## 3 Results and Discussion
### Characterization of the Surface Charge of Particles Produced Using AUPDS Surfmer  
Coalescence of particles can be prevented by means of steric and/or electrostatic stabilization and the ZP is an indicative of the electrostatic particle stability in a suspension, as previously mentioned. These values can be positive or negative, depending on the molecular configuration in the surface of the particles, but it is not quantitative. A quantitative determination of the surface charge can be achieved via polyelectrolyte titrations. This approach allows for the determination of the effective dissociated counterion charge for particles and can be used for both positively- or negatively-charged particles [63]. In order to estimate whether the AUPDS surfmer was responsible for the surface charge and if it was available for chemical reaction on the surface of the particles we investigated the ZP and the charge quantity of the NPs before and after a basic hydrolysis process of the AUPDS active ester groups on the particles. We investigated this for PMMA-_co_-AUPDS, PS-_co_-AUPDS, PMMA-_co_-EGDMA-_co_-AUPDS, and PS-_co_-DVB-_co_-AUPDS particles using 2 mol% of AUPDS in the formulations, either non- or cross-linked with 10 mol% of cross-linker. Figure 5a shows the ZP and charge quantities obtained from the polyelectrolyte titrations and Figure 5b shows the hydrodynamic diameter distributions of these particles. Figure 5: (**a**) Zeta potential and charge quantity distribution histograms obtained for non-cross-linked PMMA and PS NPs and cross-linked with 10 mol% cross-linker PMMA-_co_-EGDMA and PS-_co_-DVB NPs (all with AUPDS 2 mol%) through electrophoretic light scattering and polyelectrolyte titration. NPs were analysed before (unmodified) and after hydrolysis of the active ester group of AUPDS. The positive charge of the unmodified particles was titrated with PES and the negative charge of particles after hydrolysis was titrated using PDADMAC; and (**b**) hydrodynamic diameter distribution curves in terms of intensity (%) obtained via DLS analysis for the respective particles. DLS and PCD measurements were performed in sodium and potassium phosphate buffer (c = 5.2 mM, pH 7.0). Due to the sulfonium group on the AUPDS, the particles should present a positive surface charge and, after hydrolysis, carboxylate groups should be present and the surface charge should be negative. As expected, the ZP value was positive for unmodified particles and negative for particles after hydrolysis, within the same value range. Moreover, the change in ZP for the NPs during basic hydrolysis also shows that the possible hydrolysis of PMMA side chains to their acid counterparts is not responsible for the ZP change [64] because such a reaction is not possible for PS NPs. The charge quantities also remained in the same range before and after hydrolysis for all cases, confirming the surfmer presence at the NP surface and its importance for the stability of the NPs. The surface charge of cross-linked PMMA-_co_-EGDMA-_co_-AUPDS formulations remained in the same range as the non-cross-linked PMMA-_co_-AUPDS particles. On the other hand, the addition of cross-linker amplified the amount of surface charge from 9.9 \(\mu\)eq\(\cdot\)g\({}^{-1}\) of PS-_co_-AUPDS NPs to 48.0 \(\mu\)eq\(\cdot\)g\({}^{-1}\) of PS-_co_-DVB-_co_-AUPDS. This higher amount of surface charge is also consistent to the fact that these particles did not aggregate after the hydrolysis process and that aggregation after hydrolysis could have caused the smaller charge quantity found for PS-_co_-AUPDS particles. It is pertinent to emphasize that theZP values may also vary according to changes in the pH and in ionic strength of the dispersion media. The DLS and PCD measurements of Figure 5 were performed in pH 7.0 buffer, because measurements are required to be in buffer for PCD analysis, while the other reported DLS values (Tables 1 and 2) were performed in water.