Ferrite/curcumin hybrid nanocomposite formulation: Physicochemical characterization, anticancer activity, and apoptotic and cell cycle analyses in skin cancer cells

2.1 Materials

Silver nitrate (AgNO₃, Assay: 99%, Sigma-Aldrich, UK), anhydrous ferric chloride (FeCl₃, Assay: 98%, Alpha Chemicals, Egypt), copper nitrate hydrate (Cu(NO₃)₂·3H₂O, Assay: 98%, Laboratory Chemicals), anhydrous calcium chloride (CaCl₂, Assay: 98%, TopChem), cadmium nitrate tetrahydrate (Cd(NO₃)₂·4H₂O, Assay: 99%, Alpha Chemical,. Cairo, Egypt), magnesium chloride hexahydrate (MgCl₂·6H₂O, Assay: 98%), and sodium hydroxide (NaOH, Assay: 99%, Alpha Chemicals, Assiut, Egypt). Curcumin (98%) and doxorubicin (99%) were purchased from Sigma-Aldrich, Co., UK.

2.2 Synthesis of AgClM_0.5Fe₂O₄ nanocomposites

AgClM_0.5Fe₂O₄ nanocomposites (M = Ca, Cd, Cu, or Mg) were synthesized via a chemical co-precipitation method [30,31,32]. As an example, for the synthesis of 2 g of AgClCu_0.5Fe₂O₄, 0.97 g of AgNO₃, 1.85 g of FeCl₃, and 0.69 g of Cu(NO₃)₂·3H₂O were dissolved in 200 ml of distilled water to form a mixture solution. The solution was continuously stirred at 300 rpm using a magnetic stirrer (HS-20, LK LAB-Korea) at 80°C for 30 min. Separately, a 3 M sodium hydroxide (NaOH) solution was prepared and used as the precipitating agent. The pH of the mixture was adjusted to 12.0 by gradually adding NaOH solution. The resulting precipitate was maintained at 80°C under continuous stirring for 2 h. Then, it was cooled to room temperature, and separated using a centrifuge (10,000 rpm, BT20 bench top high-speed centrifuge, China), washed twice with distilled water, and then with ethanol. The filtered product was dried at 80°C in an oven (Binder ED53 oven, Germany) for 24 h, ground into fine powder using a mortar, and then calcined at 600°C for another 4 h in a muffle furnace (Nabertherm L-401H1TN, Germany) under a static air atmosphere. The above-mentioned steps were also used for the synthesis of AgClCa_0.5Fe₂O₄, AgClCd_0.5Fe₂O₄, and AgClMg_0.5Fe₂O₄. For the synthesis of AgClCa_0.5Fe₂O₄, 1.00 g of AgNO₃, 1.91 g of FeCl₃, and 0.33 g of CaCl₂ were used. However, for AgClCd_0.5Fe₂O₄, 0.91 g of AgNO₃, 1.73 g of FeCl₃, and 0.82 g of Cd(NO₃)₂·4H₂O were used. For the synthesis of AgClMg_0.5Fe₂O₄, 1.03 g of AgNO₃, 1.96 g of FeCl₃, and 0.61 g of MgCl₂·6H₂O were used.

2.3 Synthesis of AgClM_0.5Fe₂O₄/curcumin nanocomposites

The wet impregnation method was used to synthesize AgClM_0.5Fe₂O₄/curcumin (M = Ca, Cd, Cu, or Mg) [33]. For example, to synthesize the AgClCu_0.5Fe₂O₄/curcumin nanocomposite (50% AgClCu_0.5Fe₂O₄, 50% curcumin), 0.5 g of each component was mixed in 5 ml of distilled water and stirred at 300 rpm for 5 min at room temperature. The obtained mixture was further dried at 60°C for 24 h. The resulting dried nanocomposite powders were ground in a mortar to obtain a homogeneous powder and ensure that all AgClCu_0.5Fe₂O₄ NPs adhered to the surface of curcumin.

2.4 Characterization techniques

Crystallinity and structural properties of the nanocomposites were analyzed using an X-ray diffractometer (PW 2103, Philips, Netherlands) with CuKα radiation (λ = 1.5406 Å), collecting diffraction data over a 2θ range of 4–80° at a scanning rate of 5°/min. Functional groups in the powdered nanocomposites were analyzed using a Nicolet 6700 FT-IR spectrometer in the transmittance mode across a wavelength range of 400–4,000 cm⁻¹. JEOL JSM-IT200 scanning electron microscopy coupled with EDX analysis was performed to determine the chemical composition of the nanocomposites. Surface morphologies were observed with a JEOL transmission electron microscope (TEM, Model JSM-5400 LV, JEOL, Tokyo, Japan). The zeta potential of the nanocomposites was measured using a Malvern Zetasizer Nano ZEN 3600 [34]. To analyze the magnetic properties of the prepared nanocomposites, a VSM (Lakeshore, 7410 s, India) is used.

