Preparation and Characterization of Fluorescent Lipid Nanoparticles

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.