Co_0.85Bi_0.15Fe_1.9X_0.1O₄ (X = Ce⁴⁺, Sm³⁺, Ho³⁺, and Er³⁺) Nanoparticles with Selective Anticancer Activity: A Structural and Morphological Approach

Abstract

In this work, we synthesized the Co_0.85Bi_0.15Fe_1.9X_0.1O₄ (X = Ce³⁺, Sm³⁺, Ho³⁺, and Er³⁺) nanoparticles via the auto-combustion method. The cell viability against two breast cancer cells (MDA-MB-231 and T-47D cells) and the PC3 prostate cancer cells were carefully analyzed and correlated with the structural parameters and particle size values as well as the chemical composition. The produced compounds’ morphological and structural characteristics were performed using scanning transmission microscopy (TEM) and X-ray Diffraction (XRD). For all compounds, the analyses of the XRD experimental data revealed a structurally reversed cubic spinel with space group Fd-3m. All of the compounds had crystallites smaller than 45 nm which concorded well with the particle size values deduced from TEM images. Co_0.85Bi_0.15Fe_1.9Ho_0.1O₄ nanoparticles induced a high mortality of breast and prostate cancer cells (MDA-MB-231, T-47D, and PC3) while the Co_0.85Bi_0.15Fe_1.9Sm_0.1O₄ compound (higher particle size) reduced almost 35% of MDA-MB-231 cancer cells. With very low cytotoxicity against normal human cells, the Co_0.85Bi_0.15Fe_1.9Ho_0.1O₄ nanoparticles play a significant role in the elimination of cancer cells.

1. Introduction

In recent decades, SFNPs, spinel-ferrite nanoparticles, have drawn increased interest because of their exceptional physico-chemical properties, including the high magnetization, colossal giant dielectric constant, chemical stability, and high polarization [1,2]. They are employed in many different fields of application, such as magnetic resonance imaging (MRI), biomedicine, hyperthermia, optics, biosensors, energy storage, and electronics [3,4,5,6,7]. In addition, the hard magnetic CoFe₂O₄ compound has a high Curie-temperature (T_C equal to 517 °C) and exhibits an elevated saturation magnetization (80 emu/g), a substantial magneto-resistive coefficient, a large magneto-crystalline anisotropy constant (≈5.10⁶ erg.cm⁻³), and high coercivity (around 4.3 kOe at room temperature) [8,9,10,11].

For further enhancement of physico-chemical properties of the CFO materials, researcher have focused on doping its A or/and B-sites, which directly affects the cation distribution in these sites, which in turn improves their magnetic properties by altering the magnetic interactions that occur between both cations at the two types of sites A (tetrahedral) and B (octahedral) by way of oxygen ions [12]. Lanthanide ions possessing a magnetic moment between 0 and 10.5 µ_B (La³⁺ and Dy³⁺ ions, respectively), presented as interesting candidates for doping CFO compounds to enhance their magnetic properties [13,14,15]. However, it was proved that the insertion of Bi³⁺ ions to the Cobalt site in Co₂Fe₂O₄ material results in an intriguing and noteworthy shift in the distribution of cations, with Bismuth occupying A-sites and displacing Fe³⁺ cations to octahedral sites [16]. According to the enhancement of saturation magnetization, coercivity, and remanence magnetization, especially with 15% Bismuth ion amount insertion (Co_0.85Bi_0.15Fe₂O₄). In addition to their well-known magnetic properties, both pure CFO and Bi³⁺-doped Co_0.85Bi_0.15Fe₂O₄ (CBFO) nanomaterials have been recently investigated for their anticancer activity [12,15]. According to a prior study [15], adding 15% of Bi³⁺-cations to CBFO material increased its cytotoxicity against prostate and breast cancer cells, suggesting their potential as anticancer agents.

In this work, Co_0.85Bi_0.15Fe_1.9X_0.1O₄ (X = Ce³⁺, Sm³⁺, Ho³⁺, and Er³⁺) were made via the autocombustion preparation technique in order to evaluate the impact of substituted ions on morphological and structural characteristics. Importantly, the cytotoxicity of these materials with various types of prostate and breast cancer cells and the biocompatibility with normal human cells will be tested and linked to the particle size and the chemical substituting elements.

