, Vancouver, British Columbia) at pressures typically at the lower end of the 250–700psi range. The polycarbonate filters employed in the extrusion process were obtained from SPI Supplies (West Chester, PA). The extruded liposomes were dialyzed against

a 200-fold volume of 5% glucose solution with four changes overnight. DOX was actively loaded into the liposomes by the creation of an ammonium sulfate gradient [55, 56]. The DOX was prepared by dissolving 10mg/mL in 5% glucose. An aliquot of 250μL of this solution was then added to each 0.1mmol scale liposome batch and then incubated Inhibitors,research,lifescience,medical at 60°C for 2h. The unencapsulated doxorubicin was separated from the DOX-loaded liposomes by dialysis against a 500-fold volume of PBS with 4 solution changes over 24–48h. The size of

liposomes was evaluated by dynamic light scattering as described [23]. Dynamic light scattering analysis, using a Zetasizer Nano Series, Nano ZG with Gateway 842GM (GDC-0199 in vitro Malvren Instruments), was carried Inhibitors,research,lifescience,medical out at Louisiana State University (Department of Chemistry) to determine the mean diameter of the liposomes from each batch prepared (Table 1). Liposomes were used within 24h of preparation or stored at 4°C and used within 1 week. The liposome phospholipid content was determined by the Stewart (ammonium ferrothiocyanate) assay as described previously [57–59]. The Inhibitors,research,lifescience,medical DOX concentration was determined by the measurement of absorbance at λ = 480nm following liposome solubilization in 100% ethanol. To account for quenching effects, absorbance values were then compared to a standard curve Inhibitors,research,lifescience,medical generated using known concentrations of free DOX in the presence of empty liposomes with a drug:phospholipid ratio of 100μg/μmol phospholipid. The DOX encapsulation efficiency was usually greater than 90%. The presence of the α1(IV)1263–1277PA and DSPE-PEG-2000 in the liposomal bilayer was examined by MALDI-TOF mass spectrometry (MS) using an α-cyano-4-hydroxycinnamic acid matrix. The incorporation Inhibitors,research,lifescience,medical of the α1(IV)1263–1277PA into liposomes was quantified

by UV-visible spectroscopy using ε280 = 5579M−1cm−1 for Trp. The UV absorbance PAK6 value for Trp was recorded in ethanol/PBS using a NanoDrop spectrophotometer (Thermo Scientific) and the concentration of the peptide determined using the Beer-Lambert law where A = εlc. Table 1 Liposomal systems utilized for stability and cytotoxicity evaluations. 2.4. Liposome Stability The stability of the encapsulated doxorubicin in the various liposome systems was initially determined by monitoring DOX release from the vesicles (200μL of 0.5mg/mL vesicle solution) at 4, 25, and 37°C, over time. Briefly, a fresh batch of liposomes was prepared and loaded with DOX. The unencapsulated doxorubicin was separated from the DOX-loaded liposomes by dialysis against a 500-fold volume of PBS as described in Preparation of DOX-Loaded Liposomes.