Enhancing CNS drug delivery through direct conjugation of an A1 receptor antagonist to a retrograde transport protein

We developed a simple methodology for conjugating dipropyl-cyclopentyl xanthine (DPCPX) directly to wheat germ aggluti-nin (WGA) with minimal synthetic steps and reagents, and without a third component of a nanoparticle linker. DPCPX serves as an A1 receptor antagonist with potential to treat spinal cord injury (SCI)-associated breathing dysfunction while WGA serves as a retrograde anatomical tracer, utilizing the canonical histochemical tool for intramuscular drug delivery directly to the central nervous system (CNS). This protein–drug conjugate was first characterized chemically, revealing specific conjuga-tion predominantly at arginine residues, with some occurring at lysine residues. This insight provides an understanding of the stability of protein–aldehyde linkages more generally. The in vitro activities of the conjugate were then characterized by its drug release kinetics, cytotoxicity, and cellular internalization in a differentiated NSC-34 cell line. Our findings indicate that the conjugate exhibits a slow drug release profile, which is cru-cial for ensuring that the drug remains conjugated during slow anatomical transport until reaching the relevant neural sites, where it can then induce a compensatory recovery mechanism through adenosine receptor antagonism.