Fe3O4 Nanoparticles in Combination with 5-FU Exert Antitumor Effects Superior to Those of the Active Drug in a Colon Cancer Cell Model

(1) Background: Colon cancer is one of the most common cancer types, and treatment options, unfortunately, do not continually improve the survival rate of patients. With the unprecedented development of nanotechnologies, nanomedicine has become a significant direction in cancer research. Indeed, chemotherapeutics with nanoparticles (NPs) in cancer treatment is an outstanding new treatment principle. (2) Methods: Fe3O4 NPs were synthesized and characterized. Caco-2 colon cancer cells were treated during two different periods (24 and 72 h) with Fe3O4 NPs (6 μg/mL), various concentrations of 5-FU (4–16 μg/mL), and Fe3O4 NPs in combination with 5-FU (4–16 μg/mL) (Fe3O4 NPs + 5-FU). (3) Results: The MTT assay showed that treating the cells with Fe3O4 NPs + 5-FU at 16 µg/mL for 24 or 72 h decreased cell viability and increased their LDH release (p < 0.05 and p < 0.01, respectively). Furthermore, at the same treatment concentrations, total antioxidant capacity (TAC) was decreased (p < 0.05 and p < 0.01, respectively), and total oxidant status (TOS) increased (p < 0.05 and p < 0.01, respectively). Moreover, after treatment with Fe3O4-NPs + 5-FU, the IL-10 gene was downregulated and PTEN gene expression was upregulated (p < 0.05 and p < 0.01, respectively) compared with those of the control. (4) Conclusions: Fe3O4 NPs exert a synergistic cytotoxic effect with 5-FU on Caco-2 cells at concentrations below the active drug threshold levels.


Introduction
Colon cancer is the third most common malignancy and represents the second most common cause of cancer death despite advances in diagnosis and treatment [1]. One of the key distinguishing features of colon cancer is the loss of cellular organization and the increased ability to invade near and distant sites. The standard treatment principle encompasses chemotherapy, surgery, and radiotherapy according to the type and stage of the disease. However, the gene-type signature of the cancer tissue alters the neoplasm response to therapy regimens [2]. Thus, the 5-year survival of colon cancer patients remains at 64%, and efficient therapy, with attenuated side effects, remains a significant unmet health need [3]. Iron (III) acetylacetonate (Fe (acac)3, 1.06 g) was dissolved in a mixture of oleyl amine (15 mL) and dibenzyl ether (15 mL) under continuous stirring in a four-necked roundbottom glass reactor. The mixture was heated to 120 • C and held at the same temperature for 1 h to remove moisture under a stream of nitrogen gas. The mixing process continued throughout all stages. After one hour, the mixture temperature was rapidly increased to 300 • C, and the reaction was continued at this temperature for an 1 h. Finally, ethanol (3 × 40 mL) was added to the mixture, which was centrifuged at 8500 rpm for 12 min. After purification, Fe 3 O 4 NPs were dispersed in hexane (10 mL). Figure 1B shows a representative SEM image of the prepared Fe 3 O 4 NPs [30,31].
anticancer effects at concentrations at which the active drug, 5-FU, is ineffective. Furthermore, this article shows that the combined administration of Fe3O4 nanoparticles with 5-FU without its prior immobilization significantly increased the antitumor activity and reduced the therapeutic dose of 5-FU.

Fe3O4 Nanoparticles Synthesis
Iron (III) acetylacetonate (Fe (acac)3, 1.06 g) was dissolved in a mixture of oleyl amine (15 mL) and dibenzyl ether (15 mL) under continuous stirring in a four-necked roundbottom glass reactor. The mixture was heated to 120 °C and held at the same temperature for 1 h to remove moisture under a stream of nitrogen gas. The mixing process continued throughout all stages. After one hour, the mixture temperature was rapidly increased to 300 °C, and the reaction was continued at this temperature for an 1 h. Finally, ethanol (3 × 40 mL) was added to the mixture, which was centrifuged at 8500 rpm for 12 min. After purification, Fe3O4 NPs were dispersed in hexane (10 mL). Figure 1B shows a representative SEM image of the prepared Fe3O4 NPs [30,31].

