Green Synthesis of Cobalt Ferrite (CoFe₂O₄) Nanoparticles Utilising Co-Precipitation, Structural Features and Toxicological Evaluation against MCF-7 and HUVEC Cell Lines

Magnetic nanoparticles have garnered a lot of interest due to their unique size-dependent physical and chemical properties as well as their numerous applications in the fields of technology, medicine and the environment [1,2]. Among these materials, spinel ferrites, which have the general formula MFe₂O₄ (where M = Co, Ni, Zn and Mn), are especially intriguing due to their chemical stability, tuneable magnetic behaviour and well-defined crystal structure [3]. Cobalt ferrite can be used in targeted drug delivery and cancer treatment due to its strong crystalline magnetic contrast, mechanical durability and moderate magnetic saturation [4-6]. However, the increasing use of cobalt ferrite nanoparticles has raised concerns about potential adverse effects on cells and the environment. These nanoparticles can interact with biological systems through mechanisms such as oxidative stress, membrane rupture and the release of metal ions, depending on the particle size, the concentration of the surface chemical composition and the duration of exposure [7-9]. While endothelial cells such as HUVEC cells are commonly used to assess the biocompatibility of nanoparticles, breast cancer cell lines such as MCF-7 cells are commonly used to study antiproliferative effects [10,11]. This study used an environmentally friendly co-precipitation method to evaluate the structural, morphological and cytotoxic properties of CoFe₂O₄ nanoparticles for both normal and cancer cell lines under identical experimental conditions.

Objectives

The main objective of this study is to synthesize and characterize CoFe₂O₄ nanoparticles using an eco-friendly method and to evaluate their cytotoxic effects HUVEC and MCF-7 cell lines.

Green Synthesis of CoFe₂O₄ Nanoparticles by Co-Precipitation

The green co-precipitation method, an effective and eco-friendly technique for spinel ferrites, was used to create CoFe₂O₄ nanoparticles [12,13]. In summary, 150 mL of deionised water was used to dissolve 1 g of cobalt nitrate hexahydrate (Co (NO₃) ₂·6H₂O) and 1 g of iron nitrate nicotinate (Fe (NO₃)₃·9H₂O) with constant magnetic stirring at 25°C for 20 minutes. One gram of anhydrous citric acid was then added as a chelating agent. Ammonium hydroxide (NH₄OH) was added progressively to the reaction mixture until its pH reached 7.5. Cobalt ferrite nanoparticles were successfully co-precipitated when the mixture was heated to 135 degrees Celsius and a gelatinous precipitate developed.

Fourier Transform Infrared Spectroscopy (FTIR) of CoFe₂O₄

The functional groups and metal-oxygen linkages in the produced CoFe₂O₄ nanoparticles were investigated using Fourier transform infrared spectroscopy (FTIR). As illustrated in Figure1, the FTIR spectra of the produced sample were compared to those of Co(NO₃)₂·6H₂O and Fe(NO₃)₃·9H₂O. The Co-O and Fe-O stretching vibrations, which are distinctive characteristics of the spinel ferrite structure, were identified as the cause of the unique absorption bands at roughly 438 cm⁻¹ and 542 cm⁻¹, respectively [14]. The stretching vibrations of the C-O and C=O bonds connected to the leftover citric acid are responsible for the extra bands seen at 1384 cm⁻¹ and 1596 cm⁻¹, while the stretching vibrations of the O-H bonds are responsible for the broad band about 3417 cm⁻¹ [15].

Figure 1: FTIR spectra of main salts and CoFe₂O₄ nanoparticles

X-Ray Diffraction (XRD) of CoFe₂O₄

As seen in Figure 2, X-ray diffraction analysis was used to examine the crystal structure of the produced CoFe₂O₄ nanoparticles.

Figure 2: CoFe2O4 nanoparticles' X-ray diffraction pattern in comparison to ICDD standard data

The creation of a single-phase spinel structure was confirmed by the close match between the diffraction peaks and the conventional spinel pattern of CoFe₂O₄ (ICDD card No. 22-1086) [16]. The Debye-Scherer equation was used to determine the average crystal size and Table 1 summarises the findings.

Table 1: The Debye-Scherer equation was used to determine the crystal size of CoFe₂O₄ nanoparticles

The computed average crystal size of 20.17 nm demonstrated the synthesised material's nanocrystalline nature.

Energy - Dispersive X- ray (EDX) Spectroscopy of CoFe₂O₄

To ascertain the elemental makeup of the produced nanoparticles, EDX analysis was carried out. With weight percentages of 44.6% and 38.7%, respectively, the EDX spectrum displayed in Figure 3 verified the existence of iron and cobalt as the primary components, demonstrating the exceptional purity of the produced CoFe₂O₄ nanoparticles [17].

