Arginine-rich Intracellular Delivery Peptide Research Articles

Cellular Internalization of Quantum Dots Noncovalently Conjugated with Arginine-Rich Cell-Penetrating Peptides

Protein transduction domains comprised of basic amino acid-rich peptides, can efficiently deliver covalently fused macromolecules into cells. Quantum dots (QDs) are luminescent semiconductor nanocrystals that are finding increasing application in biological imaging. Previous studies showed that protein transduction domains mediate the internalization of covalently attached QDs. In this study, we demonstrate that arginine-rich intracellular delivery peptides (cell-penetrating peptides; CPPs), analogs of naturally-occuring protein transduction domains, deliver noncovalently associated QDs into living cells; CPPs dramatically increase the rate and efficiency of cellular uptake of QD probes. The optimal molecular ratio between arginine-rich CPPs and QD cargoes for cellular internalization is approximately 60:1. Upon entry into cells, the QDs are concentrated in the perinuclear region. There is no cytotoxicity following transport of QDs present at concentrations up to 200 nM. The mechanism for arginine-rich CPP/QD complexes to traverse cell membrane appears to involve a combination of internalization pathways. These results provide insight into the mechanism of arginine-rich CPP delivery of noncovalently attached cargoes, and may provide a powerful tool for imaging in vivo.

Arginine-Rich Intracellular Delivery Peptides Synchronously Deliver Covalently and Noncovalently Linked Proteins into Plant Cells

Protein transduction domains (PTDs) are small peptides with a high content of basic amino acids, and they are responsible for cellular uptake. Many PTDs, including arginine-rich intracellular delivery (AID) peptides, have been shown to transport macromolecules across membranes and into cells. In this study, we demonstrated for the first time that AID peptides could rapidly and efficiently deliver proteins into plant cells in both covalent and noncovalent protein transductions (CNPT) simultaneously. The optimal molecular ratio between an AID peptide carrier and cargo in CNPT was about 3:1. Fluorescence resonance energy transfer (FRET) analysis revealed protein-protein interactions between AID peptide carriers and cargos after CNPT in cells. The possible mechanisms of AID peptides-mediated cellular entry might involve a combination of multiple internalization pathways. Therefore, applications by AID peptide-mediated CNPT may provide a simple and direct transport strategy for delivering two proteins in agricultural systems.

Protein transport in human cells mediated by covalently and noncovalently conjugated arginine-rich intracellular delivery peptides

Generally, biomacromolecules, such as DNA, RNA, and proteins, cannot freely permeate into cells from outside the membrane. Protein transduction domains (PTDs) are peptides containing a large number of basic amino acids that can deliver macromolecules into living cells. Arginine-rich intracellular delivery (AID) peptides are more effective than other PTD peptides at carrying large molecules across cellular membranes. In the present study, we demonstrated that AID peptides are able to deliver cargo proteins into living cells in both covalent and noncovalent protein transductions (CNPT) synchronously. Human A549 cells were treated with a fluorescent protein (FP) that was noncovalently premixed with another AID-conjugated FP, which emitted a different color. After the delivery of carrier AID-FP and cargo FP into cells, the emission and merge of florescence were observed and recorded with a confocal microscope, while the internalization efficiency was quantitatively analyzed with a flow cytometer. The optimal molecular ratio between carrier AID-FP and cargo FP for CNPT is about 1:1/3. Fluorescence resonance energy transfer (FRET) assay further confirmed AID-conjugates can physically interact with its cargo FPs in CNPT in cells. Potential uptake mechanisms of CNPT may involve a combination of multiple internalization pathways. After delivery, intracellular distributions of AID-conjugates and FPs may possibly colocalize with lysosomes. These results will facilitate the understanding of multiple mechanisms of PTDs, and provide a powerful tool for simultaneously delivering several proteins or compounds in protein internalization.

