*3.1. Characterization of Synthesized Magnetite MNPs*

The X-ray powder diffraction patterns of the synthesized bare and PEG-coated MNPs are presented in Figure 3A,B, respectively. Characteristic peaks exhibited in the XRPD pattern are well-matched with the magnetite diffraction peaks and confirm the cubic inverse spinel structure of MNPs. The sharp diffraction of three characteristic peaks (220), (311) and (400) also indicate the spinel structure of magnetite [42]. The formation of pure cubic magnetite is confirmed by the value of the calculated lattice parameter "*a*" which has been determined to be 8.389 Å [43]. Using Scherrer's equation, the average crystallite size of bare and PEG-coated MNPs were calculated to be 25 nm and 19 nm, respectively. In addition, the decreased average crystallite size, another piece of evidence suggesting that PEG decreases the crystallinity of MNPs at a lower intensity and with broader diffraction peaks than PEG-coated MNPs.

The size and morphology of bare and PEG-coated mesoporous MNPs were observed by field-emission scanning electron microscope (FE-SEM), atomic force microscopy (AFM) and transmission electron microscopy (TEM), as shown in Figure 3A,B,G–J,K–L). Both bare and PEG-coated MNPs maintain a uniform spherical shape, some of them agglomerated due to magneto–dipole interactions between MNPs. While bare MNPs showed cluster structure with a very rough surface, the process of PEG coating revealed the flattened surface of mesoporous MNPs (Figure 3I,J). These findings have been confirmed by AFM imaging where the roughness of the MNPs surface has been increased by PEG coating, for almost 100%, from 5.58 ± 1.06 nm to 10.9 ± 2.1 nm, confirming the effective coverage of the bare MNPs by PEG [43]. The less agglomerated texture of the PEG-coated MNPs can be related to the effect of the PEG layer during the synthesis of MNPs. In addition, a size histogram of bare mesoporous MNPs obtained using SEM micrographs shows a broader size distribution than PEG-coated MNPs (*d*ave = 103.4 ± 0.7 nm and 101.0 ± 0.9 nm for bare and PEG-coated MNPs, respectively), indicating that polymer decreased the magnetic interaction among the particles and prevent their agglomeration. The cluster structure of mesoporous MNPs has been confirmed using AFM. The average size of bare MNPs and PEG-coated MNPs corresponds to results obtained by SEM, it was even 10% larger due to the convolution effect. However, in both 2D height images, it is shown that the cluster structure MNPs consists of smaller substructures, in the range of 15 to 35 nm, which roughly correspond to the dimension of the crystallite size obtained by X-ray powder diffraction. Hence, AFM revealed that only several nanometers increase in roughness is observed when PEG 4 kD is used, suggesting that the PEG layer is largely twisted the

magnetite surface, rather than stretch out linearly [44]. Furthermore, this PEG layer should decrease the magnetic interactions among the MNPs and prevents their agglomeration. The mean diameter of bare MNPs obtained by TEM is larger (143 ± 30 nm) than obtained by SEM (103.4 ± 0.7 nm), indicating a higher degree of polydispersity. However, the mean diameter of PEG-coated MNPs (96 ± 10 nm) corresponds the value obtained by SEM (101.0 ± 0.9 nm). Increasing the size of the PEG-coated MNPs can be attributed to the successful coating of PEG. As shown in Figure 3K,L, both MNPs maintain a typical spherical shape.

Nitrogen adsorption–desorption isotherms of bare MNPs confirmed their mesoporosity (see Figure 4). The surface area, pore size and total pore volume were calculated to be 20.5 m2g−1, 17.7 nm and 0. 09 cm3 g−1, respectively, strongly supporting the fact that the bare MNPs have mesoporous structure. The PEG coating of bare MNPs led to a slight decrease in surface area (19.3 m2 g−1) but an increase in pore size and total pore volume (24.6 nm<sup>1</sup> and 0.11 cm<sup>3</sup> g<sup>−</sup>1, respectively).

FTIR spectrum (Figure 5A) of bare magnetite MNPs shows bands at 585 and 395 cm−<sup>1</sup> corresponding to the symmetric stretching vibration mode of the Fe-O bond. The absorption maxima at 3452 cm−<sup>1</sup> and 1644 cm−<sup>1</sup> suggest the presence of O-H linkages. In the pure PEG, the bands at 1341 and 1100 cm−<sup>1</sup> belong to the C-O-C ether bond asymmetric and symmetric stretching vibrations. The band at 2890 cm−<sup>1</sup> is attributed to -CH2- stretching vibration in PEG. In addition, absorption bands at 1283 and 1465 cm−<sup>1</sup> attributed to the vibration of–CH2 and at 964 cm−<sup>1</sup> corresponds to the CH out-of-plane vibration [18]. The hydroxyl groups were also confirmed at 3444 and 1631 cm<sup>−</sup>1. The presence of characteristic FTIR bands of PEG in PEG-coated MNPs spectrum confirmed the successful coating of PEG on the surface of magnetite MNPs. The PEG-coated MNPs spectrum shows a strong C-O-C ether stretch at 1110 and 1381 cm−<sup>1</sup> [44]. In addition, the O-H linkages at the 1642 and 3445 cm−<sup>1</sup> bands exhibited enhanced intensity which also indicates that PEG modified the surface of MNPs. The transmittance bands at 589 and 399 cm−<sup>1</sup> confirm the symmetric stretching mode of the Fe-O bond. The results indicate that PEG is successfully functionalizing the surface of MNPs.

In order to confirm the superparamagnetic properties of synthesized bare and PEGcoated MNPs, measurements of the magnetization curve have been performed. The S-shaped hysteresis loops are a typical feature of superparamagnetic MNPs and obtained result is very similar to our previous work (77 emu g−1) on Fe3O4 MNPs [35]. The magnetization curve clearly shows that magnetization depends on the applied magnetic field, but not on the sign of the applied field. Magnetic characterization at 300 K indicates that the bare MNPs have saturation magnetization at the maximum field of 5 kOe value of 76.71 emu g−1, which is lower than obtained value for the bulk Fe3O4 (*M*<sup>s</sup> = 92 emu g−1) [42] or *M*s = 96.43 emu g−<sup>1</sup> [19].

The observed decrease in the saturation magnetization could be explained either by the use of PEG for the surface modification process [43] that causes the softening of the magnetization or by the difference in particle size [45]. In addition, the saturation magnetization of PEG-coated MNPs at the maximum field of 5 kOe is 74.75 emu g−1, somewhat lower than bare MNPs (Figure 5B). However, the magnetization measurement of synthesized MNPs confirmed their superparamagnetic properties, thus confirming their further application in release studies under applied magnetic fields.
