Modular Platform for Therapeutic Drug Delivery Using Trifunctional Bio-Orthogonal Macromolecular Conjugates

Targeted delivery of macromolecular therapeutics holds great promise for overcoming the limitations of conventional small molecules, enabling the modulation of protein-protein interactions and precise genome editing. However, efficient, safe, and cell type-specific delivery remains a major challenge. To address this, we developed a modular platform for synthesizing heterotrifunctional bio-orthogonal macromolecular conjugates (BMCs) by engineering diverse combinations of targeting ligands, cell-penetrating peptides (CPPs), and bioactive cargos. We optimized facile bioconjugation chemistries to generate BMCs with improved yields, structural integrity, and activity. Modular BMCs accommodate diverse components, including antibodies and receptor ligands for targeting, CPPs for intracellular trafficking, and optical probes, therapeutic peptidomimetics, and CRISPR-Cas9 nuclease as cargo to confer specific biological activities. We assayed their utility across multiple applications: BMCs with fluorescently labeled cargo revealed endosomal escape and intracellular accumulation; peptidomimetic MYB transcription factor inhibitor BMCs exhibited potent antileukemic activity against acute myeloid leukemia cells; and Cas9 BMCs achieved rapid delivery and cell type-specific gene editing in human cells. The BMC approach enables the customizable delivery of functional macromolecules, nominating BMCs as a broadly applicable platform for biomedical applications.