How small should nanoparticles be in order to travel freely through the cytosol similar to proteins? Answering this question remains a challenge, because the majority of nanoparticles are relatively large and their size cannot be finely tuned to match that of proteins. Here, poly(methyl methacrylate) copolymers with varied fraction and type of charged groups (carboxylate, sulfonate, and trimethylammonium) are developed, yielding nanoparticles with controlled sizes from 50 to 7 nm through nanoprecipitation. Loading these nanoparticles with a rhodamine dye/bulky counterion pair at 10wt% makes them highly fluorescent. After their coating with polyethylene glycol groups for preventing non-specific protein binding and microinjection into living cells, the first systematic study of the size effect on diffusion in the cytosol for solid nanoparticles of the same nature is realized. Single-particle-tracking data provide evidence for distinct particle sieving in the cytosol, suggesting that only nanoparticles below a critical size of 23 nm exhibit free diffusion and spreading. These findings show the size limitations imposed by intracellular crowding and compartmentalization, which is critical for applications of nanomaterials in the cytosol. The proposed concept of polymer design opens the route to organic nanoparticles of ultrasmall sizes and high loading for bioimaging and drug-delivery applications.

The inability to visualize single organic nanoparticles (NPs) in vivo is an unsolved issue in the field of nanomedicine. To enable high single particle fluorescence, we loaded polymer poly(methyl methacrylate)-sulfonate (PMMA-SO3H) NPs with octadecyl rhodamine B together with hydrophobic counter-ions as a fluorophore insulator to prevent aggregation-induced quenching. To create NPs with stealth properties we used the amphiphilic block copolymers pluronic F-127 and F-68. Fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) showed that pluronics remain at the NP surface after dialysis (density: one amphiphile per 5.5 nm 2) and protected NPs from degradation by serum proteins and surfactants. Single particle brightness was increased 150-fold as compared to commercially available preparations by increasing dye loading to 20 wt% and optimizing particle size. Added to primary cultured neurons, these NPs were stable and interacted with dendrites and axons. After intravenous injection in mice, NPs could be tracked in cerebral vessels for at least 1h by in vivo two-photon microscopy. Following brain injury or neuroinflammation, NPs extravasated from meningeal vessels, were taken up by meningeal macrophages, and entered the brain parenchyma. In summary, we developed biocompatible NPs suitable for drug delivery with in vitro and in vivo stealth properties and extremely bright fluorescence. Thus, individual NPs could be tracked in mouse brain and their dynamics visualized with subcellular resolution. Using superbright NPs may thus open new avenues for the investigation of NPs in living organisms.