*2.3. Characterization of Synthesized Magnetite MNPs*

The structural characteristics were determined by X-ray powder diffraction using Philips MPD 1880 diffractometer (Brooklyn, NY, USA) with monochromatic CuKα radiation (*λ* = 0.154 nm) at room temperature. The structural features of prepared samples were recorded at 2*θ* angles in the range of 10◦–70◦ with a step of 0.02◦ and fixed time of 10 s per step. X-ray diffraction in polycrystalline is used to confirm crystalline size and structure of bare and coated magnetite MNPs obtained by solvothermal method. Field emission scanning electron microscope, JEOL JSM-7000F (Tokyo, Japan), was used to determine the morphology, particle size distribution and surface texture of bare and PEG-coated magnetite MNPs. FE-SEM was linked to the EDS/INCA 350 (energy dispersive X-ray analyzer) manufactured by Oxford Instruments Ltd., London, UK. The morphology of the MNPs has been further investigated using atomic force microscopy (AFM). The samples for AFM imaging were prepared by deposition of a magnetite MNPs suspension on the mica substrate. The MNPs are rinsed three times with 50 μL of MiliQ water to remove all residual impurities. AFM images were obtained by scanning the magnetite MNPs on the mica surface in the air using MultiMode Scanning Probe Microscope with Nanoscope IIIa controller (Bruker, Billerica, MA, USA) with SJV-JV-130 V ("J" scanner with vertical engagement); Vertical engagement (JV) 125 μm scanner (Bruker Instruments, Inc., Bruker, Billerica, MA, USA). Tapping mode was performed for imaging using silicon tip (R-TESPA, Bruker, Nom. Freq. 300 kHz, Nom. spring constant of 40 N/m) at 25.0 ◦C, allowing thermal equilibration before each imaging. AFM images were collected using random spot surface sampling (at least two areas per sample) for each analysed sample. All the images were processed by first-order flattening only and analysed using the NanoScope Analysis software (Version 5.31r1). Morphology analysis was investigated by Transmission Electron Microscope (TEM), Zeiss EM10 (Oberkochen, Germany), operated at 100 kV. In this propose, MNPs coated with PEG were dispersed in Milli-Q H2O and bare MNPs were dispersed in ethanol, ultrasonicated and placed on carbon coated copper grids. After air drying, samples were photographed by transmission electron microscopy. Samples were analysed with ImageJ and 50 particles were counted for each image.

The MNPs mesoporosity determination has been performed using nitrogen adsorption–desorption measurements on an ASAP2020 (Micromeritics, Norcross, GA, USA) accelerated surface area analyzer at 77 K. Before measuring, the samples were degassed at reduced pressure andat 120 ◦C for at least 6 h. All measurements have been made in duplicate. The specific surface area, the pore volume and the pore size are determined using Brunauer–Emmett–Teller (BET) analysis. To confirm the superparamagnetic property of the synthesized MNPs, magnetization measurements were performed. Magnetization of the powder samples of MNPs was measured using a commercial Quantum Design MPMS-5 SQUID magnetometer (San Diego, CA, USA). The powder samples were placed into a small ampoule whose diamagnetic contribution was properly subtracted. In addition, the field dependence of the magnetization (M(H)), including magnetic hysteresis loops, was measured at 300 K under fields up to 10 kOe.
