Synthesis of Mn0.5Zn0.5SmxEuxFe1.8−2xO4 Nanoparticles via the Hydrothermal Approach Induced Anti-Cancer and Anti-Bacterial Activities

Manganese metallic nanoparticles are attractive materials for various biological and medical applications. In the present study, we synthesized unique Mn0.5Zn0.5SmxEuxFe1.8−2xO4 (0.01 ≤ x ≤ 0.05) nanoparticles (NPs) by using the hydrothermal approach. The structure and surface morphology of the products were determined by X-ray powder diffraction (XRD), transmission electron and scanning electron microcopies (TEM and SEM), along with energy dispersive X-ray spectroscopy (EDX). We evaluated the impact of Mn0.5Zn0.5SmxEuxFe1.8−2xO4 NPs on both human embryonic stem cells (HEK-293) (normal cells) and human colon carcinoma cells (HCT-116) (cancerous cells). We found that post-48 h of treatment of all products showed a significant decline in the cancer cell population as revealed by microscopically and the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assay. The inhibitory concentration (IC50) values of the products ranged between 0.75 and 2.25 µg/mL. When tested on normal and healthy cells (HEK-293), we found that the treatment of products did not produce any effects on the normal cells, which suggests that all products selectively targeted the cancerous cells. The anti-bacterial properties of the samples were also evaluated by Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays, which showed that products also inhibited the bacterial growth.


Introduction
In recent years, spinel ferrites have attracted much attention from researchers due to their versatile magnetic properties and wide range of applications. Nanosized ferrites have shown enhanced optical and magnetic properties compared to their bulk counterpart because of better magnetic coupling between the sub-lattices (tetrahedral (Td) or octahedral (Oh)) [1]. Due to improved properties, via the hydrothermal approach. Specific ratios of metals nitrates were thawed in 50 mL of DI H 2 O with forceful stirring for 45 min. The pH was attuned at 11 by the addition of sodium hydroxide (NaOH) with stirring for 30 min and then the solution was exposed to an ultrasonic water bath for 40 min. The mixture was transferred to a Teflon-lined vessel, which was heated at 180 • C for 10 h in an oven. The final powder was washed with DI H 2 O several times and left to dry at 80 • C for 5 h. Structure was confirmed using XRD (Rigaku Benchtop Miniflex XRD analyzer with Cu Kα radiation) over the 2θ range of 20 • to 70 • . The microstructure was imaged by scanning electron microscope (SEM) and (TEM) (FEI Titan ST) coupled with energy-dispersive X-ray spectroscopy (EDX).

In Vitro Testing of Cytotoxicity
In the present study, cancer cell line (human colon carcinoma cells-HCT-116) and normal healthy cell line human embryonic kidney cells (HEK-293) were used to evaluate the cytotoxicity. The cells were cultured as per the method described previously [22,24]. In brief, cells were grown in DMEM media, L-glutamine, fetal bovine serum, selenium chloride, and antibiotic penicillin and streptomycin respectively in a CO 2 incubator at 37 • C. When cells become 70% to 80% confluence, they were tested for the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (Molecules, New Zealand) assay, which was used to examine the impact of nanoparticles on cancer cell viability. Cells were treated with different concentrations (2.0 to 40 µg/mL) of Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs. In the control group, we did not add Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs. After 48 h of treatment, the culture medium was removed and 5.0-µL MTT (Sigma-Aldrich, St. Louis, MO, USA) solution (10 mg/mL) was added to each well and culture plates were incubated for 4 h. Then, culture media was removed and dimethyl sulfoxide (DMSO) was added in each well where MTT developed formazan crystals. Subsequently, culture plates were read under a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) at 570 nm. The data were analyzed with GraphPad Prism, GraphPad Software by a one-way analysis of variance (ANOVA) and p-values were calculated by Student s t-test.

