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Molecular dynamics study of the internalization of cell-penetrating peptides containing unnatural amino acids across membranes.

ABSTRACT: Peptide-based delivery systems that deliver target molecules into cells have been gaining traction. These systems need cell-penetrating peptides (CPPs), which have the remarkable ability to penetrate into biological membranes and help internalize different cargoes into cells through the cell membranes. The molecular internalization mechanism and structure-function relationships of CPPs are not clear, although the incorporation of nonproteinogenic amino acids such as α-aminoisobutyric acid (Aib) has been reported to increase their helicity, biostability and penetration efficiencies. Here, we used molecular dynamics to study two Aib-containing CPPs, poly(LysAibAla)₃ (KAibA) and poly(LysAibGly)₃ (KAibG), that previously showed high cell internalization efficiency. KAibA and KAibG displayed the lowest internalization energies among the studied CPPs, showing distinct internalization mechanisms depending on the lipid composition of the model membranes. The presence of Aib residues allows these CPPs to adopt amphipathic folding to efficiently penetrate through the membranes. Elucidating how Aib incorporation affects CPP-membrane binding and interactions is beneficial for the design of CPPs for efficient intracellular delivery.

SUBMITTER: Gimenez-Dejoz J

PROVIDER: S-EPMC9419563 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Molecular dynamics study of the internalization of cell-penetrating peptides containing unnatural amino acids across membranes.

Gimenez-Dejoz Joan J Numata Keiji K

Nanoscale advances 20211110 2

Peptide-based delivery systems that deliver target molecules into cells have been gaining traction. These systems need cell-penetrating peptides (CPPs), which have the remarkable ability to penetrate into biological membranes and help internalize different cargoes into cells through the cell membranes. The molecular internalization mechanism and structure-function relationships of CPPs are not clear, although the incorporation of nonproteinogenic amino acids such as α-aminoisobutyric acid (Aib) ...[more]

PMID: 36132688

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Project description: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).

Dataset Information

Molecular dynamics study of the internalization of cell-penetrating peptides containing unnatural amino acids across membranes.

Publications

Molecular dynamics study of the internalization of cell-penetrating peptides containing unnatural amino acids across membranes.

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