2. Experimental Details

2.1. Synthesis Method

Co_0.85Bi_0.15Fe_1.9X_0.1O₄ (X = Ce³⁺, Sm³⁺, Ho³⁺, and Er³⁺) were obtained (

Table 1. Compound abbreviations.

Chemical Formula	Abbreviation
C0.85Bi0.15Fe1.9Ce0.1O4	CBFO-Ce
C0.85Bi0.15Fe1.9Sm0.1O4	CBFO-Sm
C0.85Bi0.15Fe1.9Ho0.1O4	CBFO-Ho
C0.85Bi0.15Fe1.9Er0.1O4	CBFO-Er

), utilizing glycine as fuel for the auto-combustion reaction process. The used chemicals for these compounds are Cobalt nitrate [Co(NO₃)₂.6H₂O], Ferric nitrate [Fe(NO₃)₃.9H₂O], Cerium nitrate [Ce(NO₃)₃.6H₂O], Samarium nitrate [Sm(NO₃)₂.6H₂O], Holmium nitrate [Ho(NO₃)₂.5H₂O], Erbium nitrate [Er(NO₃)₂.5H₂O], and the glycine [C₂H₅NO₂].

We purchased these extremely pure (>99% purity) nitrate compounds from Sigma-Aldrich. We started the preparation by dissolving the stoichiometric amount of all nitrates in distilled water. For all desired compounds, the fuel to nitrate ratio utilized in this study was 1:1. It should be noted that the reaction process was conducted in an air atmosphere without inert gas protection, as shown in Figure 1. The mixed solution was heated to 100 °C while being constantly stirred on a magnetic hot plate, until it turned into brown gel of a considerable viscosity. The obtained viscous gel was heated to 200 °C until it spontaneously ignited and burned quickly, producing finely powdered black ashes. The powders were used for additional characterizations after being heat-treated for 30 min at 700 °C for all compounds.

2.2. Characterizations

Using a Bruker-8D Advance X-ray powder diffractometer equipped with Cu-Kα₁ radiation (λ = 1.5406 Å), X-ray Diffraction (XRD), experimental data were used to determine the synthesized compound’s phase purity, homogeneity, lattice structural parameter, and cell characteristics. With a step of 0.02° and an acquisition time of 1 s for each step, the acquisition was performed in the 2teta range of 10–85°. Using the FullProf software (Version 2020), the Rietveld refinement of the resulting XRD data were used to gather the lattice parameters [17].

A TESCAN VEGA3 SBH microscope (Brno, Czech Republic) operating at 20 kV and equipped with an EDS detector BrukerXFlagh 410M (Karlsruhe, Germany) had been utilized to observe the surface morphology of the grown materials. This facilitates the identification of the sample’s chemical elements and the detection of the distinctive X-rays that it emitted.

A FEI Tecnai G2 (Global EMC, Nottinghamshire, UK) was used to collect TEM images in both bright field and electronic diffractions modes, with an acceleration voltage of 2.10⁵ Volts. We worked with the Image-J program (1.52a version) to process the acquired images and estimate the average size of the nanoparticles.

2.3. In Vitro Cancer Viability Analysis

2.3.1. Cell Culture

The American Type Culture Collection (ATCC) provided the normal Human Mammary Epithelial Cell line (HuMEC) as well as the T-47D and MDA-MB-231 human breast cancer cell lines.

The MDA-MB-231 metastatic breast-cancer cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM-F12), which contained 1% PEST solution (penicillin/streptomycin) and 10% Fetal bovine serum (FBS; GibcoTM by Life Technology). While metastatic PC3 prostate cancer and non-metastatic T-47D luminal breast were cultured separately in Roswell Park Memorial Institute (RPMI) 1640 Medium in addition to 10% FBS and 1% PEST. Note that HuMECs cells were cultivated in HuMEC Basal serum free medium that had been enriched with hydrocortisone and bovine pituitary extract. All cell cultures were kept in humified environment with 5% of CO₂ at 37 °C. the TC20 automatic cell counter (Bio-Rad Laboratories, Inc., Hercules, CA, USA) or a hemocytometer were used to manually count the cells.

