Elucidating the Stability of Single-Chain Polymeric Nanoparticles in Biological Media and Living Cells

[Image: see text] The controlled folding of synthetic polymer chains into single-chain polymeric nanoparticles (SCPNs) of defined size and shape in water is a viable way to create compartmentalized, nanometer-sized structures for a range of biological applications. Understanding the relationship between the polymer’s microstructure and the stability of folded structures is crucial to achieving desired applications. Here, we introduce the solvatochromic dye Nile red into SCPNs and apply a combination of spectroscopic and microscopic techniques to relate polymer microstructure to nanoparticle stability in complex biological media and cellular environments. Our experimental data show that the polymer’s microstructure has little effect on the stability of SCPNs in biological media and cytoplasm of living cells, but only SCPNs comprising supramolecular benzene-1,3,5-tricarboxamide (BTA) motifs showed good stability in lysosomes. The results indicate that the polymer’s microstructure is vital to ensure nanoparticle stability in highly competitive environments: both hydrophobic collapse and a structured interior are required. Our study provides an accessible way of probing the stability of SCPNs in cellular environments and paves the way for designing highly stable SCPNs for efficient bio-orthogonal catalysis and sensing applications.