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Highlights: [•] We synthesized carboxylated and PEGylated bifunctionalized InP/ZnS QDs (QInP). [•] Cell-penetrating peptides (CPPs) could deliver QInP into human A549 cells. [•] Five hundred nM of CPP/QInP and QInP at or below 1μM did not reduce cell viability. [•] QInP are biocompatible nanoparticles for use in biomedical applications. [ABSTRACT FROM AUTHOR]

Abstract: Cell-penetrating peptide (CPP)-mediated intracellular drug delivery system, often specifically termed as “the Trojan horse approach”, has become the “holy grail” in achieving effective delivery of macromolecular compounds such as proteins, DNA, siRNAs, and drug carriers. It is characterized by the unique cell- (or receptor-), temperature-, and payload-independent mechanisms, therefore offering potent means to improve poor cellular uptake of a variety of macromolecular drugs. Nevertheless, this “Trojan horse” approach also acts like a double-edged sword, causing serious safety and toxicity concerns to normal tissues or organs for in vivo application, due to lack of target selectivity of the powerful cell penetrating activity. To overcome this problem of potent yet non-selective penetration vs. targeting delivery, a number of “smart” strategies have been developed in recent years, including controllable CPP-based drug delivery systems based on various stimuli-responsive mechanisms. This review article provides a fundamental understanding of these smart systems, as well as a discussion of their real-time in vivo applicability. [Copyright &y& Elsevier]

Abstract: Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA) are two of the most common inherited neuromuscular diseases in humans. Both conditions are fatal and no clinically available treatments are able to significantly alter disease course in either case. However, by manipulation of pre-mRNA splicing using antisense oligonucleotides, defective transcripts from the DMD gene and from the SMN2 gene in SMA can be modified to once again produce protein and restore function. A large number of in vitro and in vivo studies have validated the applicability of this approach and an increasing number of preliminary clinical trials have either been completed or are under way. Several different oligonucleotide chemistries can be used for this purpose and various strategies are being developed to facilitate increased delivery efficiency and prolonged therapeutic effect. As these novel therapeutic compounds start to enter the clinical arena, attention must also be drawn to the question of how best to facilitate the clinical development of such personalised genetic therapies and how best to implement their provision. [Copyright &y& Elsevier]

Abstract: Arginine-rich cell-penetrating peptides, including octaarginine (R8) and HIV-1 TAT peptides, have the ability to translocate through cell membranes and transport exogenous bioactive molecules into cells. Hydrophobic counteranions such as pyrenebutyrate (PyB) have been reported to markedly promote the membrane translocation of these peptides. In this study, using model membranes having liquid-ordered (Lo) and liquid-disordered (Ld) phases, we explored the effects of PyB on the promotion of R8 translocation. Confocal microscopic observations of giant unilamellar vesicles (GUVs) showed that PyB significantly accelerated the accumulation of R8 on membranes containing negatively charged lipids, leading to the internalization of R8 without collapse of the GUV structures. PyB displayed an alternative activity, increasing the fluidity of the negatively charged membranes, which diminished the distinct Lo/Ld phase separation on GUVs. This was supported by the decrease in fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH). Additionally, PyB induced membrane curvature, which has been suggested as a possible mechanism of membrane translocation for R8. Taken together, our results indicate that PyB may have multiple effects that promote R8 translocation through cell membranes. [Copyright &y& Elsevier]

Highlights: [•] AMPs are multifunctional molecules that selectively permeate microbial membranes. [•] Negative Gaussian curvature (NGC) enables many types of membrane permeation. [•] NGC generation by AMPs constrains their compositions in the form of a general rule. [•] We show that the sequences of known cationic AMPs are consistent with this rule. [ABSTRACT FROM AUTHOR]