2.3.2. The Test Nanomaterials and Reagents

In order to achieve a stock concentration of 100 mM, each synthesized material was dissolved in 100% DMSO (pure) solution. Serial dilutions in the culture medium were then made. To prevent the harmful effects of higher DMSO concentrations, a maximum concentration of 0.1% (v/v) was applied to the cells.

2.3.3. Cell Viability Assay

To generate dose–response curves, five-point serial dilutions were used at a range of 10 nM to 100 µM. The DMSO solvent (0.1% v/v) was employed in every experiment as a negative control. Each well had 10⁴ cells seeded in ninety-six well-plates and was allowed to remain attached for 24 h. The cells were subsequently treated with test compounds, which were diluted in the culture medium for 48 h. After this period, 100 µL of the culture medium was partially removed and replaced with a fresh test solution for another twenty-four hours. Then, in accordance with the supplier’s instructions, well viability was evaluated using the resazurin-based PrestoBlue^TM reagent (100 µL per well, Life Technologies, Foster City, CA, USA). After three hours of incubation, the values taken into consideration fell within the reading’s linear range. A microplate reader was used to automatically measure absorbance at 570 nm. Four duplicates of each concentration were tested. The cell viability is equal to $\left({\displaystyle \raisebox{1ex}{$meanAbsor.ofeachtreatment$}\!\left/ \!\raisebox{-1ex}{$meanAbsor.ofthenegativecontrol$}\right.}\right)\times 100$.

3. Results and Discussions

3.1. Structural Proprieties

The XRD pattern of the Co_0.85Bi_0.15Fe₂O₄ is presented in Figure 2. As one can see, there are seven distinct peaks found at 2θ equal to 18.40°, 30.32°, 35.73°, 43.40°, 53.80°, 57.20°, and 62.78°. There is good agreement between these diffraction peaks and the cobalt spinel ferrite’s layered structure’s (111), (220), (311), (400), (422), (511), and (440) planes (JCPDS card no. 22–1086) [18]. These findings also coincide well with the findings reported by S. K. Gore et al. and R. Kalia et al. [16,19]. They found some additional peaks that refer to BiFeO₃ cubic phase. However, in our case, the additional peaks with a very low intensity appeared at approximately 2θ ≈ 22.91°, 25.57°, 31.97°, 32.69°, and 33.19°, and were identified using the Xper’t High score (V1.1) software to belong to the Bi₂O₃ minoritarian phase. The same diffractions peaks have been detected for the five other Co_0.85Bi_0.15Fe_1.9X_0.1O₄ compounds (X = Ce³⁺, Sm³⁺, Ho³⁺, and Er³⁺) (see Figure 2) with a minor variation in the peak’s placements and the full width at half maximum-intensity of the distinctive diffraction peaks, indicating a change in the average crystallite size according to the inserted ions. In order to study the effect of substitution on the crystal-structure and on lattice parameters and the cell volume, the XRD patterns of the substances under study were subjected to Rietveld refinement using the FullProf software (Version 2020) [17]. As shown in Figure 3, the Rietveld refinement curve results fit well with the experimental XRD data of all compounds.

Table 2. Results of the refinement of XRD patterns for doped Co_0.85Bi_0.15Fe_1.9X_0.1O₄ (X = Ce⁴⁺, Sm³⁺, Ho³⁺, Er³⁺) ferrites spinel.

	Χ2	a (Å)	V (Å3)	DSC (nm)
CBFO-Ce	1.360	8.3581	583.866	25.940
CBFO-Sm	1.503	8.3797	588.423	40.203
CBFO-Ho	1.218	8.3688	586.130	31.388
CBFO-Er	1.240	8.3532	582.847	24.810

lists the values of the resultant parameters, and Figure 4 displays the fluctuation of the “a” lattice parameter. The difference in the ionic radii of Fe³⁺ and the other added ions can be used to explain the modification of the lattice parameter and the cell volume, which is in good agreement with other research [19,20,21,22,23].

