows the results of IONP, AgNP, and IO/Ag-NC characterizations by zeta potential, hydrodynamic radius, and dispersion index (DI). IONPs and AgNPs presented hydrodynamic radii of 106.7 nm and 63.27 nm and DIs of 0.159 and 0.491, respectively. Thus, the DI data of the samples indicated IONPs with homogeneous size dispersions and AgNPs with inhomogeneous size dispersions. IONPs and AgNPs also presented zeta potentials of -37.7 mV and -43.5 mV, respectively, which are highly stable (as a rule of thumb, the potentials above +30 or below -30 mV are considered highly stable) (Makowski et al., 2019).
According to the ISO 22412 (2017), the DI of the samples indicated inhomogeneous size dispersions for IO/Ag-NCs, which showed hydrodynamic radii of 443.2 and 573.9 nm and DIs of 0.645 (based on the refractive index of iron oxide) and 0.806 (based on the refractive index of silver) for iron oxide and silver analyses. IO/Ag-NCs also showed zeta potentials of -21.9 mV and -15.1 mV for iron oxide and silver analyses, respectively. The zeta potential values of IO/Ag-NCs indicate instability, as they are outside, above, or below ± 30 mV, which suggests high colloidal system stability (Makowski et al., 2019).
Table 1 – Nanocomposite characterizations by zeta potential and DLS
|
Nanocompounds |
Zeta potential (mV) |
Hydrodynamic size (nm) |
Polydispersity index - PI |
Refractive index (n) |
|
IONPs |
-37.7 ± 12.1 |
106.7 |
0.159 |
2.33 |
|
AgNPs |
-43.5 ± 14.6 |
63.27 |
0.491 |
0.54 |
|
IO/Ag-NCs |
-21.9 ± 5.70 / -15.1 ± 5.68 |
443.2 / 573.9 |
0.645 / 0.806 |
2.33 / 0.54 |
Source: authors (2023)
Figure 3 presents the transmission electron microscopy (TEM) micrographs, nanoparticle diameter distribution plots, and selected area electron diffraction (SAED) of the samples of (a) IONPs, (b) AgNPs, and (c) IO/Ag-NCs. The IONP micrographs show spherical NPs, the AgNP micrographs present spherical and oval NPs, and the IO/Ag-NC micrographs demonstrate spherical (O), oval (0), and hexagonal () NPs.
Figure 3 – Micrographs, diameter distribution histogram, and SAED of a) IONPs, b) AgNPs, and c) IO/Ag-NCs. Spherical (O), oval (0), and hexagonal () surround particles with corresponding shapes
Source: authors (2023)
Figure 3(a) of the IONPs micrograph indicates that NPs may bond potentially through NP functionalization with citric acid, as the functionalized citrate ions bonded by cross-linking on IONP surfaces.
Figure 3(b) indicates the aggregation state for AgNPs, as the image shows NPs partially dispersed and partially aggregated, confirming the DLS and zeta potential analysis data that show little homogeneity for AgNPs.
Figure 3(c) presents IO/Ag-NC NPs with a layer in different directions, suggesting NC formations. However, the growth of NCs may have increased the aggregation state of the particles, as confirmed by DLS and zeta potential analyses. IO/Ag-NC micrographs suggest a heterodimer characteristic, as IONPs are directly attached to the silver surface but do not cover it completely.
The ImageJ program found mean diameters of 11.80 ± 3 nm, 15.87 ± 4 nm, and 15.51 ± 5 nm for IONPs, AgNPs, and IO/Ag-NCs, respectively. The size distribution histograms in Figure 3 represents these values. This analysis provides size distributions of NPs from TEM images, with information about mean sizes and standard deviations. The mean diameter found was expected according to the data for iron oxide in the literature (Bhattacharjee, 2016; Kim et al., 2015; Zhao et al., 2015).