Abstract: For widening the arsenal of protein and peptide therapeutics that act within cells, their cell-entry mechanisms, intracellular trafficking and distribution need to be characterized in detail. Immunofluorescence microscopy has been a prevalent tool for these studies. However, due to the limited resolution, it is often complemented with other methods. This article focuses on the perspectives of electron microscopy in tracking the intracellular delivery and trafficking of proteins, peptides and their carriers. This review introduces the electron microscopy techniques and labeling methods currently used for studying the cellular whereabouts of peptides and proteins with a focus on their intracellular trafficking. Since cell-penetrating peptides have widely been harnessed as carriers for proteins and peptides, and their usage is rapidly expanding, a particular emphasis has been placed on their applications and cell-entry mechanisms. [Copyright &y& Elsevier]

Abstract: Cell-penetrating peptides (CPPs) are a class of short, often cationic peptides that have the capability to translocate across cellular membranes, and although the translocation most likely involves several pathways, they interact directly with membranes, as well as with model bilayers. Most CPPs attain a three-dimensional structure when interacting with bilayers, while they are more or less unstructured in aqueous solution. To understand the relationship between structure and the effect that CPPs have on membranes it is of great importance to investigate CPPs at atomic resolution in a suitable membrane model. Moreover, the location in bilayers is likely to be correlated with the translocation mechanism. Solution-state NMR offers a unique possibility to investigate structure, dynamics and location of proteins and peptides in bilayers. This review focuses on solution NMR as a tool for investigating CPP–lipid interactions. Structural propensities and cell-penetrating capabilities can be derived from a combination of CPP solution structures and studies of the effect that the peptides have on bilayers and the localization in a bilayer. [Copyright &y& Elsevier]

Abstract: Twenty years ago, the discovery of peptides able to cross cellular membranes launched a novel field in molecular delivery based on these non-invasive vectors, most commonly called cell-penetrating peptides (CPPs) or protein transduction domains (PTDs). These peptides were shown to efficiently transport various biologically active molecules inside living cells, and thus are considered promising devices for medical and biotechnological developments. Moreover, CPPs emerged as potential tools to study the prime mechanisms of cellular entry across the plasma membrane. This review is dedicated to CPP fundamentals, with an emphasis on the molecular requirements and mechanism of their entry into eukaryotic cells. [Copyright &y& Elsevier]

Abstract: Cell penetrating peptides (CPPs) are usually short, highly cationic peptides that are capable of crossing the cell membrane and transport cargos of varied size and nature in cells by energy- and receptor-independent mechanisms. An additional potential is the newly discovered anti-tumor activity of certain CPPs, including RW16 (RRWRRWWRRWWRRWRR) which is derived from penetratin and is investigated here. The use of CPPs in therapeutics, diagnosis and potential application as anti-tumor agents increases the necessity of understanding their mode of action, a subject yet not totally understood. With this in mind, the membrane interaction and perturbation mechanisms of RW16 with both zwitterionic and anionic lipid model systems (used as representative models of healthy vs tumor cells) were investigated using a large panoply of biophysical techniques. It was shown that RW16 autoassociates and that its oligomerization state highly influences its membrane interaction. Overall a stronger association and perturbation of anionic membranes was observed, especially in the presence of oligomeric peptide, when compared to zwitterionic ones. This might explain, at least in part, the anti-tumor activity and so the selective interaction with cancer cells whose membranes have been shown to be especially anionic. Hydrophobic contacts between the peptide and lipids were also shown to play an important role in the interaction. That probably results from the tryptophan insertion into the fatty acid lipid area following a peptide flip after the first electrostatic recognition. A model is presented that reflects the ensemble of results. [Copyright &y& Elsevier]

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, res [ABSTRACT FROM AUTHOR]

Abstract: 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 &y& Elsevier]

Abstract: 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 &y& Elsevier]

