Synthesis, Characterization, and Biological Activity Evaluation of Magnetite-Functionalized Eugenol

This work reports the magnetite-functionalization and biological evaluation of eugenol by the co-precipitation method employed only Fe2+ under mild conditions and control from the amount of the incorporated magnetite. Magnetic nanoparticles were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR), hydrodynamic size distribution (Zetasizer), and vibrating sample magnetometer (VSM). SEM images showed that EUG·Fe3O4 similar in shape to a nanoflower. The FTIR spectrum confirmed the presence of characteristic EUG and Fe3O4 bands in the EUG·Fe3O4 sample, the XRD analysis showed that the magnetite functionalization with eugenol slightly affected the Fe3O4 crystal structure, while the VSM measurements demonstrate that EUG·Fe3O4 1:1 shows a superparamagnetic behavior, suggesting small non-interacting particles. The in vitro safety profile and cytotoxicity of free eugenol, magnetite pristine, EUG·Fe3O4 1:1, EUG·Fe3O4 1:5, and EUG·Fe3O4 1:10 was investigated using human cell lines (keratinocytes and melanoma). The results demonstrate the high biocompatibility of EUG·Fe3O4 in HaCat cells and the greater specificity for the A375 cell line. Furthermore, the magnetite-functionalization with eugenol decreased the toxic effects of free eugenol on healthy cells. Antibacterial tests were performed in different bacterial strains. The experimental data showed that among the magnetic compounds, the microorganisms were only sensitive to treatment with EUG·Fe3O4 1:1. Regarding the antibiofilm activity assay, it can be observed that only the EUG·Fe3O4 caused a significant decrease in biomass when compared to the positive control. Finally, it can be concluded that EUG·Fe3O4 proves to be a potential candidate for future studies for drug delivery of cancer and bacterial infections treatments.


Introduction
Recently, the development of drug delivery systems has been widely investigated due to the limitations of conventional treatments, such as insolubility, toxicity, serious adverse effects, low stability, and bioavailability [1,2].
In addition, some drugs have difficulty in reaching distant organs. Thus, nanotechnology and pharmaceutical technology enable the development of drug delivery systems that are biocompatible, stable, and with high content of encapsulated drugs [3][4][5][6]. A variety of particles have been synthesized as a vehicle for drug targeting that can show different sizes, shapes, and chemical compositions [6,7]. The colloidal systems have demonstrated promising candidates for drug delivery due to ease of preparation, thermodynamic stability, and high loading capacity [8]. Khayat et al. [6] developed a eugenol microemulsion containing indomethacin. The study showed that the drug release and penetration depend on the co-surfactant and increase with eugenol percentage.
Similarly, a recent study for the doxorubicin carry was reported by Altinkok et al. [9]. The results showed that polymer micelles loaded with the antitumor had a particle diameter around 73 nm, maintaining stability for more than two weeks. However, due to the use of polymeric compounds, several polymerization reactions are necessary, increasing the time and affecting the reaction yield. In addition, the in vitro release study revealed that the ideal condition for drug delivery occurs in the acid medium (pH = 5.5), where about 80% was released after 48 h.
Regarding nanoparticulated systems, magnetic nanoparticles (MNPs) have been demonstrated as excellent nanoplatforms for drug delivery due to unique properties such as biocompatibility, ease of preparation, and functionalization [10,11].
Particularly, superparamagnetic iron oxide nanoparticles (SPIONs) (Fe 3 O 4 and γ-Fe 2 O 3 ) have been promising in biological activity studies due to their easy metabolism, biodegradability, and biocompatibility. Superparamagnetic nanoparticles have advantages over other metallic nanoparticles. The fact that SPIONs exhibit magnetic behavior only approaching a magnet decreases clot formation and biological agglomeration [12,13].
Likewise, MNPs can enhance the pharmacological effect of drugs, bioactive compounds, and essential oils. Shahabadi et al. [10] developed magnetic core-shell nanoparticlecontaining eugenol (EUG) with antitumoral and antibacterial activities superior to free EUG.
Additionally, a magnetic nanohybrid system was synthesized as a carrier of eugenol and hesperidin [14]. Nanocarriers showed particle diameter around 150 nm and low saturation magnetization (Ms) ≈ 3.00 emu/g. The maximum percentage of eugenol released was 78.36% in pH 1.2 after 24 h, whereas the hesperidin release reached a maximum of 87.44% in the same experimental conditions. The nanoformulation containing eugenol demonstrated higher compatibility in healthy cells and more significant toxic effects against breast tumor cells. However, the hesperidin-loaded nanocarrier had slightly greater cytotoxic effects when compared to nanoencapsulated eugenol [14].
Essential oils are products from the secondary metabolites of plants and are known to have several therapeutic properties. The EUG (4-allyl-2-methoxyphenol) is a phenolic compound, with hydrophilic characteristics and the majority compound of clove oil (Syzygium aromaticum), characterized for present excellent antibacterial, antiproliferative, antioxidant, and antiinflammatory activities [10,15,16]. Furthermore, there are few studies regarding the functionalization of magnetite with eugenol available.
This work reports a simple method of functionalization of the eugenol with control of the amount of incorporated magnetite, using only Fe 2+ , as the iron source by co-precipitation, under very mild conditions. Subsequently, biological properties of EUG·Fe 3 O 4 were investigated through an in vitro safety profile, antibacterial, antibiofilm, and antitumor activity.

