*3.2. Loading of Flavonoid into Magnetite MNPs*

The FTIR spectrum (Figure 6A,B) of quercetin detected OH group stretching at 3403 cm−<sup>1</sup> and 3328 cm−1. The band at 1664 cm−<sup>1</sup> corresponds to the C = O aryl ketonic stretching and the bands at 1617 cm−1, 1558 cm−<sup>1</sup> and 1520 cm−<sup>1</sup> correspond to C=C aromatic ring stretching. OH bending of the phenol functional group at 1375 cm−<sup>1</sup> and 1314 cm−<sup>1</sup> belongs to the in-plane bending of C–H in aromatic hydrocarbon. Bands at 933 cm<sup>−</sup>1, 820 cm−1, 639 cm−<sup>1</sup> and 602 cm−<sup>1</sup> correspond to aromatic C–H out-of-plane bendings. The C–O stretching in the aryl ether ring and the C–O stretching in phenol

corresponds to 1244 cm−<sup>1</sup> and 1210 cm−<sup>1</sup> transmittance maxima. The band at 1167 cm−<sup>1</sup> is attributed to the C–CO–C stretch and bending mode in ketone, respectively. The FTIR spectra of quercetin-loaded MNPs show the broadening of the OH band at 3446 cm−1, which confirms the entrapment of quercetin in MNPs [46]. The interval from 1560 to 816 cm−<sup>1</sup> matches very well with pure quercetin peaks and indicates successful loading [46].

**Figure 3.** XRD pattern of (**A**) mesoporous bare MNPs Fe3O4; (**B**) PEG-coated MNPs; SEM images of (**C**) bare Fe3O4 MNPs showing the rough rounded cluster with extensive open porosity ranging from 30 to 200 nm in diameter; (**D**) SEM image of the surface of PEG-coated Fe3O4 MNPs ranging in di-ameter from 50–130 nm; Histogram of bare MNPs Fe3O4 (**E**) and PEG-coated Fe3O4 MNPs (**F**); (2D height topographic AFM image of single (**E**) bare magnetite Fe3O4 MNPs (**G**) and (**F**) PEG-coated Fe3O4 MNPs (**H**) showing cluster structure details; 2D-amplitude AFM image of single (**G**) bare magnetite (**I**) and (**H**) PEG-coated MNPs (**J**) showing subcluster structures in size range from 10 to 30 nm; TEM images of (**K**) bare Fe3O4 MNPSs; (**L**) TEM image of the surface of PEG-coated Fe3O4 MNPs.

The loading of quercetin has been also confirmed using nitrogen adsorption–desorption isotherms of quercetin-loaded PEG MNPs. In comparison to PEG covered MNPs, the surface area has been decreased by almost 19% to 15.7 m<sup>2</sup> g−1, pore size decreased to 14.2 nm for 42.4%, while total pore volume amounting 0.06 cm<sup>3</sup> g<sup>−</sup>1, decreased for 49% to 0.06 cm3 g<sup>−</sup>1, strongly supporting the fact that the quercetin has been effectively loaded into MNPs.

The thermogravimetric study was performed to confirm the quercetin loading in MNPs. Figure 6C shows comparative weight loss for quercetin and quercetin-loaded MNPs. A thermogravimetric study of quercetin reveals that the compound undergoes a three-stage thermal decomposition. The first stage of mass loss begins at 30 ◦C and continues up to 133 ◦C. A mass loss of 3.52% is attributed to dehydration or loss of water molecules on the surface of quercetin. In the temperature range 133 ◦C to 385 ◦C, the compound experiences a weight loss of 28.5% due to the melting of quercetin. The final thermal decomposition is observed in the temperature range of 385 ◦C to 1110 ◦C, and the weight loss of quercetin is 67% [47]. In the case of quercetin-loaded MNPs, the weight loss in the temperature range from 30 ◦C to 1200 ◦C is about 12.2%, which is attributed to the decomposition of organic compounds from MNPs. This data results in the great thermal stability of quercetin when it has been encapsulated in MNPs. In the case of PEG-coated MNPs, there is an increase in the weight gain resulting from the burning of the PEG and oxygenation of Fe3O4 starting at 270 ◦C under the continuous flow of oxygen at high temperatures and finishing at 450 ◦C [48]. However, the weight loss of PEG from the PEG-coated MNPs amounts to 4%. It is assumed that the thermal decomposition of PEG occurs at both C-O and C-C bonds of the backbone chain [49]. The influence of the quercetin loading into magnetite MNPs on the size and morphology of the MNPs has been further investigated. 2D height AFM image (Figure 6D) and 2D-amplitude AFM image (Figure 6E) revealed that distinct subcluster structure containing MNPs has been retained irrespective of quercetin loading, with size grains from 20 to 50 nm in diameter. The roughness value after quercetin loading decreased from (10.9 ± 2.1) nm to (4.86 ± 1.1) nm additionally confirming the successful loading of quercetin to PEG loaded MNPs.

**Figure 4.** N2 absorption-desorption isotherm for (**A**) bare MNPs, (**B**) PEG-coated MNPs and (**C**) quercetin-loaded PEG\_MNPs.

**Figure 5.** (**A**) FTIR spectra of MNPs, PEG and PEG-coated MNPs (**B**) Magnetic behavior of MNPs. The hysteresis loop shows a slight decrease in the magnetization behavior after a thin PEG layer coating. The magnetic hysteresis loops of mesoporous MNPs at 300 K. Inset within Figure 5 (**B**) shows the magnetic coercivity *H*c = 53.3 Oe.

**Figure 6.** FTIR spectra of (**A**) quercetin and quercetin-loaded MNPs; (**B**) zoomed spectrum of quercetin-loaded MNPs in the region of quercetin reach bands; (**C**) TGA curves of PEG-coated MNPs, quercetin and quercetin-loaded MNPs. Morphology of quercetin-loaded MNPs on the 2D height AFM image (**D**) and 2D-amplitude AFM image (**E**).

In addition, UV/VIS spectroscopy is used to study quercetin loading efficiency. Compared with quercetin concentration before adding the synthesized MNPs, the concentration loss was determined using a calibration curve in pure ethanol (EtOH). The coefficient of determination was 0.9978, and the determined molar absorption coefficient of quercetin at 298 K and 374 nm is 19,131 mol−<sup>1</sup> dm3 cm<sup>−</sup>1. The loading efficiency (LE) was determined to be (20.2 ± 1.3%) calculated from 17 independent experiments. Our results suggest significant improvement of the loading efficiency of the quercetin compared with a loading efficiency of solid lipid NPs (13.20 ± 0.18%) [1]. However, the limited LE is due to the reduced specific loading site of quercetin induced by PEG coatings [50]. Despite this, the PEG –MNPs provide the capability to load antioxidant quercetin with low aqueous solubility which reflects the potential of the synthesized MNPs as drug delivery carriers.
