Radioprotective effect of nanoniosome loaded by Mentha Pulegium essential oil on human peripheral blood mononuclear cells exposed to ionizing radiation

Abstract Objective The present study aimed to assess the radioprotective effect of nanoniosomes loaded by Mentha Pulegium essential oil (MPEO-N nanoparticles) as a natural antioxidant on human peripheral blood mononuclear cells (PBMCs). Significance Despite the applications and advantages of ionizing radiation, there are many radiation risks to biological systems that are necessary to be reduced as much as possible. Methods MPEO-N nanoparticles were prepared by the lipid thin film hydration method, and its physicochemical characteristics were analyzed. PBMCs were then irradiated with X-ray using a 6 MV linear accelerator at two radiation doses in the presence of nontoxic concentrations of MPEO-N nanoparticles (IC10). After 48 and 72 h of incubation, the radioprotective effect was investigated by measuring survival, apoptosis, and necrosis of PBMCs, using MTT assay and flow cytometry analysis. Key findings The hydrodynamic diameter and zeta potential of nanoniosomes were 106.0 ± 4.69 nm and −15.2 ± 0.9 mV, respectively. The mean survival percentage of PBMCs showed a significant increase only at a radiation dose of 200 cGy compared with the control group. The percentages of apoptosis and necrosis of cells in the presence of MPEO-N nanoparticles at both radiation doses and incubation periods (48 and 72 h) demonstrated a significant reduction compared with the control. Conclusion MPEO-N nanoparticles as a natural antioxidant, exhibited a favorable radioprotective effect by a significant reduction in the percentage of apoptosis and necrosis of irradiated PBMCs.


Introduction
Many epidemiological studies show the carcinogenic risk of ionizing radiation.Occupational and medical radiation, even if it is not in the moderate and high radiation dose, can cause significant radiation risks to personnel and especially to patients with a diagnosis [1].X-rays, the most widely used in medicine and industry, have a low linear transfer rate and therefore dominate indirect interactions, so biological macromolecules are mainly damaged by interactions with water radiolysis products, such as reactive oxygen species (ROs) [2].an effective way to combat the effects of ROs is the use of antioxidant compounds in biological processes to slow down or prevent the oxidation of macromolecules through various mechanisms [3].antioxidant compounds have been shown to have radioprotective effects in some studies [4].although many studies have shown significant benefits of radioprotectors, these compounds are not usually prescribed as a dietary supplement or with food to radiation personnel or patients before or after medical imaging [5].Natural compounds meet most of the criteria required for use as radiation shielding agents.they use several mechanisms to exert radiation protective effects on living organisms [3].For example, they eliminate the destructive effect of oxidants by donating electrons to peroxy or hydroxyl radicals, and in the process can be converted into less damaging free radicals [6].Research shows that some plant-based natural antioxidant compounds are more effective than synthetic ones and less toxic.consequently, there is a growing desire to study and use natural antioxidants of plant origin in medicine [7,8].
Mentha Pulegium belongs to the lamiaceae family and contains natural antioxidants such as flavonoids, alkaloids, and polyphenols.Mentha Pulegium and its essential oil are known to scavenge free radicals [7].Despite the benefits of natural antioxidants, there are limitations to the use of these valuable substances.these limitations include low solubility, short duration of action, uncontrolled release, and instability during digestion and absorption in the gastrointestinal tract.therefore, to overcome such challenges, the use of nanocarriers has been developed nowadays [9,10].studies have shown that the addition of natural antioxidants to nanocarriers leads to their controlled release into cell membranes and improves cellular uptake, protects their compounds against premature degradation, improves biodegradability, and improves drug retention time in the bloodstream [10].Many researchers have accepted nanosomes with a flexible structure as carriers that are suitable for loading hydrophilic and hydrophobic compounds.Nanoniosomes are surfactant-based nonionic vesicles that are structurally similar to liposomes but have some advantages, such as lower production costs and higher stability than liposomes.at the same time, they are biocompatible and biodegradable and cause less toxicity due to their nonionicity [9].
the purpose of this study was to evaluate the radioprotective effect of nanoniosomes-loaded Mentha Pulegium essential oil (MPeO-N) on human peripheral blood mononuclear cells (PBMcs) as a radiosensitive cell line.For this purpose, the percentage of survival, apoptosis, and necrosis of irradiated PBMcs was evaluated in the presence of nanoparticles using Mtt assay and flow cytometry analyses.