Synthesis of Magnetite-Functionalized Eugenol with Different Proportions Magnetite (EUG·Fe 3 O 4 )
The magnetite-functionalized eugenol was carried out as described by Rhoden et al. [3]. In a 250 mL round-bottom

Cell Culture
In this study, A375 (human melanoma) and HaCat (human keratinocyte) cell lines procured from Rio de Janeiro Cell Bank were employed.

MTT Assay
The cytotoxic effects of magnetic nanoparticles were investigated through of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay using technique proposed by Mosmann [17]. After 24 h, a solution of 5 mg.mL −1 of the reagent was prepared, diluted in 1 × phosphate-buffered saline (PBS) (Prolab®) and subsequently added 20 μL per well in the 96-well plates maintained at 37 °C under CO 2 atmosphere for four hours. The supernatant was then removed and added 200 μL of dimethyl sulfoxide (DMSO) (Synth®) to solubilize the crystals formed. Finally, the absorbance was measured at a wavelength of 570 nm, using a microplate reader (SpectraMax® i3x-Molecular Devices).

Neutral Red Assay
The neutral red test was based on the initial protocol described by Borenfreund and Puerner [18], following the methodology described by Bruckmann et al. [11]. After 24 h, the supernatant was removed and a culture medium without serum was added to the fetal bovine with the neutral red reagent at concentration of 40 μg.mL −1 . The cells remained for 4 h of incubation in the CO 2 oven in contact with the reagent. Afterward, the supernatant was removed, cells were washed with 1 × PBS to remove excess unreacted reagent incorporated by them. Finally, a lysis solution with 50% ethanol (Synth®), 49% distilled water, and 1% acetic acid (Synth®) for cell lysis and dye release to read absorbance at a wavelength of 540 nm.

LDH Assay
The activity of the lactate dehydrogenase (LDH) enzyme released into the extracellular medium was measured in the supernatant of the culture wells using the non-radioactive colorimetric assay CyToTox96 according to the manufacturer's instructions. For the LDH assay, a volume of 100 µL supernatant + 100 µL substrate was transferred to another 96-well plate. After 30 min of incubation at room temperature, absorbance was measured at 490 nm in a microplate reader (SpectraMax® i3x-Molecular Devices).

Microorganisms
The following microorganisms were used in the experiments: Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 35218), Enterococcus faecalis (ATCC 29212) and Pseudomonas aeruginosa (PAO1). The strains were purchased from the American Type Culture Collection (ATCC). The samples were inoculated on Brain Heart Infusion broth (BHI) and incubated for 24 h. After that, they were seeded on Nutrient agar and incubated for 24 h at 37 ºC. From the grown colonies, the suspensions in NaCl at 0.9% corresponding to the 0.5 at McFarland scale (1.5 × 10 8 CFU/ mL −1 ) were produced.

Disc Diffusion
The antimicrobial activity was initially evaluated by the disc diffusion technique, as described previously by Bauer et al. [19]. The microorganisms were seeded in petri dishes with Mueller Hinton agar and three discs with each compound (EUG, Fe 3 O 4 , EUG·Fe 3 O 4 1:1, EUG·Fe 3 O 4 1:5, EUG·Fe 3 O 4 1:10 were added on the agar surface. The plates were incubated for 24 h at 37 ºC and, afterwards, the inhibition zones were measured in millimeters (mm). The surfactant (Tween 80) used to dissolve some compounds was also tested. The experiment was performed in triplicates and in two independent experiments.

