Synthesis of a pH-Sensitive Nitrilotriacetic Linker to Peptide Transduction Domains To Enable Intracellular Delivery of Histidine Imidazole Ring-Containing Macromolecules

Intracellular delivery of functional macromolecules using peptide transduction domains (PTDs) is an exciting technology with both experimental and therapeutic applications. Recent data indicate that PTD-mediated transduction occurs via fluid-phase macropinocytosis involving an intracellular pH drop to ∼5. Nitrilotriacetic acid (NTA)-coordinated metals avidly bind hexahistidine-tagged macromolecules, including peptides and proteins. Histidine’s imidazole ring has a p<i>K</i>_a of 6, making this an attractive target for the biological pH drop of PTD-mediated macropinocytotic delivery. The objective of this study was to develop a pH-sensitive PTD delivery peptide (NTA₃-PTD). We demonstrate the <i>in vitro</i> function of this novel peptide by delivering fluorescently labeled peptides (1.6 kDa) and functional enzymes, β-galactosidase (119 kDa) and Cre recombinase (37 kDa). Furthermore, the NTA₃-PTD peptide was able to deliver functional Cre recombinase in an <i>in vivo</i> mouse model