Spark m10 multimode plate reader

Unentrapped CHX was removed from the vesicle suspension using dialysis tubing with M_w cut-off 12–14 kDa (Spectra/Por®4, Spectrum®, VWR International, Fontenay-sous-Bois, France). An aliquot of 1 ml of vesicle suspension was dialyzed against 1 l distilled water under stirring for 4 h at room temperature. The CHX incorporated in the liposomes was quantified using Spark M10 multimode plate reader (Tecan Trading AG, Männedorf, Switzerland) at 261 nm (Hemmingsen et al., 2021a (link)).

In vitro CHX release studies were performed using a Franz cell diffusion system (PermeGear, Hellertown, PA, United States). Pre-soaked cellophane membranes (Max Bringmann KG, Wendelstein, Germany) were used as diffusion barriers with area of 1.77 cm² (Jøraholmen et al., 2014 (link)). Due to the low water solubility of CHX base (Farkas et al., 2001 (link)), the acceptor chamber was filled with PEG E 400 (10%, v/v) in distilled water (12 ml acceptor volume, Hemmingsen et al., 2021b (link)). The temperature was maintained at 32°C with heated circulating water. Vesicle suspensions (600 μl) were added to the donor chamber. Samples were withdrawn after 1, 2, 3, 4, 5, 8, and 24 h, and the sample volume was replaced with fresh medium to maintain sink conditions. The release from vesicles was compared to non-formulated CHX (dissolved in release media). Quantitative analysis was carried out using Spark M10 multimode plate reader (Tecan Trading AG, Männedorf, Switzerland) at 261 nm (Hemmingsen et al., 2021b (link)).