*2.4. The Determination of Loaded Quercetin into Magnetic Mesoporous MNPs*

The quercetin loading efficiency into mesoporous MNPs has been confirmed using Brunauer–Emmett–Teller (BET) analysis. The difference in the specific surface area, the pore volume and the pore size before and after immersing the MNPs into the quercetin solution confirmed the loading of quercetin into MNPs. FTIR spectra were obtained by Alpha-T FTIR Spectrometer (Bruker, Billerica, MA, USA). All spectra were recorded between 4000 and 350 cm−<sup>1</sup> at a nominal resolution of 4 cm−<sup>1</sup> at 25 ◦C, and the total number of recordings was 16. Dried samples were mixed with KBr powder and then they were pressed to produce pellets. TG analysis data were carried out on a TG/DTA simultaneous analyser DTG-60H with a 10 ◦C/min heating rate under a nitrogen atmosphere. The measurements were recorded in a range of room temperature up to 1200 ◦C.

UV/VIS spectrophotometer was used to study the quercetin loading efficiency of the synthesized MNPs. The loading efficiency was calculated by measuring the absorbance of the supernatant with the WTW photoLab® 7600 UV-VIS Spectrophotometer (Xylem, Rye Brook, NY, USA) at 374 nm. Measurements were performed in a rectangular quartz cuvette with a 10 mm optical path length and covers at 25 ◦C.

A total of 500 mg of quercetin was dissolved in 100 mL of ethanol and suspended in an ultrasonic bath (Bandelin Sonorex Super RK 100 H, Berlin, Germany) for 1 h at room temperature. The 25 mL aliquot of quercetin solution and 100 mg of coated MNPs were transferred into a 50 mL Falcon conical centrifuge tube. The mixture was mechanically stirred at a thermocontrol shaker (Barnstead Lab-line 4450 e-class) for 24 h at 200 rpm and 25 ◦C. Afterwards, the quercetin-loaded MNPs were separated from unloaded quercetin by centrifugation (Universal 320 Hettich Zentrifugen, Tuttlingen, Germany) at 8000 rpm for 15 min. Compared with quercetin concentration supernatant before adding the synthesized MNPs, the concentration loss was determined using a calibration curve in pure EtOH. The coefficient of determination was 0.9978, and the determined molar absorption coefficient of quercetin at temperature 298 K and 374 nm is 19,131 mol−<sup>1</sup> cm−<sup>1</sup> dm3. The loading efficiency (*LE*) was calculated by measuring the absorbance of the supernatant with the Photolab 7600 UV-VIS spectrophotometer (Xylem, New York, NY, USA) at *λ* = 374 nm. The absorbance (*λ* = 374 nm) was collected and converted to concentration by using the equation from the calibration curve. Therefore, the drug loading efficiency was calculated as the following equation:

$$LE = \frac{m\_{\text{embeddeded}}}{m\_{\text{NP}}} \times 100\% \tag{1}$$

where *m*embedded represent the mass of quercetin incorporated in nanoparticles, and *m*NP is the total mass of MNPs which is used for loading.

#### Size Distribution of Magnetic MNPs Using Dynamic and Electrophoretic Light Scattering

Hydrodynamic diameter (*d*H) and zeta potential (*ζ*) of suspended MNPs were measured by photon correlation spectrophotometer, Zetasizer Nano ZS (Malvern, UK) with green laser (*λ* = 532 nm) using the M3-PALS technique. All measurements were conducted at 25 ◦C in PBS (pH = 7.4) buffer. The hydrodynamic diameter was determined from the peak maximum of the volume size function. The zeta potential (*ζ*) was calculated from the electrophoretic mobility using a Smoluchowski approximation (*f*(κa) = 1.5). The *d*<sup>H</sup> values were obtained as an average value of 10 measurements, while the zeta potential values were reported as an average of 3 independent measurements. The results were collected by the Zetasizer software 6.32 (Malvern Instruments, Malvern, UK).