Extraction of MPEO
the preparation of Mentha Pulegium essential oil (MPeO) was carried out by the steam distillation method using a clevenger apparatus (simax, tehran, iran).Briefly, 100 g of Mentha Pulegium leaves were ground in a mortar and then mixed with 500 ml of distilled water.the oil was extracted for 4 h and then transferred to a light-block microtube until use.

Preparation of formulations
the lipid thin-film hydration method was applied to prepare MPeO-N nanoparticles [9]. to this aim, 180.0 mg of tween 60 and 22.8 mg of cholesterol (molar ratio = 9:1) were dissolved in chloroform and transferred to a round-bottom flask, and adjusted to a volume of 20 ml with chloroform.Next, 2.0 mg of MPeO was dissolved in 2 ml of methanol, and the resulting mixture as added to the balloon.after that, the contents of the flask were homogenized and the solvent was removed with a rotary device (heidolph, schwabach, Germany) for 30 min (150 rpm, 37 °c).then, in order to hydrate the lipid thin-film, 10 ml of PBs was added to the round-bottom flask and agitated at 45 °c for 30 min.the product was obtained as single and multilayer nanoparticles.then, 11.2 mg of PeG was added to the formulation and stored in the dark at 25 °c to cover the surface of vesicles.after that, to reduce the size and create small unilamellar vesicles, a sonicator probe device (ultrasonic, tehran, iran) was used for 30 min (15 s on, 10 s off ), and the product was filtered using a 0.2 µm filter.Finally, the product was transferred to a dialysis bag and placed on a heater (heidolph, schwabach, Germany) containing 150 times the sample volume of PBs to separate the unloaded MPeO.MPeO-N Nanoparticles were stored at 4 °c and protected from light for subsequent analyses.MPeO-free nanoniosomes were prepared using the same method, except that MPeO was not used in the oil phase.

Physicochemical characterization
Morphology and hydrodynamic diameter the morphology of MPeO-N nanoparticles was analyzed using atomic force microscopy (aFM) (hitachi, chiyoda city, Japan) and scanning electron microscope (seM) (NOVa NanoseM, hillsboro, OR). the hydrodynamic diameter, zeta potential, and polydispersity index (PDi) of nanoparticles were also determined using a Zetasizer instrument (hORiBa, Kyoto, Japan) using the dynamic light scattering (Dls) method.

Encapsulation efficiency (EE %) and loading capacity (LC %)
to calculate the ee % and lc %, first the maximum absorbance wavelength of MPeO was obtained by uV-vis spectrophotometry (Bio tek, Winooski, Vt) in a wavelength range between 200 and 800 nm.For this purpose, a diluted concentration of MPeO in methanol was analyzed in an absorbance range of 1, and methanol was used as a blank.then, 1 ml of the MPeO-N nanoparticle suspension (equivalent to 1 g of formulation) was dissolved in 1 ml of methanol, and the amount of MPeO in the solvent was determined by a uV-vis spectrophotometer at the wavelength of maximum absorbance of MPeO (using methanol as blank and corresponding calibration curve).to complete this step, the lc and ee values of MPeO-N nanoparticles were calculated based on the following equations [11]:

MPEO-N nanoparticles release curve
in order to measure the release rate of MPeO from MPeO-N nanoparticles, 1 ml of the formulation was poured into a dialysis bag and stirred in 10 ml of PBs buffer under body conditions (ph 7.4, 37 °c).at regular intervals of 30 min up to 72 h, 1 ml of the buffer in the container was replaced by 1 ml of a fresh buffer, and its absorption at the maximum absorption wavelength of MPeO (300 nm) was read by uV-vis spectrophotometer (using PBs as blank and related standard curve).Finally, the amount of released MPeO from MPeO-N nanoparticles was calculated at each time and the release curve.

Cell culture
For cell culture, blood samples were taken from five male volunteers, aged 20-30 years, without radiotherapy, systemic diseases, and smoking, with pre-filled heparin syringes after obtaining written consent (according to the Declaration of helsinki).human peripheral blood lymphocytes were then isolated using Ficoll density gradient centrifugation.lymphocytes were washed with PBs and centrifuged at 1500 rpm at room temperature for 5 min each time.after the removing of the supernatant, RPMi-1640 medium supplemented with 10% FBs and 1% antibiotic (pen-strep) was added to the cells and counted using trypan blue.then, 10 5 cells were seeded onto a 96-well plate and incubated in a 5% cO 2 incubator at 37 °c.

