*3.2. Zeta Potential*

For the purpose of drug loading and delivery, the ability of the nanoparticles to be coupled to molecules forming stable nanoassemblies at physiological pH and to release the drug at the precise time, as well as the stability, drug-particle or cell membrane-particle interactions, among other properties, are strongly determined by the surface charge, or, alternatively, by the zeta potential ζ, of the particles when dispersed. Calculations of the zeta potential were done from the measurements of the electrophoretic mobility of the particles BMNPs and MNPs by means of O'Brien and White's general theory [56]. As one can see in Figure 5, the isoelectric point (pHiep or pH of zero ζ) of MNPs was obtained at pH 7.0, while for BMNPs it was 4.4. This zeta potential plot reveals significant di fferences between both types of nanoparticles. They are positively charged at low pH values and negatively charged at high pH, but BMNPs change from positive to negative at quite a di fferent pH: This suggests that the MamC protein is a ffecting the surface of the particles, and it is probably located mainly at the interface. In fact, the investigations by Nudelman et al. [57], and other authors [54,58] on the biomineralization mechanism induced by this protein demonstrate that the particularity of MamC is the template e ffect for magnetite nucleation and growth that this protein exerts. The two negatively charged amino acids Asp70 and Glu66, located in MamC loop, are separated by about 8 Å, and this distance can match the disposition of iron cations on (100), (110), and (111) faces. Therefore, the crystal could grow in an orderly manner, first from the Fe cations available in solution and then, since the amount of Fe in solution is limited, at the expense of the release of the Fe cations adsorbed in other negatively charged moieties of MamC. Since the process of nucleation is kinetically favored by this template e ffect, it is precisely those exposed amino acids that act as nucleation sites. Therefore, the restricted number of nucleation sites in the presence of MamC compared to the process of homogeneous nucleation from the bulk solution allows the formation of larger crystals in the former. Moreover, since (100), (110), and (111) faces show up in the final morphology of the BMNPs [57], it can be concluded that, while exerting this template e ffect for the nucleation, the protein prevents the growth of the crystal on these specific directions.

**Figure 5.** Zeta potential of purely inorganic MNPs (-) and biomimetic magnetic nanoparticles BMNPs ( -).

The shift of the isoelectric point of the BMNPs relative to that of MNPs make the former adequate nanocarriers, a fact that is important for the sought application. Since most common drugs for cancer therapy are positively charged at physiological pH, they could be electrostatically attached to BMNPs

at such pH values, resulting in a stable nanoassembly. On the contrary, when they eventually reach the tumor (acidic pH values), the drug releases from the BMNPs, as the charge of the latter at acidic pH values is nearly zero [33]. That makes the BMNPs better drug nanocarriers compared to MNPs, as in the former the release of the drug can be controlled by an external factor, as it is a change in the environmental pH value. This feature is absent in MNPs, as their charge is positive anywhere below pH 7.
