The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction has proven to be a pivotal advance in chemical ligation strategies with applications ranging from polymer fabrication to bioconjugation. However, application in vivo has been limited by the inherent toxicity of the copper catalyst. Herein, we report the application of heterogeneous copper catalysts in azide-alkyne cycloaddition processes in biological systems ranging from cells to zebrafish, with reactions spanning from fluorophore activation to the first reported in situ generation of a triazole-containing anticancer agent from two benign components, opening up many new avenues of exploration for CuAAC chemistry.

Polymerisation reactions conducted inside cells must be compatible with the complex intracellular environment, which contains numerous molecules and functional groups that could potentially prevent or quench polymerisation reactions. Here we report a strategy for directly synthesising unnatural polymers in cells through free radical photo-polymerisation using a number of biocompatible acrylic and methacrylic monomers. This offers a platform to manipulate, track and control cellular behaviour by the in cellulo generation of macromolecules that have the ability to alter cellular motility, label cells by the generation of fluorescent polymers for long-term tracking studies, as well as the generation within cells of a variety of nanostructures. It is remarkable that free radical polymerisation chemistry can take place within such complex cellular environments and this demonstration opens up a multitude of new possibilities for how chemists can modulate cellular function and behaviour and for understanding cellular behaviour in response to the generation of free radicals.

Transition metals have been successfully applied to catalyze non-natural chemical transformations within living cells, with the highly efficient labeling of subcellular components and the activation of prodrugs. In vivo applications, however, have been scarce, with a need for the specific cellular targeting of the active transition metals. Here, we show the design and application of cancer-targeting palladium catalysts, with their specific uptake in brain cancer (glioblastoma) cells, while maintaining their catalytic activity. In these cells, for the first time, two different anticancer agents were synthesized simultaneously intracellularly, by two totally different mechanisms (in situ synthesis and decaging), enhancing the therapeutic effect of the drugs. Tumor specificity of the catalysts together with their ability to perform simultaneous multiple bioorthogonal transformations will empower the application of in vivo transition metals for drug activation strategies.

The evaluation of the role of physicochemical properties in the toxicity of nanoparticles is important for the understanding of toxicity mechanisms and for controlling the behavior of nanoparticles. The surface charge of nanoparticles is suggested as one of the key parameters which decide their biological impact. In this study, we synthesized fluorophore-conjugated polystyrene nanoparticles (F-PLNPs), with seven different types of surface functional groups that were all based on an identical core, to evaluate the role of surface charge in the cellular uptake of nanoparticles. Phagocytic differentiated THP-1 cells or non-phagocytic A549 cells were incubated with F-PLNP for 4 h, and their cellular uptake was quantified by fluorescence intensity and confocal microscopy. The amount of internalized F-PLNPs showed a good positive correlation with the zeta potential of F-PLNPs in both cell lines (Pearson’s r = 0.7021 and 0.7852 for zeta potential vs. cellular uptake in THP-1 cells and nonphagocytic A549 cells, respectively). This result implies that surface charge is the major parameter determining cellular uptake efficiency, although other factors such as aggregation/agglomeration, protein corona formation, and compositional elements can also influence the cellular uptake partly or indirectly.

We show the intracellular delivery of a homogeneous palladium–peptide catalyst able to bioorthogonally activate a profluorophore inside living prostate cancer cells.