## 4 Discussion  
The development of NP has facilitate the establishment of potential treatments for malignancy because of the ability to selectively deliver chemotherapeutic agents to malignant cells, control release, and minimize off-target toxicity. Notably, the effects of NPs, such as iron oxide, selenium, silver, and gold NPs, alone or with different therapies (e.g., chemical agents or photocatalytic therapy) on malignant cells have been also reported [17; 18; 19; 20; 21]. These therapies are mainly based on interactions with malignant cells via the generation of ROS [22]. In the present study, we examined the effect of Fe\({}_{3}\)O\({}_{4}\) NPs combined with rapamycin or carboplatin on prostate cancer cell growth in vitro in order to explore the possible applications of Fe\({}_{3}\)O\({}_{4}\) NPs for modification of chemotherapeutic agent usage in patients with CRPC. In this study, to uptake Fe\({}_{3}\)O\({}_{4}\) NPs in DU145 and PC-3 cells was analyzed using FCM. The uptake of Fe\({}_{3}\)O\({}_{4}\) NPs by both cells was recognized in a dose-dependent manner as we have previously shown cellular uptake of MNPs-Fe\({}_{3}\)O\({}_{4}\) in prostate cancer cells by TEM [12]. The physicochemical properties of the NPs including size, shape, and surface charge have various effects on their interactions with living cells such as cellular uptake, localization and cytotoxicity [23]. Small NPs have a high probability to be internalized by passive uptake than large one. In addition, culture media with/without FBS have been reported to affect the size stability in vitro systems, showing that NPs aggregated in a high-ionic-strength medium such as PBS or RPMI-1640 solution because of the suppression of the double layer, which reduces the electrostatic repulsion barrier [24; 25]. In this study, aggregation of Fe\({}_{3}\)O\({}_{4}\) NPs was observed in culture media, and induced ROS production in a dose-dependent manner. Thus, this is an interesting, leading that complex of MNPs, a chemotherapeutic agent and FBS may effect on interactions such as cellular uptake via alternative endocytosis pathways [26]. However, there remains a problem to be solved by further studies. In this study, ROS production in DU145 and PC-3 cells after exposure to Fe\({}_{3}\)O\({}_{4}\) NPs was observed in a dose-dependent manner, similar to our previous findings [12]. Thus, we predicted that Fe\({}_{3}\)O\({}_{4}\) NPs may enhance rapamycin- or carboplatin-induced prostate cancer cell death in both cell lines, similar to that in combination treatment with DTX. Fe\({}_{3}\)O\({}_{4}\) NPs have been reported to cause low toxicity or cytotoxicity at concentrations of 100 \(\upmu\)g/mL or higher via the generation of ROS, which can result in lipid peroxidation, DNA damage, and protein oxidation [27]. ROS, resulting from the transfer of energy or electrons to oxygen, act as a second messenger in cell signaling and are involved in various biological processes, including growth and survival in normal cells. Their levels in cells are controlled by enzymes such as superoxide dismutase, glutathione peroxidase, and catalase, as well as antioxidants. Oxidative stress results from an imbalance between ROS generation and elimination. The high ROS levels in cancer cells, which are a consequence of alterations in several signaling pathways, play important roles in initiation, progression, and metastasis; thus, ROS are considered oncogenic [28; 29]. However, because ROS are also implicated in triggering cell death, including that of cancer cells, their production is desirable in chemotherapy, radiotherapy, and photodynamic therapy [22]. Notably, oxidative stress has been shown to have a prominent role in the pathogenesis of prostate cancer [28]. In particular, oxidative stress is involved in the conversion of androgen-dependent prostate cancer into CRPC via regulation of androgen receptor expression [29]. Excessive accumulation of ROS may tip over a threshold, leading to cell toxicity, making some tumor cells susceptible to ROS-induced apoptosis.