X bridge beh300 c18

Encapsulated indolicidin concentrations were determined by means of HPLC (2695 eAlliance separation module, Waters Corporation, MA, USA). Indolicidin was extracted following dilution of the liposomes in PBS containing 100 mM Octyl β-D-glucopyranoside (Sigma-Aldrich) yielding a clear solution. The samples were injected to a Vydac® 218MS C18, 5 μm, 300 Å, 250 × 4.6 mm column (Grace, MD, USA) heated to 35 °C, equilibrated in 0.1% v/v aqueous trifluoroacetic acid (TFA, Sigma-Aldrich), and eluted with 0.1% v/v TFA in acetonitrile (J.T.Baker® Chemicals, PA, USA), at a flow rate of 1 ml/min. Indolicidin was detected at λ = 220 nm using a 2998 PDA detector (Waters Corporation). LL-37 liposomes were diluted 1:10 or 1:5.5 in 1% v/v TFA in ethanol, heated to 70 °C until a translucent solution was achieved, and the cooled solution was centrifuged 15 min at 4000 rpm for lipids sedimentation. The clear supernatant was injected to an XBridge™ BEH300 C18, 3.5 μm, 4.6 × 100 mm column (Waters Corporation) heated to 35 °C, equilibrated in 0.1% v/v aqueous TFA, and was eluted with 0.1% v/v TFA in acetonitrile, at a flow rate of 1 ml/min. LL-37 was detected at λ = 220 nm by means of a 2998 PDA detector (Waters).