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Genetically modified plants and crops can contribute to remarkable increase in global food supply, with improved yield and resistance to plant diseases or insect pests. The development of biotechnology introducing exogenous nucleic acids in transgenic plants is important for plant health management. Different genetic engineering methods for DNA delivery, such as biolistic methods, Agrobacterium tumefaciens -mediated transformation, and other physicochemical methods have been developed to improve translocation across the plasma membrane and cell wall in plants. Recently, the peptide-based gene delivery system, mediated by cell-penetrating peptides (CPPs), has been regarded as a promising non-viral tool for efficient and stable gene transfection into both animal and plant cells. CPPs are short peptides with diverse sequences and functionalities, capable of agitating plasma membrane and entering cells. Here, we highlight recent research and ideas on diverse types of CPPs, which have been applied in DNA delivery in plants. Various basic, amphipathic, cyclic, and branched CPPs were designed, and modifications of functional groups were performed to enhance DNA interaction and stabilization in transgenesis. CPPs were able to carry cargoes in either a covalent or noncovalent manner and to internalize CPP/cargo complexes into cells by either direct membrane translocation or endocytosis. Importantly, subcellular targets of CPP-mediated nucleic acid delivery were reviewed. CPPs offer transfection strategies and influence transgene expression at subcellular localizations, such as in plastids, mitochondria, and the nucleus. In summary, the technology of CPP-mediated gene delivery provides a potent and useful tool to genetically modified plants and crops of the future.

Cell-penetrating peptides (CPPs) are small peptides which help intracellular delivery of functional macromolecules, including DNAs, RNAs, and proteins, across the cell membrane and into the cytosol, and even into the nucleus in some cases. Delivery of macromolecules can facilitate transfection, aid in gene therapy and transgenesis, and alter gene expression. L5a (RRWQW), originally derived from bovine lactoferricin, is one kind of CPPs which can promote cellular uptake of plasmid DNA and enters cells via direct membrane translocation. The peptide complexes noncovalently with DNA over a short incubation period. DNA plasmid and L5a complex stability is confirmed by a decrease in mobility in a gel retardation assay, and successful transfection is proven by the detection of a reporter gene in cells using fluorescent microscopy. Here, we describe methods to study noncovalent interactions between L5a and plasmid DNA, and the delivery of L5a/DNA complexes into cells. L5a is the one of the smallest CPPs discovered to date, providing a small delivery vehicle for macromolecules in mammalian cells. A small vehicle which can enter the nucleus is ideal for efficient gene uptake, transfer, and therapy. It is simple to complex with DNA plasmids, and its nature allows mammalian cells to be easily transfected.

The bovine lactoferricin L6 (RRWQWR) has been previously identified as a novel cell-penetrating peptide (CPP) that is able to efficiently internalize into human cells. L6 interacts with quantum dots (QDs) noncovalently to generate stable L6/QD complexes that enter cells by endocytosis. In this study, we demonstrate a modified L6 (HL6; CHHHHHRRWQWRHHHHHC), in which short polyhistidine peptides are introduced into both flanks of L6, has enhanced cell-penetrating ability in human bronchoalveolar carcinoma A549 cells. The mechanism of cellular uptake of HL6/QD complexes is primarily direct membrane translocation rather than endocytosis. Dimethyl sulfoxide (DMSO), but not pyrenebutyrate (PB), ethanol, oleic acid, or 1,2-benzisothiazol-3(2 H)-one (BIT), slightly enhances HL6-mediated protein transduction efficiency. Neither HL6 nor HL6/QD complexes are cytotoxic to A549 or HeLa cells. These results indicate that HL6 could be a more efficient drug carrier than L6 for biomedical as well as biotechnological applications, and that the function of polyhistidine peptides is critical to CPP-mediated protein transduction.

