Identification and characterization of a novel cell-penetrating peptide of 30Kc19 protein derived from Bombyx mori

Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have attracted increasing attention due to their high potential to deliver various, otherwise impermeable, bioactive agents, such as drugs and proteins across cell membranes. A number of CPPs have been discovered since then. Recently, 30Kc19 protein has attracted attention because it was the first cell-penetrating protein that has been found in insect hemolymph. Here, we report a cell-penetrating peptide derived from 30Kc19 protein, VVNKLIRNNKMNC, which efficiently penetrates cells when supplemented to medium for mammalian cell culture. Moreover, like other CPPs, this “Pep-c19” also efficiently delivered cell-impermeable cargo proteins, such as green fluorescent protein (GFP) into cells. In addition to the in vitro system, Pep-c19 exhibited the cell-penetrating property in vivo. When Pep-c19 was intraperitoneally injected into mice, Pep-c19 successfully delivered cargo proteins into various organ tissues with higher efficiency than the 30Kc19 protein itself, and without toxicity. Our data demonstrates that Pep-c19 has a great potential as a cell-penetrating peptide that can be used as a therapeutic tool to efficiently deliver different cell-impermeable cargo molecules into the tissues of various organs.     

Human-protein-derived peptides for intracellular delivery of biomolecules

Access of therapeutic biomolecules to cytoplasmic and nuclear targets is hampered by the inability of these molecules to cross biological membranes. Approaches to overcome this hurdle involve CPPs (cell-penetrating peptides) or protein transduction domains. Most of these require rather high concentrations to elicit cell-penetrating functionality, are non-human, pathogen-derived or synthetic entities, and may therefore not be tolerated or even immunogenic. We identified novel human-protein-derived CPPs by a combination of in silico and experimental analyses: polycationic CPP candidates were identified in an in silico library of all 30-mer peptides of the human proteome. Of these peptides, 60 derived from extracellular proteins were evaluated experimentally. Cell viability and siRNA (small interfering RNA) transfection assays revealed that 20 out of the 60 peptides were functional. Three of these showed CPP functionality without interfering with cell viability. A peptide derived from human NRTN (neurturin), which contains an α-helix, performed the best in our screen and was uniformly taken up by cultured cells. Examples for payloads that can be delivered to the cytosol by the NRTN peptide include complexed siRNAs and both N- and C-terminally fused pro-apoptotic peptides.     

Cell-penetrating peptides: from molecular mechanisms to therapeutics

The recent discovery of new potent therapeutic molecules which do not reach the clinic due to poor delivery and low bioavailability have made the delivery of molecules a keystone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including CPPs (cell-penetrating peptides), which represent a new and innovative concept to bypass the problem of bioavailability of drugs. CPPs constitute very promising tools and have been successfully applied for in vivo. Two CPP strategies have been described to date; the first one requires chemical linkage between the drug and the carrier for cellular drug internalization, and the second is based on the formation of stable complexes with drugs, depending on their chemical nature. The Pep and MPG families are short amphipathic peptides, which form stable nanoparticles with proteins and nucleic acids respectively. MPG- and Pep-based nanoparticles enter cells independently of the endosomal pathway and efficiently deliver cargoes, in a fully biologically active form, into a large variety of cell lines, as well as in animal models. This review focuses on the structure-function relationship of non-covalent MPG and Pep-1 strategies, and their requirement for cellular uptake of biomolecules and applications in cultured cells and animal models.     