Human dental pulp stem cells (hDPSCs) are the only mesenchymal stem cells in pulp tissue that can differentiate into osteoblasts, odontoblasts, and adipose cells. The transcriptional co-activator with PDZ-binding motif (TAZ) protein has been reported to modulate osteogenic differentiation in mouse MSCs. Therefore, we examined whether the TAZ protein plays the same role in human pulp stem cells. In this study, TAZ was applied to cells directly with low-molecular-weight protamine (LMWP) as a cell-penetrating peptide (CPP). The LMWP-TAZ fusion proteins were expressed in an E. coli system with a pET-21b vector and efficiently transferred into hDPSCs without producing toxicity in the cells. The efficient uptake of TAZ was shown by Western blot with an anti-TAZ antibody, fluorescence-activated cell sorting, and confocal microscopy in live cells. The delivered TAZ protein increased osteogenic differentiation, as confirmed by alkaline phosphatase (ALP) staining, RT-PCR, and Western blotting. In addition, TAZ also inhibited adipogenic differentiation, regulating peroxisome proliferator-activated receptor-γ (PPAR-γ), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein (aP2) mRNA levels. These in vitro studies suggest that cell-permeable TAZ may be used as a specific regulator of hard-tissue differentiation. [ABSTRACT FROM PUBLISHER]

Abstract: 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 &y& Elsevier]

Abstract: 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 &y& Elsevier]

Abstract: Over the last decade, numerous studies have suggested that neuroglobin is able to protect against the effects of ischemia. However, such results have mostly been based on models using transgenic overexpression or viral delivery. As a therapy, new technology would need to be applied to enable delivery of high concentrations of neuroglobin shortly after the patient suffers the stroke. An approach to deliver proteins in ischemia in vivo in a timely manner is the use of cell-penetrating peptides (CPP). CPP have been used in animal models for brain diseases for about a decade as well. In a recent issue of Experimental Neurology, Cai and colleagues test the effect of CPP-coupled neuroglobin in an in vivo stroke model. They find that the fusion protein protects the brain against the effect of ischemia when applied before stroke onset. Here, a concise review of neuroglobin research and the application of CPP peptides in hypoxia and ischemia is provided. [Copyright &y& Elsevier]

Previously, we developed cell-penetrating penta-peptides (CPP5s). In the present study, VPTLK and KLPVM, two representative CPP5s, were used to characterize the cellpenetration and protein-transduction activities of these small molecules. Various inhibitors of endocytosis and pinocytosis (chlorpromazine, cytochalasin D, Filipin III, amiloride, methyl-μ- cyclodextrin, and nocodazole) were tested. Only cytochalasin D showed suppression of CPP5 entry, though the effect was partial. In addition, CPP5s were able to enter a proteoglycandeficient CHO cell line. These results suggest that pinocytosis and endocytosis may play only a minor role in the cell entry of CPP5s. By mass spectrometry, we determined that the intracellular concentration of VPTLK ranged from 20 nM to 6.0 μM when the cells were cultured in medium containing 1 μM - 1.6 mM VPTLK. To determine the proteintransduction activity of CPP5s, the Tex-LoxP EG cell line, which has a Cre-inducible green fluorescent protein (GFP) gene, was used. VPTLK and KLPVM were added to the N-terminus of Cre, and these fusion proteins were added to the culture medium of Tex-LoxP EG cells. Both VPTLK-Cre and KLPVM-Cre were able to turn on GFP expression in these cells, suggesting that CPP5s have protein-transduction activity. Since CPP5s have very low cytotoxic activity, even at a concentration of 1.6 mM in the medium, CPP5s could be utilized as a new tool for drug delivery into cells. [ABSTRACT FROM AUTHOR]