Fe3O4 Nanoparticles Characterization
The scanning electron microscope (SEM) images were obtained using an FEI Quanta 450 (USA). Dynamic light scattering (DLS) experiments were performed utilizing a Zetasizer Quinta Nano ZS90 (Malvern Instruments, Malvern, UK) at room temperature. Samples were prepared as 0.5% (w/v) solutions in DDW.
The Fourier transform infrared (FTIR) spectrum of the Fe3O4 NPs was obtained with a Shimadzu 8101 M FTIR (Kyoto, Japan) using the potassium bromide (KBr) pellet technique. The powder X-ray diffraction (XRD) pattern of the Fe3O4 NPs was obtained using a Siemens D5000 diffractometer (Aubrey, TX, USA) and an X-ray generator (CuKα radiation with λ = 1.5406 Å) at room temperature [31].

Fe 3 O 4 Nanoparticles Characterization
The scanning electron microscope (SEM) images were obtained using an FEI Quanta 450 (USA). Dynamic light scattering (DLS) experiments were performed utilizing a Zetasizer Quinta Nano ZS90 (Malvern Instruments, Malvern, UK) at room temperature. Samples were prepared as 0.5% (w/v) solutions in DDW.
The Fourier transform infrared (FTIR) spectrum of the Fe 3 O 4 NPs was obtained with a Shimadzu 8101 M FTIR (Kyoto, Japan) using the potassium bromide (KBr) pellet technique. The powder X-ray diffraction (XRD) pattern of the Fe 3 O 4 NPs was obtained using a Siemens D5000 diffractometer (Aubrey, TX, USA) and an X-ray generator (CuKα radiation with λ = 1.5406 Å) at room temperature [31].

MTT Assay
At the end of the experiment (after 24 and 72 h of treatment), MTT solution (10 µL) was added to each well, and the cell number was determined. In short, the plates were incubated for 4 h in a CO 2 incubator, to which 100 µL of DMSO solution was added to all wells. The spectrophotometer read the density at 570 nm [31].

Total Oxidant Status (TOS) and Total Antioxidant Capacity (TAC) Determination
Total oxidant status (TOS) and total antioxidant capacity (TAC) evaluations were performed spectrophotometrically (Multiskan ™ GO Microplate Spectrophotometer reader) as previously described [26]. The color density is correlated to the oxidant levels in a sample [33].

Lactate Dehydrogenase (LDH) Measurement
According to the manufacturer's instructions, the lactate dehydrogenase (LDH) was determined with an LDH detection kit. In summary, Caco-2 cells were seeded in a 96-well plate at a density of 10 3 -10 6 cells/well in 200 µL of the medium. Six wells were prepared for each concentration. Triton X-100 (10%) and the assay buffer were added, and the wells were incubated at room temperature for one hour. After centrifugation, the cell supernatant was transferred to a new 96-well assay plate. The LDH reaction solution was added to each well, and the plate was incubated with gentle shaking on an orbital shaker for 30 min at 37 • C. A microplate reader measured the absorbance OD value at 490 nm [34]. ((experimental value A490) − (spontaneous release A490))/((maximum release A490) − (spontaneous release A490)) × 100.
Maximum release: 100% dead cells by adding Triton X-100. Spontaneous release: nontoxic materials (cell medium) control group. Experiment value: application groups.

Gene Expression Determination
The total RNA from Caco-2 cells was used to synthesize complementary DNA (cDNA) using a high-capacity cDNA Reverse Transcription Kit. The sequences of the gene-specific PCR primers are listed below (forward and reverse). Results were compared with the control group and are expressed as relative fold. Gene expressions were normalized to beta actin using the ∆∆ Ct method.

Statistical Analyses
Statistical comparisons between the groups were calculated using one-way ANOVA and Tukey's HSD method. All calculations were performed using SPSS 20 software for statistical analysis, and a p < 0.05 was considered a statistically significant difference in all tests. Results are presented as mean and standard deviation (mean ± SD).

