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  • br Materials and methods br General remarks

    2020-08-12


    2. Materials and methods
    2.1. General remarks
    All of the material including solvents and reagents were commer-cially available and purchased from Sigma-Aldrich, Merck and Fluka chemical companies. X-ray diffraction (XRD) patterns were obtained on Inel French, EQUINOX KPT 185 3000 model X-ray diffractometer using CueK radiation. Scanning KPT 185 microscopy (SEM) has been investigated using SU3500 microscope with scanning range from to 20 keV. Electron dispersive X-ray spectroscopy (EDX) measurements were done with an IXRF model 550i attached to SEM. Transmission electron mi-croscopy (TEM) analyses was performed using a TEM microscope Philips CM 120 kV Netherland. Thermogravimetric analysis (TGA) was performed by thermal gravimetric analysis instrument (Shimadzu TA-60WS-TGA-50/50H) with a flow rate of 30 mL min-1 and a heating rate of 10 °C min−1 in the air. Fourier transform infrared spectroscopy (FT-IR) spectra were recorded with KBr pellets using a Bruker ALPHA FT-IR  Materials Science & Engineering C 101 (2019) 472–486
    spectrometer. X-ray photoelectron spectroscopy (XPS) was investigated using a Kratos Analytical Axis Ultra, with monochromatic aluminum and magnesium with X-ray source of 1486.6 and 1253.6 eV and Concentric Hemispherical Analyzer (CHA). A take-off angle of 90 was used on a spot size of 700 lm, and 350 lm. The instrument has Ultra High Vacuum (UHV). The magnetic property of samples was measured using a BHV-55, Riken, Japan vibrating sample magnetometer (VSM). The elemental analysis was investigated using inductively coupled plasma atomic emission spectroscopy (ICP-AES) on a SpectroCiros CCD spectrometer. The pore volume and specific surface area determination were studied by Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods, respectively, by using a Quantachrome-Autosorb 1C-VP analyzer. Prior to the measurements, the samples were degassed at 100 °C for 6 h. The dialysis bag (molecularweight cut off (MWCO) = 10 kDa) was purchased from Sigma-Aldrich Chemicals Company. The cytotoxicity assay kit was prepared from Roch Chemical Company. The optical density (OD) of wells was calculated by Bio-Tek, ELX 800, Winooski, VT microplate reader. Zeta potentials were mea-sured with a Nano ZS90 zeta sizer analyzer (Malvern, UK) with 0.001 mol L−1 NaNO3 electrolyte.
    2.2. Preparation of Fe3O4 nanoparticles
    Typically, the mixture of FeCl2·4H2O (4.3 g) and FeCl3·6H2O (11.6 g) was prepared in 350 mL of deionized water under N2 atmo-sphere. The obtained solution was heated to 80 °C while vigorous stir-ring. Then, 20 mL of 25% NH4OH was quickly added to the previous solution. The resulting dark suspension was vigorously stirred for 10 min and then, the black precipitates were separated from the mix-ture using an external magnet and washed with deionized water and ethanol frequently, then dried under vacuum conditions.
    Firstly, the uniform suspension was prepared using ultrasonically dispersion of Fe3O4 (0.3 g) into 150 mL solvent (Vmethanol/Vwater = 1/1) for 20 min. In the following, 100 mL alkaline solution of Na2CO3 and NaOH (0.32 g and 0.48 g respectively) was added dropwise into the prepared suspension until pH ca. 10.0 and kept for 5 min. Then 100 mL salt solution of 0.45 g of Al(NO3)3·9H2O (1.2 mmol) and 0.85 g of Ca (NO3)2·4H2O (3.6 mmol) was added to the previous suspension and using the alkaline solution of Na2CO3 and NaOH, the pH of above mixture was adjusted to 9.5–10.0. The obtained slurry dispersion was stirred for 5 min followed collecting by magnet and washing with deionized water and ethanol three times. Finally [email protected] dried at 60 °C overnight giving the desired product.
    The synthesis of [email protected]@L-Dopa and loading of L-Dopa on the surface and into the structure of [email protected] was carried out using anion exchange method. At first 0.01 g of [email protected] was added to 30.0 mL of 2.5 mM L-Dopa solution in double distilled water under vigorous stirring for 24 h in room temperature. In this step, the pH of the solution was adjusted to 7.4. The resulting solid was se-parated using external magnet, washed with distilled water and dried at room temperature and under vacuum conditions giving product [email protected]@L-Dopa.
    2.5. Estimation of drug loading and drug encapsulation efficiency
    In order to determining the drug encapsulation efficiency, 1 mg of [email protected] was added to 3 mL solution of L-Dopa (3 mM) under stirring for 24 h. After fully separation of solid [email protected]@L-Dopa by external magnet and filtration from the suspension, 1 mL of the filtrate was used to analyze the amount of drug residue using UV/
    N. Shahabadi, et al.
    visible spectrophotometry at 280 nm. The drug encapsulation efficiency was evaluated by the following equation [25]: r>