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Incorporation of ifosfamide into various essential oils -based nanoemulsions ameliorates its apoptotic effect in the cancers cells

The chemotherapeutic drugs, loaded in nanocarriers, have recently attracted the pharmaceutical industries due to their limited adverse side effects. The objective of the current study was to incorporate the ifosfamide (IFO) into two different essential oils-based nanoemulsions, lemon (LEM-IFO) and salvia (SAL-IFO). The antiproliferation activities of the resulted formulas were evaluated in the MCF-7 breast cancer cells and HeLa cervical cancers cells. The cytotoxic effect of the NE formulas was detected by the MTT assay, DAPI stain and light microscopy. The z-average diameters range of LEM-IFO and SAL-IFO, determined by the zetasizer, were 49.15–61.81 nm and 56.64–64.62 nm, respectively. The half maximal inhibitory concentration (IC50) of LEM-IFO and SAL-IFO, applied into the HeLa cells, were 0.165 ± 0.025 and 0.141 ± 0.035 mM, respectively, whereas the IC50 of LEM-IFO and SAL-IFO subjected into the MCF-7 cells were 0.200 ± 0.005 mM and 0.270 ± 0.025 mM, respectively. The IC50 of the free IFO was markedly larger than LEM-IFO and SAL-IFO when applied into MCF-7 cells (9.20 ± 2.01 mM) and HeLa cells (7.69 ± 1.88 mM). Among the tested formulas, LEM-IFO and SAL-IFO have the greatest apoptotic effect on the MCF-7 and HeLa cells, respectively. Solubilizing the IFO in the essential oils-based NE has ameliorated the antitumor efficacy of IFO.

In the current study, two NE formulations were produced based on SAL and LEM oils in order to evaluate their antiproliferation activities in MCF-7 breast cancer cells and HeLa cervical cancers cells.

Materials and Methods
Chemicals. Ifosfamide was purchased from Baxter, US. Span 20 and Tween 80 were obtained from Sigma (Missouri, US). The 3(4,5dimethylthiazole-2-yl)-2,5-diphyneltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and Coomassie brilliant blue were obtained from biomatik (Ontario, Canada). Ethanol and formaldehyde were purchased from Fisher Chemical (UK). The heat inactivated fetal bovine serum (FBS), and phosphate buffered saline (PBS, pH 7, 10 mM) were obtained from Lonza Walkersville (USA). Dulbeccos modified eagle medium (DMEM), trypsin and penicillin streptomycin antibiotic were obtained from Gibco life technologies (New York, US). The 4′,6-diamidino-2-phenylindole (DAPI) dihydrochloride was purchased from Invitrogen life technologies (New York, US). Construction of the phase diagrams for the NE preparations. Two pseudo ternary phase diagrams were constructed at different weight fractions of LEM or SAL oil, water, and a surfactant mixture blended at a fixed ratio of 2:1 of Tween 80 to Span 20, respectively. They were constructed to determine the emulsion (EM)

Physical characterization of NE formulas.
A key distinctive property of NE is its nanoscale particle size.
The analysis of the nanodroplets of the produced NE was performed by Zetasizer (Malvern Instruments, Malvern, UK). The sizes and charges of the NE dispersed nanodroplets were expressed as z-average diameters and zeta potentials, respectively. Measurements were performed three times at 25 °C. (v/v) penicillin-streptomycin, and incubated in a 5% CO 2 /95% humidified atmosphere at 37 °C. The media were changed every 48 h until confluence followed by washing with 2 ml of PBS, detachment by adding 2 ml of trypsin, and incubation at 37 °C.
Cytotoxicity screening using MTT assay. The toxicity of the chemotherapeutic agents against the cancerous cells is evaluated by the MTT assay. A 100 µl of culture media containing 10,000 cells, counted using a countess automated cell counter (Invitrogen, US), was seeded in each well of a 96-well plate and was incubated overnight at 37 °C in a CO 2 incubator for cell attachment. Then, cells were treated with 100 µl of different formulas, followed by incubation at 37 °C in a CO 2 incubator for 24 h. After that, a 5 µl of MTT reagent was added to each well, mixed gently for one minute and incubated for 3 to 4 h at 37 °C in a CO 2 incubator. Then, the culture medium containing MTT reagent was removed, followed by adding a 100 µl of DMSO and incubation for 2 h. Each tested concentration for all formulas ware repeated three times. The Absorbance (Abs) was read at 540 nm using a microplate reader (BioTek, US). Wells, included culture media, were considered negative control while culture media containing cells served as a positive control. The percentages of cell viabilities were determined by the following equation:

