Hek blue lps detection assay

The size distribution and zeta potential of the nanostructures were analyzed by Zetasizer Nano (Malvern Instruments) with a 4 mW He–Ne 633 nm laser at 1 mg/mL in PBS. The polydispersity index (PDI) was calculated using a two parameter fit to the DLS correlation data. The morphology of each nanostructure were determined by CryoTEM. The ICG concentration of different nanoparticles was measured by UV–vis spectroscopy (SpectraMax M3, Molecular Devices) after sample dilution in PBS solution. To characterize the stability and fluorescent properties of ICG-loaded NS, different molar ratios of polymer: ICG (1:10, 1:25, 1:33, 1:50, 1:75 and 1:100) were prepared with 10 mg/mL of PEG-bl-PPS. Solutions of ICG-loaded NS were diluted 1:50 in PBS solution prior to the generation of the absorbance spectrum. Wavelengths from 250 to 850 nm were scanned with 10 nm increments. The fluorescent spectrum was measured by the excitation of 780 nm and emission from 700 to 850 nm with 5 nm increments. In all cases, ICG-loaded NS were matched with free ICG (in PBS solution) at the same concentrations of ICG. Before animal studies, all samples were verified to be endotoxin-free (<0.01 EU/mg) by the TLR4 activation HEK Blue LPS detection assay (Invivogen).