Bioscience Journal | 2023 | vol. 39, e39029 | ISSN 1981-3163 1 Joyce Farias Louza DE SOUSA1 , Plínio Lázaro Faleiro NAVES1 , Luciana Rebelo GUILHERME1 1 Postgraduate Program in Sciences Applied to Health Products, Universidade Estadual de Goiás, Anápolis, Goiás, Brazil. Corresponding author: Luciana Rebelo Guilherme luciana.guilherme@ueg.br How to cite: DE SOUSA, J.F.L., NAVES, P.L.F. and GUILHERME, L.R. Synthesis and evaluation of toxicity and antimicrobial activity of rifampicin associated with iron oxide nanoparticles. Bioscience Journal. 2023, 39, e380329. https://doi.org/10.14393/BJ-v39n0a2023-65125 Abstract Rifampicin has broad-spectrum antimicrobial activity, but it can cause nephrotoxic and hepatotoxic damage because high doses are required. Nanosystems emerge as a perspective to improve the transport systems of this drug. In this work, iron oxide nanoparticles were synthesised, functionalized with lauric acid, and rifampicin was incorporated into the nanosystem. The samples were characterized by spectroscopic techniques: electronics in the visible ultraviolet region (UV-vis), vibrational absorption in the infrared region (IR), X-ray diffractometry (XRD), and dynamic light scattering (DSL). The toxicity of the nanocompounds and the antimicrobial activity against Staphylococcus aureus ATCC 25923 were studied by the Artemia salina lethality and disc diffusion techniques, respectively. As a result, IR analysis showed characteristic vibrations of laurate and rifampicin on the surface of the nanosystem. The presence of magnetic iron oxide was confirmed by XRD and the mean diameter of the crystallites was 8.37 nm. The hydrodynamic diameter of rifampicin associated with the nanosystem was 402 nm and that of the nanosystem without rifampicin was 57 nm. The compounds did not show toxicity to Artemia salina and the in vitro antimicrobial activity against Staphylococcus aureus was slightly decreased when rifampicin was associated with the nanosystem. In general terms, the results showed that iron oxide nanoparticles showed no toxicity and reduced the toxicity of rifampicin by 41.54% when carried compared to free rifampicin. Therefore, magnetic iron oxide nanoparticles may have the potential to act as a platform for associated drugs. Keywords: Antimicrobial. Artemia salina. Brine shrimp. Magnetic nanoparticles. Nanotoxicity. Staphylococcus aureus. 1. Introduction Rifampicin (RIF) is used in the treatment of leprosy, against Staphylococcus aureus infections, prophylaxis of meningococcal infection, and influenza (Perumal et al. 2020). It is also widely used in the treatment of tuberculosis against Mycobacterium tuberculosis (Adams et al. 2021). However, administration of high doses is necessary to exert its bactericidal activity, this can cause an increase in serum levels of bilirubin and hepatic enzymes, possibly causing hepatotoxicity and, less frequently, the development of acute interstitial nephritis and glomerulonephritis (Legout et al. 2014; Motta et al. 2020). In addition, mutations in RNA polymerase subunits confer antimicrobial resistance to RIF (Goldstein 2014). To decrease the incidence of problems like those reported above, Ruckh et al. (2012) used nanostructures obtained by poly(caprolactone) electrowinning for RIF transport. The result against SYNTHESIS AND EVALUATION OF TOXICITY AND ANTIMICROBIAL ACTIVITY OF RIFAMPICIN ASSOCIATED WITH IRON OXIDE NANOPARTICLES https://orcid.org/0000-0003-0078-7432 https://orcid.org/0000-0003-1936-1837 https://orcid.org/0000-0002-0433-5751 Bioscience Journal | 2023 | vol. 39, e39029 | https://doi.org/10.14393/BJ-v39n0a2023-65125 2 Synthesis and evaluation of toxicity and antimicrobial activity of rifampicin associated with iron oxide nanoparticles Pseudomonas aeruginosa and Staphylococcus epidermidis was minimal colonisation of the structures by both organisms compared to free RIF. Woźniak-Budych et al. (2017) presented the antimicrobial properties of copper and RIF-loaded copper nanoparticles against Staphylococcus