Toxicity test
the toxicity of MPeO and MPeO-N nanoparticles was analyzed by the Mtt assay [12].Briefly, the cells were seeded in a 96-well cell culture plate (at a density of 1 × 10 5 cells/well) and equivalent concentrations of 30-480 µg/ml of MPeO-N nanoparticles from different formulations were added to the wells with five replications for each concentration.Following 96 h of incubation, 20 µl of the Mtt solution (5 mg/ml in PBs) was added to each well.Following 4 h of incubation, the plate was centrifuged at 1800 rpm at 4 °c for 5 min.the supernatant was then discarded and 100 µl of DMsO was added to each well.after shaking for 10 min, the optical absorbance of the wells was read by an elisa reader (Bio tek, , Winooski, Vt) at a wavelength of 570 nm vs. 630 nm (as blank).
Finally, the percentage of cell survival in different groups and the ic10 of each formulation were calculated.Wells filled with PBMcs without nanoparticles were considered as a control group.

Investigation of radioprotective effect
Irradiation conditions and treatment groups the cells were divided into three main groups of control (without drug), MPeO treatment group, and MPeO-N nanoparticles treatment group.cells were irradiated in different groups with radiation doses of 0, 25, and 200 cGy using a 6 MV linear accelerator X-ray (compact-elekta, crawley, uK).irradiation was performed according to the source to axis distance (saD) technique at a depth of 5 cm of the tissue-equivalent solid phantom at a 180° gantry angle with a field size of 20 × 20 cm 2 .Monitor units were calculated by Prowess Panther treatment planning system (tPs) version 5.2 (Prowess, inc., concord, ca), according to the attenuation coefficient of the plates.in order to promote the dosimetry conditions, central plate wells were used for cell culture, while the marginal wells were filled with culture medium.

Determination of cell survival
to evaluate the radioprotective effect of formulations on the survival of irradiated PBMcs, the cells were seeded in 96-well cell culture plates (at a density of 1 × 10 5 cells/well).after 24 h of incubation, concentrations of different formulations equivalent to ic10 of MPeO-N nanoparticles were added to the wells in different treatment groups, and after 24 h of incubation, irradiation was performed as previously described.Finally, the percentage of cell survival was measured at 48 and 72 h of incubation by the Mtt assay (as described in toxicity assay section) [12].to quantify the radioprotective effect, the survival enhancement factor (seF) was defined in each radiation dose and calculated as the survival in the presence of drug to survival in the absence of drug.

Determination of apoptosis and necrosis
to investigate the radioprotective effect of the formulations on the percentage of apoptosis and necrosis of the PBMcs, as previously described, irradiation was performed in different groups.at two different incubation times of 48 and 72 h, cells were poured into tube specified for flow cytometry analysis.then, 1 ml of PBs was added to the cells and after stirring, the tubes were centrifuged at 1800 rpm for 5 min.the supernatant was then removed and the tube was centrifuged again with 0.5 ml PBs.then, the supernatant was discarded, and 4 μl of annexin-V was added to the tube.after 30 min of incubation of samples at 4 °c and in the dark, 0.5 ml of PBs was added to tube, and after a few seconds of shaking, samples were centrifuged at 1800 rpm for 5 min.Following centrifugation, the supernatant was separated.then, 4 μl of Pi was added to the cells and centrifuged at 1800 rpm for 5 min.the supernatant was removed, and the cells were incubated at 4 °c for 10 min in the dark.Finally, after brief agitation of samples, 0.5 ml of PBs was added to samples, and the percentages of apoptotic and necrotic cells were analyzed by flow cytometry (Partech, Berlin, Germany) [13].the obtained data were analyzed by FlowJo software version 7.6 (BD, ashland, OR).

Statistical analysis
GraphPad Prism version 9 (GraphPad software, la Jolla, ca) analyzed the data through descriptive statistics of mean and standard deviation as well as inferential statistics based on a 95% confidence level (p value < .050).since the data were normally distributed, the difference between groups was analyzed by one-way analysis of variance (one-way aNOVa).