2.5 Cell culture and cytotoxicity assay

Two types of cell lines were used in this study: A-431 and HSF cell lines. The investigated cell lines were chosen as A-431 cell lines to overexpress the epidermal growth factor receptor (EGFR), making it a commonly used model for the pathophysiology and therapeutic approach studies of skin cancer [35]. HSF serves as a standard model for normal skin cells, providing a baseline for determining the selective cytotoxicity. By comparing cancerous and non-cancerous cells in skin cancers, it is easier to determine the nanocomposites’ safety and specificity. The used cell lines were purchased from Nawah Scientific Inc. (Mokatam, Cairo, Egypt). Initially, cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing streptomycin and penicillin at 100 mg/ml and 100 units/ml, respectively. In addition, 10% fetal bovine serum was heat-inactivated under humidified conditions of 5% (v/v) CO₂ at 37°C.

The SRB assay method was utilized to determine cell viability. Briefly, a cell suspension (5 × 10³ cells) of an approximate 100 μL was seeded in 96-well plates and then incubated for 24 h in complete media. Further, different nanocomposite concentrations in the used medium were prepared and added to cells with a volume of 100 μL [36,37]. After NP exposure, cells were fixed by changing the media with 150 μL of 10% trichloroacetic acid (TCA) and incubated at 4°C for approximately 1 h. Then, the TCA solution was removed from the cells, followed by washing them five times with distilled water. Sulforhodamine B (SRB, 70 μL) solution (0.4% w/v) was then added, and cells were left in a dark place at room temperature for 10 min. The well plates were washed three times with 1% acetic acid and left to dry in air overnight. The protein-bound SRB stain was dissolved by adding 150 μL of Tris(hydroxymethyl)aminomethane (TRIS) (10 mM), and the absorbance was measured using a microplate reader at λ_max of 540 nm (Infinite F50 Microplate Reader, TECAN, Switzerland).

2.6 Cell apoptosis

Cell populations undergoing apoptosis and necrosis are assessed utilizing the Annexin V conjugated with FITC (fluorescein isothiocyanate), a fluorescent dye, Annexin V-FITC, Apoptosis Detection Kit (Abcam Inc., Cambridge Science Park, Cambridge, UK) in conjunction with dual fluorescence channel flow cytometry. After the treatment with AgClCd_0.5Fe₂O₄ and AgClMg_0.5Fe₂O₄ for 48 h, 10⁵ cells are collected using trypsin, followed by washing twice with ice-cold PBS with a pH of 7.4 and a final concentration of (137 mM NaCl, 10 mM Phosphate, and 2.7 mM KCl). Cells are incubated with 0.5 ml of Annexin V-FITC/PI solution for 30 min in the dark place at room temperature. After staining the cells, they are injected into the ACEA Novocyte™ flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA). Cells were analyzed for FITC and PI fluorescent signals utilizing the FL1 and FL2 signal detectors, respectively (λ _ex/em 488/530 nm for FITC and λ _ex/em 535/617 nm for PI). Each tested sample undergoes the acquisition of 12,000 events, with positive FITC and/or PI cells quantified through quadrant analysis and computed using ACEA NovoExpress™ software (ACEA Biosciences Inc., San Diego, CA, USA) [38,39].

2.7 Cell cycle assay

The tested cell lines were treated with the tested nanomaterials (AgClCd_0.5Fe₂O₄ and AgClMg_0.5Fe₂O₄) for 48 h. Then, cells (10⁵ cells) were harvested via trypsinization, followed by washing two times with ice-cold phosphate-buffered saline (PBS) at pH 7.4. Cells were resuspended in 2 ml of ice-cold ethanol at a concentration of 60%, followed by incubation at 4°C for 1 h to affect their fixation. Two additional washing steps were also performed in the fixed cells with PBS (pH 7.4) and re-suspended in 1 ml of PBS containing 50 µg/ml RNAase A and 10 µg/ml propidium iodide (PI). Cells are assessed for DNA content after their incubation for 20 min in the dark at 37°C, using flow cytometry and an FL2 (λ _ex/em 535/617 nm) signal detector (ACEA Novocyte™ flow cytometer, ACEA Biosciences Inc., San Diego, CA, USA). Each sample had 12,000 events acquired. Cell cycle distributions were determined utilizing ACEA NovoExpress™ software (ACEA Biosciences Inc., San Diego, CA, USA) [40,41].

2.8 Statistical analysis

Statistical analysis was performed via the GraphPad Prism Version 9 (GraphPad Software, San Diego, CA, USA). The results are presented using the mean standard deviation (SD). The comparison was made using ANOVA statistical tests with Turkey’s HSD post-hoc test, a multiple comparison between positive and negative control groups. Results are statistically significant when the p-value is less than 0.05.

3.1 Characterization of hybrid nanocomposites

AgClM_0.5Fe₂O₄ nanocomposites were synthesized via a chemical co-precipitation method. This method is commonly favored for synthesizing spinel ferrites because of its simplicity and cost-effectiveness. It allows for control over particle size and morphology, ensures high compositional homogeneity, and facilitates adjustments to reaction parameters such as pH to achieve desired properties. This versatility makes it an effective technique for producing tailored spinel ferrite materials with consistent characteristics [49]. AgClM_0.5Fe₂O₄ nanocomposites (50% by wt) were loaded on the surface of curcumin using the wet impregnation technique. The wet impregnation method provides numerous advantages, such as simplicity, versatility, cheap, effective control over particle size and distribution, capability to deposit active materials onto high surface area supports, and its suitability for large-scale production [50].