aureus, Escherichia coli, Bacillus pumilus, and Pseudomonas fluorescens. The results of the work indicate that the adsorption of rifampicin on the surface of copper nanoparticles can provide the reduction of the antibiotic dosage and prevent its adverse side effects. It is assumed that magnetic nanoparticles, when associated with other drugs, can direct the drug to its site of action due to their magnetic properties (Khan et al. 2022). By being functionalized to polymers or surfactants, nanoparticles can reduce side effects and toxicity by reducing interactions with other organs or tissues (Yen et al. 2013). Functionalization agents can modulate drug release in the body (Laurent et al. 2008). This characteristic can increase the interval between doses, provide greater comfort to the patient, and, consequently, greater adherence to treatment. Studies show that magnetic nanoparticles of iron oxide (MPIO) can be used in biomedical applications due to their magnetic properties and biocompatibility. These nanoparticles are formed by iron oxides of maghemite (γ-Fe2O3) and magnetite (Fe3O4) (Khalafalla and Reimers 1980; Ramezani et al. 2022). They can be used in the form of colloidal dispersions stabilised by surfactants or polymers called magnetic fluids (MF) (Khalafalla and Reimers 1980; Yen et al. 2013; Ramezani et al. 2022). Studies address improvements in the efficiency and quality of magnetic resonance imaging diagnostics when MF is used as contrast (Kermanian et al. 2021). Among other advantages, the use of these nanosystems can enable the transport of drugs known for their side effects, due to the lack of specificity of their targets to the specific site of action (Bejjanki et al. 2021). Still, further investigations are needed regarding the toxicity of iron oxide-based nanosystems. Bejjanki et al. (2021) show a nanosystem containing MPIO that uses folic acid for targeted transport of aggregated cisplatin to overcome cisplatin resistance in nasopharyngeal carcinoma. From another perspective, Zhu et al. (2021) present results demonstrating the aggravation of hepatic steatosis in liver inflammation in non-alcoholic fatty liver disease in mice and hepatocellular carcinoma (HepG2) cells, when in the presence of iron oxide. In the present work, we evaluated the toxicity of rifampicin associated with iron oxide nanoparticles, by the Artemia salina lethality method, and we evaluated the antimicrobial activity of the nanocompounds against Staphylococcus aureus, with a disc diffusion test. 2. Material and Methods Synthesis The synthesis of iron oxide nanoparticles was performed with coprecipitation of 12 grams of FeCl2.4H2O (J.T. Baker) and 24 grams of FeCl3.6H2O (Synth) in an alkaline medium (25% ammonium hydroxide - Dinâmica Ltd.) under a mechanical stirring of 500 revolutions per minute (Khalafalla and Reimers 1980; Van Ewijk et al. 1999). The solid, black-colored sample resulting from this step was decanted under a neodymium magnet and the supernatant was removed. The pellet was functionalized by adding 2.0 g of lauric acid (AL) (Sigma-Aldrich) under mechanical stirring and heating (90 °C) for 20 minutes (Van Ewijk et al. 1999; Ferreira et al. 2015). The magnetic fluid resulting from this step was taken for oxidation of the particles via bubbled oxygen under heating (90 °C) for a period of 8 h (Kang et al. 1996). Then, the fluid was dialyzed by a semipermeable membrane and sterilised in an autoclave for 15 min at 121 °C. The sample resulting from this step had a pH of 7.4. For the association process of the drug to the nanosystem, 22 mg of rifampicin were solubilized in 1 mL of previously sterilised dimethyl sulfoxide (DMSO), and then, the solution obtained was added to 10 mL of the nanosystem. This mixture remained under mechanical stirring for 48 h at room temperature. Afterward, it was submitted to magnetic separation with a neodymium magnet (400 Gauss) for 48 h under refrigeration (2 °C) and protected from light. The supernatant liquid resulting from