Physicochemical properties
the morphology of MPeO-N nanoparticles was assessed by aFM and seM.as shown in Figure 1, nanoparticles are spherical and distributed separately.according to the results of Dls analysis, the mean hydrodynamic diameter and PDi of MPeO-N nanoparticles were 106.0 ± 4.69 nm and 0.27 ± 0.04, respectively.the zeta potential of MPeO-nanoparticles was −15.2 ± 0.9 mV. the ee % and lc % of MPeO-N nanoparticles were 44.37 ± 3.62 and 3.40 ± 0.14%, respectively (Figures 2 and 3).

Release curve
the release curve of MPeO-N nanoparticles in PBs (ph = 7.4) is shown in Figure 4. after 24 h, the release percentages of Mentha Pulegium from MPeO-N nanoparticles at 37 °c and 42 °c, were 52.21% and 60.98%, respectively.

Toxicity of formulations
the toxicity of formulations on PBMcs was assessed after 96 h of incubation using the Mtt assay.as shown in Figure 4, the concentrations of MPeO and MPeO-N nanoparticles at the peak of the survival curve were 20 and 40 µg/ml, respectively.survival was then gradually reduced so that the ic10 value of MPeO was reported to be 80 µg/ml, while the ic10 value of MPeO-N nanoparticles was approximately equal to 170 µg/ml.

Radioprotective effect on survival of PBMCs
the survival of PBMcs was assessed using the Mtt assay.Figure 5 depicts the survival percentage of PBMcs in different groups at two incubation times of 48 and 72 h. the concentration of all formulations was considered to be equivalent to the ic10 value of MPeO-N nanoparticles (170 µg/ml).the mean survival percentage of PBMcs at a dose of 200 cGy and at two incubation times of 48 and 72 h was 78.80 ± 7.56 and 81.49 ± 8.53, respectively, which showed a significant increase compared to the control group (p < .05 and p < .01,respectively).at 25 cGy, despite the increase in survival percentage compared to the control group, such an increase was not statistically significant.the percentage of cell survival in the MPeO treatment group also increased compared to the control in both radiation doses and both incubation times; however, such increment was not statistically significant.
the maximum seF values for MPeO-N nanoparticles were obtained at a radiation dose of 200 cGy reported to be 1.16 and 1.26, at 48 and 72 h of incubation, respectively.

Radioprotective effect on apoptosis and necrosis of PBMCs
the mean percentage of apoptosis and necrosis of irradiated PBMcs in the presence of different formulations at the concentrations equivalent to the ic10 value of MPeO-N nanoparticles (170 µg/ml) was determined by flow cytometry.as shown in Figure 6, the mean percentage of apoptosis in all treatment groups showed a significant decrease compared with the control group at both radiation doses of 25 and 200 cGy and both incubation times of 48 and 72 h (p < .05).
the percentage of apoptosis in the MPeO-N nanoparticles treatment group at a radiation dose of 200 cGy at 48 and 72 h of incubation was 6.57 ± 0.99 and 4.56 ± 1.24, respectively, which compared to the control group (10.17 ± 2.50 and 10.78 ± 2.17, respectively) showed a significant decrease (p ≤ .001for both).also, the reduction in the apoptosis percentage was significantly higher at a dose of 200 cGy than a dose of 25 cGy (at both incubation periods).
in the MPeO treatment group compared to the control group, the highest decrease in the percentage of apoptosis in PBMcs was related to a dose of 200 cGy and 72 h incubation time, so that the percentage of apoptosis has significantly decreased from 10.16 ± 2.51 to 7.20 ± 1.40 (p < .01).
the percentage of apoptosis in the MPeO-N nanoparticles treatment group showed a greater decrease compared to MPeO treatment group.such a difference was statistically significant (p < .05)except for the treatment group with a radiation dose of 25 cGy at 72 h of incubation.
as shown in Figure 7, the percentage of necrosis in the MPeO-N nanoparticles treatment group in both radiation doses and both incubation times showed a significant decrease compared to the control group.the highest decrease (p < .001) was observed at a radiation dose of 200 cGy with an incubation time of 72 h for the MPeO-N nanoparticles treatment group (26.30 ± 4.10) compared to the control group (38.76 ± 4.15).however, the reduction in the necrosis percentage of the MPeO treatment group compared to the control group was not statistically significant in both incubation times and both radiation doses (p ≥ .05).Now the PBMcs population after 48 and 72 h of incubation in different groups as a pseudo-color plot was obtained from flow  cytometry analysis; necrotic cells (Q1), late apoptosis (Q2), apoptosis (Q3), and living cells (Q4) (Figures 8 and 9).

