Tuning pH for the Controlled Formation of Ultrasmall Nanoparticles with Direct Cytosolic Access

Direct cytosolic delivery of nanoparticles offers advantages by circumventing the endocytic pathway, thereby reducing associated intracellular trafficking barriers. Several methods facilitate translocation across the cell membrane, including passive diffusion by ultrasmall nanoparticles. However, nanoparticles under 10 nm pose challenges for drug loading due to rapid drug leaching or the need for complex, drug-specific synthesis. Here, we devise a polymer system to encapsulate a wide range of drugs in aqueous solutions; the key is adjusting the pH, leading to nanoparticle generation either below 10 nm or above 50 nm. Subsequent crosslinking ensures safe drug retention and stability. Machine learning is employed to link drug structure to its loading and nanoparticle size, identifying the pK_aH of drugs as the critical parameter determining encapsulation conditions. MD simulations reveal the compact structure of the ultrasmall nanoparticles with a mixture of hydrophobicity and hydrophilicity on the surface, which is ideal to achieve high engagement with cell membranes. Experimental evidence and computational simulations confirm the direct cytosolic delivery of these small nanoparticles, bypassing the endosomal route. In summary, this platform allows easy production of ultrasmall nanoparticles for diverse drugs, providing a robust strategy for improved intracellular delivery.