Results
Pseudo ternary phase diagrams. As exhibited in Fig. 1, the three phase region of the LEM oil was larger than SAL oil region whereas the two phase region for LEM oil was smaller than SAL oil, although the location of the two and three phases in the bottom of the phase diagrams for LEM and SAL oils were similar. The one phase regions of the O/W EM of both of LEM and SAL oils were located at the corner at which the water fraction was greater than the oil fraction and the Tween 80/Span 20 fractions. The EM solution for the selected oil, consisted of 1.8% oil, 5.5% Tween 80/Span 20 and 92.7% water, was converted to NE by heating and vortexing the solution above 70°C for one hour. Table 1, the z-average diameters of LEM-NE and SAL-NE did not markedly change when loaded with IFO. However, the absolute value of the negative zeta potential of LEM-NE was less than LEM-IFO whereas the zeta potential of SAL-NE was greater than SAL-IFO. It is worth mentioning that SAL-IFO has the largest z-average diameter and the smallest absolute value of the negative zeta potential among the tested NE formulas. The sizes of the dispersed nanodroplets of the entire NE formulas were homogeneously distributed as the polydispersity indexes (PDIs), measured through dividing the standard deviation by the mean of the z-average diameter, were less than 0.250. In terms of the cancer cells growth inhibition, it has been found that the IC 50 's of all of the tested NE formulas were markedly less than the IC 50 's of IFO, which were 7.69 ± 1.88 and 9.20 ± 2.01 mM when applied into HeLa and MCF-7 cells, respectively (P < 0.001). Among the drug-free NE formulas, the IC 50 of LEM-NE (0.219 ± 0.005 mM) was comparable to the IC 50 of SAL-NE (0.250 ± 0.001 mM) when applied into the HeLa cells. In contrast, the IC 50 of LEM-NE (0.321 ± 0.015 mM) was considerably less than the IC 50 of SAL-NE (0.355 ± 0.005 mM) (P < 0.001) when subjected into the MCF-7 cells. Regarding the NE-loaded IFO formulas, the comparable IC 50 's of LEM-IFO (0.165 ± 0.025 mM) and SAL-IFO (0.141 ± 0.035 mM), applied into HeLa cells, were significantly less than the IC 50 's of LEM-IFO (0.200 ± 0.005 mM) and SAL-IFO (0.270 ± 0.025 mM), subjected into MCF-7 cells (P < 0.001).

Morphological changes of HeLa and MCF-7 cells. As shown in Figs 3 and 4, HeLa and MCF-7 cells
exhibited morphological changes after treatment for 24 h at the IC 50 's of the tested formulas. All of the treated HeLa cells have endured different stages of apoptosis (Fig. 3). Cells treated with IFO and LEM-IFO have revealed late signs of apoptosis as their size has shrunk and membrane blebbing with collapse nucleus were seen whereas cells treated with SAL-IFO were enlarged and their shape were altered. In contrast, LEM-NE and SAL-NE Regarding the MCF-7 cells (Fig. 4), LEM-NE and LEM-IFO have markedly induced the formation of the extracellular apoptotic bodies. Cells treated with SAL-NE and SAL-IFO have endured late sign of apoptosis as ghost cells that lack nucleus were seen. On the other hand, cells treated with free IFO have revealed condensed chromatid without any alteration in their shape.  (Figs 5 and 6). Error bars represent the standard deviation for n = 3. The levels of the differences between the formulas at each concentration are ranked as very highly (***P < 0.001), highly (**0.001 ≤ P < 0.01)) and (*0.01 ≤ P < 0.05) significant.
SCientiFiC RepoRtS | (2019) 9:695 | DOI:10.1038/s41598-018-37048-x Nuclear morphological change. To determine the cell death due to apoptosis, DAPI stain was utilized to fluoresce the altered nuclei. As shown in Figs 5, 6 and 7, all of the tested formulas have decreased the cell counts as their concentrations have increased when subjected into either HeLa or MCF-7 cells. However, the shapes of the curves have differed. The counts of MCF-7 cells have declined linearly while the counts of HeLa cells have decreased exponentially when treated with the drug-free NEs and drug-loaded NEs (Fig. 7). In contrast, the increase in the IFO concentrations has caused a parabolic decrease in both of MCF-7 and HeLa cells counts. Interestingly, the decrease in both of MCF-7 and HeLa cells subjected into the drug-loaded NE was higher relative to the cells treated with drug-free NE.

