Membrane retention and cellular uptake of click conjugated cargos

Metabolic glycan labeling offers a facile yet powerful method to functionalize cell membranes with clickable chemical tags for subsequent conjugation of cargos via bioorthogonal click chemistries. However, the membrane retention and cellular uptake of covalently conjugated cargos, which is distinct from natural systems where cells interact with extracellular cargos via electrostatic or hydrophobic interactions, remain elusive. Here, we developed a dibenzocyclooctyne (DBCO)-functionalized polyacrylic acid (PAA) probe system that enables simultaneous detection of overall cellular uptake and membrane retention, and investigated how the molecular weight, valency, and hydrophobicity of DBCO-PAAs impact their membrane retention and cell uptake rates after click conjugation to azido-labeled cells. PAA with lower molecular weights exhibited enhanced cell-conjugation efficiency and prolonged membrane retention compared to higher molecular-weight counterparts. Increasing DBCO density improved the cell-conjugation efficiency and membrane retention, while altering the hydrophobicity of PAA induced minimal changes in the membrane retention and cellular internalization rates. We further uncovered energy dependent endocytosis and caveolin-dependent pathways as the main endocytic routes for the covalently conjugated cargos. We also studied the membrane retention of DBCO-functionalized phycoerythrin and allophycocyanin, two representative fluorescent proteins, on azido-labeled cells, which exhibited cargo and cell type-dependent membrane retention. Our study comprehensively assesses the membrane retention and cellular uptake of covalently conjugated cargos and could guide future design of cell-targeting clickable agents.