综述: 细胞穿透肽及其结构改造在siRNA传递中的应用

小RNA药物应用于临床的主要技术瓶颈在于如何高效、低毒地将小RNA分子传递到它发挥功能的场所．基于细胞穿透肽在小RNA透皮给药的临床应用中所取得的进展,本文系统评述了近年来细胞穿透肽在小RNA的体内、体外传递方面的研究动态,分析了细胞穿透肽的结构改造对肽/小RNA复合物转染进入细胞发挥功能的影响,展望了细胞穿透肽作为小RNA的体内药物传递载体的发展方向．     

细胞穿膜肽研究应用的新进展

细胞穿膜肽(Cell-penetrating peptides,CPPs)是一类能够穿过细胞膜或组织屏障的短肽。CPPs可通过内吞和直接穿透等机制运载蛋白质、RNA、DNA等生物大分子进入细胞内发挥其效应功能。相比于其他非天然的化学分子,CPPs具有生物相容性佳、对细胞造成的毒性小、完成入胞转运后可降解、并能与生物活性蛋白直接融合重组表达等优点,因此成为以胞内分子为靶标的药物递送技术发展的重要工具,并在生物医学研究领域具有良好的应用前景。文中针对CPPs的分类特点、入胞转运机制及其治疗应用的新近研究进展进行综述和讨论。     

Arginine-rich cell-penetrating peptides deliver gene into living human cells

Transgenesis is a process that introduces exogenous nucleic acids into the genome of an organism to produce desired traits or evaluate function. Improvements of transgenic technologies are always important pursuit in the last decades. Recently, cell-penetrating peptides (CPPs) were studied as shuttles that can internalize into cells directly and serve as carriers to deliver different cargoes into cells. In the present study, we evaluate whether arginine-rich CPPs can be used for gene delivery into human cells in a noncovalent fashion. We demonstrate that three arginine-rich CPPs (SR9, HR9, and PR9) are able to transport plasmid DNA into human A549 cells. For the functional gene assay, the CPP-delivered plasmid DNA containing the enhanced green fluorescent protein (EGFP) coding sequence could be actively expressed in cells. The treatment of calcium chloride did not facilitate the CPP-mediated transfection efficiency, but enhance the gene expression intensity. Mechanistic studies further revealed that HR9/DNA complexes mediate the direct membrane translocation pathway for gene delivery. Our results suggest that arginine-rich CPPs, especially HR9, appear to be a high efficient and promising tool for gene transfer.     

Properties of cell penetrating peptides (CPPs)

Different approaches have been developed for the introduction of macromolecules, proteins and DNA into target cells. Viral (retroviruses, lentiviruses, etc.) and nonviral (liposomes, bioballistics etc.) vectors as well as lipid particles have been tested as DNA delivery systems. However, all of them share several undesirable effects that are difficult to overcome, such as unwanted immunoresponse and limited cell targeting. The discovery of the cell penetrating peptides (CPPs) showing properties of macromolecules carriers and enhancers of viral vectors, opened new opportunities for the delivery of biologically active cargos, including therapeutically relevant genes into various cells and tissues. This review summarizes recent data about the best characterized CPPs as well as those sharing cell-penetrating and cargo delivery properties despite differing in the primary sequence. The putative mechanisms of CPPs penetration into cells and interaction with intracellular structures such as chromosomes, cytoskeleton and centrioles are addressed. We further discuss recent developments in overcoming the lack of cells specificity, one of the main obstacles for CPPs application in gene therapy. In particular, we review a newly discovered affinity of CPPs to actively proliferating cells.     

Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells

Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.     

A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides

Most bioactive macromolecules, such as protein, DNA and RNA, basically cannot permeate into cells freely from outside the plasma membrane. Cell-penetrating peptides (CPPs) are a group of short peptides that possess the ability to traverse the cell membrane and have been considered as candidates for mediating gene and drug delivery into living cells. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) are able to form stable complexes with plasmid DNA and deliver DNA into insect Sf9 cells in a noncovalent manner. The transferred plasmid DNA containing enhanced green fluorescent protein (EGFP) and red fluorescent protein (RFP) coding regions could be expressed in cells functionally assayed at both the protein and RNA levels. Furthermore, treatment of cells with CPPs and CPP/DNA complexes resulted in a viability of 84-93% indicating these CPPs are not cytotoxic. These results suggest that arginine-rich CPPs appear to be a promising tool for insect transgenesis.     

Secondary structure of cell-penetrating peptides controls membrane interaction and insertion

The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.     

