Spectrally PAINTing a Single Chain Polymeric Nanoparticle at Super-Resolution

Folding a polymer chain into a well-defined single-chain polymeric nanoparticle (SCPN) is a fascinating approach to obtaining structured and functional nanoparticles. Like all polymeric materials, SCPNs are heterogeneous in their nature due to the polydispersity of their synthesis: the stochastic synthesis of polymer backbone length and stochastic functionalization with hydrophobic and hydrophilic pendant groups make structural diversity inevitable. Therefore, in a single batch of SCPNs, nanoparticles with different physicochemical properties are present, posing a great challenge to their characterization at a single-particle level. The development of techniques that can elucidate differences between SCPNs at a single-particle level is imperative to capture their potential applications in different fields such as catalysis and drug delivery. Here, a Nile Red based spectral point accumulation for imaging in nanoscale topography (NR-sPAINT) super-resolution fluorescence technique was implemented for the study of SCPNs at a single-particle level. This innovative method allowed us to (i) map the small-molecule binding rates on individual SCPNs and (ii) map the polarity of individual SCPNs for the first time. The SCPN designs used here have the same polymeric backbone but differ in the number of hydrophobic groups. The experimental results show notable interparticle differences in the binding rates within the same polymer design. Moreover, a marked polarity shift between the different designs is observed. Interestingly, interparticle polarity heterogeneity was unveiled, as well as an intraparticle diversity, information which has thus far remained hidden by ensemble techniques. The results indicate that the addition of hydrophobic pendant groups is vital to determine binding properties and induces single-particle polarity diversity. Overall, NR-sPAINT represents a powerful approach to quantifying the single-particle polarity of SCPNs and paves the way to relate the structural heterogeneity to functionality at the single-particle level. This provides an important step toward the aim of rationally designing SCPNs for the desired application.