Catechol-linker and receptor-mediated site-specific delivery of bortezomib against non-small cell lung cancer

Angiotensin-converting enzyme-2 (ACE2) receptor-targeting bio-orthogonally conjugated bortezomib (BTZ) was site-specifically delivered against non-small cell lung cancer (NSCLC). Through rational screening, three ACE2 receptor-binding domain (RBD) variants (mutant RBD1, mutant RBD2, and mutant RBD3) were identified to introduce the genetic linker DOPA (3,4-dihydroxyphenyl-L-alanine) to bio-orthogonally load BTZ through a catechol-boronate ester with enhanced receptor binding. Extensive biophysical characterization, such as UV-Vis spectroscopy, B¹¹-NMR spectroscopy, RP-HPLC, microscale thermophoresis, and X-ray photoelectron spectroscopy, confirms the homogeneous preparation and controlled release of BTZ. In vitro analysis, including 2D monolayer and 3D spheroid models, revealed the site-specific and controlled release of BTZ through ACE2 receptor-mediated endocytosis in subcellular endosomes, which inhibits the proteasome function of NSCLC cells (A549). Finally, in vivo, animal studies (Lewis lung carcinoma (LLC1) cells-induced C57BL/6 mice) showed significant inhibition of lung tumor growth with reduced toxicity in normal ACE2-expressing organs. To our knowledge, site-specific labeling and high-potential bio-orthogonal delivery of BTZ in the NSCLC were demonstrated for the first time. More attractively, this concept strengthens future applications for delivering other boronic acid-containing therapeutics against metabolic and cardiovascular diseases.