Liposome Encapsulation Methodology

Liposomes were produced by the thin-film hydration technique, followed by subsequent agitation and freeze-thaw cycles^{18 (link),36 (link)}. A lipid mix containing Ph-DPPC, DOPC, DSPG, and cholesterol in a 3:3:2:3 molar ratio was dissolved in a solution of 90% chloroform and 10% methanol. This mixture underwent evaporation at low pressure, after which the lipid content was reconstituted in tert-butanol and subjected to freeze-drying. The resulting lipid cake was then rehydrated with different payloads, including Sulforhodamine B (SRho), tetrodotoxin (TTX), polyethylene glycol (PEG), and albumin–fluorescein isothiocyanate conjugate (FITC-Ab) in PBS buffer (pH=7.4) or bupivacaine hydrochloride (Bup), or doxorubicin hydrochloride (Dox) in saline. After 10 cycles of freezing and thawing, the liposomal solution was subjected to a 48-h dialysis against either PBS or water. The molecular mass cut-off of the dialysis devices was 1000 kDa (Spectra/Por™ Float-A-Lyzer™ G2 Dialysis Devices). The dialysis fluid was routinely replaced with fresh PBS or saline approximately every 12 h. To determine the drug and dye content across different formulations, liposomes were disrupted using octyl-β-D-glucopyranoside (100 mM, twice the volume of liposomal solution) and analyzed.