Silica spheres

Numbers and sizes of particles contained in each fraction were evaluated by the nanoparticle tracking analysis (NTA) method using the NanoSight LM10 system (Malvern Instruments Ltd., Worcestershire, UK) equipped with a 405-nm laser and a digital camera (Marlin F033B, Allied Vision Technologies). The measurements were carried out by following the reported protocol [38]. In brief, the equipment was first calibrated with silica spheres with a diameter of 100 nm (Polysciences, Inc., Warrington, PA). Then, the samples were diluted with PBS, and 30-s motion videos were captured per measurement. Ambient temperature was recorded manually. The parameters for the minimum expected particle size, minimum tracking length, and blur were automatically determined by the system. The camera level and detection threshold were set at values of 14 and 4, respectively. NTA-software version 2.3 was used for data analysis. For each sample, measurement was independently performed five times, so that more than 1,000 particles in total were analyzed. The average histogram was plotted from the data of five measurements, and diameters (mean, standard deviation, and mode) were determined.