The XRD patterns of pure curcumin, AgClCa_0.5Fe₂O₄/curcumin, AgClCd_0.5Fe₂O₄/curcumin, AgClCu_0.5Fe₂O₄/curcumin, and AgClMg_0.5Fe₂O₄/curcumin nanocomposites are shown in Figure 1. Pure curcumin displayed reflections at 2θ values of 17°, 19.3°, 21.9°, 23.8°, 26.5°, and 28.1° [51]. All the prepared nanocomposites showed diffraction peaks at 2θ values of 27.8°, 32.2°, 46.2°, 54.8°, 57.4°, and 76.7°, which are attributed to the (111), (200), (220), (311), (222), and (420) planes, respectively, of cubic AgCl (JCPDS card no. 00-006-0480). In addition, AgClCa_0.5Fe₂O₄/curcumin showed also reflections at 2θ values of 24.1°, 33.2°, 35.6°, 40.9°, 49.5°, 54.1°, 62.5°, 64.2°, and 72.1°, respectively, which correspond to the (012), (104), (110), (113), (024), (116), (214), (300), and (119) planes for rhombohedral Fe₂O₃ (JCPDS card no. 01-084-0306). Moreover, the peaks at 2θ values of 38.1° and 44.3° are related to cubic zero-valent Ag (JCPDS card no. 01-087-0717) and the peak at 67.4° belongs to CaFe₂O₄ (JCPDS card no. 00-019-0219). The XRD pattern of the AgClCd_0.5Fe₂O₄/curcumin nanocomposite showed that, besides the AgCl peaks, additional peaks at 2θ values of 24.4°, 35.5°, and 49.4° are ascribed to rhombohedral Fe₂O₃ (JCPDS card no. 01-079-1741). In addition, the peaks at 28.9°, 34.1°, 51.4°, 60.2°, 67.4°, and 70.9° are attributed to cubic CdFe₂O₄ (JCPDS card no. 00-022-1063). Moreover, the peak at 38° is related to cubic zero-valent Ag (JCPDS card no. 01-087-0597) and the peak at 74.5° belongs to cubic AgCl (JCPDS card no. 00-002-0848). The AgClCu_0.5Fe₂O₄/curcumin nanocomposite showed reflections at 2θ values of 24.1°, 33.2°, 40.9°, 49.5°, 54.1°, and 64°, which are assigned to rhombohedral Fe₂O₃ (JCPDS card no. 00-033-0664). Additionally, the peaks at 35.7° and 62.5° are assigned to CuFe₂O₄ (JCPDS card no. 01-077-0010). Moreover, the reflections at 38.8° and 67.5° are ascribed to monoclinic CuO (JCPDS card no. 00-045-0937). The AgClMg_0.5Fe₂O₄/curcumin nanocomposite exhibited signals at 2θ values of 24.2°, 33.2°, 40.7°, 49.5°, 54.1°, and 64.1° are assigned to rhombohedral Fe₂O₃ (JCPDS card no. 01-073-2234). In addition, the peaks at 35.6°, 62.5°, and 74.5° are related to cubic MgFe₂O₄ (JCPDS card no. 00-001-1120). Moreover, the peak at 38.2° is ascribed to cubic zero-valent Ag (JCPDS card no. 01-087-0717) and the signal at 67.4° is related to cubic AgCl (JCPDS card no. 00-031-1238). The average crystallite sizes for the prepared nanocomposites were estimated using the Scherrer equation [33,52,53] and were found to be 11.1, 23.5, 20.3, 26.9, and 30.3 nm for pure curcumin, AgClCa_0.5Fe₂O₄/curcumin, AgClCd_0.5Fe₂O₄/curcumin, AgClCu_0.5Fe₂O₄/curcumin, and AgClMg_0.5Fe₂O₄/curcumin nanocomposites, respectively. The relative crystallinity (RC) of pure curcumin and the prepared nanocomposites was estimated using the following equation:

Figure 1

XRD patterns of (a) pure curcumin, (b) AgClCa_0.5Fe₂O₄/curcumin, (c) AgClCd_0.5Fe₂O₄/curcumin, (d) AgClCu_0.5Fe₂O₄/curcumin, and (e) AgClMg_0.5Fe₂O₄/curcumin hybrid nanocomposites.

It was found that the RC takes the following order: curcumin (59.9%) < AgClMg_0.5Fe₂O₄/curcumin (67.5%) < AgClCu_0.5Fe₂O₄/curcumin (72.3%) < AgClCa_0.5Fe₂O₄/curcumin (74.8%) < AgClCd_0.5Fe₂O₄/curcumin (82.2%).

