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 Dp Average (nm) Dp (nm) FWHM Bsize (°) Peak position 2θ (°) λ (Å） K 20.17 8.55 0.984 18.7852 1.54178 0.94 29.15 0.2952 30.4039 14.60 0.5904 31.1387 22.17 0.3936 35.8091 17.78 0.492 36.6179 30.28 0.2952 43.4312 13.03 0.6888 44.5535 31.54 0.2952 53.8078 24.05 0.3936 57.3604 16.15 0.5904 58.8827 24.74 0.3936 62.9773 12.49 0.7872 64.7034 17.66 0.5904 74.4631   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. Concentration R1% R2% R3% Mean% SD 0 95.6133 99.06304 105.3237 100 4.922536 25 106.6014 104.1738 104.4293 105.0682 1.33394 50 94.33561 89.99149 91.39694 91.90802 2.216698 100 72.23169 77.08689 80.15333 76.49064 3.994336 200 64.31006 55.87735 59.7104 59.96593 4.222158 400 42.71721 34.15673 38.75639 38.54345 4.28421     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. r R1% R2% R3% Mean% SD 0 101.8288 98.83625 99.335 100 1.603267 25 93.84872 94.47216 98.33749 95.55279 2.431688 50 83.99834 84.24772 87.6143 85.28679 2.01954 100 59.18537 62.55196 56.56692 59.43475 3.000302 200 41.60433 36.6168 37.86368 38.69493 2.595594 400 19.40981 25.27016 22.90108 22.52702 2.948028     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.     Figure 10: Shows a 24-hour MTT test of cobalt ferrite (CoFe2O4) nanoparticles on MCF-7   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. Concentration R1% R2% R3% Mean% SD 0 100.0456 98.93628 101.0317 100.0045 1.048305 25 99.42931 96.71761 98.44324 98.19672 1.372554 50 93.38962 96.4711 97.45717 95.77263 2.121821 100 87.71971 91.91051 89.32208 89.65077 2.114649 200 71.69604 70.58671 73.05189 71.77822 1.234642 400 27.69259 24.48786 27.93911 26.71452 1.925367   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. Concentration R1% R2% R3% Mean% SD 0 98.81302 103.4969 96.96413 99.758 3.367324 25 93.1431 95.23851 100.0456 96.14241 3.538914 50 91.0477 93.26636 92.03377 92.11595 1.111611 100 89.32208 84.1452 85.99408 86.48712 2.623419 200 67.25872 68.61457 71.8193 69.23086 2.34192 400 20.42031 22.51572 24.73438 22.5568 2.157325

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.