Nuclear Staining by DAPI
The cancerous cells were stained with DAPI to examine the impact of Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs on the cell nucleus. In the first group, HCT-116 cells were divided into two types: Group one was the control (without Mn 0.5 Zn 0.5 SmEuFe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs treatment, and group two was the Mn 0.5 Zn 0.5 SmEuFe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs-treated groups. Similarly, we also tested HEK-293 cells. They were divided into two groups: One was the control (without NPs treatment), and another one was NPs-treated groups. After 48 h of treatment, both cancerous and normal cells were pre-treated with ice-cold (4%) paraformaldehyde. Then, cells were treated with (0.1%) Triton X-100 in phosphate-buffered saline (PBS) for 5 min for cell membrane permeabilization. Both control and NPs-treated cells were stained with DAPI (1 µg/mL) prepared in PBS for 5 min in a dark environment. Finally, the cells were washed with (0.1%) Triton X-100 prepared in PBS. The nuclear morphology of both control and NPs-treated cells was examined under a confocal scanning microscope (Zeiss, Germany) equipped with a digital camera.

Preparation of Test Nanomaterial and Inoculum
The NPs were homogenized and dissolved in sterile LB (Luria Bertaini) at a concentration of 16 to 0.5 mg/mL. For the preparation of the inoculum, test strains, i.e., gram negative (Escherchia coli ATCC35218) and gram positive (Staphylococcus aureus ATCC29213) were grown in LB overnight at 37 • C. The turbidity of the culture broth was adjusted to 10 6 CFUs/mL using phosphate saline buffer (PBS). The MIC of the products was tested in the concentration ranging from 16 to 0.5 mg/mL, using the broth dilution method. The freshly adjusted bacterial inoculum was added to the prepared NP solution at a cell density of 2.5 × 10 5 CFU mL −1 and further incubated at 35 ± 2 • C for 24 h with aeration. Untreated bacteria were included in the experiment as the negative control. The MIC was recorded as the lowest concentration of a drug, which visually inhibits 99% of bacterial growth [25].

Minimal Bactericidal Concentration (MBC)
In continuation to the MIC evaluation of the NPs, an aliquot of incubated suspension with no apparent bacterial growth was plated on freshly prepared Mueller Hinton Agar (MHA) plates and further subjected to incubation at 35 ± 2 • C for 24 h. The MBC was recorded as the minimum concentration of a drug that killed 100% or having less than three CFU bacterial cells on the MHA plates [25].  [25]. Precisely, ∼10 6 CFU/mL of freshly grown bacterial cells treated with NPs (at the concentration obtained as MIC) were incubated with agitation at 37 • C overnight. The untreated bacteria were included in the experiment as a negative control. After the incubation period, cells were harvested by centrifugation at 12,000 rpm for 10 min. The cell pellets were washed using PBS and subsequently fixed with 2.5% glutaraldehyde for primary fixation, which was followed by secondary fixation, i.e., 1% osmium tetroxide. The fixed cells were washed and dehydrated by varying concentrations of a series of ethanol. Later, the cells were placed on the aluminum stubs, followed by drying in a desecrator, and finally coated with gold. Samples were photocaptured and analyzed at an accelerating voltage of 20 kV by SEM.

Structural Analysis and Morphological Study
XRD powder patterns of Mn 0.5 Zn 0.5 Eu x Sm x Fe 1.5−2x O 4 (0.01 ≤ x ≤ 0.05) NPs are offered in Figure 1. It is clear that all peaks belonged to a single phase of Mn-Zn spinel ferrite and no other peaks for the extra phase could be observed. This is evidence that the substituted ions were merged successfully into the spinel lattice. The lattice parameters and crystallite size were estimated by full proof refinement [26][27][28]. The lattice parameter "a" was 8.463, 8.434, 8.428, 8.413, and 8.408, respectively. It is obvious that the lattice parameters decreased when the ratio of Sm and Eu ions increased because of the substitutions of some Fe ions by larger ionic radii ions. The average of the crystallite sizes is in the range 7-12 nm. Figure 2 presents the SEM images of Mn 0.5 Zn 0.5 Eu x Sm x Fe 1.5−2x O 4 (x = 0.01, 0.03, and 0.05). All samples exhibited an agglomerated spherical particle. EDX was used to approve the stoichiometric composition spinel ferrite. The TEM of Mn 0.5 Zn 0.5 Eu x Sm x Fe 1.5−2x O 4 (x = 0.03 and 0.05) spinel ferrites displayed a spherical particle, as seen in Figure 3. It is well known that the complex 3d-metal oxides easily allow oxygen excess and/or deficit. Oxygen nonstoichiometry greatly affects the magnetic and magnetoelectric properties of complex oxides. Oxygen nonsctoichiometry changes the oxidation degree of 3d-metalls and magnetic parameters, such as the total magnetic moment and Curie point. The intensity of the exchange interactions decreases with the oxygen vacancy concentration increase. Exchange near the oxygen vacancies is negative according to Goodenough-Kanamori empirical rules [29,30].

