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

Abstract

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 pKa 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 in vitro 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 in vivo mouse model.

Hydrodynamic Delivery of Cre Protein to Lineage-Mark or Time-Stamp Mouse Hepatocytes In situ

“…His6-tagged Cre and TAT-Cre were expressed from PET28 vectors as described previously [26], [27], [29] and were purified on Ni-NTA columns. Proteins were eluted with imidazole by standard procedures, snap-frozen in liquid nitrogen, and stored at −80°C.…”

Reversible Major Histocompatibility Complex I-Peptide Multimers Containing Ni2+-Nitrilotriacetic Acid Peptides and Histidine Tags Improve Analysis and Sorting of CD8+ T Cells

“…Although previously described tri-and tetra-NTA compounds allow diverse applications, their difficult chemical synthesis make them accessible only to specialized labs (20 -24). In only one previous study, a tri-NTA compound was synthesized as a peptide derivative (2). However, as a building block FmocAsp(␤-amido-N⑀-Lys(tri-bu t -NTA) was used, which is neither commercially available nor facile to synthesize.…”

“…The extremely strong binding of biotin to streptavidin (SA) 2 (K D ϭ ϳ1 ϫ 10 Ϫ14 M) has been extensively used for such applications (1). Although for some applications, this practically irreversible binding is desirable, for others it is not, e.g.…”

Bioreducible Polycations as Shuttles for Therapeutic Nucleic Acid and Protein Transfection

“…Such reduction-activated cytotoxins have been utilized in drug delivery; for example, listeriolysin-SS-oligocation conjugates for pDNA delivery (11,90) or numerous immunotoxins (24,25,101). Commonly used synthetic protein delivery systems include cationic lipids (13,14,62), protein transduction domains (45), such as natural penetratin (85,93) and HIV-TAT (45,103), or artificial oligoarginines (93) and cationic polymers (17,30,59).…”