Transduction and transfection of difficult-to-transfect cells: Systematic attempts for the transfection of protozoa Leishmania

Cell-penetrating peptides (CPPs) are used to internalize different cargoes, including DNA, into live mammalian and plant cells. Despite many cells being easily transfected with this approach, other cells are rather “difficult” or “hard to transfect,” including protist cells of the genus Leishmania. Based on our previous results in successfully internalizing proteins into Leishmania tarentolae cells, we used single CPPs and three different DNA-binding proteins to form protein-like complexes with plasmids covered with CPPs. We attempted magnetofection, electroporation, and transfection using a number of commercially available detergents. While complex formation with negatively charged DNA required substantially higher amounts of CPPs than those necessary for mostly neutral proteins, the cytotoxicity of the required amounts of CPPs and auxiliaries was thoroughly studied. We found that Leishmania cells were indeed susceptible to high concentrations of some CPPs and auxiliaries, although in a different manner compared with that for mammalian cells. The lack of successful transfections implies the necessity to accept certain general limitations regarding DNA internalization into difficult-to-transfect cells. Only electroporation allowed reproducible internalization of large and rigid plasmid DNA molecules through electrically disturbed extended membrane areas, known as permeable membrane macrodomains.     

Gene transport and expression by arginine-rich cell-penetrating peptides in Paramecium

Owing to the cell membrane barriers, most macromolecules and hydrophilic molecules could not freely enter into living cells. However, cell-penetrating peptides (CPPs) have been discovered that can translocate themselves and associate cargoes into the cytoplasm. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9 and PR9) can form stable complexes with plasmid DNA at the optimized nitrogen/phosphate ratio of 3 and deliver plasmid DNA into Paramecium caudatum in a noncovalent manner. Accordingly, the transported plasmid encoding the green fluorescent protein (GFP) gene could be expressed in cells functionally assayed at both the protein and DNA levels. The efficiency of gene delivery varied among these CPPs in the order of HR9 > PR9 > SR9. In addition, these CPPs and CPP/DNA complexes were not cytotoxic in Paramecium detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diohenyltetrazolium bromide (MTT) assay. Thus, these results suggest that the functionality of arginine-rich CPPs offers an efficient and safe tool for transgenesis in eukaryotic protozoans.     

Prediction of Cell-Penetrating Peptides

Cell-penetrating peptides, CPPs, are used as delivery vectors for pharmacologically interesting substances, such as antisense oligonucleotides, proteins and peptides. We present a general principle for designing cell-penetrating peptides derived from naturally occurring proteins as well as from randomly generated polyamino acid sequences. Thereby, we introduce a novel pharmacological principle for identification of cell-penetrating peptides for which the applications can be numerous, including cellular transduction vectors and mimics of intracellular protein–protein interactions. The methods of identifying a CPP comprises assessing the averaged bulk property values of the defined sequence, and ensuring that they fall within the bulk property value interval obtained from the training set. Despite this simplistic approach, the search criteria proved useful for finding CPP properties in either proteins or random sequences. We have experimentally verified cell-penetrating properties of 10–20-mer peptides derived from naturally occurring proteins as well as from random poly-amino acids. We note that since CPPs can be found in part of the protein sequences that may govern protein interactions, it is possible to produce cell-penetrating protein agonists or antagonists.     

Intelligent substance delivery into cells using cell-penetrating peptides

Cell-penetrating peptides (CPPs) are oligopeptides that can permeate the cell membrane. The use of a CPP-mediated transport system could be an excellent method for delivering cell-impermeable substances such as proteins, antibodies, antisense oligonucleotides, siRNAs, plasmids, drugs, fluorescent compounds, and nanoparticles as covalently or noncovalently conjugated cargo into cells. Nonetheless, the mechanisms through which CPPs are internalized remain unclear. Endocytosis and direct translocation through the membrane are the generally accepted routes. Internalization via both pathways can occur simultaneously, depending on cellular conditions. However, the peculiar property of CPPs has attracted many researchers, especially in drug discovery or development, who intend to deliver impermeable substances into cells through the cell membrane. The delivery of drugs using CPPs may non-invasively solve the problem of drug penetration into cells with the added benefit of low cytotoxicity. Moreover, macromolecules can also be delivered by this transport system. In this review, I discuss the possibilities and advantages of substance delivery into cells using CPPs.     

