Prodan

General chemicals were from VWR (West Chester, PA). Nile Red and Oil Red O (ORO) were from EMD (Gibbstown, NJ). Filipin was from Sigma (St Louis, MO). LipidTOX dyes and Red Phospholipidosis Detection Reagent (LipidTox-PDR, H34351) were from Molecular Probes (Eugene, OR). 4′,6-diamidino-2-phenylindole (DAPI), and concanavalin A were from Invitrogen (Temecula, CA). Alexa- and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Amersham (Piscataway, NJ). Laurdan, Prodan, 7-aminoactinomycin D (7-AAD), and diphenylhexatriene (DPH) were from Molecular Probes. The Toll-Like Receptor (TLR) ligands peptidoglycan (PGN) from Staphylococcus aureus, synthetic triacylated lipoprotein (Pam3CSK4), and standard lipopolysaccharide (LPS) from Escherichia coli were from Sigma. Phospholipidosis-inducing drugs were sourced as follows: acetaminophen, cyclosporin A (CsA) and propranolol hydrochloride from Sigma (St Louis, MO), and chlorcyclizine hydrochloride from MP Biomedicals (Santa Ana, CA). Anti-PPARγ and anti-TLR antibody were each from Abcam (Cambridge, MA). Anti-GRB2 (loading control) was from Cell Signalling Technologies (Danvers, MA).

CMC of micelle-based nanoparticle carriers (NPC) composed of p(DMAEMA)-b-p(DMAEMA-co-BMA-co-PAA) was approximated using solvatochromic shifts in fluorescence emission of PRODAN^® (Molecular Probes, Eugene, Oregon)^{50 (link), 51 (link)}. Briefly, PRODAN^® dissolved in methanol was aliquoted into black 96-well plates. After drying overnight, micelle solutions at a range of concentrations (0–2 mg/ml) were added and incubated overnight to achieve final PRODAN^® concentrations of 5.45*10⁻⁴ mg/ml. PRODAN^® emission was measured at two wavelengths (Ex/Em1: 360 nm/436 nm and Ex/Em2: 360 nm/518 nm) that corresponds to emission of PRODAN^® in hydrophobic and hydrophilic phases, respectively. The ratio of emissions (hydrophobic phase/hydrophilic phase, Em1/Em2) was plotted versus of log(micelle concentration), and CMC was determined as a concentration at which the emission ratio begins to increase with polymer concentration.

NPC critical micelle concentration (CMC) was completed using fluorescence spectral emission shifts of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN, Molecular Probes, Eugene, OR, USA) caused by partitioning differences into hydrophilic versus hydrophobic NPC regions, as described previously (38 (link), 39 , 47 (link)). PRODAN was dissolved in methanol at 24 μM, and 10 μL of this PRODAN solution was aliquoted into each well of a black 96-well plate. After drying overnight, a range of NPC solutions (0.5 ng/mL to 0.1 mg/mL) were diluted using 1X DPBS, and 100 μL/well of each sample solution was loaded into the 96-well plates containing PRODAN. All samples were loaded in triplicate, and plates were sealed and incubated at 4 °C overnight. PRODAN fluorescence was measured at two wavelengths (Ex/Em₁: 360 nm/435 nm for hydrophilic detection and Ex/Em₂: 360 nm/520 nm for hydrophobic detection) using a Tecan plate reader. The ratio of hydrophilic to hydrophobic emission (Em₁/Em₂) was plotted against the log₁₀ polymer concentration, linear regression lines were applied to each phase of the graph, and the CMC was determined as the concentration at which these regression lines intersect (see Supplemental Figure S2).