Minimal Inhibitory and Bactericidal Concentration
The minimal inhibitory concentration (MIC) was determined by microdilution method in 96 well-plates according to Clinical and Laboratory Standards Institute (CLSI) [20]  Afterwards, the plates were incubated for 24 h at 37 ºC. The assay was performed in triplicate. The assay was revealed with 2,3,5-triphenyl tetrazolium chloride, which develops a red color in the microbial growth. The lowest concentration that did not show any changes in color was considered as MIC. To determine the minimal bactericidal concentration (MBC), an aliquot of 1 μL was taken of each well, seeded on Nutrient agar plate and incubated for 24 h. After the incubation, the colonies were identified and the lowest concentration that did not demonstrate microbial growth was considered the MBC.

Biofilm Formation and Treatment
The antibiofilm potential was evaluated against the strain P. aeruginosa PAO1. The method used to this assay was described previously Manner et al. [21] with modifications.
To biofilm formation, fresh exponentially grown culture of P. aeruginosa was diluted to be 10 8 CFU/mL, and 20 μL was added to 96-well plates (Nunclon™ D surface, Nunc, Roskilde, Denmark), containing 100 μL of BHI broth. The plate was incubated in 37 ºC for 24 h. After the formation of the biofilm, the treatment was performed and incubated for 24 h in a condition of 37 ºC. The treatment was performed with MBC of tested compounds. A positive control was performed containing only BHI broth and the P. aeruginosa strain while the negative control was just BHI broth.

Quantification of Biofilm Biomass
The biomass of treated biofilm was quantified by method previously described by Lopes et al. [22]. The supernatant was removed and gently washed three times with distilled water, fixing with 95% of methanol and staining with 150 μL of 0.1% of crystal violet for 10 min at RT. After incubation, the well-plates were washed with distilled water, and ethanol 95% was added to dissolve the coloring after 15 min. After that, 100 μL were transferred into another plate to measure spectrophotometrically at 570 nm to crystal violet in spectrophotometer (TP-Reader; ThermoPlate, Goiás, Brazil).

Statistical Analysis
Cytotoxicity analysis in HaCat and A375 cell lines of magnetic nanoparticles was performed using GraphPad Prism. The treatments were compared by one-way analysis of variance (ANOVA) that was performed followed by Tukey's post hoc test. Statistically different values were considered with p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). The results of quantification of biofilm biomass were analyzed using the ANOVA, followed by the Dunnet test that was used considering values p < 0.05 (*) statistically significant compared with the Positive Control. Data were expressed as the Mean ± Standard deviation.

Fourier Transform Infrared Spectroscopy (FTIR)
The equipment used to characterize the magnetic nanoparticles and eugenol was Perkin-Elmer FTIR, model Spectro One. KBr disc method was used to record the spectra in the spectral region between 4000 and 400 cm −1 . The infrared spectra (FTIR) of the free eugenol, magnetite pristine, and EUG·Fe 3 O 4 are shown in Fig. 1.
In the FTIR spectrum of Fe 3 O 4 , it is possible to verify a band of vibration at 602 cm −1 typical of bonding Fe-O. The bands at 3443 cm −1 and 1633 cm −1 are attributed to the O-H stretching and vibration resulting from the absorption of water molecules [15].
For the spectrum of free eugenol, a characteristic band is observed around 3462 cm −1 , referring to the O-H bond in 2838 cm −1 the absorption peak corresponds to the stretching of the C-H bond. In the 1600-1500 cm −1 region, the peaks refer to the C = C bond, and the sharp peaks in the 1200-700 cm −1 region are due to the C = C bonds of the aromatic ring [10,23].
In the spectrum of EUG·Fe 3 O 4 , in addition to the peaks remaining constant, a more intense peak is observed around 600 cm −1 , referring to Fe-O binding confirming the functionalization of eugenol with Fe 3 O 4 [3].