this separation was used for quantification of free RIF, i.e., not incorporated into the nanosystem (Ferreira et al. 2015). Bioscience Journal | 2023 | vol. 39, e39029 | https://doi.org/10.14393/BJ-v39n0a2023-65125 3 DE SOUSA, J.F.L., NAVES, P.L.F. and GUILHERME, L.R. The RIF-nanosystem was collected in a dark flask and the volume was corrected with deionized and autoclaved water to 10 mL. The nanosystem was produced using the same procedure, placing a 10 mL aliquot of the magnetic fluid in 1 mL of pure DMSO. Aliquots of 100 µL from the nanosystem and the RIF - nanosystem sample were separated to quantify the concentration of iron ions. Characterization Atomic absorption spectroscopy (AAS) was used to determine the concentration of iron ions contained in the samples, nanosystem, and RIF-nanosystem, using Perkin Elmer Analyst 400 equipment. The electronic absorption spectroscopy analysis in the ultraviolet-visible (UV-vis) region was used to determine the content of rifampicin not incorporated into the nanosystem. This analysis was performed in the Perkin Elmer UV-vis Spectrometer model Lambda (Rodríguez et al. 2014). The determination of the concentration of RIF adsorbed to the nanosystem was performed by an indirect method, i.e., by analysing the supernatant resulting from the magnetic separation step. To prepare an analytical standard curve for rifampicin, rifampicin standards were prepared, followed by their absorbance reading at 474 nm wavelength. The results are in agreement with those described in the Brazilian Pharmacopoeia for rifampicin (ANVISA 2019). Fourier Transform (FTIR) vibrational absorption spectroscopy was used to identify the nature of the organic and inorganic materials contained in the studied samples. Alterations in the characteristic pattern of absorption bands clearly indicate a change in the material composition. The samples were previously lyophilized (L101 Liotop) to make KBr pellets, and in a Perkin Elmer Spectrum 400 spectrophotometer, with a spectral region between 4000 and 400 cm-1. X-ray diffractograms were obtained in a Shimadzu, model XRD 6000. The analyses were performed in the range of 10° ≤ 2θ ≤ 80°, with a Cu-Kα radiation source (λ = 1.54056 Ǻ), under a current of 30 mA, and a voltage of 40 kV, and at a scan rate of 2°/minute. The Zetasizer Nano ZS (Malvern Instruments) was used to characterise the hydrodynamic diameter of the particles, the zeta potential value (ζ), and the dispersion index (DI). Biological tests Toxicity assay with Artemia salina Eighty milligrams of brine shrimp (Artemia salina) cysts were incubated for 36 h under natural lighting, room temperature, and constant aeration for the hatching of the larvae (nauplii) in synthetic seawater culture medium prepared by dissolving 40 g.L-1 of sea salt, supplemented with yeast extract (6 mg.L-1), sterilised in an autoclave and the pH adjusted to 8.5 with a 0.1 mol.L-1 with Na2CO3 solution. After hatching the cysts, the larvae were distributed in wells of polystyrene plate, standardising 10 ± 1 individuals in each well (Ates et al. 2015; Machado et al. 2021). Then, 100 µL of each previously prepared dilution of the study compounds was added to these wells. The lethality assay was used to determine the medium lethal concentration of the compounds for 50% of the population of Artemia salina (LC50) (Machado et al. 2021). The assays were performed in 96- well polystyrene microplates with the concentrations of 800, 400, 200, 100, 50 μg.mL-1 for free RIF, of 3000, 1500, 750, 124, 62.5 μg.mL-1 of 550, 275, 135.5 and 68.76 μg.mL-1 RIF-nanosystem. In addition to these, residual solvent control tests were performed with 5% DMSO, viability control with synthetic seawater, and technique control with potassium dichromate (K2Cr2O7) at concentrations of 100, 50, 25, and 12.25 μg mL-1. After 24 h of exposure to the compounds, counts of live, dead, or immobile brine shrimp were performed in triplicate with three independent experiments. The results allowed the calculation of