Study parameters
Due to the widespread use of ionizing radiation in various areas of human life, the target population in this study was people who were exposed to occupational radiation and medical radiation.the application of ionizing radiation in medicine has been increasing in recent decades [5,6].according to NcRP Report No. 160, about half of the radiation in the united states is due to medical exposure, which is estimated at 42% worldwide, according to the 2008 aNsceaR reports [14].in diagnostic imaging, although the dose of each examination has been reduced due to improved technology and protocols, as well as the increased awareness of radiation staff, the annual effective dose has been elevated owing to the increasing number of medical examinations [5].according to reports of uNsceaR and NcRP, the dispersed radiations such as X-ray and gamma rays play a major role in medical exposure [15].
it has now been shown that the interaction of dispersed ionizing radiation such as X-rays with the biological systems leads to the production of free radicals and ROs [16].On the other hand, the ability of antioxidants to scavenge free radical has been confirmed in numerous studies [17].hence, due to the antioxidant nature of MPeO, as a result of possessing phenolic and flavonoid compounds [7], it would be expected that this essential oil can be an efficient radioprotector.the low toxicity of MPeO in comparison with synthetic compounds, as well as the possible removal of its limitations by loading on nanoniosomes, gives us hope for a desirable radioprotective agent. in this study, PBMcs were chosen to investigate the radioprotective effect of MPeO-N nanoparticles.PBMcs are considered an example of natural non-proliferating tissue cells which are at the G0 phase.these types of cells are obtained using minimally invasive methods, and a large number of cells can be collected serially in a short time [18].in addition to the above properties, due to the availability and high radio-sensitivity, they have always been considered in radiation protection studies [19].Despite Bergonié and tribondeau's law, which states the relationship between proliferation and radio-sensitivity [20], PBMcs have high radiation sensitivity, due to radiation-induced apoptosis [21].therefore, in the present study, the radioprotective effect of MPeO-N was investigated by measuring apoptotic, necrotic, and mitotic death in PBMcs by Mtt assay and flow cytometry analyses.
For this aim, the cells were irradiated at two radiation doses of 25 and 200 cGy.a radiation dose of 25 cGy was considered as approximately representative of the range of radiation dose to individuals [22] in common applications of X-rays (especially in diagnostic imaging), in such a way that it is out of the low dose range which is highly debatable in studies due to bystander effect, adaptive radiation response, etc. [23].the 200 cGy radiation dose has also been considered as the boundary between lethal dose and sub-lethal dose in radiation protection studies [24].

Characteristic of MPEO-N nanoparticles
the physicochemical properties of nanoniosomes significantly influence their behavior and systemic functioning [23].the size of nanoniosomes is one of the most important properties that directly affect cellular uptake.Nanocarriers less than 150 nm can penetrate the endothelium of liver capillaries [25,26].Nanoniosomes prepared by the thin-film hydration method are larger than those prepared by the ether-injection method; also, increasing the amount of cholesterol increases the size of the vesicles [27,28].tween 60 was used in this study.the average hydrodynamic diameter of MPeO-N nanoparticles was 106.0 ± 4.69 nm, which seems to be within the appropriate range for in vitro and in vivo treatment compared to studies using the same preparation methods [25,27].the PDi of the nanoparticles was also under 0.3, indicating an acceptable particle size distribution [29].Zeta potential is an important parameter in determining the stability of a colloidal solutions [30,31].usually, nanoparticles that have a zeta potential greater than +30 and less than −30 mV are considered completely stable [26].
however, due to the fact that the addition of large amounts of charged molecules can disrupt the mechanism of nanoniosome formation [29], the zeta potential of MPeO-N nanoparticles in this study (-15.2 ± 0.9 mV) seems to inhibit aggregation and is sufficient to create stability and this was observed in practice by keeping nanoparticles in the body's simulated environment for a long time [32].
the encapsulation efficiency and loading capacity of MPeO-N nanoparticles were 44.37 ± 3.62% and 3.4 ± 0.14%, respectively.these two quantities usually depend on various parameters such as the type of compound loaded in nanoniosomes, the preparation method, the chain length of the nonionic alkyl surfactant, and the ratio of surfactant to cholesterol.some niosome studies have shown that the use of tween60 in the formulation of nanoniosomes increases the loading of target compounds compared to other surfactants and increases stability [27,28].

