A toolbox of differently sized and labeled PMMA nanoparticles for cellular uptake investigations

The cellular internalization of defined PMMA nanoparticles was investigated. For this purpose, the biocompatible copolymer p(MMA-stat-MAA)0.91:0.09 was synthesized by RAFT polymerization and labeled with three different fluorescent dyes (λEx = 493, 557, and 653 nm). Nanoparticles were formulated from the differently labeled copolymers into samples with relatively narrow size distribution (diameter d < 100 nm, 100 to 200 nm, >300 nm) under appropriate conditions of nanoprecipitation and were subsequently characterized by DLS and SEM. Mixtures of the differently sized nanoparticle samples were applied for internalization studies using monolayer cultured HeLa cells. The localization of the nanoparticles was detected after certain time points up to 24 h by CLSM, using LysoTracker as a marker for late endosomes and lysosomes. In investigations by flow cytometry, a fast uptake of medium sized nanoparticles was found, whereas the large and small nanoparticles exhibited a slower internalization. However, small and medium sized nanoparticles were detected in the late endosomes/lysosomes, whereas the large nanoparticles exhibit little co-localization with LysoTracker. Moreover, it could be shown by using different inhibitors for clathrin-dependent (chlorpromazine), caveolin-dependent (filipin III) endocytosis and macropinocytosis (EIPA) that nanoparticles with d < 200 nm were internalized via clathrin-dependent endocytosis, whereas those with d > 300 nm were internalized via macropinocytosis.