Break on through to the other side-biophysics and cell biology shed light on cell-penetrating peptides

Cell-penetrating peptides (CPPs) have become widely used vectors for the cellular import of molecules in basic and applied biomedical research. Despite the broad acceptance of these molecules as molecular carriers, the details of the mode of cellular internalization and membrane permeation remain elusive. Within the last two years endocytosis has been demonstrated to be a route of uptake shared by several CPPs. These findings had a significant impact on CPP research. State-of-the-art cell biology is now required to advance the understanding of the intracellular fate of the CPP and cargo molecules. Owing to their presumed ability to cross lipid bilayers, CPPs also represent highly interesting objects of biophysical research. Numerous studies have investigated structure-activity relationships of CPPs with respect to their ability to bind to a lipid bilayer or to cross this barrier. Endocytosis route only relocates the membrane permeation from the cell surface to endocytic compartments. Therefore, biophysical experiments are key to a mechanistic molecular understanding of the cellular uptake of CPPs. However, biophysical investigations have to consider the molecular environment encountered by a peptide inside and outside a cell. In this contribution we will review biophysical and cell-biology data obtained for several prominent CPPs. Furthermore, we will summarize recent findings on the cell-penetrating characteristics of antimicrobial peptides and the antimicrobial properties of CPPs. Peptides of both groups have overlapping characteristics. Therefore, both fields may greatly benefit from each other. The review will conclude with a perspective of how biophysics and cell biology may synergize even more efficiently in the future.     

Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease

Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.     

D-SAP: a new, noncytotoxic, and fully protease resistant cell-penetrating peptide

Protease resistant cell-penetrating peptides (CPPs) are promising carriers for drugs unable to cross the cell membrane. As these CPPs are stable in vivo for much longer periods of time compared to other classes of therapeutic peptides, noncytotoxicity is a property sine qua non for their pharmacological development. Described herein is a fully protease resistant CPP that is noncytotoxic at concentrations up to 1 mM. Proteolytic stability was obtained by chiral inversion of the residues of a known self-assembling CPP-from all L-amino acids to all D-amino acids-and then assessed against trypsin and human serum. Circular dichroism studies confirmed the enantiomeric structure of the analogue, and transmission electron microscopy (TEM) studies indicated that the new inverso analogue retains the ability of the original peptide to self-assemble. The results of uptake experiments indicate that the protease-stable (that is, D-amino acid) analogue of the peptide is internalised by cells to the same extent as the protease-susceptible (that is, L-amino acid) parent peptide. Also reported herein are the results of studies on the cellular internalisation mechanism of the all-D analogue, which reveal the steps followed by the peptide upon its entry into the cell.     

The Efficacies of Cell‐Penetrating Peptides in Accumulating in Large Unilamellar Vesicles Depend on their Ability To Form Inverted Micelles

In this study, the direct translocation of cell‐penetrating peptides (CPPs) into large unilamellar vesicles (LUVs) was shown to be rapid for all the most commonly used CPPs. This translocation led within a few minutes to intravesicular accumulation up to 0.5 mM , with no need for a transbilayer potential. The accumulation of CPPs inside LUVs was found to depend on CPP sequence, CPP extravesicular concentration and phospholipid (PL) composition, either in binary or ternary mixtures of PLs. More interestingly, the role of anionic phospholipid flip‐flopping in the translocation process was ascertained. CPPs enhanced the flipping of PLs, and the intravesicular CPP accumulation directly correlated with the amount of anionic PLs that had been transferred from the external to the internal leaflet of the LUV bilayer, thus demonstrating the transport of peptide/lipid complexes as inverted micelles.     

Cyclic Cell-Penetrating Peptides with Single Hydrophobic Groups

A new family of cyclic cell-penetrating peptides (CPPs) has been discovered; they differ from previously reported cyclic CPPs by containing only a single hydrophobic residue. The optimal CPP structure consists of four arginine residues and a hydrophobic residue with a long alkyl chain (e.g., a decyl group) in a cyclohexapeptide ring. The most active member of this family, CPP 17 , has an intrinsic cellular entry efficiency similar to that of cyclic CPP12, the most active CPP reported to date. However, CPP 17 is 2.8 times more active than CPP12 under high serum protein concentrations, presumably because of the lower protein binding. CPP 17 enters the cell primarily by direct translocation at a relatively low concentration (≥5 μm ).     