Sonbol, Mohammed, and Korany (2022) synthesized AgNPs using the Phoma sp, Chaetomium globosum, and Chaetomium sp fungal isolates as silver-reducing agents. They calculated the particle size distribution of TEM images using the ImageJ program. Furthermore, their study showed mean sizes of 12.7, 10.7, and 16.1 nm for the three mentioned syntheses. As for the DLS analysis, the hydrodynamic radii of the particles were 98.41, 83.15, and 51.76 nm (Sonbol et al., 2022).
The SAED analysis evaluates the crystalline structure of biomaterials through the pattern of diffraction points obtained in TEM analyses. The d-spacing patterns of AgNPs and IONPs were considered to find the same patterns in NCs. Table 2 shows the peaks of the known d-spacing values representing the crystallographic plane.
Planes [220], [311], [400], [422], [511], and [440]/[214] (Figure 3(a)) and [111], [200], [220], [311], and [222] (Figure 3(b)) were found for IONPs and AgNPs, respectively. Consequently, d-spacing values showed the following planes representing silver and iron oxide in IO/Ag-NCs: [220], [311], [111], [200], [511], [220], and [311] (Figure 3(c)) (Galateanu et al., 2015; Mehtab et al., 2018; Njagi et al., 2011).
Table 2 – Miller indices of known d-spacing values for AgNPs, IONPs, and IO/Ag-NCs
|
Planes |
AgNPs |
IONPs |
IO/Ag-NCs |
|
d-spacing |
d-spacing |
d-spacing |
|
|
[220] |
2.968239834 |
2.842524161 |
|
|
[311] |
2.531966072 |
2.444390125 |
|
|
[111] |
2.251491613 |
2.232392008 |
|
|
[400] |
2.105484788 |
||
|
[200] |
1.948368242 |
1.985111663 |
|
|
[422] |
1.733102253 |
||
|
[511] |
1.630656339 |
1.583155228 |
|
|
[440] [214] |
1.487099413 |
||
|
[220] |
1.380357513 |
1.401345291 |
|
|
[311] |
1.191043354 |
1.189909567 |
|
|
[222] |
1.117505727 |
Source: authors (2023)
The AgNP planes were considered typical values of face-centered cubic structures. Silver showed no extra diffraction peaks, suggesting it is pure. These NPs with high purity and low toxicity are attractive for biological applications (Mehtab et al., 2018; Njagi et al., 2011).
The IONPs showed a d-spacing pattern of 1.48. Noval and Carriazo (2019) consider this pattern a reference to the 440 (Fe3O4) and 214 (α-Fe2O3) planes, suggesting the presence of hematite in IONP dispersions. IO/Ag-NCs showed diffraction peaks of both NPs, indicating their presence in the sample (Noval & Carriazo, 2019).
AgNPs are highly crystalline, with diffraction peaks corresponding to the face-centered cubic phase, agreeing with the reported ICDD 4-783; a = 4.0862Å. The analysis indicates a nanocrystalline sample with IONP indexing as a cubic crystalline network with surfaces according to ICDD file number 04-008-8146 (Galateanu et al., 2015; Njagi et al., 2011).
3.2 Artemia salina toxicity test
AgNPs, IONPs, and IO/Ag-NCs showed no toxicity to A. salina, as there was no LC50 at the tested concentrations. The lethal dose of NPs is higher than the highest evaluated concentration (20.5 µg mL-1). The limiting factor was that AgNPs have a maximum concentration of 100 µg mL-1, making it hard to test higher concentrations than the maximum one used in microdilution tests.
A previous study on AgNP toxicity in A. salina nauplii, under ISO TS 20787 guidelines, exposed nauplii to 0 (untreated control), 0.39, 1.56, 6.25, 25, and 100 mg L-1 for 24, 48, and 72 hours. The results indicated a low immobilization rate of nauplii within 24 hours, and their immobilization in a concentration-dependent manner occurred only after 72 hours. Thus, in 24 hours, the maximum tested concentration was insufficient to immobilize the nauplii (An et al., 2019).
