Characterization of PA@TLipo Nanoparticles

The hydrodynamic particle size, zeta potential, and polydispersity index (PDI) of PA liposomes were determined using a Malvern dynamic light scattering (DLS) instrument (Zetasizer Pro). Briefly, the purified liposomes, dispersed in PBS at a concentration of 0.5 mg/mL, were subjected to size analysis. Importantly, prior to the assessment of the zeta potential, the liposome PBS solution (0.5 mg/mL) was dialyzed in distilled water. All measurements were conducted at 25 °C and were repeated three times. The morphology of PA liposomes was observed using transmission electron microscopy (TEM) (120 kV, JEM-1400 Flash, 120 kV). Briefly, the liposome solution was dropped onto a 200-mesh copper grid and incubated for 3 minutes. The liposomes were stained with 1 wt% phosphotungstic acid and dried before imaging. Colloidal stability of the PA liposomes was characterized using DLS. Briefly, the liposomes were dispersed in PBS solution containing 10% FBS and incubated at 37 °C with shaking (200 rpm). The particle sizes of liposomes were assessed at predetermined time intervals using a DLS instrument. In order to determine the drug loading capacity (DL) of PA within liposomes, the purified liposomes underwent lyophilization, and 2 mg of the resulting lyophilized powder (total mass of liposomes) was disrupted using 1 mL of methanol. Following this, the mass of PA encapsulated in liposomes was determined by using high-performance liquid chromatography (HPLC), employing absorption measurements at a wavelength of 210 nm. The standard curve for PA quantification was constructed using free PA. Briefly, 6 mL of freshly prepared PA-encapsulated liposomes were placed in the upper chamber of the Amicon^® . Ultra centrifugal filter (100 kDa MWCO) before column purification and centrifuged at 3000 × g for 10 minutes. The liposomes collected in the upper chamber were resuspended in the original volume of PBS (6 mL) and subjected to three consecutive centrifugation cycles. The mass of PA in the filtrates was analyzed using HPLC after combining the three filtrates containing free PA. EE% was calculated as follow equation: