Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Agents

Abstract

A series of cyclic peptides, [(DipR)(WR)4], [(DipR)2(WR)3], [(DipR)3(WR)2], [(DipR)4(WR)], and [DipR]5, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as hydrophobic residues, were synthesized and evaluated for their molecular transporter efficiency. The in vitro cytotoxicity of the synthesized peptides was determined in human epithelial ovary adenocarcinoma cells (SK-OV-3), human lymphoblast peripheral blood cells (CCRF-CEM), human embryonic epithelial kidney healthy cells (HEK-293), human epithelial mammary gland adenocarcinoma cells (MDA-MB-468), pig epithelial kidney normal cells (LLC-PK1), and human epithelial fibroblast uterine sarcoma cells (MES-SA). A concentration of 5–10 µM and 3 h incubation were selected in uptake studies. The cellular uptake of a fluorescent-labeled phosphopeptide, stavudine, lamivudine, emtricitabine, and siRNA was determined in the presence of peptides via flow cytometry. Among the peptides, [DipR]5 (10 µM) was found to be the most efficient transporter and significantly improved the uptake of F’-GpYEEI, i.e., by approximately 130-fold after 3 h incubation in CCRF-CEM cells. Confocal microscopy further confirmed the improved delivery of fluorescent-labeled [DipR]5 (F’-[K(DipR)5]) alone and F’-GpYEEI in the presence of [DipR]5 in MDA-MB-231 cells. The uptake of fluorescent-labeled siRNA (F’-siRNA) in the presence of [DipR]5 with N/P ratios of 10 and 20 was found to be 30- and 50-fold higher, respectively, compared with the cells exposed to F’-siRNA alone. The presence of endocytosis inhibitors, i.e., nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin, did not completely inhibit the cellular uptake of F’-[K(DipR)5] alone or F’-GpYEEI in the presence of [DipR]5, suggesting that a combination of mechanisms contributes to uptake. Circular dichroism was utilized to determine the secondary structure, while transmission electron microscopy was used to evaluate the particle sizes and morphology of the peptides. The data suggest the remarkable membrane transporter property of [DipR]5 for improving the delivery of various small molecules and cell-impermeable negatively charged molecules (e.g., siRNA and phosphopeptide).

Cyclic and Linear Peptides Containing Alternate WW and RR Residues as Molecular Cargo Delivery Tools

“…They can transport small molecules that cannot penetrate through the cell membrane inside the cell. − These peptides use energy-independent and energy-dependent pathways to penetrate the cell membrane . CPPs are used in different ways to mediate cellular cargo delivery, which are covalent conjugating with the cargo − or creating stable noncovalent − complexes. Some of these peptides can penetrate the cell without generating significant cytotoxicity.…”

“…Before treating cells with the peptides, 8000 cells were seeded with DMEM (containing 10% FBS and 5% P/S) into 96-well plates at 100 μL per well and incubated in a warm incubator overnight at 37 °C. Different final concentrations of peptides (1,3,5,10,20,30,50, and 100 μM) were created (triplicate) and used to treat cells by adding 20 μL of different concentrations of the working solution into each well. Also, 40 μL of DMSO was added to negative control wells.…”

“…Furthermore, we recently designed hybrid cyclic-linear peptides containing alternate R and W residues and cyclic peptides composed of R and W along with unnatural hydrophobic diphenylalanine (Dip) residues . More studies are still needed to discover more optimal sequences of CPPs that can act as more efficient molecular transporters.…”

A new amino acid, hybrid peptides and BODIPY analogs: synthesis and evaluation of 2-aminotroponyl-l-alanine (ATA) derivatives

“…12 Parang has synthesized a series of cyclic peptides comprising the diphenylalanine residue and evaluated their transportation efficiency. 13 Tannenbaum has evaluated aromatic amino acids as potential biomarkers in breast cancer by Raman spectroscopy analysis. 14 Most unnatural aromatic amino acids are derived from the benzenoid aromatic ring.…”

Cell‐penetrating peptides for cancer treatment: Current state and future directions

“…2 Cell-penetrating peptides (CPPs, also known as protein transduction domains, Trojan horse peptides, and targeting polypeptides), which are extracted/prepared from natural or artificial proteins or chimeric sequences, are positively charged peptides made up of 5-30 amino acids. 3 CPPs can penetrate across biological membranes and deliver different kinds of cargoes (e.g., small molecules, biomacromolecules, and even nanoparticles) into cells. 4 Besides, some kinds of CPPs also have intrinsic anticancer properties (e.g., transactivator protein [TAT], human neutrophil peptide 1, transportan 10, and P28 [nsc745104]) and can be applied for cancer treatment alone or together with other therapeutic cargoes.…”