Cell Membrane Diversity in Noncovalent Protein Transduction

Crossing of the plasma membrane for all macromolecules without energy, receptors or any artificial methods was thought to be difficult. Our previous studies demonstrated that arginine-rich intracellular delivery (AID) peptides are able to deliver macromolecules, such as proteins, RNAs and DNAs, into either animal or plant cells. Cellular internalization could be mediated by effective and nontoxic AID peptides in either a covalent or noncovalent protein transduction (NPT) manner. AID peptides were so versatile that the procedure seemed to replace the current artificial transfection methods. However, the utilization of AID peptides has been limited to animal or plant systems so far. None has proposed that AID peptides could work in other species. Here, we select some representative organisms to screen whether NPT mediated by AID peptides works in them. They include cyanobacteria, bacteria, archaea, algae, fungi and yeasts. The results reveal that not all living beings possess this capability of protein transduction. Interestingly, all species of prokaryotes tested, which were thought to be highly diverse from the animal and plant systems, appear to be capable of NPT. The mechanism of AID-mediated NPT in cyanobacteria is in a classical endocytosis- and energy-independent pathway and may involve macropinocytosis. In contrast, green algae and multicellular fungi of the eukaryotes are impermeable to protein passage. Our results bring an interesting clue to the reexamination of the phylogeny of both algae and fungi.

Transdermal delivery of proteins mediated by non‐covalently associated arginine‐rich intracellular delivery peptides

Plasma membranes of animal cells are generally impermeable to macromolecules. Protein transduction mediated by protein transduction domains (PTDs) covalently cross-linked to cargoes for cellular internalization has previously been demonstrated. Peptides with PTDs could be an effective way to deliver proteins into living cells or tissues in vitro. In this report, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to facilitate the delivery of proteins into animal cells and to penetrate skin tissues rapidly. This cellular internalization and transdermal delivery of proteins is mediated by non-toxic AID peptides in a non-fusion protein and non-conjugation dependent manner. The efficiency of intracellular transport is further increased in the presence of chemical enhancer oleic acid. The mechanism of the AID-mediated cellular entry may involve macropinocytosis and actin rearrangement. Thus, we confirm that direct delivery of bioactive proteins into living cells and tissues mediated by non-covalent actions of AID peptides represents a useful strategy in pharmaceutics, therapeutics and cosmetics.

A Bacterial Indole-3-acetyl-L-aspartic Acid Hydrolase Inhibits Mung Bean (Vigna radiata L.) Seed Germination Through Arginine-rich Intracellular Delivery

Indole-3-acetyl-L-aspartic acid (IAA-Asp) is a natural product in many plant species and plays many important roles in auxin metabolism and plant physiology. IAA-Asp hydrolysis activity is, therefore, believed to affect plant physiology through changes in IAA metabolism in plants. We applied a newly discovered technique, arginine-rich intracellular delivery (AID), to deliver a bacterial IAA-Asp hydrolase into cells of mung bean (Vigna radiata) seeds and measured its effects on mung bean seed germination. IAA-Asp hydrolase inhibited seed germination about 12 h after the enzyme was delivered into cells of mung bean seeds both covalently and noncovalently. Mung bean seed germination was delayed by 36 h when the enzyme protein was noncovalently attached to the AID peptide and longer than 60 h when the enzyme protein was covalently attached to the AID peptide. Root elongation of mung bean plants was inhibited as much as 90% or 80%, respectively, when the IAA-Asp hydrolase was delivered with the AID peptide by covalent or noncovalent association. Further thin-layer chromatography analysis of plant extracts indicated that the levels of IAA increased about 12 h after treatment and reached their peak at 24 h. This result suggests that IAA-Asp hydrolase may increase IAA levels and inhibit seed germination of mung bean plants and that the AID peptide is a new, rapid, and efficient experimental tool to study the in vivo activity of enzymes of interest in plant cells.