Abstract: Penetratin, a 16-residue peptide, is used widely as a highly efficient delivery carrier for a wide range of poorly permeable therapeutic cargoes. The crucial structural features of penetratin remain unclear, as demonstrated by the difficulties encountered in designing new molecules. The efficiency in enhancing nasal insulin absorption was compared between l-penetratin and 20 of its analogues in rats. We also measured lactate dehydrogenase (LDH) leakage as an indicator of cytotoxicity and scored the histopathological irritation. Substitution of a cationic residue (Arg or Lys) with Leu or addition of tetra-arginine to the C- or N-terminus of penetratin caused considerable reduction in the enhancing efficiency properties of the modified analogues. Mutual exchanging of Arg and Lys in corresponding analogues produced nearly inactive analogues, although changing Arg to Lys in the same analogue produced similar penetratin activity. In addition, activity was impaired markedly upon modification of penetratin within amphiphilic (Trp) or hydrophobic (Ile and Phe) residues. Chain size-modified analogues lacked the ability to induce nasal insulin absorption. In contrast, rearrangement of the modified analogues by C,N-half-exchange and reverse analogues produced activity similar to that of the original penetratin. The enhancing activity was inhibited almost completely upon sequence arrangement of the resulting analogues. Surprisingly, a shuffle (Arg, Lys fix) 2 analogue increased insulin absorption significantly, reaching a relative bioavailability value 1.85-times that of original penetratin. This analogue caused negligible release of LDH in nasal lavage fluid and maintained the integrity of the nasal respiratory epithelium. In conclusion, modulation of amino acid sequences by fixing the cationic residue positions can augment penetratin-enhanced nasal absorption and may lead to improvements in nasal insulin absorption. [Copyright &y& Elsevier]

Abstract: We investigated the relationship between intermolecular binding and the ability of novel cell-penetrating peptides (CPPs) to enhance the nasal absorption of therapeutic peptides and proteins. The absorption-enhancing effect of a novel l-penetratin analogue, ‘shuffle (R,K fix) 2’ coadministered with different biotherapeutic peptides was evaluated after nasal administration in rats. Shuffle (R,K fix) 2 significantly increased the nasal absorption of insulin, glucagon-like-peptide-1 (GLP-1) and exendin-4, compared with the absorption seen with l-penetratin. Intermolecular binding was analyzed by surface plasmon resonance (SPR)-based binding assay. The binding characteristics implied that the higher the amount of CPP bound, the greater the nasal drug absorption. In addition, the calculated binding ratio between CPP and drug proved a critical aspect in enhancing the absorption of insulin and GLP-1. This difference in the enhancing effect of CPPs on nasal drug absorption is attributed to the degree of binding with the therapeutic macromolecule. [Copyright &y& Elsevier]

Abstract: The goal of this study was to evaluate whether cell-penetrating peptides (CPPs) affect the nasal absorption of insulin. L- or D-forms of penetratin, or the L- or D-forms of octaarginine (L- or D-R8), was used as first time for nasal insulin delivery. Furthermore, the concentration of lactate dehydrogenase (LDH) in nasal lavage fluid was determined and a histopathological study of nasal respiratory epithelium was conducted. CPPs dramatically increased nasal insulin absorption, and it was more pronounced for L- and D-penetratin than L- or D-R8. L-penetratin was the most effective promoter of insulin absorption compared with others CPPs. A dose-dependent relationship of L-penetratin and insulin bioavailability was statically significant. The pharmacological availability and bioavailability of nasally administered insulin was up to 76.7% and 50.7% relative to the subcutaneous route, respectively. In contrast, increasing the D-penetratin concentration decreased the efficiency of nasal insulin absorption. There was no significant difference in the release of LDH in nasal lavage fluid and the integrity of nasal respiratory epithelium when L-penetratin was present. In conclusion, these data demonstrate that L-penetratin markedly increased the permeability of insulin across the nasal membrane without causing detectable damage to the integrity of cells in the nasal respiratory mucosa. [Copyright &y& Elsevier]