Delivery of HIV-1 Nef Protein in Mammalian Cells Using Cell Penetrating Peptides as a Candidate Therapeutic Vaccine

The HIV-1 Nef protein expressed early in viral life cycle has been known as a potent candidate for therapeutic vaccine development. Due to different cell barriers, various cell penetrating peptides (CPPs) such as Pep-1 and CADY-2 have been known to deliver biologically active proteins to cytoplasmic compartments via the plasma membrane. In current study, we firstly evaluated the efficiency of lentiviral vector (pCDH-CMV-MCS-EF1-cGFP-T2A-puro) and eukaryotic expression vector (pEGFP-N1) for expression of HIV-1 Nef protein in HEK-293T cells using TurboFect transfection reagent. Our results showed that both vectors can effectively express the Nef proteins within the target cell. The pEGFP-N1 was more effective than pCDH-GFP for protein expression. Furthermore, Nef protein was expressed in E. coli as GST-Nef fusion and transfected by the amphipathic CPPs including Pep-1 and CADY-2 into HEK-293T cells. The size and morphology of the GST-Nef/CPP complexes were evaluated by scanning electron microscopy, and Zetasizer. Our data indicated that the recombinant GST-Nef protein generated in BL21 strain migrated as a clear band of ~50 kDa in SDS-PAGE. The CPP/GST-Nef nanoparticles were formed with a diameter of below 200 nm and notably delivered into HEK-293T cells. Generally, the Nef protein was expressed in prokaryotic and eukaryotic expression systems using different vectors and efficiently transfected in mammalian cells using various delivery systems. The in vitro efficient delivery of HIV-1 Nef gene and also its protein supports the potential of Nef DNA constructs and CPPs as potent carriers of Nef protein for HIV vaccine design in Future.     

Protein transduction of an antioxidant enzyme: subcellular localization of superoxide dismutase fusion protein in cells

In protein therapy, it is important for exogenous protein to be delivered into the target subcellular localization. To transduce a therapeutic protein into its specific subcellular localization, we synthesized nuclear localization signal (NLS) and membrane translocation sequence signal (MTS) peptides and produced a genetic in-frame SOD fusion protein. The purified SOD fusion proteins were efficiently transduced into mammalian cells with enzymatic activities. Immunofluorescence and Western blot analysis revealed that the SOD fusion proteins successfully transduced into the nucleus and the cytosol in the cells. The viability of cells treated with paraquat was markedly increased by the transduced fusion proteins. Thus, our results suggest that these peptides should be useful for targeting the specific localization of therapeutic proteins in various human diseases.     

siRNA delivery using amphipathic cell-penetrating peptides into human hepatoma cells

Cell-penetrating peptides (CPPs) are an attractive tool for delivering membrane-impermeable compounds, including anionic biomacromolecules such as DNA and RNA, into living cells. Amphipathic helical peptides composed of hydrophobic amino acids and cationic amino acids are typical CPPs. In the current study, we designed amphipathic helical 12-mer peptides containing α,α-disubstituted α-amino acids (dAAs), which are known to stabilize peptide secondary structures. The dominant secondary structures of peptides in aqueous solution differed according to the introduced dAAs. Peptides containing hydrophobic dAAs and adopting a helical structure exhibited a good cell-penetrating ability. As an application of amphipathic helical peptides, small interfering RNA (siRNA) delivery into living human hepatoma cells was investigated. One of the peptides containing dAAs dipropylglycine formed stable complexes with siRNA at appropriate zeta-potential and size for intracellular siRNA delivery. This peptide showed effective RNA interference efficiency at short peptide length and low concentrations of peptide and siRNA. These findings will be helpful for the design of amphipathic helical CPPs as intracellular siRNA delivery.