Mentha Pulegium release rate
the release curve (Figure 4) of MPeO-N nanoparticles showed that the release rate of Mentha Pulegium from nanoniosomes is continuous and controlled so that a 50% release (T1/2) occurred during 24 h under the normal body condition (37 °c, ph = 7.4).a longer alkyl surfactant chain is able to lower the drug release rate, reduce drug leakage, and improve the stability of nanoniosomes [27,33].at 42 °c, the release rate was achieved with a steeper slope (T1/2 = ⁓7 h), and after 10 h, an almost constant trend was achieved.it is clear that the increase in temperature had a direct effect on the release rate.according to previous studies, PeGylation of nanoniosomes also improves the hydrophilicity of their molecular surface and thus prevents their recognition and elimination by phagocytic systems, which increases stability and half-life of PeGylated nanoniosomes [33].

Toxicity of formulations
a nontoxic concentration of nanoparticles was used to assess the radioprotective effect of MPeO-N nanoparticles on PBMcs [34], which in this study was considered ic10.to evaluate the toxicity of different formulations, MPeO-N equivalent concentrations of each formulation with 96 h of incubation were used, which was proportional to the incubation time used to determine the radioprotective effect of formulations by Mtt assay and flow cytometry analyses.as the results showed, MPeO and MPeO-N nanoparticles showed no toxicity up to a concentration of 170 µg/ml.

Radioprotective effect of MPEO-N nanoparticles
MTT assay as illustrated in Figure 5, 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 nanoparticles treatment group, the survival of PBMcs was significantly higher than the control, as the highest increase was observed at 72 h of incubation (p < .01).Notably, the increase in PBMcs survival within the MPeO-N nanoparticles treatment group was not statistically significant compared to the 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) [35].since the radioprotective effect of MPeO-N nanoparticles was examined at two radiation doses of 25 and 200 cGy, the quantity of seF [36] was defined which is the ratio of cell survival in the presence and absence of radioprotector in each radiation dose [37].according to this definition, the maximum seF value was 1.29 associated with the MPeO-N nanoparticles treatment group at a dose of 200 cGy in the 72 h incubation.to explain the reasons for these results, it has previously been stated that apoptosis is the main mechanism underlying radiation damage to PBMcs, and in fact, PBMcs are considered resistant to radiation-induced mitotic death due to their differentiation and non-proliferation [20,21].consequently, the lack of the optimal radioprotective effect of MPeO-N nanoparticles in enhancing the survival of PBMcs may be related to this issue.

Flow cytometry analysis
according to Figure 7, 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 [5,17].it should be noted that such an increase was moderated in the presence of MPeO and MPeO-N nanoparticles. in the MPeO-N treatment group, the percentage of apoptosis and necrosis of irradiated PBMcs was significantly decreased in both radiation doses and both incubation times compared to the control group (p < .01 for apoptosis and p < .05for necrosis).such a decrease in the MPeO treatment group was significant only for the percentage of apoptosis (p < .05,compared to control group).
comparing the radioprotective effect of MPeO-N nanoparticles and MPeO, the results showed that the reduction of apoptosis percentage of PBMcs in MPeO-N nanoparticles treatment group was more than MPeO treatment group, but this difference was significant only in 200 cGy radiation dose (p < .05).also, the percentage of necrosis in PBMcs showed a significant decrease only in the MPeO-N nanoparticles treatment group compared to the control, and this clearly indicates that the MPeO-N nanoparticles are more effective than the MPeO on the radioprotective effect.
in some useful studies, the radioprotective effect of curcumin on PBMcs has been investigated [38,39].the results of which may be generalized to MPeO. a study conducted on PBMcs irradiated with a radiation dose of 2 Gy, showed that dendrosomal nanoformulation of curcumin, by modulating the NF-κB and Nrf-2 pathways, affect expression of genes whose products are involved in cell cycle regulation, DNa damage detection and apoptosis, thereby increasing cell survival [39].
amifostine is the first FDa-approved radioprotector to reduce the incidence of moderate to severe xerostomia after radiation therapy of head and neck cancer [40].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. in addition, the short half-life of this drug in patients diminishes its effectiveness [41].another group of radioprotectors that have been discussed and extensively studied over the past two decades 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 low-let ionizing radiations [42].
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 [43].One of the biggest challenges in the development of radioprotectors is the lack of a comprehensive system to biologically examination of these compounds [44].the radioprotective effect of a radioprotector candidate depends on diverse 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.as a consequence, it seems that a single system for measuring the radioprotective effect is necessary to select a radioprotector with the optimal performance for additional analyses.