FT-IR spectroscopy is a powerful tool for understanding and optimizing material surfaces by identifying and quantifying functional groups crucial to their performance and application. Figure 2 shows the FT-IR spectra of pure curcumin, AgClCa_0.5Fe₂O₄/curcumin, AgClCd_0.5Fe₂O₄/curcumin, AgClCu_0.5Fe₂O₄/curcumin, and AgClMg_0.5Fe₂O₄/curcumin nanocomposites.

Figure 2

FT-IR spectra of (a) pure curcumin, (b) AgClCa_0.5Fe₂O₄/curcumin, (c) AgClCd_0.5Fe₂O₄/curcumin, (d) AgClCu_0.5Fe₂O₄/curcumin, and (e) AgClMg_0.5Fe₂O₄/curcumin hybrid nanocomposites.

The curcumin spectrum showed FT-IR bands at 3,417 cm⁻¹ (phenolic hydroxyl (–OH) groups), 2,924 cm⁻¹ (aliphatic C–H groups), 2,857 cm⁻¹ (aliphatic C–H groups), 1,737 cm⁻¹ (C═O stretching), 1,640 cm⁻¹ (C═C stretching [54,55], 1,515 cm⁻¹ (C═O and C═C vibrations) [54], 1,455 cm⁻¹ (aromatic C–H groups), 1,372 cm⁻¹ (aromatic C–H groups), 1,160 cm⁻¹ (C–O–C stretching for methoxy groups), 1,078 cm⁻¹ (C–O stretching mode), 1,031 cm⁻¹ (C–O–C stretching vibrations), and 520 cm⁻¹ (aromatic C–C–C out of plane bending) [55]. The FT-IR spectra of the nanocomposites revealed, in addition to the characteristic bands of curcumin, additional bands in the range of 670–430 cm⁻¹, corresponding to metal–oxygen vibrations. These findings confirm the successful loading of AgClM_0.5Fe₂O₄ onto the surface of curcumin. The FT-IR spectra of the nanocomposites also show distinct vibrational bands corresponding to various bonding modes and structural features in the region below 1,000 cm⁻¹. In all samples, a band around 855 cm⁻¹ (843 cm⁻¹ for AgClMg_0.5Fe₂O₄/curcumin) is attributed to the Fe–O–H bending mode [56]. The Fe–O, Fe–O–Fe, and O–Fe–O vibrations appear in the range of 568–573 cm⁻¹ for AgClCa_0.5Fe₂O₄/curcumin, AgClCd_0.5Fe₂O₄/curcumin, and AgClCu_0.5Fe₂O₄/curcumin, while AgClMg_0.5Fe₂O₄/curcumin shows this band at 574 cm⁻¹ [57]. The Fe–O bond at tetrahedral sites is observed at 533–555 cm⁻¹, with a prominent band at 555 cm⁻¹ for AgClCd_0.5Fe₂O₄/curcumin [58], while the octahedral Fe–O bond is highlighted at 545 cm⁻¹ for AgClCd_0.5Fe₂O₄/curcumin. The Fe³⁺–O bond vibration is detected in all samples at 549–565 cm⁻¹. Additionally, the intrinsic stretching vibration of metal cations in tetrahedral regions appears at 526–533 cm⁻¹. The bands in the range of 452–462 cm⁻¹ are assigned to oxygen stretching vibrations at the B sites [58], while Fe–O bending and stretching vibrations are observed at 446–452 cm⁻¹ and 432–438 cm⁻¹, respectively [59,60]. Unique bands include the Cu–O vibration at 476 cm⁻¹ for AgClCu_0.5Fe₂O₄/curcumin [61] and the Mg–O vibration at 410 cm⁻¹ for AgClMg_0.5Fe₂O₄/curcumin [59]. Lattice vibrations of Ca–O are also identified at 426 cm⁻¹ for AgClCa_0.5Fe₂O₄/curcumin [62]. Overall, these findings reflect the various bonding environments, structural properties of the nanocomposites, and the successful synthesis of these nanocomposites.

To determine the elemental composition of the AgClM_0.5Fe₂O₄/curcumin nanocomposites, EDX analysis was performed. The EDX spectra of the synthesized composites are shown in Figure 3. The EDX spectra of the synthesized AgClM_0.5Fe₂O₄/curcumin nanocomposites confirmed the presence of key elements. Specifically, the spectrum of AgClCa_0.5Fe₂O₄/curcumin (Figure 3a) indicates the presence of C, O, Cl, Ca, Fe, and Ag. For AgClCd_0.5Fe₂O₄/curcumin (Figure 3b), the detected elements include C, O, Cl, Cd, Fe, and Ag. Similarly, AgClCu_0.5Fe₂O₄/curcumin (Figure 3c) shows the presence of C, O, Cl, Cu, Fe, and Ag, while AgClMg_0.5Fe₂O₄/curcumin (Figure 3d) reveals C, O, Cl, Mg, Fe, and Ag. These findings confirm the successful synthesis of the respective nanocomposites. As summarized in Table 1, the AgClCa_0.5Fe₂O₄/curcumin nanocomposite contains 35.03% carbon (C), 50.79% oxygen (O), 2.19% chlorine (Cl), 1.94% calcium (Ca), 5.35% iron (Fe), and 4.70% silver (Ag). The AgClCd_0.5Fe₂O₄/curcumin nanocomposite consists of 25.80% C, 38.17% O, 3.91% Cl, 13.12% cadmium (Cd), 11.45% Fe, and 7.55% Ag. The AgClCu_0.5Fe₂O₄/curcumin nanocomposite contains 38.29% C, 43.02% O, 2.16% Cl, 2.27% copper (Cu), 3.48% Fe, and 5.78% Ag, and the AgClMg_0.5Fe₂O₄/curcumin nanocomposite shows 15.50% C, 36.87% O, 2.19% Cl, 6.96% magnesium (Mg), 19.68% Fe, and 18.80% Ag.

