Incorporation of etoposide (ETP) into nanoemulsion (NE) containing polyunsaturated fatty acids (PUFAs) may potentially augment its antiproliferation effect on the cancer cells. The current study aimed to examine the
Cancer incidence and mortality are growing rapidly worldwide. According to the Global Cancer Statistics of 2018, there will be 9.6 million deaths caused by cancer (
Etoposide (ETP) is a chemotherapeutic drug that belongs to the podophyllotoxin family. It affects the cell cycle at the G2 phase (
Among different nano-delivery systems, nanoemulsions are one of the most promising approaches that have been established to overcome the main hurdles linked with the standard drug delivery systems (
Black currant seed oil (BC) and organic evening primrose (EP) oil were purchased from Chateau Cosmetics Botanical Beauty (FL, USA). Etoposide (ETP) was acquired from Ebewe Pharma GmbH (Unterach, Austria). Formaldehyde, Tween 80, and span 20 were purchased from Sigma (MO, USA).
Fetal bovine serum (FBS) was obtained from Gibco™ Life Technologies (NY, USA).Trypsin-EDTA (0.25%), phosphate-buffered saline (PBS), penicillin-streptomycin, and Dulbecco’s modified eagle’s medium (DMEM) were acquired from UFC biotechnology, Inc., (Riyadh, KSA). 4’,6-diamidino-2-phenylindole (DAPI) dihydrochloride was procured from Invitrogen life technologies (NY, USA). Coomassie brilliant blue was acquired from Fisher Scientific (PA, USA). The ovarian cancer cell line (SK-OV-3) was obtained from the Tissue Culture Bank at King Fahd Center for Medical Research, King Abdulaziz University (Jeddah, KSA).
Annexin V-FITC Apoptosis Detection Kit (Cat. No. MBS668896) was acquired from MyBioSource, Inc. (San Diego, USA). Cell counting kit- 8 (CCK-8) (Lot. No. LE612) was purchased from Dojindo Molecular Technology, Inc. (Kumamoto, Japan). Cell Death Detection ELISA plus kit (Lot. No. 19315700) was procured from Roche (Mannheim, Germany). Mitochondria Staining Kit (Cat. No. CS0390) was obtained from Sigma-Aldrich (MO, USA). QCM™ collagen cell invasion assay (Cat. No. ECM551) was purchased from Merck KGaA (Darmstadt, Germany).
The drug-free, oil-in-water nanoemulsion (BC/EP-NE), was prepared by the high energy method. In brief, volume fractions of different components of 2% BC, 2% EP, 7% Tween 80, 3% Span 20, and 86% distilled water were blended at 25°C. After that, the mixture was sonicated with the Omni Sonic Ruptor 4000 Ultrasonic Homogenizer (NY, USA). The ultrasonic power supply transforms the voltage to high-frequency (20 kHz) electrical energy. The probe, in turn, received the produced electrical energy and converted to mechanical energy to be directed to the titanium tip, which has a 4.0 mm diameter. The tip was immersed in the solution and used to radiate the energy into the emulsion. This emulsion was sonicated for 20 min until it became clear and transparent. It should be mentioned that the drug-loaded formulations (ETP-BC/EP-NE) were prepared by directly solubilizing 0.29 μL of 20 mg/mL ETP in 99.7 μL BC/EP-NE.
Absorption spectra for droplet size distributions of BC/EP-NE and ETP-BC/EP-NE were plot at a wavelength ranging from 200 to 600 nm using GENESYS 10S UV-Vis spectrophotometer (Thermo Scientific™, USA). The particle sizes and zeta potentials of the dispersed nanodroplets for BC/EP-NE and ETP-BC/EP-NE were measured using the Zetasizer (Malvern Instruments Ltd., UK) at 25°C.
The release profiles of free-ETP and ETP-BC/EP-NE were determined by the dialysis procedure. In brief, 1.0 mL of the tested formula was transferred into the dialysis bag (Cut-off 3.5 Da, Spectra Lab., CA, USA). Then, the bag was submerged into phosphate buffer (200 mL, pH 7.4) and stirred at 100 rpm. Approximately 1.0 mL of the buffer was taken at a determined interval (1, 2, 3, 4, 5, 6, and 24 h) and replaced by another 1.0 mL of fresh buffer. The absorbance of the removed buffer at a specific time (A1) and wavelength of 300 nm was determined using the GENESYS 10S UV-Vis spectrophotometer (Thermo Scientific™, USA). The percentage release of the tested formula at each time was measured by dividing the absorbance of the removed solution (A1) by A0, which is the absorbance of the initial drug sample added to the dialysis bag, and then multiplying by 100.
