EGFR-Functionalized Nanolipid Particle Assembly

To form the EGFR-NLPs, we used a protocol modified from the literature [39 (link), 47 (link)]. Briefly, 100 μg egg PC (Avanti Polar Lipids, Alabaster, AL) was dried in a glass vial under nitrogen stream. The lipids were solubilized with 10 mM Triton X-100 in PBS with gentle vortexing. To the lipids, 50 μg ApoA1 (Athens Research and Technology, Athens, GA) was added and briefly vortexed. Except where noted, 15 μg EGF (Gold Biotechnology, Olivette, MO) was mixed with approximately 30 μg FLAG-EGFR isolated from insect cells on ice. The EGF/EGFR mixture was added to the lipid/ApoA1 mixture and briefly vortexed and incubated at room temperature for 1–2 hours. To self-assemble the NLPs, the detergent was removed by incubating the assembly mixture with Bio-Beads SM-2 Resin (Bio-Rad, Hercules, CA), which has been pre-hydrated with methanol, on a nutator for 1–2 hours at 4°C. The NLP mixture was removed from the Bio-Beads with a spin column. For egg PC:ApoA1 and EGFR:ApoA1 optimization experiments, assemblies were performed as above with a constant amount of ApoA1 and varying the amount of egg PC or EGFR.