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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 cargos 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 anti-leukemic 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 customizable delivery of functional macromolecules, nominating BMCs as a broadly applicable platform for biomedical applications.<\/jats:p>\n                <jats:sec>\n                  <jats:title>One-Sentence Summary<\/jats:title>\n                  <jats:p>The establishment of modular platform for synthesizing bio-orthogonal macromolecular conjugates (BMC) enabled fast and targeted delivery of membrane-impermeant macromolecular drugs.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.07.24.666613","type":"posted-content","created":{"date-parts":[[2025,7,27]],"date-time":"2025-07-27T05:10:12Z","timestamp":1753593012000},"source":"Crossref","is-referenced-by-count":0,"title":["Modular platform for therapeutic drug delivery using trifunctional bio-orthogonal macromolecular conjugates"],"prefix":"10.64898","author":[{"given":"Danmeng","family":"Luo","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ning","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hannah","family":"Major-Monfried","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Ralls","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sophia","family":"Rha","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stacy A.","family":"Maitland","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karthikeyan","family":"Ponnienselvan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Makiko","family":"Yamada","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daniel E.","family":"Bauer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Scot A.","family":"Wolfe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8063-9191","authenticated-orcid":false,"given":"Alex","family":"Kentsis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2025072906000637000_2025.07.24.666613v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-022-01582-x"},{"key":"2025072906000637000_2025.07.24.666613v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/nrd2399"},{"key":"2025072906000637000_2025.07.24.666613v1.3","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-38039-x"},{"key":"2025072906000637000_2025.07.24.666613v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.tips.2016.10.005"},{"key":"2025072906000637000_2025.07.24.666613v1.5","doi-asserted-by":"crossref","unstructured":"H. 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