LC 50 by the probit program StatplusPro V5 2015 professional (AnalystSoft) (Arulvasu et al. 2014; Machado et al. 2021). Nguta et al. (2011) established that compounds with LC50 less than 100 µg.mL-1 are classified as highly toxic compounds to artemia, those with LC50 between 100 µg.mL-1 and 500 µg.mL-1 with moderate Bioscience Journal | 2023 | vol. 39, e39029 | https://doi.org/10.14393/BJ-v39n0a2023-65125 4 Synthesis and evaluation of toxicity and antimicrobial activity of rifampicin associated with iron oxide nanoparticles toxicity, those with LC50 between 500 µg.mL-1 and 1000 µg. mL-1 with low toxicity and those with LC50 greater than 1000 µg.mL-1 are classified as non-toxic. Microscopic analysis The brine shrimp were observed with a Leica DMRB microscope (Leica, Switzerland). Images were obtained using a Leica DFC420C CCD camera and Leica software (Leica Application Suite LAS EZ) and imagens of A. salina were taken at 100x magnification. The resulting images were stored and viewed in the Leica software program using the TIFF image format with a resolution of 1600 x 1200. Disc Diffusion Test The disk diffusion test was used to evaluate and compare the inhibitory activity of free RIF, nanosystem, and RIF-nanosystem. The assay followed the Clinical and Laboratory Standard Institute (CLSI 2011) recommendations for antimicrobial susceptibility testing of bacteria growing in aerobiosis (M7 2018) and the Staphylococcus aureus ATCC 25923 was tested. Succinctly, bacterial suspensions were adjusted to 1.5 x 108 CFU.mL-1 with a 0.5 McFarland scale, and the inocula were plated on Mueller Hinton agar. Subsequently, filter paper discs previously impregnated with free rifampicin 5 μg.mL-1 and 500 μg.mL-1, nanosystem 6000 μg.mL-1, and iron and rifampicin 5 μg.mL-1 and 500 μg.mL-1 were placed on the surface of the inoculated medium. In addition to these compounds, gentamicin discs were used as a control technique. The plates were incubated at 35 °C for 24 h. The diameters of the inhibition zones were measured in millimetres. All experiments were performed in independent triplicates (Rodríguez et al. 2014). 3. Results Characterization Figure 1 presents the results obtained using vibrational absorption spectroscopy in the infrared region. The technique was used to obtain information regarding the functional groups and bonds present in the compounds (Casillas et al. 2012). In Figure 1a it is possible to observe a band at 587 cm-1, which can be attributed to the Fe-O stretching vibration at tetrahedral and octahedral Fe-O-Fe sites in the maghemite (γ-Fe2O3) phase (Ishii et al. 1972; Nasrazadani and Raman 1993; Namduri and Nasrazadani 2008; Khan et al. 2022). And a band at 634 cm-1, which can be attributed to Fe-O bond deformation at octahedral sites in the maghemite phase (Ishii et al. 1972; Nasrazadani and Raman 1993; Namduri and Nasrazadani 2008; Casillas et al. 2012). It is also observed in Figure 1b at the 1710 cm-1 band that corresponds to the symmetric and asymmetric C=O stretching of the COO- bond of the protonated carboxylic acid used to perform the functionalization of the sample (Arulvasu et al. 2014). In Figure 1b, obtained for the RIF-nanosystem sample, it can be seen that the bands at 635 and 588 cm-1, attributed to Fe-O bond deformation and Fe-O stretching at tetrahedral and octahedral Fe-O-Fe sites of γ-Fe2O3, did not undergo displacement when RIF is associated with the nanosystem. It can be further noted in Figure 1b that some characteristic bands of the free RIF, Figure 1c, did not undergo significant displacements, such as the bands 1643 cm-1 and 1457 cm-1. These rifampicin bands can be attributed to asymmetric and symmetric stretching vibrations of carboxylate COO- groups, confirming the presence of rifampicin in the proposed nanosystem (Arulvasu et al. 2014; Parumasivam et al. 2016). The presence of the bands at 1726 cm-1 and 1645 cm-1 in Figure 1c suggests that the crystalline structural form of rifampicin used in the experiments is not the polymorphic