Protein‐Sized Dye‐Loaded Polymer Nanoparticles for Free Particle Diffusion in Cytosol

Abstract

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.

Fighting Aggregation‐Caused Quenching and Leakage of Dyes in Fluorescent Polymer Nanoparticles: Universal Role of Counterion

“…Then, generated cationic R18 dye being adsorbed at the NPs surface could decrease their negative surface charge (Figure ) and, thus, compromise their colloidal stability as observed by DLS. Indeed, we have shown previously that the amount of charged groups on the polymers used for NP formation is crucial for the formation of small particles . The small size of polymer NPs containing R18 with bulky hydrophobic and medium‐sized counterions suggests that these ion pairs probably do not dissociate in water and do not neutralize surface carboxylates.…”

“…On the other hand, NPs with dispersed dyes (using counterions of medium hydrophobicity, such as B(CF 3 ) 4 and F5‐3PB‐OH), which exhibit stable emission, will be of high interest as robust labels for single molecule/particle tracking and imaging applications. It is well established that brightness, photo‐stability and continuous non‐blinking emission of NPs are of key importance for single‐particle tracking in live cells ,…”

“…Thec ounterion plays the role of both spacer,w hich prevents dyes from ACQ, and "glue" that brings the dyest ogether and ensures fast EET. [16,17,20] In these conditions, any transientd ark species inside ap article can quencht he entire particle. Here, we showed that this unique ON/OFFs witching behavior of al arge dyee nsemble is realized for different typeso fc ounterions:t hree from fluorinated TPB and one from the borohydride family.Aremarkable observation in this respect is ac ompletea bsence of the ON/OFF switching behavior forB (CF 3 ) 4 and F5-3PB-OH.…”

“…[8,16] We have shown that the bulky counterion tetrakis(pentafluorophenyl)borate (F5-TPB) in the ionp air with rhodamine Bo ctadecyle ster (R18) enables dye encapsulationw ith minimal ACQ, resulting in 40 nm polymer NPs that are 5-100-fold brighter than corresponding quantum dots. [8,[16][17] This approach was proven to be compatible with other cationic dyes, such as cyanines, leading to the development of am ethodf or long-term rainbow cell barcoding. [18] Moreover,F 5-TPB counterion can be replaced with another fluorinated tetraphenylborate [18] and af luorinated aliphatic aluminate.…”

Light‐Harvesting Nanoparticle Probes for FRET‐Based Detection of Oligonucleotides with Single‐Molecule Sensitivity

“…The primary approach to improve FRET donor efficiency of a particle together with its light‐harvesting capacity is to minimize its size while keeping the largest possible number of encapsulated donor dyes. Our earlier studies showed that smaller polymer NPs can be obtained by increasing the pH during nanoprecipitation or increasing the charge on the polymer . In the first approach, we performed nanoprecipitation of PMMA‐AspN3‐1.6 % at pH 9, which yielded NPs of 30 nm (Figure S7 A,B).…”