Cell Proliferation Testing by MTT Assay
Antiproliferative activities of Mn0.5Zn0.5SmxEuxFe1.8−2xO4 (0.01 ≤ x ≤ 0.05) NPs on cancerous cells were done by the MTT assay. After 48 h of treatment, the cytotoxic effects of Mn0.5Zn0.5SmxEuxFe1.8−2xO4 (0.01 ≤ x ≤ 0.05) NPs were observed and we found that Mn0.5Zn0.5SmxEuxFe1.8−2xO4 (0.01 ≤ x ≤ 0.05) NPs showed inhibitory action on HCT-116 cancerous cells. The inhibitory concentration (IC50) values of different compounds were calculated as depicted in Table 1. We also examined the effects of NPs on normal cells (HEK-293) to check whether they produced any cytotoxic effects. We found that NPs did not produce any significant cytotoxic effects on HEK-293 cells after 48 h of treatment.   Table 1. We also examined the effects of NPs on normal cells (HEK-293) to check whether they produced any cytotoxic effects. We found that NPs did not produce any significant cytotoxic effects on HEK-293 cells after 48 h of treatment. The cancer cell nuclear morphology was evaluated by confocal scanning microscopy, which revealed that treatment of NPs showed strong inhibitory action on HCT-116 cells ( Figure 4B,C) as compared to control group cells ( Figure 4A). The cancer cell nuclear morphology was evaluated by confocal scanning microscopy, which revealed that treatment of NPs showed strong inhibitory action on HCT-116 cells ( Figure 4B,C) as compared to control group cells ( Figure 4A). There are several reports where magnetic nanoparticles have shown potential applications in drug delivery and other diagnostic assays [31][32][33][34]. These results suggest that NPs selectively targeted both colon and breast cancerous cells and do not cause any harm to normal and healthy cells. There are reports of involvement of nanoparticles in cancer cell death where nuclear fragmentation and nuclear disintegration were prominent features [35][36][37][38][39][40]. We recommend that Mn0.5Zn0.5SmxEuxFe1.8−2xO4 (0.01 ≤ x ≤ 0.05) NPs possess a selective targeting capability to cancerous cells and could be a potential candidate for cancer treatments.  Figure 5B). The activity of the broth culture was determined as the effectiveness of the content of element Fe (x = n) in the test material. The obtained results demonstrated that the effectiveness of the test material showed little improvement with the increasing ratio of the Fe content, i.e., the minimum MIC/ MBC was obtained by Fe x = 0.05 and 0.06. However, an enhanced activity was found against E. coli as compared to S. aureus; this slight difference could be attributed to the varying cell wall composition among these bacterial strains [41]. In some earlier studies, various metal-substituted NPs, like copper, zinc, nickel, and manganese, were recorded as possessing antibacterial activities [42,43], although the antibacterial activity of the current There are several reports where magnetic nanoparticles have shown potential applications in drug delivery and other diagnostic assays [31][32][33][34]. These results suggest that NPs selectively targeted both colon and breast cancerous cells and do not cause any harm to normal and healthy cells. There are reports of involvement of nanoparticles in cancer cell death where nuclear fragmentation and nuclear disintegration were prominent features [35][36][37][38][39][40]. We recommend that Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs possess a selective targeting capability to cancerous cells and could be a potential candidate for cancer treatments.  Figure 5B). The activity of the broth culture was determined as the effectiveness of the content of element Fe (x = n) in the test material. The obtained results demonstrated that the effectiveness of the test material showed little improvement with the increasing ratio of the Fe content, i.e., the minimum MIC/ MBC was obtained by Fe x = 0.05 and 0.06. However, an enhanced activity was found against E. coli as compared to S. aureus; this slight difference could be attributed to the varying cell wall composition among these bacterial strains [41]. In some earlier studies, various metal-substituted NPs, like copper, zinc, nickel, and manganese, were recorded as possessing antibacterial activities [42,43], although