form II of RIF, due to the absence of the double signal around 1740 - 1710 cm-1 , attributed to the C=O bond of the acetyl group (Ibiapino et al. 2014). The double signal is also not observed for the RIF-nanosystem sample. It is thus expected that polymorphic I and/or amorphous forms may be present in the RIF-nanosystem sample, known to be thermodynamically more stable (Pelizza et al. 1977). Bioscience Journal | 2023 | vol. 39, e39029 | https://doi.org/10.14393/BJ-v39n0a2023-65125 5 DE SOUSA, J.F.L., NAVES, P.L.F. and GUILHERME, L.R. Figure 1. FTIR results for: A - nanosystem, B - RIF-nanosystem and C - free RIF. The X-ray diffraction (XRD) technique was used to characterise the crystallographic phase of the iron oxide nanoparticles. It is possible to observe in Figure 2 the diffractogram of the nanosystem sample. The diffractogram shows peaks at 30.12°; 35.80°; 43.46°; 57.34°; 63.08° and 74.60° reflections that can be indexed to the planes of the crystal lattice determined by (220), (311), (400), (422), (511), and (440); which correspond to the intensity of the diffracted waves as a function of angle 2θ for an inverse spinel-type structure, characteristic of the iron oxides phase: magnetite Fe3O4 (ICCD 019-0629) and/or maghemite γ- Fe2O3 (ICCD 39-1346). The majority phase present in the nanosystem is expected to be maghemite since the samples underwent an eight-hour oxidation process. The Debye-Scherrer equation (CULLITY and STOCK 2001) was used to estimate the average diameter of the particles present in the nanosystem samples. Where D is the crystallite diameter; 0.9 is the correction factor; λ is the X-ray wavelength; θ is the diffraction angle of the most intense peak and β is the half-height width of the most intense diffraction peak. In this study, the value of β used was obtained for the most intense peak (311). The calculated value for the average diameter of the particles comprising the nanosystem was 8.37 nm. A B C Bioscience Journal | 2023 | vol. 39, e39029 | https://doi.org/10.14393/BJ-v39n0a2023-65125 6 Synthesis and evaluation of toxicity and antimicrobial activity of rifampicin associated with iron oxide nanoparticles Figure 2. X-ray diffractometry of the nanosystem. Through the dynamic light scattering technique, it was possible to characterise the nanosystem and RIF-nanosystem samples regarding hydrodynamic size, zeta potential, and dispersion index (DI). The result found for the hydrodynamic diameter of the nanosystem was 56.89 mm and a DI of 0.212. While for RIF - nanosystem the hydrodynamic diameter was 406 mm and the was DI 0.615. It is observed that the RIF - nanosystem showed a hydrodynamic size larger than the nanosystem. It is suggested that it is due to the adsorption of RIF to the nanosystem (Santos et al. 2011) and that a higher DI value for RIF -nanosystem is a consequence of the formation of possible aggregate during the adsorptive process. A schematic proposal for RIF-nanosystem can be consulted in previous works of the research group (Santos et al. 2011). The colloidal stability of the samples was evaluated based on the determination of the zeta potential (ζ). It was observed that the value of ζ for the RIF-nanosystem was -22.0 mV while for the nanosystem was -15.2 mV. These values of ζ suggest the occurrence of the repulsive force between the particles, favouring their stability (Riddick 1968; Arulvasu et al. 2014). The addition of RIF to the nanosystem reduced the ζ values suggesting that the colloidal dispersion disfavors the colloidal stability of the nanosystem. The iron ion concentration obtained by AAS was 6 mg.mL-1 for both samples, this value does not differ from the other results obtained in other studies (Santos et al. 2011). The concentration of rifampicin in the RIF-nanosystem was determined by UV-vis was 1.1 mg.mL-1. Lethality assay to Artemia salina Rifampicin showed LC50 to artemia at 312.94 µg.mL-1, and 535.38 µg.mL-1 to rifampicin - associated with the nanosystem, with classification to the toxicity or