Modular and Orthogonal Post-PEGylation Surface Modifications by Insertion Enabling Penta-Functional Ultrasmall Organic-Silica Hybrid Nanoparticles

Multifunctional nanoparticles (NPs) combining different functional components into a single NP platform are of great interest in the fields of nanobiotechnology and nanomedicine. Here, we report a versatile surface modification approach to modularly and orthogonally functionalize Cornell prime dots (C′ dots), ultrasmall sub-10 nm PEGylated fluorescent core–shell silica nanoparticles that have been translated into the clinic, with up to four types of different functional ligands on the NP surface. It enables the synthesis of penta-functional C′ dots integrating a variety of properties into a single NP, i.e., fluorescence detection, specific cell targeting, radioisotope chelating/labeling, ratiometric pH sensing, and drug delivery, while the overall NP size remains below 7 nm. This is achieved by taking advantage of the fact that the PEG layer of C′ dots is penetrable to small molecules. Amine- and/or thiol-functionalized silane molecules can be inserted between PEG chains and onto the silica surface of C′ dots, to which additional functional ligands can subsequently be attached. This post-PEGylation surface modification by insertion (PPSMI) approach only requires a few extra steps sandwiched between C′ dot PEGylation and purification in the one-pot type water-based synthesis without diminishing high-quality NP generation. The resulting C′ dots with additional functionalities exhibit physicochemical properties like their size and PEG density close to clinically translated C dots, opening a gate to the diversification of their clinical applications. We further demonstrate a modification of the C′ dot synthesis enabling large numbers of targeting peptides per particle as well as a facile and versatile spectroscopic approach to quantitatively assess the specific numbers of the different surface ligands by deconvolution of absorption spectra into individual components. Insights gained from this study of synthetic PEGylation and post-PEGylation surface modification methods may be transferred to the development of other PEGylated NP platforms for biomedical applications and clinical translation.