X-ray Diffraction (XRD)
Bruker Optics D2 Advance USA equipment was used for the characterization using X-ray diffraction (XRD) to determine the crystalline phases of the samples. Figure 2 shows the XRD referring to the magnetite, and magnetite-functionalized eugenol, which demonstrates the characteristic peak of these nanoparticles.
The figure above shows XRD patterns of magnetic nanoparticles synthetized in this work. In the diffractogram of the Fe 3 O 4 pristine, it is possible to verify the crystal planes at 2θ ≈ 30º, 35º 45º, 57º, and 62º, which corresponds to the characteristic diffraction interference of magnetite [10]. For the XRD of EUG Fe 3 O 4 , it can be observed that the functionalization of eugenol with magnetite caused a decrease in the intensity of the diffraction peaks. The partial suppression of the peaks indicates that eugenol presence slightly affected the crystal structure of the magnetite [3,11].

Scanning electron spectroscopy (SEM) and Energy dispersive X-ray spectroscopy (EDS)
The morphology of magnetite functionalized eugenol and the element analysis of the nanoparticle was obtained by Scanning electron microscope (SEM) (Sigma 300 VP Carl Zeiss), and Energy dispersive X-ray spectroscopy (EDS) (Quantax 200-Z10, Bruker). Figure 3 and Figs. S1 to S3 (see supporting information) shows the SEM images of the eugenol and magnetic nanoparticles (EUG·Fe 3 O 4 ).  The morphological structure and size of the magnetic eugenol nanoparticles were measured according to the SEM image.
As can be observed in the SEM images, the Fe 3 O 4 deposited on the eugenol surface has a spherical shape, and the nanocomposite produced exhibited a morphology nanoflower-like [3,24,25].
Through the EDS analysis (details see supporting information, Fig. S4), it was possible to verify the presence of Fe (11.15%), C (46.97%), and O (41.31%) as the main chemical elements in the sample. Furthermore, the low relatively S percentage (0.57%) can be attributed to the residue of the iron salt (FeSO 4 ) used in the reaction. The histogram of the particle size distribution of EUG·Fe 3 O 4 was obtained from the SEM images is shown in Fig. 4.
As shown in Fig. 4, it was possible to observe that the average particle size was around 90 nm. Maheswari et al. [14] obtained a EUG carried on the magnetic nanohybrid system, whose size particle was 145 nm. Similarly, Wang et al. [26] developed gelatin-chitosan nanoparticles containing eugenol with size distribution exhibiting an average diameter of 229.09 nm. Corroborating these results, the eugenol microemulsion prepared for transdermal indomethacin delivery showed a size of 184.1 nm [6].

Hydrodynamic Size of the Magnetite-Functionalized Eugenol
The size distribution of EUG·Fe  Fig. 5a-c. From hydrodynamic size profiles, it is possible to assume that the average size of nanoparticles rises directly with the amount of the Fe 2+ employed in synthesis. The increase of the size distribution of magnetite-functionalized eugenol could be related to the tendency of formation aggregates due to the critical diameter of nanoparticles [27].

Vibrating-Sample Magnetometer (VSM) Analysis
The hysteresis loops were obtained in a vibrating sample magnetometer (VSM), under magnetic fields varying within ± 1 T, at room temperature (Fig. 6).
In contrast, for melanoma lineage from the lowest concentration, the cell viability rate decreases, in a

Colorimetric Viability Assay from the Neutral Red
Vital Dye Figure 9a, b shown the results of the cellular toxicity of the free eugenol and magnetite-functionalized eugenol employing the neutral red colorimetric assay under the same treatment conditions of the HaCat and A375 cell lines described in the cell culture item of the materials and methods assignment. Through the neutral red (NR) colorimetric assay, a significant decrease in the viability of HaCat cells was observed at the highest concentrations (30 and 100 µg.mL −1 ) in all treatments, except for EUG·Fe 3 O 4 1:1, which showed cytotoxic effects only in the concentration of 100 µg.mL −1 .
However, in human melanoma cells, the action of nanoparticles was more evident, demonstrating that the materials offer better specificity against the tumor lineage, for instance, the highest concentration of free eugenol, EUG·Fe 3 O 4 1:1, EUG·Fe 3 O 4 1:5, and EUG·Fe 3 O 4 1:10 reduced the number of viable cells to 46, 64, 54, and 44%, respectively. Figure 9a, b shows the viability of HaCat and A375 cells treated with magnetite pristine after 24 h of contact using the NR assay, respectively.
Confirming the MTT results (Fig. 8), only the highest concentrations of magnetite pristine shows a decrease in the cell viability of HaCat lineage. In contrast, Fe 3 O 4 nanoparticles exhibited lower cell viability and the largest cytotoxicity to melanoma cells when compared to healthy cells. For these in vitro assays, all dilutions of magnetite caused the decrease of viability with statistical significance (p < 0.05*, and p < 0.001***, respectively), as can observed in the graph (Fig. 10b).