moderate and low, respectively. The nanosystem was considered non-toxic and no artemia mortality was observed in wells with 5% DMSO and viability controls with synthetic seawater. Potassium dichromate showed LC50 of 45 µg.mL-1. The results of the toxicity assay of the compounds to Artemia salina are shown in Table 1. Bioscience Journal | 2023 | vol. 39, e39029 | https://doi.org/10.14393/BJ-v39n0a2023-65125 7 DE SOUSA, J.F.L., NAVES, P.L.F. and GUILHERME, L.R. Table 1. Medium lethal concentration of the compounds to Artemia salina. Compounds LC50 (µg.mL-1) Classification Toxicity Rifampicin 312.94 Moderate toxicity Rifampicin - nanosystem 535.38 Low toxicity Nanosystem ≥ 3,000 Non-toxic Antimicrobial activity of the compounds In Table 2 is possible to observe that the nanosystem did not show a halo of inhibition on the growth of Staphylococcus aureus and that the RIF-nanosystem at the concentration of 5 µg.mL-1 showed a halo 11.76% smaller than free RIF 5 µg.mL-1. The same phenomenon was observed for the concentration of 500 µg.mL-1, with a decrease of 8.82% of the halo compared to free RIF. Table 2. Diameters of inhibition halos of Staphylococcus aureus ATCC 25923 by the tested compounds. Compounds Concentration (µg.mL-1) Halo (mm) Rifampicin 500 34,33±0,58 Rifampicin 5 16,67±0,58 RIF-nanosystem 500 30,67±1,15 RIF-nanosystem 5 14,67±2,08 Nanosystem 6,000 0 Microscopic analysis of Artemia salina The microscopy image in Figure 3 shows the artemia after exposure to the evaluated compounds. Figure 3. Microscopy image (100x magnification) of Artemia salina after exposure to the compounds: A - unexposed control, B - Rifampicin, C - Nanosystem and D - Rifampicin - Nanosystem. Bioscience Journal | 2023 | vol. 39, e39029 | https://doi.org/10.14393/BJ-v39n0a2023-65125 8 Synthesis and evaluation of toxicity and antimicrobial activity of rifampicin associated with iron oxide nanoparticles 4. Discussion The characterization techniques confirm the presence of iron oxide in the maghemite phase. The techniques used show that it was possible to functionalize the synthesised iron oxide with lauric acid. Furthermore, it was confirmed that rifampicin is associated with the nanosystem and that this association indicates that the nanosystem can transport the drug. The considerable increases in the hydrodynamic diameter and the dispersivity index for RIF-nanosystem compared to the nanosystem without the drug, suggest that the drug associated with the nanosystem can promote particle aggregation. The addition of RIF to the nanosystem reduced the ζ values suggesting that the colloidal dispersion does not favour the colloidal stability of the nanosystem (Santos et al. 2011; Arulvasu et al. 2014). The nanosystem fluid was non-toxic and showed LC50 greater than 3,000 µg.mL-1 as observed by Gambardella et al. (2014) and Rajabi et al. (2015). In the present study, we observed that there was a 41.54% reduction in the toxicity of rifampicin when associated with the nanosystem to the A. salina. Xiong et al. (2015) found that nanoparticles could inactivate free radicals and decrease injury to lipid membranes in guinea pigs. One of the problems concerning rifampicin is nephrotoxicity (Min et al. 2013) and hepatological (Legout et al. 2014) side effects. Rajabi et al. (2015) evidenced that the results obtained with the toxicity assay in Artemia salina showed no statistical difference (p > 0.05) compared to those obtained with the MTT assay. These findings suggest that the Artemia salina assay can accelerate toxicity studies, decrease costs, and therefore be considered a viable alternative to the in vitro cell culture assay (Ntungwe et al. 2020). Artemia salina is considered a non-selective filterer and can easily ingest particles up to 50 µm in diameter (Ates et al. 2013; Rodd et al. 2014). However, it was observed that the presence of the compounds inside the microcrustaceans did not induce mortality after 24 h of exposure (Cornejo -Garrido et al. 2011). Furthermore, artemias treated with