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Mar. Drugs 2020, 18, 217 7 of 11 3.4. Membrane Fluidity The membrane fluidity of all samples was measured by fluorescence anisotropy measurements. TMA–DPH was used as the fluorescent probe. The measurement was carried out according to the method described by Maherani et al. [54]. In brief, the solution of TMA–DPH (1 mM in ethanol) was added to the liposome suspension to reach a final concentration of 4 μM and 0.2 mg/mL for the probe and the lipid, respectively. The mixture was lightly stirred for 1 h at ambient temperature in the dark. Then, 180 μL of the solution was distributed into each well of a 96-well black microplate. The fluorescent probe was vertically and horizontally oriented in the lipid bilayer. The fluorescent intensity of the samples was measured with a Tecan INFINITE®200 PRO (Grödig, Austria) equipped with fluorescent polarizers. Samples were excited at 360 nm and emission was recorded at 430 nm under constant stirring at 25 ◦C. The Magellan 7 software was used for data analysis. The polarization value (P) of TMA–DPH was calculated using the following equation: P= III−GI III + 2GI where III is the fluorescent intensity parallel to the excitation plane, I⊥ the fluorescent intensity perpendicular to the excitation plane, and G is the factor that accounts for transmission efficiency. Membrane fluidity was defined as 1/P. The results were measured in triplicate. 3.5. Transmission Electron Microscopy (TEM) The morphology of the nanoliposomes was monitored via TEM, using a negative staining method, as previously described [21]. In brief, the liposomal formulation was diluted in distilled water (1:10 ratio) and then mixed with 2% ammonium molybdate solution with a ratio of 1:1. The mixture was reserved at room temperature for 3 min. Then, one drop was placed and dried on a Formvar carbon-coated copper grid (200 mesh, 3 mm diameter HF 36). Then, the morphology of the nanoliposomes was examined using a Philips CM20 TEM equipped with an Olympus TEM CCD camera at 200 kV. 3.6. In Vitro Evaluation of the Anti-cancer Activity of Encapsulated Curcumin 3.6.1. Cell Culture Human breast cancer MCF-7 cells (NCCS Pune) were cultured in an RPMI 1640 medium without phenol red (GibcoTM, ThermoFisher Scientific, Grand Island, NY, USA), supplemented with 10% (v/v) fetal bovine serum, 1% penicillin/streptomycin and 2mM l-Glutamine, at 37◦C in a humidified atmosphere of 5% CO2. 3.6.2. Evaluation of Drug Toxicity The in vitro cellular effect on MCF-7 cells of the curcumin-loaded nanoliposome was studied using the xCELLigence system (Roche Diagnostics GmbH, Mannheim, Germany), as described previously [23]. In brief, 1 x 104 cells per well were cultured overnight in 96-well E-PlatesTM. Three different concentrations of curcumin, encapsulated in nanoliposomes (5, 12 and 20 μM), were mixed with the culture medium. The concentrations of nanoliposomes and chitosan corresponding to 5, 12, and 20 μM of curcumin are presented in Table 2. The xCELLigence system (real-time cell analyzer single plate, RTCASP®) allows the real-time monitoring of cell proliferation based on impedance measurement. The technology uses specific 96-well cell culture E-PlatesTM with bottoms covered with microelectrodes as an electrical impedance cell sensor. The analysis is based on the measurement of electrical impedance created by attached cells across the high-density electrode array coating the bottom of the wells [55]. The impedance value is automatically converted to a dimensionless parameter called a cell index, which is defined as the relative change in electrical impedance created by the attached cells. As a quantitative measure of cellular status, the CI value represents the extent of thePDF Image | Growth-Inhibitory Effect of Chitosan-Coated Liposomes Encapsulating Curcumin
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