Figure 3

EDX spectra of (a) AgClCa_0.5Fe₂O₄/curcumin, (b) AgClCd_0.5Fe₂O₄/curcumin, (c) AgClCu_0.5Fe₂O₄/curcumin, and (d) AgClMg_0.5Fe₂O₄/curcumin hybrid nanocomposites.

TEM analysis provides unparalleled high-resolution imaging and detailed structural, chemical, and morphological information at the atomic scale, making it essential for advanced research in materials science, biology, and nanotechnology. Figure 4 shows the TEM micrographs of AgClCd_0.5Fe₂O₄/curcumin, AgClMg_0.5Fe₂O₄/curcumin, and their particle size distribution (nanocomposites with lower IC₅₀ values). As shown in Figure 4a, the AgClCd_0.5Fe₂O₄/curcumin nanocomposite has connected semi-spherical particles with an average diameter of 62.0 nm (Figure 4b). The TEM image of the AgClMg_0.5Fe₂O₄/curcumin nanocomposite (Figure 4c) revealed agglomerate particles, semi-spherical in shape with an average NP diameter of 114 nm, as shown in Figure 4d. It was observed that the AgClCd_0.5Fe₂O₄/curcumin nanocomposite, with an average particle size of 62.0 nm, was more aggregated than the AgClMg_0.5Fe₂O₄/curcumin nanocomposite, which had an average particle size of 114 nm, which can be justified based on the well-established relationship between the NP size and aggregation behavior. Smaller NPs generally exhibit a significantly higher surface-to-volume ratio, leading to increased surface energy and stronger van der Waals forces, which drive the particles to aggregate to reduce the overall system energy [33,63]. The impact of the observed particle aggregation on the biological properties is investigated in the cytotoxicity study.

Figure 4

TEM images and particle size distribution curves of AgClCd_0.5Fe₂O₄/curcumin (a) and (b) and AgClMg_0.5 Fe₂O₄/curcumin (c) and (d) nanocomposites.

The stability of the AgClCd_0.5Fe₂O₄/curcumin and AgClMg_0.5Fe₂O₄/curcumin nanocomposites (hybrid nanocomposites with lower IC₅₀ values) was assessed by measuring their zeta potential using Malvern Zetasizer nano ZEN 3600 (Malvern, LO, UK). The zeta potential, which indicates the stability of colloidal dispersions and reflects the surface charge of the nanocomposites, was found to be −28.1 ± 0.5 and −27.7 ± 0.9 mV for AgClCd_0.5Fe₂O₄/curcumin and AgClMg_0.5Fe₂O₄/curcumin nanocomposites, respectively, as shown in Figure 5. These values demonstrate the good physical stability of the synthesized curcumin hybrid nanocomposites. According to a published report, zeta potential values that are higher than +25 mV or less than −25 mV are favored because they provide a significant repulsion force between the NPs, which inhibits aggregation [64]. The AgClMg_0.5Fe₂O₄/curcumin nanocomposite has a lower zeta potential negative value (−36.8 mV) than that obtained from pure MgFe₂O₄ [65].

Figure 5

Zeta potential spectra of (a) AgClCd_0.5Fe₂O₄/curcumin and (b) AgClMg_0.5 Fe₂O₄/curcumin nanocomposites.

Figure 6 presents the hysteresis loop measured at room temperature for the prepared nanocomposites. The figure shows that the AgClCa_0.5Fe₂O₄/curcumin and AgClCd_0.5Fe₂O₄/curcumin nanocomposites exhibit a paramagnetic behavior because of the linear increase with the applied magnetic field. The AgClCu_0.5Fe₂O₄/curcumin and AgClMg_0.5Fe₂O₄/curcumin nanocomposites showed hysteresis loops with S-shaped response without a clear hysteresis loop, indicating superparamagnetic behavior. The obtained values, including saturation magnetization (Mₛ), coercivity (Hₒ), retentivity (Mᵣ), and the squareness ratio (M _r/M _s) of the synthesized hybrid nanocomposites, are calculated and listed in Table 2. The saturation magnetization according to an analysis of the data that was obtained follows the order curcumin AgClMg_0.5Fe₂O₄ > AgClCu_0.5Fe₂O₄ > AgClCd_0.5Fe₂O₄ > AgClCa_0.5Fe₂O₄/curcumin. Differences in the magnetic moment, ionic radius, super exchange contact strength, and structural stability decrease the saturation magnetization in the latter order. According to reports, a single-domain magnetic structure is indicated by a squareness ratio (Mᵣ/Mₛ) of ≥0.5, whereas the construction of a multidomain structure is suggested by a ratio of <0.5 [66]. All of the prepared nanocomposites have a multidomain structure, as shown in Table 2, and their squareness ratios ranged from 0.0061 to 0.1557. Owing to the observed magnetic properties of these materials, they are used to improve contrast for magnetic resonance imaging (MRI) and X-ray computed tomography (CT) imaging, which are both useful for detecting cancer cells. Additionally, they offer a powerful platform for cancer treatment guided by multimodal imaging [67].