Lactate Dehydrogenase Assay
The effect of magnetic nanoparticles on cell membrane integrity was measured using the lactate dehydrogenase assay, as shown in Fig. 11a, b.
The LDH assay allows the assessment of cell damage caused by the release of the enzyme lactate dehydrogenase from lysed cells by rupture of the cell membrane. For the HaCat lineage, the LDH assay confirmed low cytotoxicity for all treatments and dilutions with the maintenance of membrane integrity similarly the negative control, with a significant difference only at the highest concentration of EUG·Fe 3 O 4 1:10 (p < 0.01**) [29].
The lactate dehydrogenase results on the A375 cell line demonstrated that only the highest concentrations of all treatments caused slightly enzyme release for the extracellular medium (considered statically significant, p < 0.05* and p < 0.01**) [30].
The release of LDH by cell lines (HaCat and A375) after 24 h of contact with different dilutions of magnetite pristine is shown in Fig. 12 (a-b).
The results of the LDH assay show that treatment of magnetite does not induce the release of enzyme LDH from the intracellular to the extracellular medium maintaining the cell integrity of the two cell lines tested.
A significant decrease in survival after the incubation time with the different treatments was found only at the highest concentrations tested, and even more expressive results can be observed in the tumor cell line. However, A375 cells demonstrate sensitivity to treatments, even at the lowest concentration tested.
Farcas et al. [32] developed a study to assess the cytocompatibility/cytotoxicity of Fe 3 O 4 microparticles. The results reveal that the toxic effects on healthy cell lines and melanoma occur in a dose-dependent manner, and the mechanism involved in the decrease in cell viability is the induction of the apoptotic pathway.
It is shown in the literature that eugenol exhibits antitumor effects against several cell lines through different mechanisms. Regarding the cytotoxicity of eugenol, as it can be observed a decrease in the cell viability of a dosedependent manner.
Al Wafai et al. [33] reported that MCF-7 cell lines are chemosensitive at low concentrations of eugenol. Furthermore, it was observed that cytotoxicity occurred not only due to mitochondrial dysfunction but also due to loss of plasma membrane integrity. Additionally, the apoptotic  Fig. 11 a Cytotoxicity of free eugenol and magnetite-functionalized eugenol against HaCat and b A375 cells assessed through lactate dehydrogenase assay molecular mechanism of EUG for melanoma also has been supported and mentioned by several studies [34,35].

IC 50 Values of HaCat and A375 Cell Lines
The IC 50 is the concentration of nanoparticles required for resulting in a 50% decrease in cell numbers compared to untreated controls. The results of IC 50 for the different treatments and cell lines are shown in Table 1.
According to the IC 50 values (Table 1), the free EUG was considered more toxic against the A375 cell line (55.65 μg. mL −1 ) than for the keratinocyte cell lines (79.08 μg.mL −1 ). Furthermore, it is also observed that tumor cells were more susceptible to treatments with magnetic nanoparticles when compared to normal cells, except for EUG·Fe 3 O 4 1:1, which showed higher cytotoxicity for the HaCat cell line (IC 50 = 184.11 µg.mL −1 ). In addition, it is possible to verify that the magnetitefunctionalization with eugenol raised the IC 50 value of free eugenol, suggesting that the synergistic effect of the compounds decreases the cytotoxicity of the free oil to healthy cells.
Several studies have been proposing some mechanisms of cytotoxicity of the eugenol in tumor cells, for instance, Jaganthan et al. [36] and Ghost et al. [37] reported that effect against colon cancer and melanoma occurs by antiproliferative effect and induction of apoptosis. Regarding iron oxide nanoparticles, the mechanisms described are the generation of reactive oxygen species, inhibition of chemoresistant proteins, impairment of mitochondrial function, damage of DNA, and increase of apoptotic signals [38,39].
The safety profile study performed using the HaCat lineage demonstrates that the nanoparticles proved to be biocompatible due to cell viability remaining around 80% according to ISO 10993-12 2009 [40].