the antibacterial activity of the current combination of Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs is the first of its kind to the best of the author s knowledge. The morphological alteration in E coli and S. aureus cells caused by Mn0.5Zn0.5SmxEuxFe1.8−2xO4 (0.01 ≤ x ≤ 0.05) NPs was further studied by SEM. The untreated (control) cells of both organisms were normal in shape, and intact with a regular and smooth cell surface (Figure 6-I). However, cells treated with Mn0.5Zn0.5SmxEuxFe1.8−2xO4 (x ≤ 0.05) NPs were not found intact, i.e., the cells started appearing abnormal in shape with irregularities at the cell surfaces (Figure 6-I b-f). During the examination, untreated cells had no obvious visible damage, but the treated cells were observed as moderately damaged to severely damaged, with the increasing ratio of Fe(x=n), i.e., the maximum damage was caused by x = 0.04 and x = 0.05. Furthermore, it was observed that the effect of Mn0.5Zn0.5SmxEuxFe1.8−2xO4 (x ≤ 0.05) NPs on both gram positive and gram negative were somehow similar, although a slightly enhanced activity was observed against E. coli (Figure 6-I and II). This difference in activity can be attributed to the varying cell wall composition between the two organisms, which in turn may play an important role in the attachment of Mn0.5Zn0.5SmxEuxFe1.8−2xO4 (x ≤ 0.05) NPs to the bacterial cell surface, and is therefore essential in obtaining enhanced antibacterial activity [44]. The morphological alteration in E coli and S. aureus cells caused by Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs was further studied by SEM. The untreated (control) cells of both organisms were normal in shape, and intact with a regular and smooth cell surface ( Figure 6I). However, cells treated with Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (x ≤ 0.05) NPs were not found intact, i.e., the cells started appearing abnormal in shape with irregularities at the cell surfaces ( Figure 6(Ib-If)). During the examination, untreated cells had no obvious visible damage, but the treated cells were observed as moderately damaged to severely damaged, with the increasing ratio of Fe (x=n) , i.e., the maximum damage was caused by x = 0.04 and x = 0.05. Furthermore, it was observed that the effect of Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (x ≤ 0.05) NPs on both gram positive and gram negative were somehow similar, although a slightly enhanced activity was observed against E. coli ( Figure 6I,II). This difference in activity can be attributed to the varying cell wall composition between the two organisms, which in turn may play an important role in the attachment of Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (x ≤ 0.05) NPs to the bacterial cell surface, and is therefore essential in obtaining enhanced antibacterial activity [44].

Conclusions
In the present study, we synthesized five different derivatives of Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs hydrothermally. The structure and surface morphology of Mn 0.5 Zn 0.5 SmEuFe 1.8−2x O 4 spinel hydrothermal NPs were characterized by the XRD, SEM, TEM, and EDX methods, respectively. We examined the impact of Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs on both normal (HEK-293) and cancerous (HCT-116) cells. We found that after 48 h of treatment, Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs showed a significant decline in the cancer cell population. The IC 50 value of Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs ranged between 0.75 to 2.25 µg/mL. When tested on normal and healthy cells (HEK-293), we found that the treatment of Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs did not produce any effects on normal cells, which suggests that Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs selectively targeted cancerous cells. The anti-bacterial properties of Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs were also evaluated by MIC and MBC assays. We conclude that Mn 0.5 Zn 0.5 Sm x Eu x Fe 1.8−2x O 4 (0.01 ≤ x ≤ 0.05) NPs produced via the hydrothermal route possess potential anti-cancer and anti-bacterial abilities.
Author Contributions: All authors equally contributed to this study.