Acridine cell-penetrating peptide conjugates are an extremely important family of compounds in antitumor chemotherapy. These conjugates are not so widely analysed in antimicrobial therapy, although bioactive peptides could be used as nanocarriers to smuggle antimicrobial compounds. An octaarginine conjugate of an imidazoacridinone derivative (Compound 1-R8) synthetized by us exhibited high antifungal activity against reference and fluconazole-resistant clinical strains (MICs ≤ 4 μg mL−1). Our results clearly demonstrate the qualitative difference in accumulation of the mother compound and Compound 1-R8 conjugate into fungal cells. Only the latter was transported and accumulated effectively. Microscopic and flow cytometry analysis provide some evidence that the killing activity of Compound 1-R8 may be associated with a change in the permeability of the fungal cell membrane. The conjugate exhibited low cytotoxicity against human embryonic kidney (HEK-293) and human liver (HEPG2) cancer cell lines. Nevertheless, the selectivity index value of the conjugate for human pathogenic strains remained favourable and no hemolytic activity was observed. The inhibitory effect of the analysed compound on yeast topoisomerase II activity suggested its molecular target. In summary, conjugation with R8 effectively increased imidazoacridinone derivative ability to enter the fungal cell and achieve a concentration inside the cell that resulted in a high antifungal effect. [ABSTRACT FROM AUTHOR]

Abstract: Cell-penetrating peptides (CPPs) are a useful tool for delivering therapeutic macromolecules across cell membranes. We previously devised an approach using CPPs without intermolecular cross-linking and showed the efficient delivery of insulin from the intestine to the systemic circulation using a typical CPP, oligoarginine. However, this approach required relatively high doses of the CPP. Therefore, this study aimed to identify CPPs that are more effective for the delivery of insulin and do not induce toxic effects on the intestine. In this study, we examined the effects of various types of CPPs including arginine-rich peptides and amphipathic peptides that aid insulin absorption from rat ileal segments. Among these peptides, coadministration of insulin with R8, penetratin, pVEC, and RRL helix significantly increased ileal insulin absorption compared with insulin administration alone. In the case of R8, the d-form of the peptide had stronger absorption enhancing ability than the l-form. In contrast, the other three peptides exerted a more significant effect when the l-forms were applied, and l-penetratin had the strongest ability to enhance intestinal insulin absorption. Meanwhile, in a physical mixture of CPP and insulin, aggregates formed in the solution when high concentrations of CPPs were present. l-penetratin enhanced insulin absorption even when administered in an aggregated solution. We then showed that aggregates of l-penetratin and insulin were broken down in the presence of intestinal degradation enzymes. Thus, among CPPs used in this study, l-penetratin had the strongest ability to improve insulin intestinal absorption. [Copyright &y& Elsevier]

Abstract: Cell-penetrating peptides (CPPs), such as the one derived from the human immunodeficiency virus Tat protein, facilitate the delivery of cargoes across cellular membranes. However, questions about the therapeutic potential of CPP-mediated delivery remain. For instance, the impact of the purification procedure on the functionality of Tat-fusion proteins has not been systematically examined. Here, we isolated fusion proteins of the chaperone heat shock protein 70 (Hsp70) and the Tat CPP under denaturing or native conditions. To investigate the therapeutic potential of different recombinant protein preparations, we examined Tat-Hsp70 transduction efficiency and quantified Tat-Hsp70-mediated folding of a chaperone-dependent yellow fluorescent protein in vitro. Transduction efficiency and chaperone activity of Tat-Hsp70-treated cells was significantly higher compared to cells treated with Hsp70. The application of native isolated Tat-Hsp70 had the strongest effect. This chaperone activity correlates with increased viability of cells treated with the recombinant protein after cell death induction with 6-hydroxydopamine. This suggests that the method of recombinant Tat-fusion protein purification influences its functionality. For Tat-Hsp70, the method of choice seems to be isolation under native conditions, for which we present a purification protocol. Our results may contribute to improve Tat-fusion protein application in basic research and may facilitate its use as therapeutic tool, for instance in Parkinson''s disease. [Copyright &y& Elsevier]

