Dimethyldioctadecylammonium

Dimethyldioctadecylammonium was obtained from Avanti Polar Lipids (Alabaster, AL, United States). TDB was purchased from Niels Clauson-Kaas A/S (Farum, Denmark). Xenolight 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR) near infra-red fluorescent dye was purchased from Perkin Elmer (Waltham, MA, United States). H56 protein was produced recombinantly in E. coli as previously described (15 (link)), reconstituted in 20 mM glycine buffer (pH 8.8), checked for purity, and validated for residual DNA, endotoxins and bioburden following internal good manufacturing practice standards at Statens Serum Institut as described previously (16 (link)). Alexa Fluor^® 647-labeling of H56 was performed commercially (Thermo Fischer Scientific, Eugene, OR, United States). 2,5-dihydroxybenzoic (DHB), 1,5-diaminonaphthalene (DAN), trifluoroacetic acid (TFA), and Meyer’s hematoxylin solution and eosin (H&E) solution were purchased from Sigma-Aldrich (St. Louis, MO, United States). Methanol was obtained from Th. Geyer (Renningen, Germany). Water was prepared by using a Millipore Direct-Q3 UV system (Billerica, MA, United States). All other chemicals and reagents were of analytical grade and were acquired from commercial suppliers.

Liposomes were generated by lipid film hydration as described (Kallert et al., 2015 (link)). Briefly, dimethyldioctadecylammonium (DDA; 0.3 mg/ml; Avanti Polar Lipids), D-(+)-trehalose 6,6′-dibehenate (TDB; 0.25 mg/ml; Avanti Polar Lipids, Sigma Aldrich) and L-α-phosphatidylcholine (PC; 0.9 mg/ml; Avanti Polar Lipids) were mixed in chloroform (VWR): methanol (Sigma-Aldrich; 9:1, v/v). The organic solvents were evaporated under nitrogen flow. Liposomes were formed by hydrating the lipidfilm in 500 μl 10 mM Tris-buffer (pH 7.4; Sigma-Aldrich) for 25 min at 57°C within between vortexing, as previously described (Kennerknecht et al., 2020 (link)). Angie1 or Angiogenin were added in a 1:1 mixture to the liposomes at a final concentration of 2.7 mM. The size of liposomes was determined by Nanoparticle Tracking Analysis (NTA) using a ZetaView TWIN (Particle Metrix, Inning, Germany). Samples were diluted in TRIS-buffer and videos of the light-refracting particles were recorded with the following settings: 25°C fixed temperature, 11 positions, 1 cycle, sensitivity 85, shutter 100, 15 fps, 2 s videos/position, and six measurements. The number and size distribution were evaluated by ZetaView Analyze (Version 08.05.05 SP2), as previously described (Conzelmann et al., 2020 (link)).