Design, synthesis and characterization of a new anionic cell-penetrating peptide: SAP(E)

Cell-penetrating peptides (CPPs) are powerful tools to transport cell-impermeable cargoes into the cytoplasm without damaging the cell membrane. The vast majority of these peptides described to date share several features, among others, they are positively charged at physiological pH. In several cases a clear correlation between an increasing number of positive charges and internalization properties has been reported. Here, we describe what, to the best of our knowledge, is the first anionic CPP. This new compound SAP(E) internalizes into a range of cell lines with good efficiency and it shows low toxicity. We also report on the internalization mechanism. The discovery of this new class of CPP opens the way to the intracellular delivery of new molecular cargoes.     

Novel cell-penetrating peptides based on α-aminoxy acids

The remarkable ability of cell-penetrating peptides (CPPs) to deliver cell-impermeable compounds into living cells makes them attractive transporters for use in biology and medicine. Despite their highly efficient cellular uptake, CPPs consisting of natural amino acids always suffer from degradation and endosomal entrapment, thereby greatly limiting their application in vivo. Here, we describe the preparation of novel CPPs incorporating α-aminoxy acid residues and their cellular uptake behavior. We demonstrate that introducing α-aminoxy acids into the backbones of CPPs enhances their diffuse cytosolic distribution after direct membrane translocation. We also reveal a hybrid peptide, consisting of D-α-aminoxy acids and L-α-amino acids, that achieves efficient diffuse distribution in the cytosol, is stable toward serum, and possesses low cytotoxicity, thus making it a possible vector candidate for in vivo applications. Our results confirm that α-aminoxy acids are useful building blocks when designing novel CPPs possessing favorable properties.     

Intracellular Delivery of Molecular Cargo Using Cell-Penetrating Peptides and the Combination Strategies

Cell-penetrating peptides (CPPs) can cross cellular membranes in a non-toxic fashion, improving the intracellular delivery of various molecular cargos such as nanoparticles, small molecules and plasmid DNA. Because CPPs provide a safe, efficient, and non-invasive mode of transport for various cargos into cells, they have been developed as vectors for the delivery of genetic and biologic products in recent years. Most common CPPs are positively charged peptides. While delivering negatively charged molecules (e.g., nucleic acids) to target cells, the internalization efficiency of CPPs is reduced and inhibited because the cationic charges on the CPPs are neutralized through the covering of CPPs by cargos on the structure. Even under these circumstances, the CPPs can still be non-covalently complexed with the negatively charged molecules. To address this issue, combination strategies of CPPs with other typical carriers provide a promising and novel delivery system. This review summarizes the latest research work in using CPPs combined with molecular cargos including liposomes, polymers, cationic peptides, nanoparticles, adeno-associated virus (AAV) and calcium for the delivery of genetic products, especially for small interfering RNA (siRNA). This combination strategy remedies the reduced internalization efficiency caused by neutralization.     

Efficient intracellular delivery of nucleic acid pharmaceuticals using cell-penetrating peptides

Over the last 20 years, researchers have designed or discovered peptides that can permeate membranes and deliver exogenous molecules inside a cell. These peptides, known as cell-penetrating peptides (CPPs), typically consist of 6-30 residues, including HIV TAT peptide, penetratin, oligoarginine, transportan, and TP10. Through chemical conjugation or noncovalent complex formation, these structures successfully deliver bioactive and membrane-impermeable molecules into cells. CPPs have also gained attention as an attractive vehicle for the delivery of nucleic acid pharmaceuticals (NAPs), including genes/plasmids, short oligonucleotides, and small interference RNAs and their analogues, due to their high internalization efficacy, low cytotoxicity, and flexible structural design. In this Account, we survey the potential of CPPs for the design and optimization of NAP delivery systems. First, we describe the impact of the N-terminal stearylation of CPPs. Endocytic pathways make a major contribution to the cellular uptake of NAPs. Stearylation at the N-terminus of CPPs with stearyl-octaarginine (R8), stearyl-(RxR)(4), and stearyl-TP10 prompts the formation of a self-assembled core-shell nanoparticle with NAPs, a compact structure that promotes cellular uptake. Researchers have designed modifications such as the addition of trifluoromethylquinoline moieties to lysine residues to destabilize endosomes, as exemplified by PepFect 6, and these changes further improve biological responsiveness. Alternatively, stearylation also allows implantation of CPPs onto the surface of liposomes. This feature facilitates "programmed packaging" to establish multifunctional envelope-type nanodevices (MEND). The R8-MEND showed high transfection efficiency comparable to that of adenovirus in non-dividing cells. Understanding the cellular uptake mechanisms of CPPs will further improve CPP-mediated NAP delivery. The cellular uptake of CPPs and their NAP complex involves various types of endocytosis. Macropinocytosis, a mechanism which is also activated in response to stimuli such as growth factors or viruses, is a primary pathway for arginine-rich CPPs because high cationic charge density promotes this endocytic pathway. The use of larger endosomes (known as macropinosomes) rather than clathrin- or caveolae-mediated endocytosis has been reported in macropinocytosis which would also facilitate the endocytosis of NAP nanoparticles into cells.     