The SK-OV-3 cells (2 × 106), generously supplied by the Cell Regenerative Unit (Jeddah, KSA), were plated in a culture flask (25 cm2) containing 5 mL of medium, supplemented with 10% FBS, 1% penicillin/streptomycin, and incubated at the culture conditions, which were 5% CO2 humidified atmosphere and 37°C. For subculture, every 48 h, cell media (DMEM) was discarded, and cells were rinsed with PBS, followed by detachment from flasks by adding 1.0 mL of 0.25% trypsin. The cells were nourished with DMEM until 90% confluence was obtained.
The CCK-8 assay was used for the determination of the antiproliferation and cytotoxicity by measuring the number of viable cells subjected to the tested formulation. The 1 × 104 of SK-OV-3 cells were cultured in 100 μL of a medium in each well of 96 well-plates and incubated at 37°C for 24 h in a CO2 humidified atmosphere incubator. Then, cells were treated with 200 μL of ETP, BC/EP-NE and ETP-BC/EP-NE at different micromolar concentrations (5, 3, 1, 0.1, 0.01, 0.005, and 0.0001), which were serially diluted with DMEM and incubated for 24 h at the same culture conditions. To measure the cell viability, 5 μL of CCK-8 reagent was added in the dark into each well, mixed gently, and incubated for 3 h at culture conditions. After that, the absorbance (A) of treated cells, media containing cells (control), and media (blank), was measured at λ = 450 nm in a BioTek Microplate Reader (VT, USA). The calculations were implemented as follows:
In a six well-plate, cells (3 × 105/well) were cultured in each well containing 2 mL of growth media and incubated for 24 h at 37°C. Then, 2 mL of IC50 for the tested formula was added to each well and incubated at culture conditions. Following 24 h incubation, cells were harvested, rinsed two times with pre-cold PBS, spun down (300 g), re-suspended in 100 μL of 1X binding buffer, and transferred to a flow cytometry tube. Then, 5 μL of each Fluorescein isothiocyanate (FITC) and propidium iodide (PI) supplemented to the cell suspension, incubated at 25°C in the dark (20 min), and then completed with 400 μL of binding buffer. Finally, cells were evaluated within one hour using a flow cytometer (FACS AriaTM III, BD Biosciences, CA, USA).
Cells were seeded at 1 × 104 per well into a 96-well plate at which each well contained 100 μL of the growth medium, followed by incubation at 37°C for 24 h. Then, 200 μL of treatments, ETP, BC/EP-NE, and ETP-BC/EP-NE were added to each well and incubated at culture conditions. After removing the media, cells were rinsed with PBS (100 μL), fixed with formaldehyde (200 μL, 4%), and stained by the addition of 100 μL of 5% Coomassie blue dye for 10 min. Finally, cells were cleaned several times with tap water, followed by drying overnight at 25°C. The morphological changes of treated SK-OV-3 cells were detected using a phase-contrast inverted microscope (Olympus, Japan).
DAPI is a blue-fluorescent DNA stain that fluoresces brightly once binding to A-T regions of dsDNA. Cells (5 × 104/well) were cultured in 500 μL of DMEM in 24 well-plates and incubated for 24 h, at 37°C. Then, 500 μL of the IC50 of ETP, BC/EP-NE, and ETP-BC/EP-NE were added and incubated at cultured conditions. Following incubation, cells were rinsed with PBS (300 μL), fixed via 4% formaldehyde (200 μL), stained for 2 min with DAPI stain (300 μL, 300 nM) and visualized using a fluorescent microscope at 437 μm (Leica CRT6000, Germany).
JC-1 is a cytofluorimetric, lipophilic, cationic dye used to evaluate SK-OV-3 cells’ mitochondrial membrane potential. Briefly, cells (1 × 104/well) were cultured into 100 μL of DMEM in 96 well-plates and incubated for 24 h at 37°C. Then, 200 μL of IC50 of ETP, BC/EP-NE, and ETP-BC/EP-NE were added to each well and incubated at culture conditions. Following incubation, SK-OV-3 cells were stained by JC-1 solution (JC-1 stain, JC-1buffer solution, and DMEM media) and incubated at 37°C for 30 min. Finally, the fluorescence was examined at excitation (Ex): 525 nm/emission (Em): 590 for red fluorescence and at Ex: 490 nm/Em: 530 for green fluorescence using a fluorescence microplate reader (Synergy™ HTX, BioTek, USA).