Figure 6

VSM spectra of the prepared nanocomposites.

3.2 Cytotoxicity study

The anticancer potential of the prepared spinal ferrite/curcumin hybrid nanocomposite systems was evaluated in normal and cancer skin cell lines, HSF and A-431, compared to free curcumin and Dox as a positive control anti-tumor. The study aimed to assess the IC₅₀ values of the prepared curcumin hybrid nanocomposite and Dox on HSF and A-431 cell lines. Concentration-dependent plots for each of the cell lines, along with the respective IC₅₀ values and statistical tests, are provided in the Supporting Information. These additional details provide a complete analysis of the nanocomposites’ cytotoxic effects (Figures S1 and S2). The data revealed a significant, dose-dependent reduction in the viability of A-431 cells upon treatment with the nanocomposites, while HSF cells exhibit minimal sensitivity under similar conditions. This differential cytotoxicity underscores the selective efficacy of the prepared hybrid curcumin nanocomposites against cancerous cells. The IC₅₀ value, which is the concentration of a substance necessary to inhibit 50% of cell growth, is an important metric. Table 3 and Figure 7 present the IC₅₀ values of the prepared compounds. Among the metal hybrid nanocomposites, AgClMg_0.5Fe₂O₄/curcumin and AgClCd_0.5Fe₂O₄/curcumin exhibited remarkable anticancer potential against the A-431 cell line, with IC₅₀ values of 34.83 and 26.83 µg/ml, respectively. In contrast, AgClCu_0.5Fe₂O₄/curcumin and AgClCa_0.5Fe₂O₄/curcumin showed reduced efficacy, with IC₅₀ values exceeding 100 µg/ml for the A-431 cell line. Particularly, the free curcumin at the tested concentrations revealed minimal anticancer activity and toxicity, with IC₅₀ values exceeding 100 µg/ml for both cell line types.

Figure 7

Percentage cell viability of Dox, curcumin, and the prepared doped ferrite/curcumin hybrid nanocomposites on HSF: Human skin fibroblast versus A-431: Human epidermoid skin carcinoma. Cu refers to (AgClCu_0.5Fe₂O₄/curcumin), Mg (AgClMg_0.5Fe₂O₄/curcumin), Ca (AgClCa_0.5Fe₂O₄/curcumin), Cd (AgClCd_0.5Fe₂O₄/curcumin), while Dox (doxorubicin) is used as a positive control.

Dox, a well-known anticancer medication, demonstrated a strong cytotoxic effect against the A-431 cell line (IC₅₀ = 0.15 µg/ml). In addition, it also showed higher activity against the HSF cell line, with an IC₅₀ value of 2.85 µg/ml, which proved their toxicity on normal cells at very low concentrations. These findings demonstrated the distinct sensitivity of malignant and non-cancerous skin cells to Dox, underlining the importance of tailored treatment options to reduce the negative effects on healthy cells [39]. The toxicity of Dox in the normal HSF cell line has also been reported previously [68,69]. Interestingly, all of the investigated nanocomposites showed a safe pattern on normal skin fibroblasts, as they showed IC₅₀ values of >100 µg/ml, hence a better biocompatibility. Although TEM images delineated an observed particle aggregation, especially with AgClCd_0.5Fe₂O₄/curcumin nanocomposite, than that of AgClMg_0.5Fe₂O₄/curcumin nanocomposite, they have a small particle size of about 62.0 nm, which does not affect the biological behavior as depicted from the observed lower IC₅₀. Many studies have shown that the NPs between 50 and 100 nm have high cellular internalization and absorption, as previously investigated [70].

The comparison of IC₅₀ values between A-431 and HSF cells reveals important information about the selectivity and potential toxicity of the tested hybrid nanocomposites. The large variance in IC₅₀ values for the investigated materials emphasizes the necessity of creating targeted medicines that specifically target cancer cells while causing minimal harm to healthy organs [71].

3.3 Cell apoptosis and cell cycle

The mechanisms underlying the potential toxicity of the prepared materials could be investigated by studying cell apoptosis and cell cycle, utilizing the flow cytometry assay.

