4.5.1 MTT assay
As illustrated in Fig. 3, the results showed that despite an increase in the survival of PBMCs in all treatment groups compared with the control, such an increase in a radiation dose of 25 cGy at both incubation times was not statistically significant. Only at a radiation dose of 200 cGy and for the MPEO-N treatment group, the survival of PBMCs was significantly higher than the control, as the highest increase was observed at 72 h of incubation (P < 0.01). Notably, the increase in the survival of PBMCs in MPEO-N treatment group was not statistically significant compared to MPEO treatment group. In radiation protection studies, to quantify the radioprotective effect of an agent, the well-known quantity of DRF is introduced, which is defined at a radiation dose that results in 50% survival (D50) . Since the radioprotective effect of MPEO-loaded nanoniosomes was examined at two radiation doses of 25 and 200cGy, the quantity of survival enhancement factor (SEF)  was defined which is the ratio of cell survival in the presence and absence of radioprotector in each radiation dose . According to this definition, the maximum SEF value was reported to be 1.29, related to the MPEO-N treatment group, at a dose of 200 cGy in 72 h of incubation.
To explain the reasons for these results, it has previously been stated that the main mechanism underlying radiation damage in PBMCs is apoptosis, and in fact, PBMCs are considered resistant to radiation-induced mitotic death due to their differentiation and non- proliferation [19, 18]. Consequently, the lack of the optimal radioprotective effect of MPEO-N in the increase of the survival of PBMCs may be related to this issue.
4.5.2 Flow cytometry analysis
According to the Fig. 5, the results of the flow cytometry analysis showed that the percentages of apoptosis and necrosis of PBMCs are increased in response to irradiation which is consistent with previous studies [16, 5]. Of note, such an increase was moderated in the presence of MPEO and MPEO-N. In the MPEO-N treatment group, the percentage of apoptosis and necrosis of irradiated PBMCs was significantly reduced in both radiation doses and both incubation times compared to the control group (P < 0.01 for apoptosis and P < 0.05 for necrosis). Such a decrease in the MPEO group was significant only for the percentage of apoptosis (P < 0.05, compared to control).
Comparing the radioprotective effect of MPEO-N and MPEO, the results showed that the reduction of apoptosis percentage of PBMCs in MPEO-N treatment group was more than MPEO treatment group, but this difference was significant only in 200 cGy radiation dose (P < 0.05). Also, the percentage of necrosis in PBMCs showed a significant decrease only in the MPEO-N treatment group compared to the control, and this clearly indicates that the MPEO-N is more effective than the MPEO on the radioprotective effect.
Among the natural compounds, useful studies have been performed on the radioprotective effect of curcumin on PBMCs [35, 36], the results of which may be generalized to MPEO. In a study carried out on PBMCs irradiated with a radiation dose of 2 Gy, it was shown that dendrosomal nanoformulation of curcumin, by modulating the NF-κB and Nrf-2 pathways, affects the expression of genes whose products are involved in cell cycle regulation, DNA damage detection, and apoptosis, thereby increasing cell survival .
Amifostine is the first FDA-approved radioprotector to reduce the incidence of moderate to severe xerostomia after radiation therapy of head and neck cancer . Despite the favorable radioprotective effect, its use is usually discontinued in 15–20% of patients due to severe side effects, such as hypotension, fatigue, and drowsiness. Also, the short half-life of this drug in patients diminishes its effectiveness . Another group of radioprotectors discussed in the last two decades, which have been studied extensively, are Fullerenol nanoparticles or other water-soluble derivatives of Fullerenol (C60). However, there is much controversy about their toxicity, and most of the effectiveness of these agents is limited to ionizing radiations with low LET .
Although numerous studies have been conducted from the past to the present to analyze the radioprotective effects of chemical and natural compounds, it appears that we are still far from introducing an ideal radioprotector agent with versatile clinical applications . One of the major challenges in the development of radioprotectors is the lack of a comprehensive system to biologically examination of these compounds . The radioprotective effect of a radioprotector candidate depends on various parameters, the most important of which are the radiation dose, cell line, and the mechanism to study. These variables make it difficult to compare the radioprotective effect of different mediators. Hence, it seems that a single system for measuring the radioprotective effect is necessary to select a radioprotector with the optimal performance for additional analyses.