## 3 Results and Discussion
### Bioactivity of pDNA
#### 3.3.2 Analysis of the p53 Protein Expression  
To better understand the bioactivity of the sc p53-pDNA isoform purified by the different chromatographic strategies, an ELISA assay was performed to evaluate the expression of p53 protein, after 48 h of HeLa cells transfection with the different samples (Figure 6). For the control group, non-transfected HeLa cancer cells were used and basal p53 expression levels were obtained. The low p53 levels presented by this kind of cells are described as a result of the degradation of this protein by the E6 and E7 oncoproteins from the HPV (human papillomavirus) [44]. Also, a native p53-pDNA sample extracted through a commercial kit was used in this experiment to evaluate the bioactivity performance of a native sample (containing both isoforms, \(\mathrm{oc}+\mathrm{sc}\)) and compare with the efficiency found for the different sc pDNA samples obtained from different chromatographic supports used in this research work. The p53 expression induced by the cell transfection with the plasmid extracted through the commercial kit also presents satisfactory results. However, this kind of sample is not suitable for in vivo use, since the procedure applied uses RNase from an animal source, which is not acceptable for the regulatory agencies and, in addition, it increases the costs of the process. Figure 6 shows that the bioactivity of the sc p53 encoding plasmid remains independent of the chromatographic method chosen and cells transfected with the isolated sc isoform presented higher p53 expression than cells transfected with a native sample (\(\mathrm{oc}+\mathrm{sc}\)). This kind of behavior presented by HeLa cells when transfected with the sc pDNA isoform has been already explored and reported by other authors [44; 45], which reinforces the importance of working with the sc topology, the most active form of pDNA. This ability of sc pDNA isoforms to promote a higher protein expression has been related to the maintenance of the genetic information integrity [46], which can be different when a native pDNA sample is delivered. Furthermore, sc pDNA samples obtained from different chromatographic strategies induced different p53 protein expression levels. A higher value was achieved for the sc pDNA purified with the arginine-macroporous, followed by the histidine-monolith, and finally the pDNA recovered from the histidine-agarose support. Actually, the sc p53-pDNA purified by the arginine-macroporous matrix induced an expression of the p53 protein 4 times higher than the control. In addition, this sample was statistically more relevant when compared, for example, with the p53 protein expression promoted by the histidine-agarose sample. Considering these results and relating them with the quality control tests (Table 1), it is verified that, although the sc p53-pDNA sample purified by the arginine-macroporous matrix presents the lower purity level (92%), it induced a higher p53 protein expression. These results suggest that the kind of gradient or general conditions used during the plasmid purification process is of extreme importance for pDNA activity. The fact that milder conditions have been used, with a lower salt concentration and using a salt with lower environmental impact (NaCl), resulted in a non-significant effect on pDNA structure, which can be reflected in the pDNA bioactivity. Also, the low salt concentration used in the elution step (500 mM NaCl) required less manipulation of the sample during the desalting of the purified pDNA, possibly favoring the pDNA stability. Otherwise, samples purified through histidine-monolith and histidine-agarose, even with a higher purity level (100%) when compared with the samples recovered from arginine-macroporous support, showed lower efficiency at inducing the p53 expression. This result was even more pronounced for the sc p53-pDNA obtained by the histidine-agarose support, as the pDNA purified by the histidine-monolith resulted in a slightly higher p53 expression.