Development of Cell-Penetration PG-Surfactants and Its Application in External Peptide Delivery to Cytosol

Abstract

Recently, development of techniques to deliver pharmacologically active biomacromolecules such as peptides and proteins to cytosol has gained much interest. Here, we applied the peptide gemini (PG)-surfactants to a novel platform to design cell penetration lipopeptides (CP-PGs), which can deliver exogenous peptides and proteins to cytosol. Among the number of candidate CP-PGs having different peptide sequences at the X-, Y-, and Z-positions, we focused on those having two C12 alkyl chains appended to the side chain of two Cys residues, the betaine sequence -Asp-Lys-Asp-Lys-between the alkylated Cys residues (i.e., at the X-position), and having different cationic peptide sequences of oligo-Lys or oligo-Arg at the Y-and/or Z-positions. With respect to cytotoxicity for mammalian cells such as NIH3T3 cells upon 1 h exposure, those having (Lys) 3 (K 3 -DKDKC 12 and DKCK 12 -K 3 ) showed lower cytotoxicity (IC 50 = 241 and 198 μM) among those having oligo-Lys, (Lys) n (n = 1, 3, 5; IC 50 = 88−197 μM). Similar lower cytotoxicity was also observed for the CP-PG having two (Lys) 3 at both N-and C-terminal sides (K 3 -DKDKC 12 -K 3 ) (IC 50 = 225 μM). In contrast, the CP-PG having (Arg) 3 at the N-terminal side (R 3 -DKDKC 12 ) showed higher cytotoxicity (IC 50 = 88 μM). Carrier abilities of the CP-PGs for exogenous peptides were evaluated using the proapoptotic domain (PAD) peptide, which induces apoptosis by disturbing mitochondrial membranes after delivery into cytosol. As a result, the CP-PGs of K 3 -DKDKC 12 , DKCK 12 -K 3 , K 3 -DKDKC 12 -K 3 , DKCK 12 -K 5 , and R 3 -DKDKC 12 exhibited micromolar range carrier ability (the necessary half concentration to induce cell death (EC 50 ) by delivering PAD peptide to cytosol was 10, 6.2, 8.5, 5.8, and 11.5 μM, respectively). Especially, the carrier abilities of DKCK 12 -K 3 and DKCK 12 -K 5 were superior to the well-established cell penetration Arg-rich R8 peptide (EC 50 = 6.8 μM). Together, our results indicate that the PG-surfactant molecular framework could be a potential new platform to design efficient cell penetration carrier materials.

Structural Modifications to the Internal Oligoguanidinium Transporter Uncover Two Potent Analogues that Effectively Deliver the Proapoptotic Peptide in Multiple Cancer Cell Lines

“…Further research is underway for screening these two analogues for PMO delivery via a detailed mechanistic pathway analysis and development of more analogues to further enhance the efficacy of IGTs. IGT can certainly deliver a membrane-impermeable PAD peptide into cells by conjugation and is different from the CPP-based delivery system for the PAD peptide. ,,,− Among the nonpeptidic cellular transporters, − IGT is different because it requires as minimum as four guanidinium groups that are in organized form to minimize the interaction with serum . Furthermore, the IGT can be synthesized very easily.…”

Protein Delivery: If Your GFP (or Other Small Protein) Is in the Cytosol, It Will Also Be in the Nucleus

“…11,12 Direct entry into the cytosol is generally barred to proteins by the selectively permeable cell membrane. 13 A number of nanocarriers 14,15 and cell-penetrating peptides 16,17 have been engineered for delivery of proteins into the cell. However, access to the cytosol is often limited in efficiency, because these systems are generally taken up by endocytosis 18 through a range of mechanisms.…”