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  • br Drug entrapment e ciency and loading capacity br Twenty

    2020-07-06


    2.2.4. Drug entrapment efficiency and loading capacity
    Twenty-five mg of lyophilized FNPs were dispersed in ethanol, so-nicated at 40% amplitude for 1 min, and centrifuged at 31,514 × g for 30 min. The α-mangostin content in the supernatant was measured using UV–vis spectrophotometry (Genesis 10S, Thermo Fisher) at λmax
    The initial amount of α − mangostin
    The total amount of particles (2)
    Particle morphology was observed using the transmission electron microscopy (TEM, Tecnai G TF20, 200 kV, Philips, USA). Twenty μL of sample dispersed in DI water was dropped onto the carbon-coated 300-mesh copper grid. Then, the samples were negatively stained with 10 μL uranyl acetate 2% (w/v), followed by air drying, and observed under TEM nitrogen atmosphere.
    2.2.6. Particle structure and crystallinity
    Particle structure and crystallinity were determined using Fourier transformed-infrared spectrophotometry (FT-IR), X-ray diffractometry (XRD), and differential scanning calorimetry (DSC).
    FT-IR analysis was performed using a Tensor 27 spectrophotometer (Bruker Optics, Germany), equipped with a Deuterated Triglycine Sulfate (DTGS) detector, by an attenuated total reflection (ATR) method, with a Zinc Selenide (ZnSe) crystal cell. Spectra were obtained under a dry air purge at co-addition of 256 interferograms collected at 4.0 cm−1 resolution, and a wavenumber range of 4000–400 cm−1. XRD was conducted by the wide-angle X-ray diffractometer (D2 Phaser, Bruker, USA) using Cu Kα radiation generated at 45 kV and 36 mA (λ = 0.15418 nm). The samples (approx. 0.5 g) were mounted onto a quartz substrate and scanned from 10 to 30° (2θ) at a scan speed of 2°/min.
    DSC analysis was performed using DSC 1 STAR System (Mettler Toledo, USA) with the main sensor composed of ceramic:FRS6. The machine was run at a temperature range of 0–380 °C, with a scanning rate of 10 K/min, and in nitrogen gas flow of 100 mL/min.
    % Cumulative release =
    Sheep whole blood was centrifuged at 2,432 × g for 5 min, washed with PBS thrice, and followed by re-dispersion the red blood CORM-3 in PBS at a hematocrit of 1%. Approximately 50 μL of the blank FNPs, α-mangostin loaded FNPs, and the free α-mangostin, containing 3.125–100 μg/mL α-mangostin, were incubated with 1 mL of red blood cells, at 37 °C for 30 min. The cell lysis was halted by immersion the sample in an ice bath for 5 min. The unlysed cells were removed by centrifugation at 2432 × g for 5 min, and the hemoglobin in the su-pernatants was measured UV–vis spectrophotometrically at 540 nm. 100% lysis was induced with DI water. The percentage of hemolysis was calculated following Eq. (4).
    Where At, An, Ap are the absorbance values of the test samples, the negative control (PBS), and the positive control (DI water), respec-tively.
    Colon cancer cell line Caco-2 and breast cancer cell line MCF-7 were cultured in DMEM-F12, pH 7.4 supplemented with 10% FBS and 1% penicillin/streptomycin in humidified atmosphere of 5% CO2, at 37 °C. (3) Trypsin-EDTA (0.25%) was used to sub-culture the cells weekly.
    Where: Ct, Ci: concentrations of released α-mangostin at the time point t and i
    V0: total volume of dissolution buffer (30 mL)
    V: withdrawal sample volume at each time point (1 mL) M0: initial amount of α-mangostin Mi: total amount of α-mangostin withdrawal at the time point i
    2.2.9. Impact of intravenous diluent on the FNP properties
    The lyophilized samples were stored in darkness for 6 months at 4 °C and room temperature. After 6 month storage, the particle size and surface charge were determined using ZetaPALS® analyzer. For the chemical stability, α-mangostin in samples was extracted with ethanol and the percentage of α-mangostin remaining was examined with HPLC analysis at 320 nm (LD10A, Shimadzu, Kyoto, Japan). The method was developed using VertiSep™, C18 column (4.6 mm × 250 mm ×5 μm) as a stationary phase and a mobile phase composed of 0.2% (w/v) H3PO4:ACN (1:9 v/v). The system was run at 25 °C, flow rate 1 mL/min, and a concentration range of 3.125-100 μg/mL. This method was also validated followed ICH guidelines and specified for α-mangostin with a retention time of 8.38 min; the linearity was confirmed with equation y = 64992x – 11197, R2 = 1, in the range of 3.125–100 μg/mL. For 
    Mitochondrial functional MTT assay was used to measure the cell viability. Caco-2 and MCF-7 cells were seeded in 24-well plate, with an initial 25,000 cells/well. The cells were growth until 90% confluence when applying the samples. All particles were sterilized by filtration through a 0.45-μm membrane. The cells were then incubated at 37 °C, 5% CO2 for 24 h for Caco-2 or 48 h for MCF-7, followed by washing thrice with PBS. Finally, MTT (1 mg/mL) was added to the wells and incubated for 4 h. The formazan product was then dissolved by EtOH:DMSO (1:1 v/v) for 1 h, and UV–vis measured at 570 nm. The untreated cells and the solvent were used as a negative control and the blank, respectively. The percentage of cell viability was calculated by Eq. (5).