The A. salina lethality test correlates well with more complex tests in determining antifungal, antiviral, and antimicrobial activities, among others. It is also an inexpensive and easily interpreted test extensively used to screen bioactive compounds (Krishnaraju et al., 2005; Luna et al., 2005; Pisutthanan et al., 2004).
3.3 Nanocomposite activity on biofilm formation of P. aeruginosa
All 13 P. aeruginosa formed biofilms, of which nine (69.23%) were classified as strong biofilm formers and four (30.77%) as moderate biofilm formers. The impact of nanocomposites on P. aeruginosa biofilm formation occurred variably and depended on clinical isolates.
IONPs reduced biofilm formation for Pa3, Pa11, Pa22, and Pa23 isolates; AgNPs decreased it for Pa3, and Pa11; and IO/Ag-NCs reduced it for Pa3, Pa11, Pa22, Pa23, and Pa28. However, IONPs increased biofilm formation for Pa5, Pa13, Pa ATCC 9027, and Pa ATCC 27853; AgNPs increased for Pa13, Pa20, Pa28, Pa29, and Pa ATCC 9027; while IO/Ag-NCs increased for Pa10, Pa13, Pa20, Pa29, Pa ATCC 9027, and Pa ATCC 27853. For the other bacteria, under the same treatment conditions, biofilm formation did not significantly decrease or increase (Figure 4).
Figure 4 – Biofilm formation of P. aeruginosa clinical isolates in the presence of a) IONPs (0.258 µg mL-1), b) AgNPs (1 µg mL-1), and c) IO/Ag-NCs (0.258/1 µg mL-1) compared to untreated controls (UC)
|
a b c |
Source: authors (2023)
The size of AgNPs is crucial in antibiofilm activity against multidrug-resistant P. aeruginosa because smaller particles have a more significant surface area contact with the microorganism. Antibiofilm activity occurs through Bi structure disruption and oxidative stress. Using AgNPs is promising for developing new antimicrobial systems against P. aeruginosa strains (de Lacerda Coriolano et al., 2021).
IONP diameters significantly influence the inhibition of bacterial biofilm formation (Sathyanarayanan et al., 2013). Our study found that IONPs with a diameter of 11.80 ± 3 nm at a concentration of 0.258 µg mL-1 inhibited biofilm formation in four of the analyzed isolates (Pa3, Pa11, Pa22, and Pa23).
A previous study showed that IONPs with diameters smaller than 10 nm, besides the dispersion concentration, significantly inhibited the growth of bacterial biofilms. IONPs at 10 µg mL-1 can impact P. aeruginosa biofilm growth, but only concentrations higher than 50 µg mL-1 significantly inhibited biofilm formation (Sathyanarayanan et al., 2013).
Zinc oxide NPs (~20 nm) at 350 μg mL-1 reduced biofilm formation by more than 94% in 15 P. aeruginosa clinical isolates ATCC 9027 (da Silva Bruckmann et al., 2022). In another study, AgNPs showed minimum inhibitory concentrations (MICs) of 1.406 - 5.625 µg mL-1 against multidrug-resistant P. aeruginosa in a concentration-dependent manner (Liao et al., 2019).
NCs can improve the stability and antimicrobial activity of AgNPs (known and extensively studied in antimicrobial testing) (Prabhu & Poulose, 2012; Zhang et al., 2020). Contrarily, our results indicate that IO/Ag-NCs did not increase the inhibition of P. aeruginosa biofilm formation. However, isolated AgNPs inhibited identical isolates, except for Pa28, which was affected only by IO/Ag-NCs.