Characterization of Fe 3 O 4 NPs
The synthesized Fe 3 O 4 NPs were characterized using SEM and DLS analysis, as presented in Figure 1. The SEM image showed that the shape of the synthesized Fe 3 O 4 Pharmaceutics 2023, 15, 245

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NPs was spherical with an average diameter of 35 ± 5 nm. The DLS analysis revealed that the Fe 3 O 4 NPs had an average size of~32 nm. In addition, the polydispersity index (PDI) of the synthesized NPs was found to be 0.24, which indicated their relatively monodisperse synthesis.
The FTIR spectrum and XRD pattern of the Fe 3 O 4 NPs are shown in Figure 2. The most prominent absorption bands in the FTIR spectrum are the stretching vibration of the metal-oxygen (Fe-O) group at 576 cm −1 and the stretching and bending vibrations of the surface hydroxyl groups at 3420 and 1608 cm −1 , respectively ( Figure 2a).

Characterization of Fe3O4 NPs
The synthesized Fe3O4 NPs were characterized using SEM and DLS analysis, as presented in Figure 1. The SEM image showed that the shape of the synthesized Fe3O4 NPs was spherical with an average diameter of 35 ± 5 nm. The DLS analysis revealed that the Fe3O4 NPs had an average size of ~32 nm. In addition, the polydispersity index (PDI) of the synthesized NPs was found to be 0.24, which indicated their relatively monodisperse synthesis.
The FTIR spectrum and XRD pattern of the Fe3O4 NPs are shown in Figure 2. The most prominent absorption bands in the FTIR spectrum are the stretching vibration of the metal-oxygen (Fe-O) group at 576 cm −1 and the stretching and bending vibrations of the surface hydroxyl groups at 3420 and 1608 cm −1 , respectively ( Figure 2a).

Evaluation of Caco-2 Cell Viability by MTT and LDH Assay
The effect of various treatments on Caco-2 cell viability was determined by the MTT assay after 24 and 72 h of treatment ( Figure 3). Cell viability was considered as 100% in the control (negative control) and is expressed as a percentage of that of the control for all other treatments. Notably, DMSO and the nonloaded Fe 3 O 4 NPs at 6 µg/mL did not affect the viability of Caco-2 cells. Treating the cells for 24 h with 5-FU 16 µg/mL exerted a nonsignificant 10% decrease in viability (p = NS), and the reduction (34%) was statistically significant after 72 h of treatment (p < 0.05). The effects of the Fe 3 O 4 -NPs + 5-FU combination on cell viability were more prominent. Thus, treating cells for 24 h with Fe 3 O 4 NPs + 5-FU (16 µg/mL) decreased their viability to 31% (p < 0.05), whereas treating Caco-2 cells with Fe 3 O 4 NPs + 5-FU (16 µg/mL) for 72 h resulted in a substantial decrease in their viability (41%) (p < 0.01). other treatments. Notably, DMSO and the nonloaded Fe3O4 NPs at 6 µ g/mL did not affect the viability of Caco-2 cells. Treating the cells for 24 h with 5-FU 16 µ g/mL exerted a nonsignificant 10% decrease in viability (p = NS), and the reduction (34%) was statistically significant after 72 h of treatment (p < 0.05). The effects of the Fe3O4-NPs + 5-FU combination on cell viability were more prominent. Thus, treating cells for 24 h with Fe3O4 NPs + 5-FU (16 µ g/mL) decreased their viability to 31% (p < 0.05), whereas treating Caco-2 cells with Fe3O4 NPs + 5-FU (16 µ g/mL) for 72 h resulted in a substantial decrease in their viability (41%) (p < 0.01). Because LDH is released by necrotic cells, it is an excellent metabolic marker of cell viability. The effect of various treatments on Caco-2 cell LDH activity was determined by utilizing an LDH kit ( Figure 3). The measured LDH activity of treated cells expressed as a percent of the standard (designated as 100%) is presented in Figure 4. Treating the cells with only Fe3O4-NPs and different concentrations of 5-FU did not affect their LDH activity. However, an increase in LDH activity, correlated with cell death, was demonstrated in cells treated with a combination of Fe3O4-NPs + 5-FU (8 µ g/mL) for 72 h (p < 0.05) and cells treated with Fe3O4-NPs + 5FU (16 µ g/mL) for 24 and 72 h, (p < 0.05 and p < 0.01), respectively. These data demonstrate that combining 5FU with Fe3O4 -NPs significantly increased the active drug cytotoxic effect, even at concentrations below the active drug range.  The Caco-2 cell TAC values, determined spectrophotometrically, were 12.01 and 13.84 mmol Trolox equiv/L, respectively ( Figure 5). Treatment with Fe3O4, NPs, and different concentrations of 5-FU did not affect these cells' TAC. However, treatment with the loaded Fe3O4-NPs + 5-FU significantly decreased the Caco-2 cell antioxidant status in a time-and concentration-dependent manner ( Figure 5).

