Recyclable cell-surface chemical tags for repetitive cancer targeting

Metabolic glycan labeling provides a facile yet powerful tool to install chemical tags to the cell membrane via metabolic glycoengineering processes of unnatural sugars. These cell-surface chemical tags can then mediate targeted conjugation of therapeutic agents via efficient chemistries, which has been extensively explored for cancer-targeted treatment. However, the commonly used in vivo chemistries such as azide-cyclooctyne and tetrazine-cyclooctene chemistries only allow for one-time use of cell-surface chemical tags, posing a challenge for long-term, continuous cell targeting. Here we show that cell-surface ketone groups can be recycled back to the cell membrane after covalent conjugation with hydrazide-bearing molecules, enabling repetitive targeting of hydrazide-bearing agents. Upon conjugation to ketone-labeled cancer cells via a pH-responsive hydrazone linkage, Alexa Fluor 488-hydrazide became internalized and entered endosomes/lysosomes where ketone-sugars can be released and recycled. The recycled ketone groups could then mediate targeted conjugation of Alexa Fluor 647-hydrazide. We also showed that doxorubicin-hydrazide can be targeted to ketone-labeled cancer cells for enhanced cancer cell killing. This study validates the recyclability of cell-surface chemical tags for repetitive targeting of cancer cells with the use of a reversible chemistry, which will greatly facilitate future development of potent cancer-targeted therapies based on metabolic glycan labeling.