Apoptotic DNA fragmentation (mono- and oligonucleosomes) was analyzed by utilizing a cell death detection ELISA kit, which was carried out as stated by the manufacturer’s instructions. Briefly, SK-OV-3 cells (1 × 104/well) were cultivated into 96 well-plates at which each well contained 100 μL of growth medium followed by incubation at 37°C for 24 h. Following incubation, 200 μL of IC50 of ETP, BC/EP-NE, and ETP-BC/EP-NE were added and incubated for 24 h. After that, the 96 well-plate was centrifuged (200 × g), and the cells were re-suspended in lysis solution (200 μL/well). The 20 μL of lysates were relocated into the streptavidin-coated microplate, followed by the addition of 80 μL of an immunoreagent. After that, the microplate was covered and incubated at 300 rpm on a shaker for 2 h. Then, 100 μL of ABTS stain was added to each well and incubated on a plate shaker at 250 rpm for 10 min. After that, the ABTS stop solution (100 μL) was added, and the optical densities (A) were determined at 405 nm. The specific enrichment of mono- and oligo nucleosomes (Enrichment factor) was considered according to the subsequent equation:
The invasion of SK-OV-3 cells was evaluated by using a Chemicon QCM™ 24-well collagen-based invasion assay kit. Briefly, SK-OV-3 cells at (3 × 105/well) were cultured in 2 mL of DMEM into 6 well-plates and incubated at culture conditions. Then, SK-OV-3 cells were treated with 2 mL of formulation and incubated for 24 h. After that, 300 µL of media without FBS was supplemented to each insert and incubated at 25°C for 30 min to rehydrate the collagen layer. After that, 250 µL of media were extracted, and 250 µL of cell suspension was added into each insert suspended in 500 µL of the media with 10% FBS in the lower chamber. Then, the plate was covered and incubated for 24 h in a CO2 incubator. After that, all non-invaded cells were detached, and the insert was placed in 400 µL of cell stain for 20 min at 25°C. After that, the insert was rinsed, gently cleaned with a cotton-tipped swab, transferred into 200 µL of extraction buffer, and left for 15 min at 25°C. Finally, 100 µL of the extracted buffer was shifted to a 96-well culture plate for colorimetric measurement at 560 nm. The invasion ability of the cells was measured according to the following equation:
Statistical analyses between results were evaluated by one-way ANOVA. The significant differences between the two variables were assessed by pairwise
The influence of droplet size distributions on the absorption spectrum was displayed in
The droplet sizes and surface charges were measured by the zetasizer. As illustrated in
The release of ETP-BC/EP-NE and ETP in phosphate buffer (0.5 mM, pH 7) was determined at different periods of time as shown in
The anti-proliferation activities of the tested treatments against the cancer cellular growth were determined, as shown in
Cells treated with various formulations were discriminated according to their stages of apoptosis, as shown in
The effect of the tested treatments on the DNA fragmentation was displayed in
The mitochondrial membrane potential of treated SK-OV-3 cells was displayed in
The percentages of invading SK-OV-3 cells when subjected to the IC50 of ETP, BC/EP-NE, and ETP-BC/EP-NE were clarified in
The particle sizes of the drug nanocarriers, which are smaller than 200 nm, play a major role in the cellular penetration and uptake by the cancer cells (
In the present study, all the ETP, BC/EP-NE, and ETP-BC/EP-NE displayed a dose-dependent antiproliferation effect on the SK-OV-3 ovarian cancer cells. In agreement with our results,
In fact, the ETP-BC/EP-NE showed a notable reduction in the % of cellular growth, which could be ascribed to the potent anticancer effect of PUFAs and the chemotherapeutic effect of the ETP. It has been found that the essential oils, black currant seed, and organic evening primrose oils with a high content of gamma-linolenic acid (GLA) were potential in supporting the anti-cancer therapy (
The apoptosis of SK-OV-3 cells was evaluated by mitochondrial membrane potential, Annexin V-FITC, DNA fragmentation, light, and fluorescence microscopy. The changes in the mitochondrial membrane potential (Δψm) can be optically measured by the red/green fluorescence intensity ratio occurring during the process of apoptosis (
Regarding the invasion assessment of the treated cells, the percentages of the invaded SK-OV-3 cells were lower when cells were treated with ETP-BC/EP-NE. This could be due to the potential effect of GLA included in the nanoemulsion. The previous study of
Formulating ETP in a nanoemulsion based on mixing two oils rich in polyunsaturated fatty acid, black currant seed oil (BC) and organic evening primrose (EP) oils, had immensely inhibited the growth of SK-OV-3 ovarian cancer cells by stimulating apoptosis and impeding the cellular invasions ability. This novel formulation, ETP-BC/EP-NE, has a potent therapeutic effect on the ovarian cancer cells that need to be confirmed in animal models.
This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under Grant No. DF-606-247-1441. The authors, therefore, acknowledge DSR’s technical and financial support.