Chitosan (CS) and zinc oxide (ZnO) nanocomposites with and without gentamicin were tested at concentrations lower than the MIC against P. aeruginosa PAO1 biofilms and provided significant reductions (p < 0.05). The MIC of 128 μg mL-1 of CS-ZnO NCs showed a 63% biofilm reduction, and CS-ZnO NCs with gentamicin at 0.5 μg mL-1 dramatically reduced biofilm formation by 84% (Hemmati et al., 2020).
The tested nanocomposites did not reduce biofilm formation for all isolates: AgNPs did not inhibit biofilm formation for Pa28 and Pa ATCC 9027 isolates (Figure 4a), IO/Ag-NCs did not reduce it for Pa20, Pa ATCC 9027, and Pa ATCC 27853 (Figure 4b), and IONPs did not inhibit it for Pa5 and Pa ATCC 9027 (Figure 4c).
However, concentrations higher than the one tested in our study (1 µg mL-1) significantly reduced P. aeruginosa biofilm formation (da Silva Bruckmann et al., 2022; Hemmati et al., 2020; Sathyanarayanan et al., 2013).
The prepared IO/Ag-NCs were particles with spherical morphology commonly presenting aggregates, suggesting that functionalization caused the aggregate state. However, the formed colloid was stable under the tested conditions, showing a high zeta potential value. The synthesized IONPs and AgNPs were particles with spherical morphology, a few aggregates, and a high zeta potential value, suggesting particle stability.
NPs showed no toxicity at the maximum LC50 of 20.5 µg mL-1 for A. salina. They inhibited biofilm formation in some P. aeruginosa clinical isolates. AgNPs inhibited biofilm formation in three isolates, IONPs reduced it in four, and IO/Ag-NCs inhibited it in three P. aeruginosa isolates.
The non-toxicity of NPs and biofilm formation results indicate that further research should be performed with higher silver concentrations and using IO/Ag-NCs as nanocarriers for a controlled and targeted drug release by combining the magnetic property of IONPs with the antimicrobial property of AgNPs.
The authors would like to thank the Universidade Estadual de Goiás (UEG) for the master scholarship’s financial support. Also, to the Centro de Análises, Inovação e Tecnologia Universidade Estadual de Goiás (CAITec-UEG), the Multi-user Analysis Center (CAM-UFG), and Multi-user Laboratory of High-Resolution Microscopy (Labmic – UFG) for the TEM images. For the financial support to Universidade Estadual de Goiás Edital PRÓ-PROJETOS PESQUISA n.005/2021.
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Authorship contributions
1 – Aline Fernandes Barcelos
Master in Sciences Applied to Health Products
https://orcid.org/0000-0002-8561-5586 • fernandesbaline@gmail.com
Contribution: Data curation, Formal Analysis, Investigation, Writing – original draft, and review & editing.
2 – Alliny das Graças Amaral
PhD in Animal Science
https://orcid.org/0000-0002-1418-9698 • alliny.amaral@ueg.br
Contribution: Data curation, Formal Analysis, Investigation, Methodology, Software, Supervision, Validation, Writing – original draft, and review & editing.
PhD in Cellular and Molecular Biology
https://orcid.org/0000-0003-4067-1506 • liliancarla@ufg.br
Contribution: Investigation, Writing – review & editing
4 - Plínio Lázaro Faleiro Naves
PhD in Microbiology and Parasitology
https://orcid.org/0000-0003-1936-1837 • plinionaves@ueg.br
Contribution: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, and review & editing.
5 - Luciana Rebelo Guilherme
https://orcid.org/0000-0002-0433-5751 • luciana.guilherme@ueg.br
Contribution: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, and review & editing.
How to quote this article
Barcelos, A. F., Amaral, A. das G., Carneiro, L. C., Naves, P. L. F., & Guilherme, L. R. (2025). Synthesis and antimicrobial activity of iron oxide/silver nanocomposites against Pseudomonas aeruginosa biofilms. Ciencia e Natura, 47, e84264. DOI: https://doi.org/10.5902/2179460X84264. Available in: https://doi.org/10.5902/2179460X84264