Switchable Nanoparticle Systems Enabled by Bioorthogonal Inverse Electron-Demand Diels-Alder Reactions

In modern pharmaceutical research, numerous drug candidates with high therapeutic potential have been identified. Nevertheless, the clinical efficacy of many compounds falls short of expectations, frequently due to unfavorable biodistribution that prevents the establishment of therapeutically relevant concentrations at the target site. Nanoparticle-based drug delivery systems offer a promising strategy to address this challenge by protecting active agents and enabling targeted delivery. However, many targeted nanotherapeutics fail in vivo due to biological barriers such as plasma protein adsorption, rapid clearance by the mononuclear phagocyte system, and insufficient discrimination between target and off-target cells. The aim of this work was therefore to develop polymeric nanoparticles with switchable functionality, enabling controlled activation of target cell recognition directly within the biological environment. To achieve this, bioorthogonal chemistry was employed as a tool for additive nanoparticle functionalization in situ. In particular, the inverse electron-demand Diels–Alder (iEDDA) reaction was identified as a suitable approach due to its exceptional selectivity and fast reaction kinetics under physiological conditions. This work demonstrates that the iEDDA reaction proceeds efficiently on the surface of polymeric NPs and even exhibits accelerated kinetics compared to reactions in solution. Importantly, the reaction remained efficient in complex biological media, and stability studies confirmed sufficient robustness of the reactive partners, thereby laying the foundation for potential in vivo applications. Building on this foundation, a concept for switchable target cell recognition was developed in which ligands are not pre-installed on the nanoparticle surface but are generated on demand within the biological environment. As a proof of concept, angiotensin I as an inactive pro-ligand was conjugated to NPs via iEDDA and subsequently converted by the ectoenzyme angiotensin-converting enzyme (ACE) into the active angiotensin II ligand. This resulted in a two-stage activation and recognition system that requires both enzymatic processing by ACE and the presence of the AT1 receptor on the target cell, thereby providing an additional level of specificity. Finally, the impact of switchable receptor recognition on cellular uptake was investigated. While ACE-mediated ligand activation enabled receptor-specific binding, enhanced cellular internalization was not observed, highlighting the influence of additional biological and experimental parameters on nanoparticle uptake. Overall, this work demonstrates that the combination of bioorthogonal chemistry with enzyme-mediated ligand activation represents a promising strategy for the development of adaptive nanoparticles.