PEG-coated PS nanoparticles were formulated as previously described (21, 36). Briefly, 1 mg of 40-nm dark red fluorescent (660 nm/680 nm) PS-COOH was mixed with a different proportion of methoxy-PEG5k-amine in 100 mM phosphate buffer (pH 7.2; 150 mM NaCl), followed by addition of excess sulfo–N-hydroxysuccinimide (~5 to 6 mg) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (~3 to 4 mg) to a volume of 500 μl. Particle suspensions were placed on a rotary incubator, and the reaction was allowed to proceed for 4 hours at 25°C. After the reaction, particles were purified by ultracentrifugation (Amicon Ultra-15 ml, 100 kDa MW cutoff) with ultrapure water (three washes in total). Nanoparticles were resuspended in ultrapure water and stored at 4°C until use.

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