## 3 Results and Discussion
### Structural Analysis of pDNA
#### 3.2.2 Zeta Potential  
The zeta potential of sc pDNA solutions purified using the distinct chromatographic supports were evaluated using the Zetasizer Nano-ZS aiming to determine if the chromatographic conditions exert any change on the electric charge of sc pDNA. Thus, the zeta potential, which is established through the molecule surface charges and the concentration and types of ions present in the solution, was assessed. Through light scattering, the charge density was measured, and the result is shown in Figure 4. The pDNA molecules present negative charges on their surface and in solution they interact electrostatically with the available ions, resulting in repulsion or agglutination of these molecules. For the interaction and the equilibrium of the system, the tendency is the formation of a layer of counterions, in this case positive ions that adhere strongly to the negatively charged groups on the surface of the pDNA molecule, resulting in a compact layer or stern layer where the ions are immobilized [40]. This layer is then surrounded by ions with opposite charges that agglomerate together to neutralize the charged molecules, and these ions are in motion and hence the layer is called the diffuse layer [40]. The stability of the suspended molecule is dependent on both van der Waals forces and the double electric layer formed by ions and counterions, which in the case of therapeutic products also can influence the cellular uptake and intracellular traffic [41]. The zeta potential, which is determined by the composition of ions involved in these layers, can be altered by both the ionic strength and pH of the solution [42]. In Figure 4,  
Figure 3: Circular dichroism spectra of p53-pDNA samples. Native pDNA open circular (oc) + supercoiled (sc) recovered from a commercial kit used as control, to confirm the stability of sc pDNA purified by: (**A**) histidine-agarose and arginine–macroporous supports; (**B**) histidine–monolith support. The pDNA control samples were prepared in the same buffer used for the purification of sc pDNA in each strategy (10 mM Tris–HCl, pH 8.0 or 10 mM citrate–EDTA buffer, pH 5.0).  
it is possible to observe that the samples obtained by the commercial kit (oc + sc) clearly demonstrate a difference in the zeta potential values, mainly influenced by the pH difference of the buffers, 10 mM Tris-HCl buffer, pH 8.0 (\(-\)22 mV) and 10 mM citrate-EDTA buffer, pH 5.0 (\(-\)55 mV). Regarding the pDNA samples purified by the chromatographic strategies where the Tris-HCl buffer was used, it is also verified that there was a slight difference between them, even considering the same pH 8.0. This fact can be attributed to the salts used in the elution gradients established for the purification procedures, that even after the sample desalination can be present in trace amounts or can be responsible for some structural changes on pDNA which influence the charged group exposure, being sufficient to alter the ionic constitution of the electric double layer of the colloid. A more significant difference was achieved for the pDNA purified samples, present in buffers with different pH, namely Tris-HCl (pH 8.0) and citrate-EDTA buffer (pH 5.0), where the zeta potential values for pH 8 varied between \(-\)20 and \(-\)25 mV and \(-\)40 mV to pH 5, respectively (Figure 4). Also, these results indicate that a decrease of pH can increase the negative zeta potential, improving the stability of pDNA on the formulation. In the case of this work, the protons in acidic pH can alter the electrical double layer, leading to a reduction of the attraction forces and consequently the interaction between therapeutic molecules in the medium. As reported in literature, the agglomeration of biopharmaceuticals on the medium are associated with the low quality of product [42] and this agglomeration could occur mainly due to van der Waals forces, according the DLVO theory [43].