Cell-penetrating peptides (CPPs) containing a preponderance of basic amino acids are able to deliver biologically active macromolecules and nanomaterials into live cells. Quantum dots (QDs) are nanoparticles with unique fluorescence properties that have found wide application in biomedical imaging. In this study, we demonstrate transduction of an L6 CPP (RRWQWR) derived from bovine lactoferricin (LFcin) into human lung cancer cells. L6 noncovalently interacts with QDs to form stable complexes. L6/QD complexes enter cells most efficiently when prepared at a nitrogen/phosphate ratio of 60. Mechanistic studies indicate that L6/QD complexes enter cells by endocytosis. Treatment with 1,2-benzisothiazol-3(2 H )-one (BIT), an industrial preservative that enhances uptake of certain CPPs, does not affect L6 CPP-mediated protein transduction efficiency. L6 and L6/QD complexes are not cytotoxic. These results indicate that L6 LFcin might be an efficient and safe nanoshuttle for nanoparticles or nanomedicines in biomedical applications.

Background: Development of effective drug delivery systems (DDS) is a critical issue in health care and medicine. Advances in molecular biology and nanotechnology have allowed the introduction of nanomaterial-based drug delivery systems. Cell-penetrating peptides (CPPs) can form the basis of drug delivery systems by virtue of their ability to support the transport of cargoes into the cell. Potential cargoes include proteins, DNA, RNA, liposomes, and nanomaterials. These cargoes generally retain their bioactivities upon entering cells., Method: In the present study, the smallest, fully-active lactoferricin-derived CPP, L5a is used to demonstrate the primary contributor of cellular internalization., Results: The secondary helical structure of L5a encompasses symmetrical positive charges around the periphery. The contributions of cell-specificity, peptide length, concentration, zeta potential, particle size, and spatial structure of the peptides were examined, but only zeta potential and spatial structure affected protein transduction efficiency. FITC-labeled L5a appeared to enter cells via direct membrane translocation insofar as endocytic modulators did not block FITC-L5a entry. This is the same mechanism of protein transduction active in Cy5 labeled DNA delivery mediated by FITC-L5a. A significant reduction of transduction efficiency was observed with structurally incomplete FITC-L5a formed by tryptic destruction, in which case the mechanism of internalization switched to a classical energydependent endocytosis pathway., Conclusion: These results support the continued development of the non-cytotoxic L5a as an efficient tool for drug delivery., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

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.

Bacterial and archaeal cell envelopes are complex multilayered barriers that serve to protect these microorganisms from their extremely harsh and often hostile environments. Import of exogenous proteins and nanoparticles into cells is important for biotechnological applications in prokaryotes. In this report, we demonstrate that cell-penetrating peptides (CPPs), both bacteria-expressed nona-arginine peptide (R9) and synthetic R9 (SR9), are able to deliver noncovalently associated proteins or quantum dots into four representative species of prokaryotes: cyanobacteria (Synechocystis sp. PCC 6803), bacteria (Escherichia coli DH5α and Arthrobacter ilicis D-50), and archaea (Thermus aquaticus). Although energy-dependent endocytosis is generally accepted as a hallmark that distinguishes eukaryotes from prokaryotes, cellular uptake of uncomplexed green fluorescent protein (GFP) by cyanobacteria was mediated by classical endocytosis. Mechanistic studies revealed that macropinocytosis plays a critical and major role in CPP-mediated protein transduction in all four prokaryotes. Membrane damage was not observed when cyanobacterial cells were treated with R9/GFP complexes, nor was cytotoxicity detected when bacteria or archaea were treated with SR9/QD complexes in the presence of macropinocytic inhibitors. These results indicate that the uptake of protein is not due to a compromise of membrane integrity in cyanobacteria, and that CPP can be an effective and safe carrier for membrane trafficking in prokaryotic cells. Our investigation provides important new insights into the transport of exogenous proteins and nanoparticles across the complex membrane systems of prokaryotes.

