Inhibition of hepatocellular carcinoma cells through ferroptosis therapy using Fe3O4/Ag nanocomposites

In the current study, Fe3O4/Ag nanohybrids demonstrating high efficiency in liver cancer ferroptosis therapy and MR imaging were synthesized using a facile one-pot approach. Subsequently, their morphology, chemical composition, structure, Fenton reaction activity, and magnetic resonance (MR) contrast effect were systemically assessed. Importantly, the in vitro studies demonstrated that the Fe3O4/Ag nanohybrids had a high therapeutic efficiency in treating liver cancer cells and performed exceptionally well during T2-weight MR imaging. Correspondingly, these Fe3O4/Ag nanohybrids demonstrate significant potential for use in the field of biomedicine.


Introduction
Hepatocellular carcinoma (HCC) ranks as the fourth most common cause of cancer-related death worldwide [1].It is the second most significant contributor to years of life lost globally due to cancer, emphasizing the substantial disease burden it carries [2].In the United States, liver cancer has experience the most significant increase among all other forms of cancer [3].However, there is a lack of effective approaches for the clinical management of liver cancer.Conventional cancer therapies generally comprise of surgical intervention, chemotherapy, and radiotherapy.However, each of these approaches possesses its own limitations, including poor target specificity and significant adverse effects.Consequently, the development of novel strategies for effective cancer treatment is imperative.In recent years, ferrotherapy has emerged as a novel approach to cancer treatment [4][5][6].Ferrotherapy employs Fenton reactions, where iron ions (Fe 3+ /Fe 2+ ) catalytically dissociate hydrogen peroxide (H 2 O 2 ) into hydroxyl radicals (•OH) to eliminate cancer cells.Currently, various Fe-based nanoparticles have demonstrated Fenton reaction activities [7][8][9][10][11][12][13][14][15][16].These involve the decomposition of H 2 O 2 into •OH radicals, owing to which, they have been rationally engineered to induce ferroptosis [7][8][9][10][11][12][13][14][15][16].Importantly, Fe-based nanoparticles can be used as MRI contrast agents to enhance the accuracy and dependability of cancer diagnosis.Thus, Fe-based nanoparticles have gained significant attention in the current field of cancer therapy research.
The present study presents a facile, one-pot approach to synthesize Fe 3 O 4 /Ag nanohybrids for the purpose of cancer ferroptosis therapy and MR imaging [17,18].The resulting Fe 3 O 4 /Ag nanohybrids were characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), powder x-ray diffraction (PXRD), and relaxivity measurements.The nanohybrids were also examined for their Fenton reaction activity.In addition, the cytotoxicity of the substance was examined in vitro using MTT assay and flow cytometry.5-Dimethylthiazol-2-yl)−2, 5-diphenyltetrazolium bromide) (MTT) and trypan blue were purchased from Beyotime (China).The reagents and chemicals used were of high purity, suitable for analytical purposes.

Synthesis of Fe 3 O 4 /Ag nanohybrids
The Fe 3 O 4 /Ag nanohybrids were synthesized using a one-pot hydrothermal approach.Briefly stating, a mixture of FeCl 3 •6H 2 O (270 mg, 1.0 mmol) and NaOAc (328 mg, 4.0 mmol) was dissolved in 20 ml of ethylene glycol.Subsequently, AgNO 3 (136 mg, 0.8 mmol) was added to the solution while stirring continuously until complete dissolution was achieved.The solution was then sealed in a 25 ml Teflon-lined autoclave and heated at 200 °C for 10 h.The resulting Fe 3 O 4 /Ag nanohybrids were then collected using a magnet and subsequently washed with ethanol and water.

In vitro hydroxyl radical (•OH) detection
The •OH radicals generated from the Fenton reaction using the Fe 3 O 4 /Ag nanohybrids would cause the oxidation of the colorless TMB to form a blue-colored compound called oxTMB.This transformation can be detected using UV-vis spectra at a wavelength of 650 nm.Briefly stating, Fe 3 O 4 /Ag nanohybrids (20 μg ml −1 ) and TMB (0.1 mg ml −1 ) were added to HAc buffer (0.1 M, pH = 4.8).The catalytic kinetics were monitored promptly after adding a range of H 2 O 2 concentrations (40, 60, 80, and 100 mM) into the aforementioned solutions.The data were obtained by measuring the absorption of the solutions at a wavelength of 650 nm using a UV-vis-NIR spectrophotometer in time-scan mode.

