3.1.3. Zeta Potential

Zeta-potential results of PS9 and G30 at different concentrations are presented in Figure 2. Regarding ζ potential of minerals in pH 7 buffer, PS9 and G30 results were in agreement with other sepiolite and palygorskite samples previously studied [94–96]. In particular, the zeta potential of PS9 in aqueous pH 7 solution was more negative (higher) than that of G30.

**Figure 2.** Zeta potential variations of PS9 and G30 (mean values ± s.d.; n = 3) in pH 7 buffer and complete DMEM culture medium at different concentrations.

It has been demonstrated that surface charge of nanoparticles has the potential to influence cell viability [97]. The importance of clay particle zeta potentials during biocompatibility and wound healing tests lies in the fact that cells possess a negative surface zeta potential. Negative zeta potential is one of the most decisive factors of biocompatible materials, showing higher cell viability [98,99]. It is known that particles with positive zeta potentials interact and/or penetrate cells easily due to their opposite charge, thus being one of the main strategies to improve transfection efficiency [100,101]. Negatively charged particles have also proved to interact with cells up to a certain extent and even be able to enter by endocytosis-mediated mechanisms [102,103], although this happens with higher difficulty for negative particles than for positive ones. The uncontrolled entrance of certain substances into the cells could jeopardize their viability, thus inferring that positively charged materials are more likely to put the cell viability at risk.

Another factor by which nanoparticle surface charge demonstrated to influence cell viability is due to their agglomeration state [99]. Berg and co-workers revealed that hepatocytes showed less viability when exposed to metal nanoparticles with zeta potentials close to their isoelectric point [97]. This change in zeta potential was also strongly related to the agglomeration state of the very same particles. That is, the more neutral the nanoparticles are, the more easily they aggregate and, subsequently, the faster their precipitation over the cell tapestry and/or their interaction with the negatively charged cell membranes.

Cell media, such as DMEM, have demonstrated to significantly modify zeta potential of different suspended particles [104–106]. These changes are ascribed to the presence of a wide variety of charged molecules such as amino acids and vitamins, among others. The interaction of clay particles with these molecules changes the zeta potential of the former ones. In fact, PS9 and G30 suffered a significant change of zeta potential when added to full DMEM culture medium (Figure 2). In this occasion, no significant differences were found between PS9 and G30 values, thus confirming that the resultant surface charges of the particles is governed by the culture medium. Therefore, during biocompatibility and wound healing tests, clay particles should maintain a −10 mV zeta potential. The reduction of zeta potential strength would reduce the stability of the clay suspensions, making them less flocculated and, consequently, more prone to precipitation phenomena. Precipitation of clay particles could hinder cell viability by cell suffocation. Nonetheless, the fact that they still showed a negative surface charge would hinder the entrance of clay particles inside cells. These results will be confirmed by MTT analysis.
