B16 f10 murine melanoma cell

B16-F10 murine melanoma cells (ATCC, Manassas, VA, no. CRL-6475) were plated at 2000 cells in 100 µL media per well in 96-well tissue culture plates 24 h prior to peptide and particle addition. Free peptide or particles were added to each well in 100 µL of complete media. Cells were incubated continuously with either free peptide (0.5, 0.25, 0.1, 0.05 mg/mL) or particles (1, 0.5, 0.25, and 0.1 mg/mL) for either 24 or 48 h at 37 °C in a 5% CO₂ incubator. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reagent (Invitrogen, Carlsbad, CA) was added to media at a concentration of 0.5 mg/mL, and cells were incubated 2 h at 37 °C in a 5% CO₂ incubator. Media was removed, and formazan crystals dissolved in DMSO (100 µL/well, Sigma-Aldrich, St. Louis, MO). Absorbance was measured using a Tecan (San Jose, CA) Infinite M1000 microplate reader.

PD-1 siRNA and negative control siRNA were from Integrated DNA Technologies (IDT) (Coralville, IA). Cholesterol, Lugol’s solution, sodium dodecyl sulfate (SDS), Triton X-100, and Amicon Ultra-15 centrifugal filters were from Sigma-Aldrich (St. Louis, MO). Lecithin was from Alfa Aesar (Tewksbury, MA). Block-iT^™ fluorescent oligo was from ThermoFisher Scientific (Waltham, MA). TopFluor^®-labeled Cholesterol and 1, 2-dioleoyl-3-trimethyl ammonium-propane chloride (DOTAP) were from Avanti Polar Lipids (Alabaster, AL). PEG2000-hydrazone-stearic acid (C18) conjugate (PHC) was synthesized as previously described (31 (link)). Phycoerythrin (PE)-labeled anti-mouse PD-1 antibody and cell staining buffer were from BioLegend (San Diego, CA). Anti-mouse PD-1 antibodies were from Bio X-cell (West Lebanon, NH). All solvents were from Sigma-Aldrich. J774A.1 murine macrophages and B16-F10 murine melanoma cells were from the ATCC (Manassas, VA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin (1%)/streptomycin (100 U/ml), all from Thermo Fisher Scientific (Grand Island, NY).