MTT assay
HepG2 cells were inoculated in 24-well plates at a concentration of 5 × 10 4 cells per well.Following an overnight culture, the samples were subjected to treatment with H 2 O 2 , various concentrations of Fe 3 O 4 /Ag nanohybrids, or a combination of H 2 O 2 and Fe 3 O 4 /Ag nanohybrids for a duration of 24 h.After a 24 h period, the ability of the cells to proliferate was evaluated by adding an MTT solution (50 μg/well) and allowing it to incubate for 1 h.Following the removal of the liquid above the cells, the MTT solution was combined with dimethyl sulfoxide.The optical density (OD) at a wavelength of 570 nm was measured using the microplate reader.The experiment was repeated three times.

Apoptosis assay in vitro
In order to assess the impact of H 2 O 2 on Fe 3 O 4 /Ag nanohybrids in preventing cell apoptosis, we conducted flow apoptosis experiments.HepG2 cells were uniformly seeded into 6-well plates at a concentration of 5 × 10 5 cells per well.Once the cells were adhered to the wall, H 2 O 2 , Fe 3 O 4 /Ag nanohybrids with or without H 2 O 2 were added to the experimental group and the control group, respectively.and incubated in an incubator for 24 h.Subsequently, the medium was removed and the cells were rinsed thrice with warm PBS.Thereafter, the cells were digested with trypsin, excluding EDTA, rinsed twice with cold PBS, and subsequently suspended in the Annexin V binding solution.The Annexin V-FITC dye was then added, and the cells were incubated at 4 °C in the absence of light for a duration of 15 min.Subsequently, propidium iodide (PI) dye was added and subjected to incubation at a temperature of 4 °C in the absence of light for 5 min.Cell apoptosis was identified utilizing a flow cytometer.

Results and discussion
The morphology of the Fe 3 O 4 /Ag nanohybrids was analyzed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM).The SEM and TEM images in figures 1 and 2(A) revealed that the synthesized nanoparticles exhibit a spherical morphology, with an average particle size of 303 nm and a good size dispersity (standard deviation (SD) = 70) (figure 2(E)) [19].Furthermore, as illustrated in figures 2(B)-(D), the elemental mapping results revealed that the Fe 3 O 4 /Ag nanohybrids were primarily composed of Fe, Ag and O.In addition, the elemental maps confirm that the Ag element was well distributed on the Fe 3 O 4 nanoparticles.
It is widely recognized that Fe-based nanoparticles exhibit Fenton reaction activity under acidic conditions, which enables them to catalyze the conversion of H 2 O 2 into free radicals (•OH) and kill cancer cells [22,23].Herein, in order to evaluate the Fenton reaction activity of the Fe 3 O 4 /Ag nanohybrids, the radical generation was monitored using 3′,3',5,5′-tetramethyl-benzidine (TMB).Correspondingly, the hydroxyl radicals generated are likely to oxidize the colorless TMB to blue-colored oxTMB (oxTMB), which features a characteristic absorbance at 650 nm.Accordingly, the production of the blue-colored oxTMB was monitored using UV-vis absorption spectroscopy.As observed in figure 4   percent even at a concentration of 200 μM (figure 6).Furthermore, we verified that the application of H 2 O 2 alone did not impact the proliferation activity of HepG2 cells (figure 6).However, the combination of H     7). Furthermore, our study demonstrated that the simultaneous application of Fe 3 O 4 /Ag nanohybrids and H 2 O 2 resulted in large morphological changes in HepG2 cells, when compared to the control group [24].The alterations in cell morphology were more evident when comparing Fe 3 O 4 /Ag nanohybrids or H 2 O 2 alone to each other (figure 8).