Moreover, we used Sherer’s formalism to approximate the mean value of the crystallite size (D_SC) according to [24] (Equation (1)) and we found that, for all studied nanomaterial, the average crystallite values were less than 40.2 nm, confirming the nanosize criteria of the prepared compounds. Also, there is a good agreement between average crystallite size values and the lattice parameter (a) ones as function of the inserted ions in B-site.

$$D_{SC}={\displaystyle \frac{k\times \lambda }{\beta \times \cos \left(\theta \right)}}$$

(1)

where k is 0.9, λ equals 15.406 nm, θ denotes the Bragg angle of the most intense peak, and β represents the full width at half maximum (FWHM) of the Bragg peak.

3.2. Morphological Study

Figure 5a–d depicts the Co_0.85Bi_0.15Fe_1.9X_0.1O₄ (X = Ce⁴⁺, Sm³⁺, Ho³⁺, and Er³⁺) compounds elemental composition, respectively, which were obtained using the Energy-dispersive X-ray analysis. All compounds are confirmed by EDX pictures to have no extra peaks beyond those of the initially employed elements, such as Co, Fe, Bi, O, Ce, Sm, Ho, and Er. These results demonstrate that there was no contamination throughout the synthesis process. In addition, the obtained SEM micrographs of the prepared samples are presented in the inset of Figure 5. These pictures show the development of an uneven, dense morphology with many agglomerations, where the added chemical element in the synthesized samples’ B-site essentially controls the grain size.

The exanimated Co_0.85Bi_0.15Fe_1.9X_0.1O₄ (X = Ce⁴⁺, Sm³⁺, Ho³⁺, and Er³⁺) nanoparticles’ surface morphology has been analyzed by transmission electron microscopy (TEM) images are presented in Figure 6a–d, respectively. For all compounds, the dense morphology of particles with a combination spheroidal shape (with slight aspect ratio changes), is visible in the TEM images of all compounds. In order to evaluate the average particle size of these compounds, we have used the “Image-J” software.

The counts of particles have been adjusted with the Lorentzian equation, providing the average particle size for all compounds as results. The corresponding particle size histogram of each compound is depicted in Figure 6a–d, as well as the Lorentzian adjustment results. The average particle size was found to be between 28.394 and 42.573 nm, which confirms the nanosize criteria for all Co_0.85Bi_0.15Fe_1.9X_0.1O₄ (X = Ce⁴⁺, Sm³⁺, Ho³⁺, and Er³⁺) nanoparticles. Furthermore, the highest and lowest values of particle size were found for Co_0.85Bi_0.15Fe_1.9Sm_0.1O₄ and Co_0.85Bi_0.15Fe_1.9Er_0.1O₄, respectively. Additionally, it is important to mention that these values are consistent with the crystallite size values previously calculated by Scherrer’s formula, which confirm that each crystallite presents almost one particle.

3.3. Cancer Cells Activity

In this section of the study, we began examining the in vitro cytotoxic effects of 100 µM of the Co_0.85Bi_0.15Fe_1.9X_0.1O₄ (X = Ce⁴⁺, Sm³⁺, Ho³⁺, and Er³⁺ noted as CBF-Ce, CBF-Sm, CBF-Ho, and CBF-Er, respectively) nanoparticles on the three cancer cells, MDA-MB-231, T-47D, and PC3 (the third is a prostate cancer, and the first two are human breast cancer). For such a study, the nanoparticles have been incubated for 72 h with the cancer cells. The cell viability results of PC3, T-47D, and MDA-MB-231 cell lines for different studied compounds are shown in Figure 7. We notice that the Co_0.85Bi_0.15Fe_1.9Ho_0.1O₄ (CBF-Ho) compound presents the lowest values of cell viability of all PC3, T47D, and MDA-MB-231 cancer cells. Furthermore, one can see that, for such nanoparticle concentration, the moderate low cell viability value against MDA-MB-231 and PC3 cancer cells was found when using the Co_0.85Bi_0.15Fe_1.9Sm_0.1O₄ compound. It was reported that the cell viability of Cobalt ferrites depends on particle size where a drop in cell viability is accompanied by a decrease in particle size as a result of the ions’ increased solubility [25]. In this work, according to TEM and XRD analyses, the lowest values of particle size were found for CBF-Ce and CBF-Er compounds, where they did not show important anticancer activity with all the used cells as compared to the other two CBF-Sm and CBF-Ho compounds. This confirms that cell viability essentially depends on the chemical composition, not only on the particle size.