The Effect of Fe3O4-NPs, 5-FU and Fe3O4-NPs + 5-FU on Caco-2 Cells Redox State
The Caco-2 cell TAC values, determined spectrophotometrically, were 12.01 and 13.84 mmol Trolox equiv/L, respectively ( Figure 5). Treatment with Fe3O4, NPs, and different concentrations of 5-FU did not affect these cells' TAC. However, treatment with the loaded Fe3O4-NPs + 5-FU significantly decreased the Caco-2 cell antioxidant status in a time-and concentration-dependent manner ( Figure 5). In correlation with the TAC results, the combined Fe3O4-NPs + 5-FU treatments (Figure 6) was found to increase Caco-2 cell TOS levels, dependent on time and concentration. The most pronounced effects were obtained after 72 h of treatment with Fe3O4-NPs + 5U 8 µ g/mL/5-FU 16 µ g/mL (p < 0.01).

Discussion
Over the past decades, remarkable advances have occurred in nanotechnology, particularly nanomedicine, focusing, among others, on novel cancer therapeutics [35]. NPs can accumulate in cells without being recognized by p-glycoproteins, one of the primary mediators of multidrug resistance, resulting in increased intracellular concentrations of drugs [36]. Notably, NP carriers exhibit intrinsic abilities affecting cancer and immune cell biological functions [37]. Therefore, our study examined the synergistic effect of Fe 3 O 4 -NPs and 5-FU on Caco-2 colon cancer cell viability, oxidative stress, and oncogene expression.
Previous studies have shown an ambiguous effect of iron oxide NPs on cell biological functions, dependent on cell type and concentration utilized. Thus, it was shown that iron oxide NPs could induce the cellular inflammatory response and increase the secretion of proinflammatory cytokines in human or mouse cells [37,38]. Lately, they have been approved by the FDA, and their beneficial effects on cell physiology have been suggested [13]. However, Fe 3 O 4 /composites were also shown to facilitate various active drugs' cytotoxic and immunomodulatory properties [39]. Thus, the peroxidase-like activity of Fe 3 O 4 and carbon NPs was found to facilitate ascorbic-acid-induced oxidative stress and to incur specific damage to PC-3 prostate cancer cells [40]. Furthermore, increased ROS production generates oxidative stress within the cells and cell apoptosis, resulting in PC-3 tumor cell growth inhibition [40]. Moreover, composite NPs can induce mitochondrial membrane alteration, DNA damage, cytokine production associated with oxidative stress, and apoptosis-correlated cell death [41].
A separate research direction is the regulation of magnetic fields, as various studies have shown that the discrete modulation of these fields can inhibit the proliferation of cancer cells and tumor growth [42,43]. Furthermore, due to the promoting effect of iron metabolism on ROS production, increased concentrations of iron-based NPs in cancer cells enhance their exposure to the local magnetic field and cellular death [44,45].
Our study showed that Fe 3 O 4 NPs did not negatively affect Caco-2 cell viability, oxidative stress, or oncogene expression. However, together with 5-FU, Fe 3 O 4 NPs acted synergistically, and the combination exerted cytotoxic, immunomodulatory, and oxidativestress-promoting effects at a concentration at which the active drug does not affect these cell functions. Notably, LDH is a cytotoxic marker as its release is enhanced due to cell necrosis. In this study, combined Fe 3 O 4 _NPs + 5-FU strongly increased LDH activity, correlated with the upregulation of cell death. Furthermore, TOS increase and TAC attenuation were evident after combined Fe 3 O 4 _NPs + 5-FU treatment. Notably, the effects were exerted only after combined Fe 3 O 4 _NPs + 5-FU treatment, as treating cells with only 5-FU at the same concentrations did not affect these parameters of cell homeostasis.
PTEN is a well-established tumor suppressor, and alterations in its expression/activity are correlated with tumorigenesis [46]. Moreover, as PTEN controls polarity in normal epithelial cells, loss of this protein plays a critical role in the invasion and metastasis of various cancer types, including colon cancer [47,48]. Several studies have established a negative correlation of PTEN expression with colon cancer progression due to its vital role in inhibiting the malignant transformation of intestinal epithelial cells [49]. A similar association was determined with the dysregulation of PTEN-binding partners [50][51][52].