Discussion
Nanocarriers, like NEs, can be involved in combining two or more anticancer agents with different physical properties in order to assure their simultaneous delivery into the target cells and thereby a synergistic effect of the incorporated agents may develop. Compared to the IC 50 of IFO, the IC 50 's of the entire NE formulas were markedly reduced in both of MCF-7 and HeLa cells which can be attributed to the small sizes and negative zeta potentials of the NEs nanodroplets that facilitate their permeation into the cancer cells 20 . Additionally, the incorporation of the IFO and ESSOs, SAL and LEM oils, into the NEs may enhance their accumulation in the cancer cells and thereby potentiate their anticancer activity 21 . Interestingly, the produced nanoemulsions formulations in the present study have the narrowest average size as compared with the previous studies [3][4][5][6][7] . In fact, the IC 50 's subjected to HeLa cells of the SAL-IFO and LEM-IFO was reduced three times relative to the formulation produced in previous work at self-microemulsifying drug delivery systems 6 . The response of the MCF-7 cells to the NE formulas has differed from the HeLa cells. In general, HeLa cells were more sensitive than the MCF-7 cells as the IC 50 's of the tested formulas subjected into the HeLa cells were less than the IC 50 s administered into the MCF-7 cells. The growth of the MCF-7 cells was greatly impeded when subjected into the LEM-IFO meanwhile SAL-IFO has the best inhibition effect on the growth of the HeLa cells. The great apoptotic effect of LEM-IFO on the MCF-7 cells may stem from the extracellular vesicle formation, seen under the light microscope, indicating that LEM-IFO has got attached into the cell membrane and induced apoptosis integrated with autophagocytosis 22 . In contrast, the light microscopy images revealed that the apoptotic effect of SAL-IFO on the HeLa cells has enlarged the cancer cells and damaged the nucleus without vesicle formation which implies that SAL-IFO has passed the cell membrane. Although both of LEM and SAL oils have antiproliferation activity, they have different mechanism of action in inducing cell apoptosis. LEM oil was found to impede the growth of the colon cancer cells through suppressing PI3K/Akt pathway that lead to the mitochondrial death 13 . On the other hand, many research studies have reported the antiangiogenic activity of SAL oil that prevents the tumor growth and invasion in various types of cancers [16][17][18][19]23 .
According to the analysis of the DAPI images, the growth of the MCF-7 cells was linearly decreased whereas the growth of the HeLa cells was exponentially decreased as the concentration of the subjected NE increased. This inverse relation between the growth of the cells and the NE concentration was first-order reaction which means that the inhibition process relies on one component. In other words, the nanodroplets of the NE formulas did not get degraded in the cancer cell and may act as a one metabolite. In addition, the inhibition of the LEM-IFO and SAL-IFO has reduced the growth of the MCF-7 and HeLa cells with similar trends when compared to the LEM-NE and SAL-NE. This implies that there has been a synergistic effect caused by the incorporation of the IFO into the ESSOs-based NE 24 . On the other hand, the free IFO applied into either MCF-7 or HeLa cells has caused a parabolic decrease in the growth of the cancer cells as its concentration increase indicating that the inhibition process is a second order reaction which depends on two metabolites. In fact, IFO may get degraded in the cell and form the two active metabolites, IFO mustard and choroacetaldehyde, that may induce apoptosis in the cancer cells 25 .

Conclusion
Incorporating the IFO into the ESSOs-based NE has markedly improved its cytotoxicity in the HeLa and MCF-7 cells. HeLa cells were more sensitive to the tested formulas than the MCF-7 cells. Among the NE formulas, LEM-IFO has the best apoptotic and inhibition effect on the MCF-7 cells while SAL-IFO has the best cytotoxic effect on the HeLa cells. Further studies have to be performed on the mechanism of apoptosis of LEM-IFO and SAL-IFO.