Conclusion
the present study evaluated for the first time the radioprotective effect of MPeO-N.MPeO-N nanoparticles show their optimal radioprotective effect by reducing the percentage of apoptosis and necrosis of PBMcs.Due to the unique properties of MPeO-N nanoparticles, such as low toxicity, biocompatibility, biodegradability, and controlled release, it can be a useful candidate for further studies aimed to developing functional food products or daily supplements for radiation staff, as well as patients who are exposed to low let radiation.Further studies are needed to better elucidate the mechanism of radioprotective effect of MPeO-N nanoparticles. in this regard, the evaluation of other cell lines that are sensitive to radiation-induced mitotic death would be recommended.

Ethical approval
the experimental procedures of the present research study were confirmed by the ethics committee of shahid sadoughi university of Medical sciences in yazd (iR.ssu.MeDiciNe.Rec.1398.326).

Figure 2 .
Figure 2. hydrodynamic size and zeta potential of mentha pulegium essential oil.

Figure 3 .
Figure 3. (a) maximum absorption wavelength of mentha pulegium essential oil, (b) standard calibration curves of mentha pulegium essential oil in isopropanol, and (c) standard calibration curves of mentha pulegium essential oil in pBs.

Figure 4 .
Figure 4. (a) release curve of nanoniosome-loaded mentha pulegium essential oil at 37 °c and 42 °c.(b) mtt assay-toxicity of different formulations on pBmcs after 96 h of incubation.

Figure 5 .
Figure 5. mtt assay -survival percentage of irradiated pBmcs with different radiation doses of X-ray in the presence of different formulations with concentrations equivalent to the Ic10 of mpeo-n at 48 and 72 h of incubation.*p < .05 and **p < .01.

Figure 6 .
Figure 6. the mean percentage of apoptosis in irradiated pBmcs at radiation doses of 0, 25, and 200 cgy was determined by flow cytometry analysis in different treatment groups with concentrations equivalent to Ic10 of mpeo-n nanoparticles (170 µg/ml) at 48 and 72 h of incubation.*p < .05,**p < .01,and ***p < .001.

Figure 7 .
Figure 7. the mean percentage of necrosis in irradiated pBmcs at radiation doses of 0, 25, and 200 cgy was determined by flow cytometry analysis in different treatment groups with concentrations equivalent to Ic10 of mpeo-n nanoparticles (170 µg/ml) at 48 and 72 h of incubation.*p < .05,**p < .01,and ***p < .001.

Figure 8 .
Figure 8. Flow cytometry analyses on irradiated pBmcs after 48 h of incubation with an equivalent concentration of Ic10 of mpeo-n nanoparticles.In each graph, the lower and left quadrants represent the percentage of living cells, and the lower and right quadrants represent the apoptotic cells.(a) control group without radiation dose.(b) treatment group with mpeo without radiation dose.(c) treatment group with mpeo-n nanoparticles without radiation dose.(d) control group with 25 cgy radiation dose.(e) mpeo treatment group with 25 cgy radiation dose.(f) mpeo-n nanoparticles treatment group with 25 cgy radiation dose.(g) control group with 200 cgy radiation dose.(h) mpeo treatment group with 200 cgy radiation dose.(i) mpeo-n nanoparticles treatment group with 200 cgy radiation dose.

Figure 9 .
Figure 9. Flow cytometry analyses on irradiated pBmcs after 72 h of incubation with an equivalent concentration of Ic10 of mpeo-n nanoparticles.In each graph, the lower and left quadrants represent the percentage of living cells, and the lower and right quadrants represent the apoptotic cells.(a) control group without radiation dose.(b) treatment group with mpeo without radiation dose.(c) treatment group with mpeo-n nanoparticles without radiation dose.(d) control group with 25 cgy radiation dose.(e) mpeo treatment group with 25 cgy radiation dose.(f) mpeo-n nanoparticles treatment group with 25 cgy radiation dose.(g) control group with 200 cgy radiation dose.(h) mpeo treatment group with 200 cgy radiation dose.(i) mpeo-n nanoparticles treatment group with 200 cgy radiation dose.