Conclusions
To conclude, we have effectively synthesized the Fe 3 O 4 /Ag nanohybrids for the purpose of conducting both cancer ferroptosis therapy and MR imaging simultaneously.The experimental results demonstrated that the Fe 3 O 4 /Ag nanohybrids exhibited a high therapeutic efficacy against cancer cells and demonstrated excellent performance in T 2 -weighted MR imaging.Correspondingly, the magnetic nanohybrids obtained offer a novel opportunity for the combined diagnosis and treatment of cancer.Moreover, the combination of Fe 3 O 4 /Ag  nanohybrids and H 2 O 2 was found to exhibit a synergistic effect in suppressing the growth of liver cancer cells.The mechanism by which it operates was found to involve the decomposition of hydrogen peroxide (H 2 O 2 ) into hydroxyl radical (•OH) induced by iron ions (Fe 3+ /Fe 2+ ), resulting in the destruction of liver cancer cells.Accordingly, the potential therapeutic applications of the synthesized Fe 3 O 4 /Ag nanohybrids is highly suggested.
, no absorbance was detected in the absence of the TMB-H 2 O 2 mixture solution.Conversely, the addition of the Fe 3 O 4 /Ag nanohybrids to the TMB-H 2 O 2 mixture solution (pH = 6.5) for 5 min resulted in the formation of a blue-colored solution, indicating the production of •OH by the Fe 3 O 4 /Ag nanohybrids and H 2 O 2 .In order to verify the accuracy of the MR imaging, we measured the transverse relaxivity (r 2 ) of the Fe 3 O 4 /Ag nanohybrids in aqueous media.Additionally, we obtained T 2 -weighted MR images using 9.4 T MR systems at various concentrations of Fe ions.As shown in figure 5(A), the r 2 value was measured to be 164.5 mM −1 S −1 .In addition, figure 5(B) illustrates a concentration-dependent darkening effect on T 2 -weighted MR images.This indicates that the Fe 3 O 4 /Ag nanohybrids have the potential to serve as an effective T 2 contrast agent for MR imaging.MTT tests were also conducted to further evaluate the cytotoxicity of Fe 3 O 4 /Ag nanohybrids.Accordingly, HepG2 liver cancer cell lines were treated with different concentrations of Fe 3 O 4 /Ag nanohybrids.The findings indicate that nanomaterials exhibit cytotoxicity at lower concentrations (50 μM), with cell viability exceeding 50

Figure 2 .
Figure 2. SEM image (A) and Fe/Ag/O (B, C, and D) elemental mapping of the Fe 3 O 4 /Ag nanohybrids; E) The particle size distribution of the Fe 3 O 4 /Ag nanohybrids.
2 O 2 and Fe 3 O 4 /Ag nanohybrids resulted in a notable suppression of cell proliferation, as shown in figure 6.These results clearly demonstrate the enhanced efficacy of the Fe 3 O 4 /Ag nanohybrids, which were synthesized through a biological process involving H 2 O 2 , in targeting cancer cells.The iron ion (Fe 3+ /Fe 2+ ) may catalyze the decomposition of hydrogen peroxide (H 2 O 2 ) into hydroxyl radical (•OH), thereby causing the death of HepG2 cells.

Figure 3 .
Figure 3. X-ray diffraction patterns of the Fe 3 O 4 nanoparticles and the Fe 3 O 4 /Ag nanohybrids.

Figure 4 .
Figure 4. (A) UV-vis-NIR absorption spectra of the catalyzed oxidation of TMB (oxTMB) as catalyzed by the Fe 3 O 4 /Ag nanohybrids at different concentrations of H 2 O 2 (25, 50, 100 mM) in the reaction buffer (pH = 6.5); (B) Time-dependent absorbance changes at 650 nm as a result of the catalyzed oxidation of TMB at different H 2 O 2 concentrations.

Figure 5 .
Figure 5. (A) Relaxation rate r 2 (1/T 2 ) versus different Fe concentrations of the Fe 3 O 4 /Ag nanohybrids at room temperature; (B) T 2weighted MR images of the Fe 3 O 4 /Ag nanohybrids at different Fe concentrations.

Figure 7 .
Figure 7. HepG2 cells were treated with H 2 O 2 , Fe 3 O 4 /Ag nanohybrids, or H 2 O 2 and Fe 3 O 4 /Ag nanohybrids for 24 h and then analyzed for apoptosis by flow cytometry for Annexin-V+ and PI+ stained cells.