Transfection and expression of plasmid DNA in plant cells by an arginine-rich intracellular delivery peptide without protoplast preparation

The delivery and expression of exogenous genes in plant cells have been of particular interest for plant research and biotechnology. Here, we present results demonstrating a simple DNA transfection system in plants. Short arginine-rich intracellular delivery peptide, a protein transduction domain, was capable of delivering plasmid DNA into living plant cells non-covalently. This peptide-mediated DNA delivery conferred several advantages, such as nuclear targeting, non-toxic effect, and ease of preparation without protoplast formulation. Thus, this novel technology shall provide a powerful tool to investigate gene function in vivo, and lay the foundation for the production of transgenic plants in future.

An intracellular delivery method for siRNA by an arginine-rich peptide

RNA interference has recently become a useful research tool for the studies of gene functions, regulations, and therapies. The double-stranded RNA is utilized to induce the sequence-specific gene silencing. To achieve this goal of specific gene silencing, a proper delivery system of siRNA is highly demanded. A number of approaches for delivering siRNA have been explored over the last few years. In the present study, we demonstrated a simple peptide-based siRNA delivery system in mammalian cells. A GC-EGFP cell line stably expressing enhanced green fluorescent protein was established from stable transfection of human gastric carcinoma cells. The synthetic nona-arginine peptide, an arginine-rich intracellular delivery peptide, or called protein transduction domain peptide, could noncovalently form stable complexes with EGFP siRNA and deliver these mixtures into cells. After entry, siRNA appeared to stay in perinuclear regions within cell, and ultimately fulfilled its targeted egfp gene silencing. These data were in consonance with that RNA-induced silencing complex components could be also localized to these perinuclear regions, creating a focal point for RNA interference factories. In the future, this non-toxic peptide may be proved to be a useful tool for the delivery of exogenous siRNA in RNA interference research.

Noncovalent protein transduction in plant cells by macropinocytosis

* Protein delivery across cellular membranes or compartments is primarily limited by low biomembrane permeability. * Many protein transduction domains (PTDs) have previously been generated, and covalently cross-linked with cargoes for cellular internalization. * An arginine-rich intracellular delivery (AID) peptide could rapidly deliver fluorescent proteins or beta-galactosidase enzyme into plant and animal cells in a noncovalent fashion. The possible mechanism of this noncovalent protein transduction (NPT) may involve macropinocytosis. * The NPT via a nontoxic AID peptide provides a powerful tool characterized by its simplicity and quickness to have active proteins function in living cells in vivo. This should be of broad utility for functional enzyme assays and protein therapies in both plant biology research as well as biomedical applications.

Arginine-rich intracellular delivery peptides noncovalently transport protein into living cells

Plasma membranes of plant or animal cells are generally impermeable to peptides or proteins. Many basic peptides have previously been investigated and covalently cross-linked with cargoes for cellular internalization. In the current study, we demonstrate that arginine-rich intracellular delivery (AID) peptides are able to deliver fluorescent proteins or β-galactosidase enzyme into animal and plant cells, as well as animal tissue. Cellular internalization and transdermal delivery of protein could be mediated by effective and nontoxic AID peptides in a neither fusion protein nor conjugation fashion. Therefore, noncovalent AID peptides may provide a useful strategy to have active proteins function in living cells and tissues in vivo.

Cellular Internalization of Fluorescent Proteins via Arginine-rich Intracellular Delivery Peptide in Plant Cells

The protein delivery across cellular membranes or compartments is limited by low biomembrane permeability because of the hydrophobic characteristics of cell membranes. Usually the delivery processes utilize passive protein channels or active transporters to overcome the membrane impediment. In this report, we demonstrate that arginine-rich intracellular delivery (AID) peptide is capable of efficiently delivering fused fluorescent proteins unpreferentially into different plant tissues of both tomato (a dicot plant) and onion (a monocot plant) in a fully bioactive form. Thus, cellular internalization via AID peptide can be a powerful tool characterized by its simplicity, non-invasion and high efficiency to express those bioactive proteins in planta or in plant cells in vivo. This novel method may alternatively provide broader applications of AID chimera in plants without the time-consuming transgenic approaches.