## 3 Results and Discussion
### Nanoparticles Characterization  
Average values of particle size, polydispersity index (PDI), zeta-potential, DL and EE for different preparations of pDNA-NPs are shown in Table 1. Three different sets of experiments were performed by changing the initial amount of pVAX1-NH36 used in preparations, while the amount of polymers was maintained constant and related in the form of TDL. pDNA-NPs average diameter and average PDI could be observed in Figure 3A, where it is noted that the average diameters of three formulations are in the range of 170-230 nm and a correlation with TDL was also observed. In the range of study, diameter of pDNA-NPs increases with an increase in TDL. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**pDNA-NP TDL (\%)** & **Diameter (nm)** & **PDI** & **Zeta Potential (mV)** & **DL (\%)** & **EE (\%)** \\ \hline
0.2 & 177.2 \(\pm\) 18.2 & 0.131 \(\pm\) 0.020 & \(-\)26.6 \(\pm\) 4.7 & 0.0309 \(\pm\) 0.003 & 15.437 \(\pm\) 1.56 \\
0.5 & 202.1 \(\pm\) 12.1 & 0.144 \(\pm\) 0.047 & \(-\)24.0 \(\pm\) 4.1 & 0.0794 \(\pm\) 0.004 & 15.881 \(\pm\) 0.74 \\
0.8 & 229.3 \(\pm\) 20.0 & 0.142 \(\pm\) 0.053 & \(-\)29.5 \(\pm\) 2.8 & 0.1874 \(\pm\) 0.024 & 23.429 \(\pm\) 2.96 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Characteristics of pDNA-NP as a function of theoretical pVAX1-NH36 loadings (pVAX1-NH36 TDL). Data represent mean \(\pm\) SD (\(n=3\)). Figure 2: FT-IR absorption peaks of PLGA-PEG-FA polymer. Characteristic bands for aromatic ring present in folic acid at 1452 and 1608 cm\({}^{-1}\) and PLGA-PEG-FA representative absorption peaks at 1623 and 1571 cm\({}^{-1}\) due to the presence of –CONH– linkage. In previous works, [PERSON] et al. performed plasmid encapsulation using PLGA with different molecular mass and obtained spherical microspheres in the range of 0.4 to 2.0 \(\upmu\)m [28]. Similarly, [PERSON] et al. encapsulated plasmid for gene therapy using chitosan and obtained particles in the range of 157-198 nm [33]. [PERSON] et al. and [PERSON] et al. have encapsulated different cancer chemotherapues using modified polymer PLGA-PEG-FA, obtaining particles in the range of 216-220 nm with polydispersity index of 0.225-0.113 [12; 13]. [PERSON] et al. also encapsulated an anticancer drug by conjugating the drug to PEG-FA, resulting in nano-aggregates with an average size of 200 nm [44]. Therefore, size range obtained in present research is appropriate for the type of particles being synthetized. Figure 3A also exhibits average polydispersity index for rDNA-NPs ranges from 0.130 to 0.145. Surface charge is an important indication for stability of a colloidal nanoparticle system suspended in medium. Electrostatic repulsion among nanoparticles with the same type of surface charge provides extra stability [59]. Figure 3B show zeta potential obtained for diverse sets of experiments which vary between \(-26\) to \(-30\) mV, indicating good stability. In the present work, variations in zeta potential with respect to different TDL do not present a significant statistical difference. Nanoparticle zeta potentials from previous works with similar polymer results in same kind of values (\(-28.0\) mV) are reported [13]. Other similar works, in which positively charged polymers such as polyethylenimine (PEI) 25 K or chitosan were being used, reported zeta potentials in the range of 20 to 50 mV [11; 33]. EE and DL are represented in Figure 3C. DL ranged between 0.03% and 0.19% as TDL increase. EE was 15.437%, 15.881% and 23.429% for corresponding TDL of 0.2%, 0.5% and 0.8%. This trend could be explained if the double emulsification process employed, water/oil/water (W/O/W) in the preparation of nanoparticles is considered. After the first emulsification, plasmid is mostly in the  
Figure 3: Characteristics of rDNA-NP prepared at different theoretical pVAX1-NH36 loadings (pVAX1-NH36 TDL). (**A**) Particle diameter and polydispersity index; (**B**) zeta potentials; (**C**) EE and DL for different pVAX1-NH36 with TDL of 0.2% (\(\equiv\)), 0.5% (\(\upmu\)) and 0.8% (\(\upmu\)) were evaluated.