Nanoparticles, such as semiconductor quantum dots (QDs), have been found increasing use in biomedical diagnosis and therapeutics because of their unique properties, including quantum confinement, surface plasmon resonance, and superparamagnetism. Cell-penetrating peptides (CPPs) represent an efficient mechanism to overcome plasma membrane barriers and deliver biologically active molecules into cells. In this study, we demonstrate that three arginine-rich CPPs (SR9, HR9, and PR9) can noncovalently complex with red light emitting QDs, dramatically increasing their deliv- ery into living cells. Zeta-potential and size analyses highlight the importance of electrostatic interactions between positive-charged CPP/QD complexes and negative-charged plasma membranes indicating the efficiency of transmembrane complex transport. Subcellular colocalization indicates associations of QD with early endosomes and lysosomes following PR9-mediated delivery. Our study demonstrates that nontoxic CPPs of varied composition provide an effective vehicle for the design of optimized drug delivery systems.

Many viral and nonviral systems have been developed to aid delivery of biologically active molecules into cells. Among these, cell-penetrating peptides (CPPs) have received increasing attention in the past two decades for biomedical applications. In this review, we focus on opportunities and challenges associated with CPP delivery of nucleic acids and nanomaterials. We first describe the nature of versatile CPPs and their interactions with various types of cargoes. We then discuss in vivo and in vitro delivery of nucleic acids and nanomaterials by CPPs. Studies on the mechanisms of cellular entry and limitations in the methods used are detailed.

Cellular and nuclear delivery of biomolecules is limited by low membrane permeability. Cell-penetrating peptides (CPPs) can be covalently linked to cargos to improve cellular internalization. Our work indicates that arginine-rich CPPs are also able to interact with a variety of cargos, including DNA, RNA, proteins and nanomaterials, in a noncovalent manner and subsequently effect their delivery into cells. The advantages of noncovalent attachment in CPP-mediated transduction are multiple: ease of use, ease of production, and versatility with respect to both cargo composition and functional delivery (i.e., the cargo is not chemically modified). We have extended this approach to achieve simultaneous transduction of covalently and noncovalently associated complexes, opening a new method for delivering multiple types of cargos, including proteins, fluorescent nanomaterials, nucleic acid and others. These novel variations of CPP-mediated transport should be of broad utility in the transport of genes, small interfering RNAs, proteins and nanoparticles in biomedical research and therapeutic intervention.

Semiconductor nanoparticles, also known as quantum dots (QDs), are widely used in biomedical imaging studies and pharmaceutical research. Cell-penetrating peptides (CPPs) are a group of small peptides that are able to traverse cell membrane and deliver a variety of cargoes into living cells. CPPs deliver QDs into cells with minimal nonspecific absorption and toxic effect. In this study, water-soluble, monodisperse, carboxyl-functionalized indium phosphide (InP)/zinc sulfide (ZnS) QDs coated with polyethylene glycol lipids (designated QInP) were synthesized for the first time. The physicochemical properties (optical absorption, fluorescence and charging state) and cellular internalization of QInP and CPP/QInP complexes were characterized. CPPs noncovalently interact with QInP in vitro to form stable CPP/QInP complexes, which can then efficiently deliver QInP into human A549 cells. The introduction of 500nM of CPP/QInP complexes and QInP at concentrations of less than 1μM did not reduce cell viability. These results indicate that carboxylated and polyethylene-glycolylated (PEGylated) bifunctionalized QInP are biocompatible nanoparticles with potential for use in biomedical imaging studies and drug delivery applications., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cell-penetrating peptides (CPPs) are a group of short, membrane-permeable cationic peptides that represent a nonviral technology for delivering nanomaterials and macromolecules into live cells. In this study, two arginine-rich CPPs, HR9 and IR9, were found to be capable of entering rotifers. CPPs were able to efficiently deliver noncovalently associated with cargoes, including plasmid DNAs, red fluorescent proteins (RFPs), and semiconductor quantum dots, into rotifers. The functional reporter gene assay demonstrated that HR9-delivered plasmid DNAs containing the enhanced green fluorescent protein and RFP coding sequences could be actively expressed in rotifers. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan assay further confirmed that CPP-mediated cargo delivery was not toxic to rotifers. Thus, these two CPPs hold a great potential for the delivery of exogenous genes, proteins, and nanoparticles in rotifers.