Therapeutic Potential of Cell Penetrating Peptides (CPPs) and Cationic Polymers for Chronic Hepatitis B

Chronic hepatitis B virus (HBV) infection remains a major health problem worldwide. Because current anti-HBV treatments are only virostatic, there is an urgent need for development of alternative antiviral approaches. In this context, cell-penetrating peptides (CPPs) and cationic polymers, such as chitosan (CS), appear of particular interest as nonviral vectors due to their capacity to facilitate cellular delivery of bioactive cargoes including peptide nucleic acids (PNAs) or DNA vaccines. We have investigated the ability of a PNA conjugated to different CPPs to inhibit the replication of duck hepatitis B virus (DHBV), a reference model for human HBV infection. The in vivo administration of PNA-CPP conjugates to neonatal ducklings showed that they reached the liver and inhibited DHBV replication. Interestingly, our results indicated also that a modified CPP (CatLip) alone, in the absence of its PNA cargo, was able to drastically inhibit late stages of DHBV replication. In the mouse model, conjugation of HBV DNA vaccine to modified CS (Man-CS-Phe) improved cellular and humoral responses to plasmid-encoded antigen. Moreover, other systems for gene delivery were investigated including CPP-modified CS and cationic nanoparticles. The results showed that these nonviral vectors considerably increased plasmid DNA uptake and expression. Collectively promising results obtained in preclinical studies suggest the usefulness of these safe delivery systems for the development of novel therapeutics against chronic hepatitis B.     

Development of viral nanoparticles for efficient intracellular delivery

Viral nanoparticles (VNPs) based on plant viruses such as Cowpea mosaic virus (CPMV) can be used for a broad range of biomedical applications because they present a robust scaffold that allows functionalization by chemical conjugation and genetic modification, thereby offering an efficient drug delivery platform that can target specific cells and tissues. VNPs such as CPMV show natural affinity to cells; however, cellular uptake is inefficient. Here we show that chemical modification of the CPMV surface with a highly reactive, specific and UV-traceable hydrazone linker allows bioconjugation of polyarginine (R5) cell penetrating peptides (CPPs), which can overcome these limitations. The resulting CPMV-R5 particles were taken up into a human cervical cancer cell line (HeLa) more efficiently than native particles. Uptake efficiency was dependent on the density of R5 peptides on the surface of the VNP; particles displaying 40 R5 peptides per CPMV (denoted as CPMV-R5H) interact strongly with the plasma membrane and are taken up into the cells via an energy-dependent mechanism whereas particles displaying 10 R5 peptides per CPMV (CPMV-R5L) are only slowly taken up. The fate of CPMV-R5 versus native CPMV particles within cells was evaluated in a co-localization time course study. It was indicated that the intracellular localization of CPMV-R5 and CPMV differs; CPMV remains trapped in Lamp-1 positive endolysosomes over long time frames; in contrast, 30-50% of the CPMV-R5 particles transitioned from the endosome into other cellular vesicles or compartments. Our data provide the groundwork for the development of efficient drug delivery formulations based on CPMV-R5.     

Comparing Agent-Based Delivery of DNA and PNA Forced Intercalation (FIT) Probes for Multicolor mRNA Imaging

Fluorogenic oligonucleotide probes allow mRNA imaging in living cells. A key challenge is the cellular delivery of probes. Most delivery agents, such as cell-penetrating peptides (CPPs) and pore-forming proteins, require interactions with the membrane. Charges play an important role. To explore the influence of charge on fluorogenic properties and delivery efficiency, we compared peptide nucleic acid (PNA)- with DNA-based forced intercalation (FIT) probes. Perhaps counterintuitively, fluorescence signaling by charged DNA FIT probes proved tolerant to CPP conjugation, whereas CPP–FIT PNA conjugates were affected. Live-cell imaging was performed with a genetically engineered HEK293 cell line to allow the inducible expression of a specific mRNA target. Blob-like features and high background were recurring nuisances of the tested CPP and lipid conjugates. By contrast, delivery by streptolysin-O provided high enhancements of the fluorescence of the FIT probe upon target induction. Notably, DNA-based FIT probes were brighter and more responsive than PNA-based FIT probes. Optimized conditions enabled live-cell multicolor imaging of three different mRNA target sequences.     

