Ovarian cancer cells (MCF-7 and T47D human breast adenocarcinoma cells) were prepared from the Pasteur Institute (Tehran, Iran). Tween 80 was bought from DaeJung Chemicals & Metals (Seoul, Korea). DOTAP and cholesterol (1, 2-dioleoyl-3-trimethylammonium-propane) were obtained by Sigma-Aldrich (MO, USA), respectively. PBS tablets, DMSO (dimethyl sulfoxide), dialysis bag (MW¼12 kDa), MTT (5-diphenyl tetrazolium bromide; 3-(4, 5-dimethylthiazol-2-yl)-2) and paraformaldehyde solution were supplied by Sigma-Aldrich (MO, USA). DAPI (40, 6- diamidino-2-phenylindole) was obtained from Thermo Fisher Scientific (MA, USA). No additional purification was considered for other chemicals, salts and solvents with analytical grades, unless specified. The sequences of the miRNA-34a Primers were synthesized as follows: forward, CTTGAACTCCTGGGGCCTGAAG; reverse, GCCAAAGAAACACTCACAGCT. Eurofins Genomics Ebersberg (Ebersberg bei München, Germany) was used to synthesize the sequences of the miRNAs. Fluorescence microscopy was used to label the 50th end with FAM to allow for tracking.
The thin-film hydration technique was used to make niosomes (Matsumura and Maeda, 1986). Tween 80 (DaeJung Metals and Chemicals, South Korea) cholesterol (Sigma-Aldrich, MO, USA) was accurately calculated and dissolved in 100 𝜇L of choloform C. A thin lipid layer was formed under reduced pressure on a rotary flash evaporator (Ultrasonics GmbH, Heidolph, Germany). Then, the film was hydrated using 3 mL of phosphate buffered saline (PBS) at 60 °C and at a pH of 7.4. A microtip probe sonicator (Ultrasonics GmbH, Hielscher Germany) was used to sonicate the hydrated thin lipid for 30 min to reduce the mean size of vesicles. Niosomal formulations were screened for physical characterization.
Then, the polyethylene glycol (Lipoid GmbH, DSPE-mPEG 2000, Lipoid PE 18:0/18:0-PEG2000, Darmstadt, Germany) and cationic lipid DOTAP (1,2-dioleoyl- 3-trimethylammonium-propane, Sigma-Aldrich, MO, USA) were inserted to improve the stability of niosomal formulations. We used a rotary evaporator (Ultrasonics GmbH, Heidolph, Germany) to remove the organic solvent at 45 °C. Then, the layers were hydrated by the addition of PBS (pH 7.4) for 45 min at 60 °C to achieve the niosomal suspensions. A microtip probe sonicator (Ultrasonics GmbH, Hielscher, Germany) was used to sonicate the niosome suspensions for 15 min to decrease the mean size of the vesicles (Bader et al., 2011; Whitehead et al., 2010; Fernandez-Piñeiro, 2017; Babaei et al., 2020).
Physical characterization of niosomal vesicles
The PDI (Poly-Dispersity Index), zeta potential, and the size distribution of the noisome particles were calculated by the dynamic light scattering method, using a ZetaPALS particle size and zeta potential analyzer (Holtsville, Brookhaven Instruments, NY, USA). The dispersed light was found at an angle of 90° and at room temperature, and specimens in 1700 µL of deionized water (0.1 mg/mL) were made and calculated after preparation. All measurements were triplicated to calculate the mean value. Photomicrography representing the surface morphology of niosomes were taken using a scanning electron microscope (SEM) (model sm-5510, JELO Company, Tokyo, Japan). A drop of the Nanoniosome solution dissolved in water was placed on a mesh copper grid 400 and then placed in a vacuum desiccator to evaporate the solvent. Finally, specimens were covered with a gold coating to make them conductive, after assessing the surface morphology using SEM with a 100 W power instrument (model KYKY-EM3200-30 kV, Peking, China). To use AFM (Atomic Force Microscopy), similar samples were also prepared (Chiche et al., 2010).
Physical stability examination
After 60 days of storage, the physical stability of the niosomal was determined. Changes in zeta potential, particle size, and PDI were evaluated at14, 28, and 60 days (Seo et al., 2019).
Cell lines and culture conditions
T47D cells (the Iranian Biological Resource Center, Tehran, Iran) and human breast cancer MCF-7 were cultured in the combination of Ham RPMI1640 (InoClon, Tehran, Iran) supplemented with 1 mg/mL penicillin/ streptomycin (Gibco, MA, USA), 2 mM GlutaMAX™-I (100X, Gibco, MA, USA), and 15% of FBS (Fetal Bovine Serum, Gibco, MA, USA). The non-tumorigenic human breast epithelial cell lines MCF-10a (Iranian Biological Resource Center, Tehran, Iran) were developed in a mixture of Ham DMEM/F12, supplemented with 5% horse serum (Gibco, MA, USA), 2 mM GlutaMAX™-I, and EGF (Epithelial growth factor, Sigma-Aldrich, MO, USA) hydrocortisone 0.5 μg/mL (Sigma-Aldrich, MO, USA), 20 ng/mL, 1 mg/mL penicillin/streptomycin, and insulin 10 μg/mL (Sigma-Aldrich, MO, USA), 100 ng/mL cholera toxin (Sigma-Aldrich, MO, USA).
The cytotoxicity of different concentrations of miRNA34-a was determined through MTT analysis (Sigma, USA) (Mohammady et al., 2019; Uchegbu and Vyas, 1998). MCF-7, MCF-10a, and T47D cells were cultured in 96-wells plates at 10,000 cells per well. After attaching for 24 h, 200 μL of fresh medium comprising serial dilutions of the various miRNA-34a/niosome formulations were used to treat the cells, including free- miRNA-34a solution miRNA-34a/noisome, and empty noisome. The addition of 20 μL MTT or 5 mg/mL in PBS in each 96 well plate was performed, followed by incubation for 24 and 48 h, and incubation at 37 °C for 3 h. The medium was carefully eliminated during the introduction of 180 μL of DMSO into each well to dissolve the formed formazan crystals. The EPOCH Microplate Spectrophotometer (synergy HTX, BioTek, VT, USA) was used to record the absorption of each well at 570 nm.
Nanoniosomal cellular uptake
For each well, 5×104 cells of MCF-7, MCF-10a and T47D were planted in a 6-well plate and incubation was performed for 24 h for attaching them. Then, the empty noisome, miRNA-34a/noisome, and free- miRNA-34a solutions were used to treat the cells. Rinsing the cells with cold PBS was performed 3 times after 4 and 8 h of incubation and fixed with a mixture of citric acid and methanol (Sigma, USA). To stain the cells, DAPI (0.125 μg/mL, Thermo Fisher Scientific, MA, USA) was used, moreover, a fluorescence microscope was used to photograph (BX61, Olympus, Japan) (Uchegbu and Vyas, 1998; Ertekin et al., 2015; Mohammady et al., 2019).
All data were analyzed by using the GraphPad Prism (version 6.00 for Windows GraphPad Software, San Diego California USA, www.graphpad.com) and the data were expressed as mean ± standard deviation (SD). To compare two independent groups, a student t-test was used, and multiple samples were compared using an ANOVA test. A p value <0.05 was considered significant.