Abstract: The aim of this study was to evaluate whether oligoarginine, a cell-penetrating peptide (CPP), can improve intestinal absorption of insulin in rats. Peptides composed of six (R6), eight (R8) and 10 (R10) residues of arginine were used as the CPP. No insulin absorption was observed following administration of insulin solution alone; however, insulin absorption increased dramatically after coadministration of the d-form of R6 (d-R6) and the l-form of R6 (l-R6) in a dose-dependent manner. The effects on insulin absorption were more pronounced for d-R6 than for l-R6. Among oligoarginines composed of six, eight, or 10 arginine residues, d-R8 showed the strongest enhancing effects on insulin intestinal absorption. In contrast, intestinal absorption of other model hydrophilic macromolecules, interferon-β and fluorescein isothiocyanate-labeled dextran 4400, was not affected by coadministration with oligoarginine. Pretreatment by the effective dose of l-R6 did not induce lactate dehydrogenase leakage or histological damage, suggesting that oligoarginine has no untoward effect on the intestinal mucosa. Our data demonstrate that coadministration of oligoarginine increases intestinal insulin absorption markedly without causing detectable damage in cellular integrity and that the covalent binding between insulin and oligoarginine is not necessary for this effect. We conclude that oligoarginines are likely to become powerful tools for overcoming the low permeability of insulin through the epithelial cell membrane, the major barrier to oral insulin delivery. [Copyright &y& Elsevier]

Abstract: Glial-cell-line-derived neurotrophic factor (GDNF) promotes mesencephalic dopaminergic neuronal survival in several in vitro and in vivo models. As the demise of dopaminergic neurons is the cause for Parkinson's disease (PD) symptoms, GDNF is a promising agent for its treatment. However, this neurotrophin is unable to cross the blood–brain barrier, which has complicated its clinical use. Therefore, ways to deliver GDNF into the central nervous system in an effective manner are needed. The HIV-1-Tat-derived cell-penetrating peptide (CPP) provides a means to deliver fusion proteins into the brain. We generated a fusion protein between the 11 amino acid CPP of Tat and the rat GDNF mature protein to deliver GDNF across the blood–brain barrier. We showed previously that Tat-GDNF enhances the neuroprotective effect of GDNF in in vivo models for nerve trauma and ischemia. Here, we tested its effect in a subchronic scheme of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) application into the mouse as a model for PD to evaluate the effect of Tat-GDNF fusion protein in dopaminergic neuron survival. We showed that the fusion protein did indeed reach the dopaminergic neurons. However, the in vivo application of Tat-GDNF did not provide neuroprotection of dopaminergic neurons, as revealed by immunohistochemistry and counting of the number of tyrosine-hydroxylase-immunoreactive neurons in the substantia nigra pars compacta. Possibly, GDNF does protect nigro-striatal projections of those neurons that survive MPTP treatment but does not increase the number of surviving dopaminergic neurons. A concomitant treatment of Tat-GDNF with an anti-apoptotic Tat-fusion protein might be beneficial. [Copyright &y& Elsevier]

Abstract: During the last decade, small peptides (10 to 15 amino acids) derived from the HIV-1 Tat protein and from the drosophila Antennapedia homeodomain have been used to internalize various types of molecules into the cells. The way these peptides enter cells is still under investigation and the object of strong controversy. The main discussions rely on whether these peptides are internalized or not in an energy-independent fashion, and, depending on the situation, whether they follow one pathway instead of another. At present, we find in the literature a very large number of data with, at times, some contradictory results. Indeed the diversity of employed peptide sequences, the cell type used, the attachment or not of a cargo molecule, the chemical nature of this cargo itself, and the followed protocol during the experimental process do not simplify the comparison and hence final conclusions about the mechanism of cell entry. However, one common feature emerges with these cell-penetrating peptides: most of them do not show any cell specificity. Despite their demonstrated efficiency in delivering biologically active molecules in in vitro experiments, their use for a therapeutic application in vivo has been the object of a relatively little number of studies, probably because of the quite important amounts of CPP–cargo that needs to be prepared for an accurate and complete in vivo study, but more likely, because of the massive spreading of the cargo all around the body. However, it appears from recent studies that an increased targeting ability of these CPPs is possible, making the use of CPP mediated delivery compatible with an in vivo therapeutic approach. [Copyright &y& Elsevier]