Cell-penetrating peptides (CPPs) can traverse cellular membranes and deliver biologically active molecules into cells. In this study, we demonstrate that CPPs comprised of nona-arginine (R9) and a penetration accelerating peptide sequence (Pas) that facilitates escape from endocytic lysosomes, denoted as PR9, greatly enhance the delivery of noncovalently associated quantum dots (QDs) into human A549 cells. Mechanistic studies, intracellular trafficking analysis and a functional gene assay reveal that endocytosis is the main route for intracellular delivery of PR9/QD complexes. Endocytic trafficking of PR9/QD complexes was monitored using both confocal and transmission electron microscopy (TEM). Zeta-potential and size analyses indicate the importance of electrostatic forces in the interaction of PR9/QD complexes with plasma membranes. Circular dichroism (CD) spectroscopy reveals that the secondary structural elements of PR9 have similar conformations in aqueous buffer at pH 7 and 5. This study of nontoxic PR9 provides a basis for the design of optimized cargo delivery that allows escape from endocytic vesicles.

Cell-penetrating peptides (CPPs) comprised of basic amino residues are able to cross cytoplasmic membranes and are able to deliver biologically active molecules inside cells. However, CPP/cargo entrapment in endosome limits biomedical utility as cargoes are destroyed in the acidic environment. In this study, we demonstrate protein transduction of a novel CPP comprised of an INF7 fusion peptide and nona-arginine (designated IR9). IR9 noncovalently interacts with quantum dots (QDs) and DNAs to form stable IR9/QD and IR9/DNA complexes which are capable of entering human A549 cells. Zeta-potentials were a better predictor of transduction efficiency than gel shift analysis, emphasizing the importance of electrostatic interactions of CPP/cargo complexes with plasma membranes. Mechanistic studies revealed that IR9, IR9/QD and IR9/DNA complexes may enter cells by endocytosis. Further, IR9, IR9/QD and IR9/DNA complexes were not cytotoxic at concentrations below 30, 5 and 20.1 µM, respectively. Without labor intensive production of fusion proteins from prokaryotes, these results indicate that IR9 could be a safe carrier of genes and drugs in biomedical applications.

Background: The plasma membrane plays an essential role in selective permeability, compartmentalization, osmotic balance, and cellular uptake. The characteristics and functions of cyanobacterial membranes have been extensively investigated in recent years. Cell-penetrating peptides (CPPs) are special nanocarriers that can overcome the plasma membrane barrier and enter cells directly, either alone or with associated cargoes. However, the cellular entry mechanisms of CPPs in cyanobacteria have not been studied., Results: In the present study, we determine CPP-mediated transduction efficiency and internalization mechanisms in cyanobacteria using a combination of biological and biophysical methods. We demonstrate that both Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 strains of cyanobacteria possess red autofluorescence. Green fluorescent protein (GFP), either alone or noncovalently associated with a CPP comprised of nine arginine residues (R9/GFP complexes), entered cyanobacteria. The ATP-depleting inhibitor of classical endocytosis, N-ethylmaleimide (NEM), could block the spontaneous internalization of GFP, but not the transduction of R9/GFP complexes. Three specific inhibitors of macropinocytosis, cytochalasin D (CytD), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and wortmannin, reduced the efficiency of R9/GFP complex transduction, indicating that entry of R9/GFP complexes involves macropinocytosis. Both the 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) and membrane leakage analyses confirmed that R9/GFP complexes were not toxic to the cyanobacteria, nor were the endocytic and macropinocytic inhibitors used in these studies., Conclusions: In summary, we have demonstrated that cyanobacteria use classical endocytosis and macropinocytosis to internalize exogenous GFP and CPP/GFP proteins, respectively. Moreover, the CPP-mediated delivery system is not toxic to cyanobacteria, and can be used to investigate biological processes at the cellular level in this species. These results suggest that both endocytic and macropinocytic pathways can be used for efficient internalization of regular protein and CPP-mediated protein delivery in cyanobacteria, respectively.

