Polycarbonate membrane

The liposomes utilized in this study were prepared using E. coli lipids (Avanti) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-glycerol (POPG). The lipids were initially dissolved in dichloromethane and subsequently evaporated under a stream of nitrogen to remove the solvent [10 (link)]. The resulting dry lipid layers were dissolved in lipid buffer (50 mM Tris-HCl [pH 7.4] and 150 mM NaCl). To mimic the membrane-lipid composition of E. coli, a 7:3 ratio of PE to PG was used. To obtain a homogeneous size distribution, unilamellar liposomes with an average diameter ranging from 250 to 300 nm were generated by extrusion (Mini extruder, Avanti Polar Lipids, Inc., Alabaster, AL, USA) through a 0.4 µm polycarbonate membrane (Whatman) [10 (link),11 (link)]. Subsequently, dynamic light scattering (DLS) was used to measure the size distribution of the lipid vesicles.

Patients provided one sample of urine of ∼50 ml volume, collected at the time of the interview. The entire micturition volume was filtered through a polycarbonate membrane of 14 µm mesh size and 25 mm diameter (Whatman plc, Springfield Mill, UK). Thereafter the membrane was stained with Trypan blue (Sigma-Aldrich Corp, St. Louis, MO). Schistosome ova retained on the membrane were identified with the aid of a light microscope, as described [22] (link).

Membrane compositions used in this study were DOPC:DOPS:PI(4,5)P₂ at 80:15:5 or DOPC:DOPS:PI(4,5)P2:Rh-PE at 79:15:5:1 for fluorescent-tagged membrane. For liposome preparation, lipid mixtures were dried, rehydrated in buffer containing 20 mM Hepes (pH 7.5) and 150 mM KCl, and then subjected to three rapid freeze–thaw cycles followed by extrusion through a 0.1-µm polycarbonate membrane (Whatman) using Avanti mini extruder. SUPER templates were generated by incubating 2.5-µm silica beads in a solution containing 100 µM fluorescent-tagged liposomes and 1 M NaCl for 30 min at room temperature with intermittent flicking. Excess unbound liposomes were washed four times with filtered water after incubation (Neumann et al., 2013 (link)).

TPL-loaded liposomes (TPL-Lips) were prepared by the ethanol injection method^{44 (link)}. Briefly, lipids containing SPC (360 mg) and DSPE-PEG₂₀₀₀ (40 mg) with or without TPL (4 mg) (at a lipid-to-drug weight ratio of 100:1) were dissolved in 0.5 mL of ethanol as organic phase and mixed thoroughly. The mixture was rapidly injected into 5 mL phosphate buffered saline (PBS, pH 7.4) under magnetic stirring at 60 °C for 1 h using a syringe needle. The obtained suspension was further passed once through a 0.2 μm pore size polycarbonate membrane and then five times through a 0.1 μm pore size membrane (Whatman, Maidstone, Kent, UK) under nitrogen gas using an extruder (Northern Lipids Inc., Burnaby, BC, Canada) to remove unincorporated drug aggregates and generate unilamellar vesicles of low polydispersity. NBD-DPPE/DiR-labeled liposomes were prepared as above, but with fluorescent dye instead of TPL.

DMU-214-loaded liposomes were prepared by a thin lipid film hydration method followed by extrusion as described here (Skupin-Mrugalska et al., 2021 (link)) by using chloroform solutions of DMPC, DPPC, POPC (50 mg/ml), POPG (50 mg/ml), DMU-214 (10 mg/ml). DMU-214 was loaded passively into vesicles during hydration. Appropriate volumes of stock solutions were mixed, and then chloroform was evaporated under gradually reduced pressure at 40 °C in a round-bottomed flask. The resulting lipid film was then hydrated in PBS buffer pH 7.4. The resulting liposome suspension was passed 21 times through the polycarbonate membrane (Whatman, Kent, UK) with pore diameters of 100 nm using a syringe extruder (Avanti Polar Lipids Inc., Alabaster, AL, USA). Unbound material was separated from liposomes by fast ultrafiltration using Amicon Ultra 2 ml centrifugal filters with molecular weight cutoff (MWCO) 50 kDa (Merck KGa, Darmstadt, Germany). The amount of DMU-214 incorporated into liposomes was determined by the chromatographic method (paragraph 2.5). Encapsulation efficiency EE (%) was calculated according to Eq. (1): EE (%) = (C_m/C_i) x 100 (1), where C_m is the concentration of DMU-214 loaded into liposomes, determined by HPLC, C_i is the initial (maximum) concentration of DMU-214 added in the liposomal formulation. The experiment was performed in triplicates.