# Sildenafil Citrate Liposomes for Pulmonary Delivery by Ultrasonic Nebulization
## Materials and Methods
### Characterization of Liposomes  
The liposomes were characterized in terms of size (hydrodynamic diameter = Dh), polydispersity index (PDI), zeta potential, drug entrapment efficiency (EE%), and drug loading (mg of SC/g lipid). The Dh, PDI, and zeta potential were determined by dynamic light scattering (DLS) in a Zetasizer Nano ZS (Malvern Instruments, CO., UK). The analysis was performed at 25 \({}^{\circ}\)C and with a scattering angle of 173\({}^{\circ}\) after the appropriate dilution (\(\times\)100 or \(\times\)1000) with Milli-Q water or buffer solution (pH = 3.2) to avoid the phenomenon of multiple scattering. Liposome morphology was characterized through transmission electron microscopy (TEM) using a Hitachi HT7700, Japan. The samples were diluted in Milli-Q water (1:100) and then were placed on a formvar-coated copper grid and allowed to dry at room temperature overnight. The images were captured using an accelerating voltage of 80 kV at the magnification of \(\times\)10,000 to \(\times\)20,000.  
To determine the EE%, the liposome suspensions were centrifuged at 14.000 rpm for 45 min at 6 \({}^{\circ}\)C to separate the unentrapped drug in the supernatant from the drug loaded into liposomes. The amount of drug in the supernatant was determined by high pressure liquid chromatography (HPLC), using a Purosphere STAR rpE18, with a 3 \(\upmu\)m column of 50 cm \(\times\) 4.0 mm. A mixture of 0.1% formic acid in water and acetonitrile (70/30 \(v/v\)) adjusted to pH 4.2 with triethanolamine was used as the mobile phase at a flow rate of 1.5 mL/min. The UV detector was set at 292 nm (HPLC system with Waters Alliance 2695 separation module, 2998 photodiode array detector, and empower processor system), and the calibration range was 25\(-\)500 \(\upmu\)g/mL. EE (%) was estimated from the following equation:  
\[\text{EE (\%)}=\text{[($Qt$-$Qs$)/$Qt$]}\times 100,\]  
where \(Qt\) is the total drug amount in the liposome suspension and \(Qs\) is the drug amount quantified in the supernatant.  
Drug loading (DL) was estimated from the _Qsc_/_Qlip_ ratio, where _Qsc_ and _Qlip_ represent the amount of SC (mg) and the amount of lipids (g) in the liposomes, respectively.  
The Dh, PDI, zeta potential, and EE% were determined for the liposomes obtained without and with applying the transmembrane pH gradient. In the latter case, these characteristics were determined for fresh, lyophilized, and nebulized liposomes.  
The density (\(\rho\)) and kinematic viscosity (\(\eta\)) of the liposome suspensions at 25 \({}^{\circ}\)C were measured before and after adding the lyoprotective agent. A capillary viscometer was used, and \(\eta\) (mm\({}^{2}\)/s) was estimated by measuring the time (second) it took for the sample to flow through the capillary under the influence of gravity. The instrument was calibrated with Milli-Q water (\(\rho\) = 1 g/mL; \(\eta\) = 1 mm\({}^{2}\)/s) and the viscometer constant (K, mm\({}^{2}\)/s\({}^{2}\)) was estimated. Then, the viscosity of liposome suspensions was calculated as follows:  
\[\eta=\text{K}\times t,\]  
where \(t\) is the time it took for the liposome suspension to flow from the lower to upper mark.  
The following scheme summarizes the above-described methodology (Figure 2).