Single-trigger dual-responsive nanoparticles for controllable and sequential prodrug activation.

Here we have developed a novel approach where two synergistically acting drugs were completely inactivated upon chemical immobilization on a nanoparticle template and activated in response to a chemical stimulus. The activation rate of each drug payload is controlled using a biologically inert bioorthogonal chemistry approach. By exploiting the subtle differences in the 'click-to-release' bioorthogonal reaction, we engineered a single delivery platform capable of releasing the payloads in a time-staggered manner in response to a single dose of a highly specific, yet reactive, small molecule. Incorporation of both di-axial, 'fast release', and di-equatorial, 'slow release', TCO linkers into our nanodrug assembly inhibited the activity of the drug molecules and enabled us to control the timing and activation of each payload. This single-trigger dual-responsive nanoparticle construct and its release kinetics were characterized using two molecular fluorescent probes and tested in vitro for efficient delivery of molecular payloads. In this manuscript we show that this approach was also successful in the treatment of triple negative BT-20 breast cancer cells. Our nanodrug loaded with the slow-releasing doxorubicin and fast-releasing PAC-1 prodrugs displayed a greater therapeutic response than the nanodrug which released both payloads simultaneously.