*3.3. Homogeneity and Stability of Synthesized Mesoporous MNPs*

One of the essential features for successful drug delivery is stability and homogeneous dispersion of NPs in buffer. Zeta potential was used to determine the stability of the colloidal suspension of bare MNPs, PEG-coated MNPs and quercetin-loaded MNPs. It has been shown that PEG-stabilized NPs exhibit longer bloodstream circulation time and higher resistance to protein binding [44]. Due to its unique properties and its biocompatibility, PEG is selected as the stabilizing agent in this study. The surface charge of NPs has an important effect on the blood circulation time, the pharmacokinetics of NPs and the zeta potential above ±30 mV indicated to be relevant for stability of NPs in aqueous suspensions [12,51,52]. The zeta potential of MNPs (Table 1) was determined in phosphatebuffered solution (PBS). Bare MNPs exhibited a zeta potential of (−30.6 ± 0.7) mV, while PEG coating increased the absolute zeta potential value to (−35.1 ± 1.5) mV. Indeed, the higher negative zeta potential value indicated that PEG-coated MNPs possessed higher stability after PEG coating. The zeta potential of quercetin-loaded MNPs (−31.3 ± 0.8) mV decreased slightly in comparison to PEG-coated MNPs indicating quercetin adsorption on the PEG layer of MNPs. However, the observed change in zeta potential value did not decrease the stability of MNPs. Moreover, the successful loading of quercetin into PEG-coated MNPs has been confirmed also by electrophoretic measurements.

Furthermore, the hydrodynamic diameter is lower which could be attributed that PEG coating while quercetin loading provides better colloidal stability and reduces aggregation. The volume size distributions of all samples, i.e., bare MNPs, PEG-coated MNPs and quercetin-loaded MNPs were unimodal. In Table 1, it can be seen that the highest polydispersity index was observed to be 0.54 ± 0.1 for the bare synthesized MNPs suspension. This result is consistent with the results obtained from the size distribution of bare MNPs using SEM where the size distribution of bare MNPs was broader than the size distribution of the PEG-coated MNPs. The observed discrepancy between SEM and DLS data, particularly in the polydispersity index (PDI), can be explained by the fact that the SEM micrographs were taken in a dried state, whereas the DLS experiment was carried out in suspension. The PDI results reported in Table 1 also support the conclusion that PEG coatings, even presented in suspension, decreased the process of aggregation of bare MNPs and break down the cluster. The average hydrodynamic diameter of both, bare and PEG-coated MNPs also supports the above findings. In Figure 7, it is shown the volume size distributions of bare MNPs, PEG-coated MNPs and quercetin-loaded PEG\_MNPs. The PDI of PEG-coated MNPs is lower than PDI of loaded MNPs (PDI = 0.47 ± 0.1 and 0.52 ± 0.07, respectively) indicating narrower size distribution of the PEG-coated MNPs than quercetin-loaded MNPs (see Figure 7). However, the cluster sizes of quercetin-loaded MNPs were within the range of (681 ± 73) nm, which is the smallest range of all analysed MNPs. Thus, the PEG coating on the surface of Fe3O4 decreased the size compared to the bare MNPs and indicates that the PEG coating prevents or reduces aggregation to some extent.

**Table 1.** Zeta potential and hydrodynamic diameter of bare MNPs, PEG-coated MNPs and quercetinloaded MNPs.