nanosystem alone remained alive after the 72 h incubation period, while artemias exposed to rifampicin, rifampicin - nanosystem, and the DMSO control did not survive after the same exposure period. The toxicity of Fe3O4 nanoparticles was evaluated in ethological parameters of A. salina and was related to oxidative stress, as ROS species, malondialdehyde content, total antioxidant capacity and antioxidant activities increased significantly. In addition, mitochondrial malformation was observed after exposure to Fe3O4-NPs (Zhu et al. 2017). Behaviour changes were visualised in the artemia exposed to the nanosystem and rifampicin- nanosystem, which were more agitated than those in the untreated control. These observations were also described by Gambardella et al. (2014) in the presence of Fe3O4 nanoparticles. These authors also demonstrated an increase in biochemical responses, catalase and cholinesterase activities that may be correlated with inflammation and that the stress recorded by the larvae was due to an increase in cholinesterase and not ROS. In other studies, increases in cholinesterase and catalase were also identified in artemias. It is known that cholinesterase can function as a regulator of inflamed tissue (Falugi et al. 2012), suggesting that iron oxide nanoparticles have antioxidant mechanisms in preventing oxidative damage (Huang et al. 2013). 5. Conclusions In the present study, a magnetic colloidal dispersion consisting of iron oxide nano particles stabilised with lauric acid and associated rifampicin was obtained and characterised by spectroscopic techniques, confirming the functionalization of lauric acid on the surface of the particles and the adsorption of rifampicin to the system. In addition, the analytical methods provided an estimate of the average diameter of the nanoparticles and determined the presence of maghemite in the nanosystem. Regarding the biological activities evaluated, it was possible to observe that there was a reducti on of rifampicin toxicity when associated with iron oxide nanoparticles, as detected by lethality assay with Artemia salina and that the in vitro antimicrobial activity of rifampicin, when associated with Bioscience Journal | 2023 | vol. 39, e39029 | https://doi.org/10.14393/BJ-v39n0a2023-65125 9 DE SOUSA, J.F.L., NAVES, P.L.F. and GUILHERME, L.R. nanoparticles, was slightly reduced when compared to free rifampicin against Staphylococcus aureus ATCC 25923. Overall, the results demonstrated that the nanosystems showed no toxicity to Artemia salina and may have the potential for use as a platform for reducing the toxicity of associated drugs. Therefore, we suggest that additional studies should be conducted to corroborate the results found in this study. Authors' Contributions: DE SOUSA, J.F.L.: acquisition of data, analysis and interpretation of data, drafting the article, and critical review of important intellectual content; NAVES, P.L.F.: conception and design, analysis and interpretation of data, drafting the artic le, and critical review of important intellectual content; GUILHERME, L.R.: conception and design, analysis and interpretation of data, drafting the article, and critical review of important intellectual content. All authors have read and approved the final version of the manuscript. Conflicts of Interest: The authors declare no conflicts of interest. Ethics Approval: Not applicable. Acknowledgments: We thank to Professor Sebastião William da Silva of the Institute of Physics of the Federal University of Goiás, for laboratory support. We also thank the Coordination for the Improvement of Higher Education Personnel (CAPES) for granting a scholarship to the first author. References ADAMS, R.A., et al. 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Nanotoxicology. 2021, 15(6), 761–778. https://doi.org/10.1080/17435390.2021.1919329 Received: 19 March 2022 | Accepted: 30 September 2022 | Published: 24 February 2023 This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank about:blank