As the highest activity was exhibited by the Co_0.85Bi_0.15Fe_1.9Ho_0.1O₄ compound, the in vitro cytotoxic effects at the following concentrations 0, 10 nanomolar, 100 nanomolar, 1 micromolar, 10 micromolar and 100 micromolar have been carried out. We presented in Figure 8 the cell viability results of PC3, T-47D, and MDA cell lines as a function of nanoparticle concentrations. As one can see in this figure, the cell viability found to be dependent on nanoparticle concentration, it gradually decreases when increasing the concentration for the CBF-Ho compound. It can also be seen that, when the concentration is below 1 µM, the viability of all cancer cells slightly decreases, while for concentrations over 1 µM/mL, the cell mortality increases significantly with the concentration. The lowest values of cell viability were found for 100 µM concentration of both MDA-MB-231 and T47D Cancer cells (see

Table 3. In vitro Cytotoxicity studies of Co_0.85Bi_0.15Fe_1.9X_0.1O₄ (X = Ce⁴⁺, Sm³⁺, Ho³⁺, Er³⁺) ferrites nanoparticles against human breast cancer (T-47D and MDA-MB-231) and prostate cancer (PC3) cell lines, determined by a resazurin-based assay after 72 h of compound incubation.

	Cell Viability at 100 µM (%)
	CBF-Ce	CBF-Sm	CBF-Ho	CBF-Er
MDA-MB-231	81.28 ± 0.57	62.79 ± 0.21	58.02 ± 0.24	66.12 ± 0.50
PC3	90.53 ± 1.48	81.14 ± 1.14	69.50 ± 0.91	89.12 ± 1.76
T47D	65.36 ± 0.31	74.34 ± 0.58	53.01 ± 0.70	83.15 ± 0.77
IC50 MDA (µM)			102.730 ± 0.524
IC50 PC3 (µM)			166.892 ± 0.986
IC50 T47D (µM)			118.704 ± 0.635

). Importantly, we deduce from these results that cell viability depends on the nanoparticle concentration; the insertion of Holmium ions (Ho³⁺) enhances the MDA-MB-231, T-47D, and PC3 cell mortality, which further confirms the importance of this work. For the CBF-Ho compound, we have calculated the concentration values required to inhibit 50% of cell growth, known as the IC₅₀ values for all cancer cells (Table 3). The CBF-Ho compound showed very significant IC50 values in between 102 and 166 µM.

It is crucial to evaluate the CBF-Ho compound’s cytotoxicity against normal human cells since it has the highest activity in breast and prostate cancer cell lines. In Figure 9, we showed the percent cell viability of the CBF-Ho nanoparticles in opposition to HuMECs normal human cells. Even at high concentrations of CBF-Ho nanoparticles, its values were determined to be over 99 percent, meaning that the cytotoxicity to human normal cells is less than 1 percent. In conclusion, CBF-Ho and CBF-Sm nanoparticles exhibited cytotoxic activity against PC3, MDA-MB-231 and T-47D cell lines without showing toxicity against normal human mammary epithelial cells (HuMECs), suggesting potential for a good therapeutic index.

4. Conclusions

In the present work, Co_0.85Bi_0.15Fe_1.9X_0.1O₄ (X = Ce⁴⁺, Sm³⁺, Ho³⁺, and Er³⁺) nanoparticles were effectively produced utilizing glycine as fuel in the auto-combustion method. All synthesized compounds with a particle size value less than 45 nm satisfied the nanosize criterion, according to the structural and morphological examination. Reduced particle size presents suitable criteria for biomedical application. The cytotoxic effects of the prepared compounds to on breast (MDA-MB-231 and T-47D) and prostate (PC3) human cancer cells were evaluated in vitro. The Co_0.85Bi_0.15Fe_1.9Ho_0.1O₄ nanoparticles show excellent anticancer activity against MDA-MB-231, PC3, and T-47D cancer cells with no toxicity to normal cells up to 100 µM. These results indicate that Co_0.85Bi_0.15Fe_1.9Ho_0.1O₄ nanomaterial is a promising candidate for breast and prostate anticancer treatment.