Controlling Surface Chemical Heterogeneities of Ultrasmall Fluorescent Core–Shell Silica Nanoparticles as Revealed by High-Performance Liquid Chromatography

Ultrasmall (diameter below 10 nm) fluorescent core–shell silica nanoparticles have garnered increasing attention in recent years as a result of their high brightness and favorable biodistribution properties important for applications including bioimaging and nanomedicine. Here, we present an in-depth study that provides new insights into the physical parameters that govern full covalent fluorescent dye encapsulation within the silica core of poly­(ethylene glycol)-coated core–shell silica nanoparticles referred to as Cornell prime dots (C′ dots). We use a combination of high-performance liquid chromatography (HPLC), gel-permeation chromatography, and fluorescence correlation spectroscopy to monitor the result of ammonia concentration in the synthesis of C′ dots from negatively and positively charged versions of near-infrared dyes Cy5 and Cy5.5. HPLC, in particular, allows the distinction between cases of full versus partial dye encapsulation in the silica particle core leading to surface chemical heterogeneities in the form of hydrophobic surface patches, which, in turn, modulate biological response in ferroptotic cell death experiments. Our results demonstrate that there is a complex interplay between dye–dye and dye–silica cluster interactions originally formed in the sol–gel synthesis governing optimal dye encapsulation. We expect that the reduced surface chemical heterogeneities will make the resulting nanoparticles attractive for a number of applications in biology and medicine.