# Sildenafil Citrate Liposomes for Pulmonary Delivery by Ultrasonic Nebulization
## 3 Results  
As reported in previous studies [39; 41; 42], the direct sonication of the components produced drug-loaded liposomes without the use of organic solvents, which results in an environmentally friendly approach. The characteristics of the SC-loaded liposomes obtained in this study are summarized in Table 1.  
Figure 2: Schematic description of applied methodology. EE% values under 18% were obtained when Milli-Q water was used as the hydration medium, and this value was moderately increased when citrate buffer (pH = 3.2) was used instead. However, the most relevant increase in the EE% was observed after applying the transmembrane pH gradient, irrespective of whether the liposomes contained Vit E TPGS or not (EE = 89.77 \(\pm\) 7.64% and 80.30 \(\pm\) 11.03%, respectively). The difference between these two values was not statistically significant (\(p\) = 0.1495). However, when these values were compared to those obtained prior to the application of the pH gradient, the differences were statistically significant for both types of liposomes (\(p\) = 0.0039). The particle size and charge were determined using the Zetasizer Nano ZS device, as described in the Material and Methods Section. The results showed the effects of pH on liposome size and zeta potential. At pH = 3.2, the Dh values were 304.3 nm (PDI = 0.413) and 303.2 nm (PDI = 0.452) for liposomes without and with Vit E TPGS, respectively. Smaller liposomes were obtained when the pH was adjusted to 7.0 for the transmembrane gradient (209.7 nm; PDI = 0.537 or 219.8 nm; PDI = 0.534 for liposomes without or Vit E TPGS, respectively). Changes were also observed for the zeta potential, which showed values of \(-\)2.10 mV or \(-\)2.05 mV at the acidic pH and \(-\)20.90 mV or \(-\)21.30 mV at pH = 7.0. The liposomes prepared by active loading were lyophilized with sucrose, trehalose, or sucrose and trehalose, and homogeneous cakes were obtained in all cases. After the cakes were hydrated, the aspect and characteristics of the fresh suspensions were immediately restored, and the EE% values determined for rehydrated liposomes did not show statistical differences when compared to fresh liposomes (\(p\) = 0.1390). By contrast, the lyophilized liposomes exhibited a greater diameter and more negative charge as compared to the fresh liposomes (see data in Table 2), irrespective of whether sucrose, trehalose, or sucrose and trehalose was used as the lyoprotective agent. \begin{table}
\begin{tabular}{c c c c c c c} \hline \multicolumn{6}{c}{**Influence of the Transmembrane pH Gradient on the Characteristics of the Liposomes**} \\ \hline \multicolumn{2}{c}{**Liposomes**} & \multicolumn{1}{c}{**Dh (nm)**} & \multicolumn{1}{c}{**PDI**} & \multicolumn{1}{c}{**Zeta Potential (mV)**} & \multicolumn{1}{c}{**EE (\%)**} & \multicolumn{1}{c}{**DL (mg/g lipid)**} \\ \hline
**Without** & **pH = 6.7** & - & - & - & \textless{18\%} & \textless{11} \\
**Vit** & **pH = 3.2** & 304.3 & 0.413 & \(-\)2.10 & 49.47 \(\pm\) 9.78 & 29.71 \(\pm\) 5.87 \\
**E TPGS** & **pH gradient** & 209.7 & 0.537 & \(-\)20.90 & 89.77 \(\pm\) 7.64 & 53.92 \(\pm\) 4.59 \\
**With** & **pH = 6.7** & - & - & - & \textless{15\%} & \textless{9} \\
**Vit** & **pH = 3.2** & 303.2 & 0.452 & \(-\)2.05 & 22.67 \(\pm\) 11.32 & 13.62 \(\pm\) 11.00 \\
**E TPGS** & **pH gradient** & 219.8 & 0.534 & \(-\)21.30 & 80.30 \(\pm\) 11.03 & 48.23 \(\pm\) 6.62 \\ \hline \end{tabular}
\end{table} 
Table 1: Characteristics of the liposomes prepared using Milli-Q water (pH = 6.7), citrate buffer (pH = 3.2), or the transmembrane gradient (pH gradient). Abbreviations: Vit E TPGS: d-a-tocopherol polyethylene glycol 1000 succinate; EE: entrapment efficiency; Dh: hydrodynamic diameter; PDI: polydispersity index; DL: drug loading. The morphology observed under the TEM showed that the liposomes present a circular or elliptical shape typical of the classical vesicular bilayer structure. Although the lyophilization increased the Dh of the liposomes, they maintained their typical morphology, as illustrated in Figure 3.