3.4 Cell apoptosis

Results indicated notable differences in the cytotoxic effects of Dox and the nanocomposite hybrid treatments (AgClMg_0.5Fe₂O₄/curcumin and AgClCd_0.5Fe₂O₄/curcumin) in comparison to control untreated cells (CT). In the untreated control (CT) group, a high proportion of cells remained viable (95.61%), exhibiting minimal signs of apoptosis or necrosis, consistent with expectations for untreated cells. The low percentages of cells in early apoptosis (0.97%) and late apoptosis (3.06%) suggest a minimal induction of cell death under normal conditions. Our study demonstrated that AgClMg_0.5Fe₂O₄/curcumin and AgClCd_0.5Fe₂O₄/curcumin induce apoptosis and cause cycle arrest in A-431 cells. These effects are likely mediated through mechanisms like those observed with curcumin and its derivatives in various cancer models [72].

The administration of Dox showed a significant increase in necrosis (53.15%, P < 0.001), consistent with the compound’s strong cytotoxic effects, which cause considerable cellular damage and membrane disruption. Furthermore, a significant increase in late apoptosis (34.92%, P < 0.001) indicated that Dox effectively activates apoptotic pathways, presumably via DNA intercalation and topoisomerase II inhibition [73]. The diminished live cell population (11.56%) further corroborates the significant cytotoxicity of Dox. The initial apoptosis rate (0.37%, P < 0.001) was comparatively low, suggesting that cells predominantly advanced to late-stage apoptosis or necrosis following Dox treatment rather than undergoing an earlier apoptotic phase. AgClCd_0.5Fe₂O₄/curcumin exhibited a moderate cytotoxic effect, with necrosis at 8.53% and late apoptosis at 14.52%, both significantly elevated in comparison to the untreated control cells (P < 0.001). This suggests that AgClCd_0.5Fe₂O₄/curcumin can induce significant apoptosis, likely resulting from the combined effects of cadmium and curcumin in promoting oxidative stress and DNA damage [74]. The live cell population (76.19%, P < 0.001) was significantly higher than that of Dox, indicating that AgClCd_0.5Fe₂O₄/curcumin exhibited lower overall toxicity, although they remain considerably more cytotoxic than the control group. Early apoptosis (0.75%) was marginally elevated compared to the control, indicating the commencement of apoptotic pathways.

AgClMg_0.5Fe₂O₄/curcumin exhibited cytotoxicity, albeit to a lesser extent than AgClCd_0.5Fe₂O₄/curcumin. The necrosis rate (6.07%) and late apoptosis (10.20%) were lower than those observed with AgClCd_0.5Fe₂O₄/curcumin, yet remained significantly (P < 0.001) higher than the control group. The live cell percentage (83.03%) exceeded that of both Dox and AgClCd_0.5Fe₂O₄/curcumin, indicating that AgClMg_0.5Fe₂O₄/curcumin may exhibit lower toxicity, possibly attributable to variations in NP characteristics and cellular uptake mechanisms [75]. The early apoptosis rate of 0.70% was comparable to that observed with AgClCd_0.5Fe₂O₄/curcumin, suggesting some activation of apoptosis pathways at an early stage, albeit to a lesser extent [76].

The results indicated that Dox produces the highest cytotoxicity, characterized by a substantial degree of necrosis and late apoptosis, which is in accordance with the cytotoxicity study. Both AgClCd_0.5Fe₂O₄/curcumin and AgClMg_0.5Fe₂O₄/curcumin exhibited promising apoptosis-inducing effects; however, at a lower level than Dox, with AgClCd_0.5Fe₂O₄/curcumin demonstrating the highest potency among the two treatments. The relatively mild effect of AgClMg_0.5Fe₂O₄/curcumin may indicate its potential for lower toxicity while still achieving effective cell death, suggesting it as a promising nanomaterial for further investigation in cancer therapies (Figure 8) [75].

Figure 8

Percentage of cells (mean ± SD) of living, early, and late apoptotic, along with dead cells, in non-treated control cells CT (a), doxorubicin Dox (b), AgClCd_0.5Fe₂O₄/curcumin (c), AgClMg_0.5Fe₂O₄/curcumin (d) treated A-431: Human epidermoid skin carcinoma for 48 h. The percentage of the cell of different living cells (e), early apoptosis (f), late apoptosis (g), and dead cells (h) (n = 3 biological replicates/group). Data were analyzed by one-way ANOVA with Tukey’s HSD post-hoc test; *P < 0.05, **P < 0.01, and ***P < 0.001 indicate the significant difference compared with CT, and ^#iP < 0.05, ^## P < 0.01, and ^### P < 0.001 indicate the significant difference compared with Dox.

