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.

Intracellular applications of fluorescent nanoparticles (NPs) as probes and labels are currently limited by the significant molecular crowding and the high level of complexity encountered inside living cells. The solution is to develop very small, bright, and non-interacting (stealth) NPs. Combining these properties requires to implement the stealth behavior through the thinnest possible hydrophilic shell. Here, we propose a one-step process for preparing ultrasmall and bright stealth nanoparticles (NPs) based on a zwitterionic methacrylate based copolymer.Dye-loaded polymer nanoparticles are assembled through nanoprecipitation of the copolymer together with the salt of a rhodamine B derivative and a bulky hydrophobic counterion to achieve high particle brightness. We found that 10 mol% zwitterionic groups in the polymer yields NPs of less than 15 nm that are stable in physiological salt conditions and practically resistant to protein adsorption, as suggested by fluorescence correlation spectroscopy. The combination of the very small size with the non-fouling nature of these particles enables spreading of zwitterionic polymer NPs in the whole cytosol after their microinjection into living cells. In addition, single-particle tracking showed an up to 4 times faster diffusion of zwitterionic NPs in the cytosol compared to PEGylated nanoparticles. The obtained dye-loaded zwitterionic polymer NPs open the route to intracellular single-particle tracking and biosensing applications.