Interaction between Cell-Penetrating Peptides and Acid-Sensitive Anionic Oligopeptides as a Model for the Design of Targeted Drug Carriers

Abstract

Overcoming the nonspecific cellular uptake of cell-penetrating peptides (CPPs) is a major hurdle in their clinical application. Using pH as the activation switch, histidine-glutamic acid (HE) dipeptide repeats were fused to CPPs to trigger the membrane-penetrating activity at mildly acidic pH environments (i.e., pH 6.5 or below) while masking the internalization at neutral pH (i.e., pH 7.0 or above). In this study, a series of recombinant GST-fusion proteins containing an HE oligopeptide sequence (i.e., (HE)n with n = 8, 10, or 12) and a cationic CPP (i.e., YG(RG)6, YGR6G6, or Tat) were engineered for a pH-sensitive study comparing their cellular uptake and surface binding in cultured HeLa cells. Circular dichroism (CD) spectroscopy was performed to correlate differences between CPPs in secondary structure with the pH sensitivity. YGR6G6 with clustered arginine residues exhibited greater pH sensitivity in cellular uptake than YG(RG)6 with separated arginine residues. Increasing the stretch of HE repeats decreased cellular uptake and surface binding for both YG(RG)6 and YGR6G6. The ratio of cellular internalization at pH 7.5 vs 6.0 was not changed by the presence of serum. CD spectral data revealed that both (HE)10-Tat and (HE)10-YGR6G6 exhibited an unordered secondary structure, whereas (HE)10-YG(RG)6 adopted an antiparallel β-sheet conformation. This β-sheet conformation presumably stabilized the association of (HE)10 with YG(RG)6, leading to weakened pH sensitivity of (HE)10-YG(RG)6. On the other hand, the random-coiled structures, that is, (HE)10-YGR6G6 and (HE)10-Tat, both showed higher pH sensitivity as determined in cell experiments. The data presented in this study provide a basis for the future design of pH-sensitive HE-CPP carrier for targeted drug delivery.

Anti-metastatic Effects of Cationic KT2 Peptide (a Lysine/Tryptophan-rich Peptide) on Human Melanoma A375.S2 Cells

“…Lysine/tryptophan-rich KT2 (NGVQPKYKWWKWWKK WW-NH 2 ), an amphipathic cationic peptide, can be internalized into A375.S2 cells ( Figure 1C). It is well known that arginine and lysine, positively charged amino acids, are involved in the cell surface binding and uptake of cellpenetrating peptides in mammalian cells (33). KT2 can also penetrate the bacterial membrane of Escherichia coli O157: H7 cells (34).…”

Characterization of Polyelectrolyte Complex Formation Between Anionic and Cationic Poly(amino acids) and Their Potential Applications in pH-Dependent Drug Delivery

“…The pH-responsive (HE) 20 peptide was produced recombinantly as a glutathione S-transferase (GST)-(HE) n fusion protein containing a thrombin cutting site between the GST and (HE) n domains as previously described [ 23 ]. Briefly, plasmids containing the GST-(HE) 20 gene were transformed into Escherichia coli expression strain BL21 and the recombinant protein was expressed as previously described [ 21 , 23 , 24 ]. The resultant bacterial pellets were resuspended in phosphate buffered saline (PBS) (pH 7.4) containing 0.25 mg/mL lysozyme.…”

“…Additionally, our lab has previously developed a pH-responsive peptide composed of repeats of glutamic acid and histidine, (HE) n [ 21 ]. The pH-responsiveness of this copolymer has been demonstrated at several repeat lengths including (HE) 15 [ 22 ], (HE) 10 [ 23 ], and (HE) 8–12 [ 24 ]. At physiologic pH of ~7.4, glutamic acid is negatively charged, while histidine remains unprotonated, giving (HE) n a net negative charge.…”

Advances in intracellular delivery through supramolecular self-assembly of oligonucleotides and peptides

“…To overcome the lack of specificity of CPPs, a variety of targeting moieties have been conjugated. Sun et al complexed CPPs with the acid-sensitive anionic oligopeptides to block the internalization at neutral pH 184. In acidic pH, the oligopeptide changed its conformation, exposing the CPP moiety to cells.…”