Disc Diffusion
After the incubation, the inhibition zones were measured with a digital caliper. The results of free eugenol, magnetite pristine, EUG·Fe 3 O 4 1:1, EUG·Fe 3 O 4 1:5 and EUG·Fe 3 O 4 1:10 are shown in Table 2 and Fig S7 (details see supporting  information).
According to the results, it is possible to verify that free eugenol has a broad spectrum against pathogenic microorganisms. In this regard, the bacterial strain S. aureus demonstrated the most susceptibility, and E. faecalis is the most resistant. On the other hand, among the magnetic compounds, only EUG·Fe 3 O 4 1:1 showed antimicrobial activity. The presence of an inhibition zone indicates a biocidal effect of the compounds that may implicate disruption of the bacterial cell membrane [41].  The sensitivity to treatments depends on some factors, such as the concentration of the substance, particle size, solubility, and bacterial concentration. As demonstrated on the topic of hydrodynamic size of the magnetite-functionalized eugenol (Fig. 5), the amount of the iron precursor employed in the reaction increased the size of magnetic nanoparticles [3], which is possibly related to the results obtained in the study of antibacterial activity. A larger surface area and smaller diameter particle enable better interaction with bacterial cells, increasing permeability and adhesion in the cell membrane, facilitating rupture and release of intracellular content [42].
Previous studies reported different mechanisms to the antibacterial activity of eugenol, which includes alteration of fatty acids, changes in morphology, and the cytoplasmic membrane. Further, it can also cause ion transport modification, production of reactive oxygen species [43], and binding of the hydroxyl group with proteins, inhibiting bacterial enzymatic action [44,45].
While the action mechanisms of MNPs can be attributed to the production of free radicals, such as hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2˙) , causing intense oxidative stress, resulting in the degradation of vital substances for bacterial survival, such as lipids, proteins, and nucleic acids [46,47].

Minimal Inhibitory and Bactericidal Concentration
With the addition of the reagent, it was possible to verify visible microbial growth and determine the MIC. The MBC was observed after the incubation of seeded Nutrient agar plates. The MBC and MIC were demonstrated in Table 3.
The MIC values of free EUG for bacterial strains ranged from 0.62 mg.mL −1 to 2.5 mg.mL − After the treatment, the biomass biofilm was quantified by crystal violet assay. The results inhibit the growth of biofilm using free eugenol and EUG·Fe 3 O 4 1:1 is shown in Fig. 13.
The experiments demonstrate that free eugenol exhibits a low antibiofilm effect, while for EUG·Fe 3 O 4 1:1 causes a significant decrease in biofilm biomass when compared to the positive control. A synergistic effect of magnetite and eugenol nanoparticles can be observed for P. aeruginosa, corroborating the results presented in Table 3.  The antibacterial and antibiofilm activity of eugenol is widely known in the literature. The antibacterial effects are associated with increased cell permeability, disruption of the cytoplasmic membrane, and modification of shape cells [49,50]. Meanwhile, antibiofilm activity occurs through cell lysis, disruption of the cell-cell junction, and inhibition of the quorum detection system [51,52].

Conclusions
This work reported the preparation of EUG·Fe 3 O 4 with different proportions of Fe 3 O 4 incorporated via the coprecipitation method employing only one iron precursor (FeSO 4 ). Through a simple methodology and under very mild conditions, the eugenol incorporated magnetite was fully characterized by infrared spectroscopy, X-ray diffractogram, and scanning electron microscopy, as well as by approximation of an external magnetic field. It was also found that the magnetite-functionalization eugenol caused changes in crystallinity of Fe 3 O 4 and average particle size. Also the VSM measurements reveals a superparamagnetic behavior for the EUG·Fe 3 O 4 1:1, indicating small particles, corroborating XRD results. The evaluation of the safety and cytotoxic profile performed by the MTT, NR, and LDH assay showed that the magnetic compounds demonstrate cytocompatibility and exhibit higher toxicity in tumor cells compared to the non-magnetic analogue. Moreover, increasing the proportion and concentration of iron increases cell toxicity, as verified from the IC 50 values. The antibacterial assay evidenced the antibacterial activity of free eugenol and EUG·Fe 3 O 4 1:1 against S. aureus, E. coli, E. faecalis, and P. aeruginosa were similar. In the meantime, the EUG·Fe 3 O 4 1:1 demonstrates more effectiveness in inhibiting the bacterial biofilm when compared to free eugenol. Nevertheless, the controlled incorporation of magnetite on the eugenol surface shows to be an effective tool for future studies for biological applications in essential oil drug derivatives.