Endocytosis has been proposed as one of the primary mechanisms for cellular entry of cell-penetrating peptides (CPPs) and their cargoes. However, a major limitation of endocytic pathway is entrapment of the CPP-cargo in intracellular vesicles from which the cargo must escape into the cytoplasm to exert its biological activity. Here we demonstrate that a CPP tagged with an endosomolytic fusion peptide derived from the influenza virus hemagglutinin-2 (HA2) remarkably enhances the cytosolic delivery of proteins in human A549 cells. To determine the endosome-disruptive effects, recombinant DNA plasmids containing coding sequences of HA2, CPPs and red fluorescent proteins (RFPs) were constructed. The fusion proteins were purified from plasmid-transformed Escherichia coli, and their effects on protein transduction were examined using live cell imaging and flow cytometry. Our data indicate that endocytosis is the major route for cellular internalization of CPP-HA2-tagged RFP. Mechanistic studies revealed that the fusogenic HA2 peptide dramatically facilitates CPP-mediated protein entry through the release of endocytosed RFPs from endosomes into the cytoplasm. Furthermore, incorporating the HA2 fusion peptide of the CPP-HA2 fusion protein improved cytosolic uptake without causing cytotoxicity. These findings strongly suggest that the CPP-HA2 tag could be an efficient and safe carrier that overcomes endosomal entrapment of delivered therapeutic drugs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The piggyBac (PB) transposable element has recently accumulated enormous attention as a tool for the transgenesis in various eukaryotic organisms. Arginine-rich cell-penetrating peptides (CPPs) are protein transduction domains containing a large amount of basic amino acids that were found to be capable of delivering biologically active macromolecules into living cells. In this study, we demonstrate a strategy, which we called "transposoduction", which is a one-plasmid gene delivery system mediated by the nontoxic CPP-piggyBac transposase (CPP-PBase) fusion protein to accomplish both protein transduction and transposition. CPPs were proven to be able to synchronously deliver covalently linked PBase and noncovalently linked a cis plasmid into human cells. The expression of promoterless reporter genes coding for red (dTomato) and yellow (mOrange) fluorescent proteins (RFP and YFP) with PB elements could be detected in cells treated with the PBase-expressing plasmid after 3 days indicating transposition of coding regions to downstream of endogenous promoter sequences. An enhanced green fluorescent protein (EGFP) plasmid-based excision assay further confirmed the efficiency of the bifunctional CPP-PBase fusion protein. In conclusion, this strategy representing a combinational concept of both protein transduction and mobile transposition may provide tremendous potential for safe and efficient cell line transformation, gene therapy and functional genomics., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Functional peptides that transfer biomaterials, such as semiconductor quantum dots (QDs), into cells in biomaterial research have been developed in recent years. Delivery of QDs conjugated with cell-penetrating peptides (CPPs) into cells by the endocytic pathway was problematic in biomedical applications because of lysosomal trapping. Here, we demonstrate that histidine- and arginine-rich CPPs (HR9 peptides) stably and noncovalently combined with QDs are able to enter into cells in an extremely short period (4 min). Interrupting both F-actin polymerization and active transport did not inhibit the entry of HR9/QD complexes into cells, indicating that HR9 penetrates cell membrane directly. Subcellular colocalization studies indicated that QDs delivered by HR9 stay in cytosol without any organelle capture. Dimethyl sulphoxide, ethanol and oleic acid, but not pyrenebutyrate, enhanced HR9-mediated intracellular delivery of QDs by promoting the direct membrane translocation pathway. HR9 and HR9/QDs were not cytotoxic. These findings suggest that HR9 could be an efficient carrier to deliver drugs without interfering with their therapeutic activity., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Quantum dots (QDs) are luminescent semiconductor nanocrystals that are widely used as fluorescent probes in biomedical applications, including cellular imaging and tumor tracking. Cell-penetrating peptides (CPPs), also called protein transduction domains (PTDs), are short basic peptides that permeate cell membranes and are able to deliver a variety of macromolecule cargoes, such as DNAs, RNAs, proteins, and nanomaterials. Here we review strategies to couple QDs to CPPs, by either covalent linkages or noncovalent interactions, to provide a tool to study intracellular delivery. This facilitated transport of QDs by CPPs into cells is both simple and efficient. Accordingly, CPP-QD nanoparticles are likely to be of broad utility in biological research and advance the development of medical and pharmaceutical therapeutics.