Figure 3: EDX spectrum showing the elemental composition of CoFe₂O₄ nanoparticles

Scanning Electron Microscopy (SEM) of CoFe₂O₄

The surface morphology of the CoFe₂O₄ nanoparticles was investigated using SEM analysis. The particles were mostly in the nanoscale range, as seen in Figure 4 and partial agglomeration was ascribed to the electrostatic and magnetic interactions that are frequently seen in magnetic nanoparticles [18]. According to SEM scans, the average particle diameter was roughly 55.75 nm.

Figure 4: SEM micrographs of CoFe₂O₄ nanoparticles

Atomic Force Microscopy (AFM) of CoFe₂O₄

The surface topography and roughness of the produced nanoparticles were thoroughly examined using atomic force microscope analysis. The average particle diameter was 124 nm, the average surface roughness (Sa. Roughness) was 178 picometres and the average root-mean-square roughness (Sq. Root mean square) was 471 picometres, according to the AFM data, which are displayed in Figure 5.

Figure 5: Atomic Force Microscopy Examination of CoFe₂O₄ Nanoparticles' Surface Topography and Roughness

Cytotoxicity Assay (MTT Test)

The MTT test, a well-used technique for evaluating cell viability based on mitochondrial metabolic activity, was used to investigate the cytotoxic effects of CoFe₂O₄ nanoparticles [19]. HUVEC and MCF-7 cells were exposed to CoFe₂O₄ nanoparticles at concentrations ranging from 25 to 400 µg/ml for 24 and 48 hours. Each experiment was performed three times and the results were expressed using the mean ± standard deviation.

Cytotoxicity towards HUVEC Cells

After 24 hours of exposure, CoFe₂O₄ nanoparticles showed a concentration-proportional decrease in HUVEC cell viability. Cell viability exceeded 100% at a concentration of 25 µg/ml but decreased to 38.54% at a concentration of 400 µg/ml, as shown in (Table 2, Figure 6).

Table 2: Toxic effects of CoFe₂O₄ nanoparticles on HUVEC cells after 24 hours.

Figure 6: Cobalt ferrite (CoFe2O4) nanoparticles' 24-hour MTT test on HUVEC

Statistical analysis revealed a significant difference between concentrations (P < 0.05), with an IC50 value of 221 µg/ml (Figure 7).

Figure 7: 24-hour IC50 for cobalt ferrite (CoFe2O4) nanoparticles

After 48 hours of exposure, a more pronounced cytotoxic effect was observed, with cell viability decreasing to 22.53% at a concentration of 400 µg/ml (Table 3, Figure 8).

Table 3: Toxic effects of CoFe₂O₄ nanoparticles on HUVEC cells after 48 hours.

Figure 8: 48-hour MTT test of cobalt ferrite (CoFe2O4) nanoparticles on HUVEC

The IC₅₀ value decreased to 147 µg/ml (Figure 9), indicating an increasing cytotoxic response over time.

Figure 9: Half-inhibition concentration (IC50) of cobalt ferrite (CoFe2O4) nanoparticles after 48 hours

Cytotoxicity of MCF-7 Cells

MCF-7 cells exhibited a clear cytotoxic reaction to CoFe₂O₄ nanoparticles, which was influenced by both concentration and duration.

According to (Table 4 and Figure 10), cell viability dropped to 26.71% at a dose of 400 µg/ml after a 24-hour exposure. After 24 hours, the IC50 for MCF-7 cells was determined to be 294.3 µg/ml (Figure 11).

Figure 11: shows the 24-hour IC50 for cobalt ferrite (CoFe2O4) nanoparticles on MCF-7

Table 4: Shows the harmful effects of CoFe₂O₄ nanoparticles on MCF-7 cells over a 24-hour period.

Cell viability dropped to 22.56% at the same concentration after 48 hours of treatment, indicating a more severe cytotoxic effect (Table 5, Figure 12).

Figure 12: MTT test of cobalt ferrite (CoFe2O4) nanoparticles on MCF-7 in 48 hours

After 48 hours, the IC50 value dropped to 257.6 µg/ml (Figure 13), suggesting a growing cytotoxic effect. Overall, the findings demonstrate that MCF-7 cells were more sensitive to CoFe₂O₄ nanoparticles than HUVEC cells, suggesting that under equal experimental settings, cancer cells and normal cells respond differently.

Figure 13: shows the IC50 of cobalt ferrite (CoFe2O4) nanoparticles on MCF-7 after 48 hours

Table 5: Shows the harmful effects of CoFe₂O₄ nanoparticles on MCF-7 cells over a period of 48 hours.

An environmentally acceptable co-precipitation approach was used to synthesize and systematically characterize cobalt-iron oxide (CoFe₂O₄) nanoparticles. While cytotoxicity experiments showed concentration- and time-dependent effects on both HUVEC and MCF-7 cell lines, structural investigations verified the creation of a crystalline nano-spinel phase. These results provide a strong experimental basis for further research on cobalt ferrite nanoparticles with a focus on their biosafety and potential biological applications.

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