3.5 Cell cycle

The sub-G1 phase, indicative of apoptosis through fragmented DNA, revealed a significant increase in apoptosis with treatments. Untreated cells exhibited baseline apoptosis (3.55%), while Dox treatment increased significantly (7.7-fold, P < 0.001), reflecting its cytotoxic effects via DNA intercalation and topoisomerase II inhibition. AgClCd_0.5Fe₂O₄/curcumin showed the highest apoptosis (10-fold of control, P < 0.001), surpassing Dox, likely due to synergistic oxidative stress and DNA damage from cadmium and curcumin. AgClMg_0.5Fe₂O₄/curcumin induced moderate apoptosis (6.2-fold of control, P < 0.001), lower than AgClCd_0.5Fe₂O₄/curcumin but at the same time higher than Dox, suggesting reduced toxicity. In the G0/G1 phase, Dox caused a significant reduction (24.07%), consistent with its known efficacy to perform cell cycle arrest in S and G2/M phases. AgClCd_0.5Fe₂O₄/curcumin induced a moderate decrease in G0/G1 (2-fold of Dox, P < 0.001), while AgClMg_0.5Fe₂O₄/curcumin had a smaller reduction (2.02-fold of Dox, P < 0.001), suggesting partial cell cycle arrest. The S-phase analysis showed that Dox significantly reduced S-phase cells (7.13%), while AgClCd_0.5Fe₂O₄/curcumin had little effect (13.03%), and AgClMg_0.5Fe₂O₄/curcumin increased S-phase population (16.66%), indicating enhanced proliferation or delayed progression. Dox induced significant G1/M arrest (41.61%), while AgClCd_0.5Fe₂O₄/curcumin and AgClMg_0.5Fe₂O₄/curcumin caused mild-to-moderate G1/M arrest, with AgClMg_0.5Fe₂O₄/curcumin exhibiting a slightly stronger effect, suggesting differences in bioactivity or uptake (Figure 9). These results are in agreement with the previous finding that the inhibition of A-431 cells in the G2/M phase of the cell cycle after treatment with curcumin. The increase in the number of cells in the G2/M phase corresponded with a decrease in the number of cells in the G0/G1 phase. The impact of curcumin on cell cycle progression has been previously reported in different types of cell lines [77,78]. Incorporation of Cd²⁺ and Mg²⁺ into curcumin in the prepared nanocomposites can synergistically improve the cell cycle arrest of curcumin [75]. Curcumin has been shown to induce apoptosis, possibly by causing the production of ROS, disruption of mitochondrial membrane potential, release of cytochrome c, and activation of caspase cascades. Curcumin also affects important signaling pathways, including the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, causing cell cycle arrest at the G2/M phase and apoptosis [79]. Hence, it is possible that the prepared curcumin hybrid nanocomposites could exert their cytotoxic effects through the same mechanisms. In addition, the presence of metal ions such as Mg²⁺ and Cd²⁺ may enhance these effects by regulating ROS production and apoptotic pathways. However, we are aware that extensive mechanistic studies should be pursued to elucidate the specific pathways mediating our nanocomposite cytotoxicity. Future research will focus on interrogating these mechanisms for a complete understanding of their anti-cancer potential.

Figure 9

Percentage of cells (mean ± SD) in the different phases of the cell cycle in non-treated control cells CT (a), doxorubicin Dox (b), AgClCd_0.5Fe₂O₄/curcumin (c), AgClMg_0.5Fe₂O₄/curcumin (d), treated A-431: Human epidermoid skin carcinoma for 48 h. The percentage of the cell of different cell cycle sub-G1 (e), G0/G1 phase (f), S phase (g), and G2/M phase (h) (n = 3 biological replicates/group). Data were analyzed by one-way ANOVA with Tukey’s HSD post-hoc test; *P < 0.05, **P < 0.01, and ***P < 0.001 indicate the significant difference compared with CT, and ^# P < 0.05, ^##iP < 0.01, and ^### P < 0.001 indicate the significant difference compared with Dox.

The cell cycle and apoptosis data collectively highlight the differential cytotoxic effects of Dox, AgClCd_0.5Fe₂O₄/curcumin, and AgClMg_0.5Fe₂O₄/curcumin on the investigated cancer cells. Dox treatment caused significant apoptosis, with a marked increase in necrosis and late apoptosis, consistent with its established mechanism of inducing DNA damage and cell cycle arrest [80]. This resulted in a dramatic reduction in live cell populations, primarily through G1/S arrest and subsequent apoptosis. Both Cd and Mg-curcumin nanocomposites induced apoptosis as well, with AgClCd_0.5Fe₂O₄/curcumin showing the most potent cytotoxic effects, surpassing Dox in late apoptosis and necrosis. This suggests that the combination of cadmium and curcumin in hybrid NPs may enhance the apoptotic response through synergistic oxidative stress and DNA damage. AgClMg_0.5Fe₂O₄/curcumin exhibited a less pronounced cytotoxic effect, though still inducing apoptosis and cell cycle arrest, particularly in the S and G2/M phases. The lower overall toxicity of AgClMg_0.5Fe₂O₄/curcumin compared to AgClCd_0.5Fe₂O₄/curcumin nanocomposites suggests that they may offer a more controlled therapeutic option, with fewer adverse effects.

There are certain limitations of our study, such as the exclusive use of only two cell lines as skin models, which may not be capable of exhaustively exploring the variety of skin cancers. Future studies should be conducted to encompass different skin cancer models for enhancing the generalizability of the work conducted so far. Additionally, detailed mechanistic studies are required to determine the specific pathways through which oxidative stress and DNA damage caused the observed apoptotic effects of the prepared nanocomposites. Even though earlier reports have indicated possible tumor imaging applications of such nanocomposites with MRI and CT, it is vital to perform certain imaging studies to empirically verify such effects as well as determine their clinical implications.