Biological properties of iron oxide nanoparticles for cellular and molecular magnetic resonance imaging

Superparamagnetic iron-oxide particles (SPIO) are used in different ways as contrast agents for magnetic resonance imaging (MRI): Particles with high nonspecific uptake are required for unspecific labeling of phagocytic cells whereas those that target specific molecules need to have very low unspecific cellular uptake. We compared iron-oxide particles with different core materials (magnetite, maghemite), different coatings (none, dextran, carboxydextran, polystyrene) and different hydrodynamic diameters (20-850 nm) for internalization kinetics, release of internalized particles, toxicity, localization of particles and ability to generate contrast in MRI. Particle uptake was investigated with U118 glioma cells und human umbilical vein endothelial cells (HUVEC), which exhibit different phagocytic properties. In both cell types, the contrast agents Resovist, B102, non-coated Fe(3)O(4) particles and microspheres were better internalized than dextran-coated Nanomag particles. SPIO uptake into the cells increased with particle/iron concentrations. Maximum intracellular accumulation of iron particles was observed between 24 h to 36 h of exposure. Most particles were retained in the cells for at least two weeks, were deeply internalized, and only few remained adsorbed at the cell surface. Internalized particles clustered in the cytosol of the cells. Furthermore, all particles showed a low toxicity. By MRI, monolayers consisting of 5000 Resovist-labeled cells could easily be visualized. Thus, for unspecific cell labeling, Resovist and microspheres show the highest potential, whereas Nanomag particles are promising contrast agents for target-specific labeling.     

Biological Properties of Iron Oxide Nanoparticles for Cellular and Molecular Magnetic Resonance Imaging

Superparamagnetic iron-oxide particles (SPIO) are used in different ways as contrast agents for magnetic resonance imaging (MRI): Particles with high nonspecific uptake are required for unspecific labeling of phagocytic cells whereas those that target specific molecules need to have very low unspecific cellular uptake. We compared iron-oxide particles with different core materials (magnetite, maghemite), different coatings (none, dextran, carboxydextran, polystyrene) and different hydrodynamic diameters (20–850 nm) for internalization kinetics, release of internalized particles, toxicity, localization of particles and ability to generate contrast in MRI. Particle uptake was investigated with U118 glioma cells und human umbilical vein endothelial cells (HUVEC), which exhibit different phagocytic properties. In both cell types, the contrast agents Resovist, B102, non-coated Fe₃O₄ particles and microspheres were better internalized than dextran-coated Nanomag particles. SPIO uptake into the cells increased with particle/iron concentrations. Maximum intracellular accumulation of iron particles was observed between 24 h to 36 h of exposure. Most particles were retained in the cells for at least two weeks, were deeply internalized, and only few remained adsorbed at the cell surface. Internalized particles clustered in the cytosol of the cells. Furthermore, all particles showed a low toxicity. By MRI, monolayers consisting of 5000 Resovist-labeled cells could easily be visualized. Thus, for unspecific cell labeling, Resovist and microspheres show the highest potential, whereas Nanomag particles are promising contrast agents for target-specific labeling.     

A Facile Cyclization Method Improves Peptide Serum Stability and Confers Intrinsic Fluorescence

Peptides are a growing class of macromolecules used in pharmaceutics. The path toward the clinical use of candidate peptides involves sequence optimization and cyclization for stability and affinity. For internalized peptides, tagging is also often required for intracellular trafficking studies, although fluorophore conjugation has an impact on peptide binding, permeability, and localization. Herein, a strategy based on cysteine arylation with tetrafluoroterephthalonitrile (4F‐2CN), which simultaneously cyclizes peptides and imparts fluorescence, is reported. The 4F‐2CN cyclization of an M2 macrophage‐targeting peptide yields, in a single step, a peptide with improved serum stability, intrinsic fluorescence, and increased binding affinity. In a murine breast cancer model, it is demonstrated that the intrinsic fluorescence from the cyclized peptide is sufficient for monitoring biodistribution by whole‐organ fluorescence imaging and cell internalization by flow cytometry.     