In the present study, the combined Fe 3 O 4 _NPs + 5-FU significantly increased this gene expression. Notably, PTEN acts as a negative regulator of the PI3K/Akt signaling pathway and was shown to affect many processes deregulated in tumorigenesis, such as cell survival, proliferation, migration, and invasion [53]. Indeed, inhibition of the PI3K/Akt pathway induces programmed cell death in different cell lines [54]. Recently, patients presenting PTEN hamartoma tumor syndrome were advised to employ earlier surveillance for colon cancer due to an increased risk of early onset [55]. In the present study, Fe 3 O 4 -NPs in combination with 5-FU increased PTEN gene expression. Moreover, in an in vivo prostate cancer model, a NP-mediated increase in PTEN led to disease remission, highlighting the importance of this gene in tumorigenesis and defining it as a promising therapeutic target and progression marker [56].
IL-10 is a versatile immunosuppressive cytokine with immunomodulatory functions [20]. Thus, IL-10 increases tumor cell survival, proliferation, and metastasis by controlling antitumor immunity. Indeed, IL-10 exerts suppressive effects on effector immune cells, including potent antitumor cytotoxic NK and CD8 T cells [21]. The immunosuppressive functions of IL-10 are exercised through the Jak1/STAT3 pathway. Moreover, IL-10 suppresses the level of proinflammatory cytokines, including IL-1β [57]. IL-10 exhibits a role in colon cancer progression as increased levels of IL-10 facilitated liver metastasis in a mouse model [55]. Reprogramming the colon cancer tumor environment by silencing IL-10 expression resulted in dendritic-cell-dependent activation of the antitumor response [56]. This is a significant achievement, especially as dendritic cells (DCs) have a key role in triggering antitumor immune responses [58]. Madhubala et al. [59], in their study on titanium dioxide NPs' effects in a leukemia cell line, observed that the expression of IL-10 significantly decreased. In the present study, Fe 3 O 4 -NPs + 5-FU treatment significantly reduced the IL-10 release of colon cancer cells.
Thus, this study shows that the combined administration of Fe 3 O 4 and 5-FU NPs will reduce the dose of the drug required to achieve pronounced antitumor activity. The latter effect is a significant result since 5-FU has a pronounced toxicity, which can be reduced due to its immobilization, for example, using metal-organic frameworks [60][61][62]. Therefore, the role of NPs is not only to provide suitable dynamics for 5-FU release in the event of its immobilization but also to eliminate the barrier associated with penetration through the cell membrane. A similar synergistic effect was described when platinum NPs were administered together with nonimmobilized doxorubicin to U2OS osteosarcoma cells. In this model, cotreatment significantly increased the drug's effectiveness compared with pure doxorubicin at a similar dose [63]. This was explained by an increase in oxidative stress in the presence of platinum NPs [63], which we also noted with the combined introduction of Fe 3 O 4 and 5-FU nanoparticles in the present study. On the other hand, NP treatment promotes the activation of endocytosis [64], which can also promote the penetration of 5-FU through cell membranes. Therefore, administering anticancer drugs, even without their preliminary immobilization, together with NPs, can significantly increase their cytostatic activity. Furthermore, in vivo and in vitro experiments for the characterization of Fe 3 O 4 nanoparticles/active drug effects on specific cells/tissues are in order.

Conclusions
Magnetite NPs penetrate (passive delivery) due to increased vascular permeability and weakened lymphatic drainage of cancer tissues. Moreover, magnetite NPs are easily uptaken and accumulate in cancer cells due to their small size. Our study showed that the combined Fe 3 O 4 _NPs + 5-FU, through a synergistic effect, significantly reduced Caco-2 cell viability at a concentration at which the active drug did not induce an effect. Likewise, the combined treatment, but not the solitary components, facilitated oxidative stress correlated with the decreased viability of Caco-2 cells. Moreover, we determined that combined