Role of charge and hydrophobicity in translocation of cell-penetrating peptides into Candida albicans cells

Although the interactions of cell-penetrating peptides (CPPs) with mammalian cells have been widely studied, much less is known about their interactions with fungal cells. To study how the properties of CPPs affect translocation into fungal cells, we designed variants of the peptides pVEC and SynB with altered levels of charge and hydrophobicity and evaluated the translocation of the variants into the important human fungal pathogen Candida albicans. Charge played a greater role in translocation efficacy of the peptides than hydrophobicity, with a higher net positive charge leading to higher level of translocation into C. albicans and a higher level of cytosolic localization. Hydrophobicity had little effect on translocation efficacy, but a low level of hydrophobicity did lead to increased vacuolar localization and an energy-dependent translocation mechanism. Our results suggest that CPPs can be designed for desired levels of cargo delivery into fungal cells and for desired translocation mechanisms.     

Peptide vectors for the nonviral delivery of nucleic acids

Over the past two decades, gene therapy has garnered tremendous attention and is heralded by many as the ultimate cure to treat diseases such as cancer, viral infections, and inherited genetic disorders. However, the therapeutic applications of nucleic acids extend beyond the delivery of double-stranded DNA and subsequent expression of deficient gene products in diseased tissue. Other strategies include antisense oligonucleotides and most notably RNA interference (RNAi). Antisense strategies bear great potential for the treatment of diseases that are caused by misspliced mRNA, and RNAi is a universal and extraordinarily efficient tool to knock down the expression of virtually any gene by specific degradation of the desired target mRNA. However, because of the hurdles associated with effective delivery of nucleic acids across a cell membrane, the initial euphoria surrounding siRNA therapy soon subsided. The ability of oligonucleotides to cross the plasma membrane is hampered by their size and highly negative charge. Viral vectors have long been the gold standard to overcome this barrier, but they are associated with severe immunogenic effects and possible tumorigenesis. Cell-penetrating peptides (CPPs), cationic peptides that can translocate through the cell membrane independent of receptors and can transport cargo including proteins, small organic molecules, nanoparticles, and oligonucleotides, represent a promising class of nonviral delivery vectors. This Account focuses on peptide carrier systems for the cellular delivery of various types of therapeutic nucleic acids with a special emphasis on cell-penetrating peptides. We also emphasize the clinical relevance of this research through examples of promising in vivo studies. Although CPPs are often derived from naturally occurring protein transduction domains, they can also be artificially designed. Because CPPs typically include many positively charged amino acids, those electrostatic interactions facilitate the formation of complexes between the carriers and the oligonucleotides. One drawback of CPP-mediated delivery includes entrapment of the cargo in endosomes because uptake tends to be endocytic: coupling of fatty acids or endosome-disruptive peptides to the CPPs can overcome this problem. CPPs can also lack specificity for a single cell type, which can be addressed through the use of targeting moieties, such as peptide ligands that bind to specific receptors. Researchers have also applied these strategies to cationic carrier systems for nonviral oligonucleotide delivery, such as liposomes or polymers, but CPPs tend to be less cytotoxic than other delivery vehicles.     

A TAT-streptavidin fusion protein directs uptake of biotinylated cargo into mammalian cells

The HIV-1 TAT peptide has been used extensively for directing the intracellular delivery of an assortment of cargo, including DNA, liposomes and macromolecules. For protein delivery, a variety of TAT-fusion proteins have been described which link the TAT coding sequence to the protein coding sequence of interest. Streptavidin represents a potentially useful TAT-fusion protein because it could be used to deliver a wide array of biotinylated cargo. Here we have characterized a TAT-streptavidin (TAT-SA) fusion protein, which retains the ability to bind biotinylated cargo while directing their efficient cellular uptake. Fluorescence activated cell sorting (FACS) analysis and confocal microscopy characterization showed that TAT-SA is internalized by Jurkat T-cells and NIH 3T3 cells alone and when complexed to phycoerythrin, whereas the native streptavidin is not. Additionally, biotinylated alkaline phosphatase is successfully internalized and retains its activity when complexed to TAT-SA and incubated with Jurkat T-cells. Confocal microscopy suggested, however, that internalized TAT-SA and TAT-SA complexes were largely compartmentalized in vesicular compartments, rather than freely diffusing in the cytoplasmic compartment. To effect cytoplasmic delivery, the endosomal releasing polymer, poly(propylacrylic acid) (PPAA), was biotinylated and complexed to TAT-SA. Endosomal release and cytoplasmic delivery of fluorescently labeled TAT-SA complexes with PPAA was shown by the diffuse distribution of fluorescent protein in the cytoplasm. Taken together, these results demonstrate that TAT-SA can be used to direct intracellular delivery of large biotinylated cargo to intracellular compartments and that biotinylated PPAA can direct cytoplasmic delivery where desired.