Cell penetrating peptides (CPPs) have been postulated to carry macromolecules across cell plasma membranes without the need of receptors, transporters, endocytosis or any energy-consuming mechanism.We developed an assay to study lipid bilayer permeation of CPPs. HIV-1 TAT peptides were conjugated to N-(4-carboxy-3-hydroxyphenyl)maleimide (SAM) and incubated with Tb3+-containing liposomes. Upon chelation of Tb3+ by an aromatic carboxylic acid, the fluorescence of Tb3+ increases many fold. The CPP TAT(44–57)-SAM and TAT(37–53)-SAM, as a negative control, were unable to enter liposomes consisting of phosphatidylcholine (PC) or a mix of PC, negatively charged lipids and cholesterol.In parallel, cell entry of fluorescein-labeled TAT peptides was studied using confocal laser scanning microscopy (CLSM). TAT(44–57)-fluorescein did not enter Madin Darby canine kidney (MDCK) cells with intact plasma membranes but accumulated at their basal side. Only cells with impaired plasma membranes, as identified by nuclear staining with ethidium homodimer-1 (EthD-1), showed accumulation of TAT(44–57).Our findings change the perspectives of the potential use of TAT peptides as carriers for intracellular targeting. SAM- and fluorescein-labeled TAT(44–57) cannot penetrate lipid bilayers and intact plasma membranes of MDCK cells, respectively. [Copyright &y& Elsevier]

The most commonly studied of the cell-penetrating peptides (CPP) is “penetratin” (pAntp), which functions as a carrier (vector), even for large hydrophilic (cargo) molecules. pAntp originates from the third helix of the Antennapedia homeodomain protein. The peptide is known to interact with negatively charged phospholipid vesicles, which leads to induction of secondary structure. In the present study, circular dichroism (CD) spectroscopy has been used to characterize the different secondary structures induced upon interaction with small unilamellar vesicles (SUVs) from mixtures of zwitterionic 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG). The interaction was monitored using an electron paramagnetic resonance (EPR) spin probe attached to the peptide, and the intrinsic fluorophore (tryptophan). We measured the secondary structure as a function of surface charge density, total lipid-to-peptide (L/P) molar ratio, and salt concentration, for completely bound peptide. With vesicles from POPG/POPC in a molar ratio below 30:70, at a high L/P, the peptide adopts a mainly helical conformation. Increasing the charge density, at the same L/P, promotes a higher degree of β-structure. At a fixed charge density, reducing the L/P also results in an α→β structure conversion. Hence, low membrane surface charge density and low pAntp concentration both favor a mainly helical conformation, while high charge density and pAntp concentration promote a dominating β-structure. We conclude that pAntp, when residing at the surface of a membrane, is chameleon-like in terms of its induced structure. [Copyright &y& Elsevier]

The emergence and spread of multiple drug-resistant bacteria strains caused the development of new antibiotics to be one of the most important challenges of medicinal chemistry. Despite many efforts, the commercial availability of peptide-based antimicrobials is still limited. The presented study aims to explain that immobilized artificial membrane chromatography can support the characterization of antimicrobial peptides. Consequently, the chromatographic experiments of three groups of related peptide substances: (i) short cationic lipopeptides, (ii) citropin analogs, and (iii) conjugates of ciprofloxacin and levofloxacin, with a cell-penetrating peptide were discussed. In light of the discussion of the mechanisms of action of these compounds, the obtained results were interpreted. [ABSTRACT FROM AUTHOR]

The ability to deliver or transduce proteins into cells allows for the manipulation of cell biology in culture, preclinical models, and potentially human disease. Fusion proteins containing the TAT peptide transduction domain (PTD), also known as cell-penetrating peptide (CPP), allow for delivery of a wide variety of proteins, including enzymes, transcription factors, tumor suppressor proteins, and many more. TAT-fusion proteins are generated cloning in-frame into the pTAT-HA plasmid, then transformed into E. coli for expression, and purified by the 6-His affinity tag over Ni-NTA column, followed by a final IEX FPLC purification step., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The African swine fever virus (ASFV) is a huge and complex DNA virus that can lead to the acute death of pigs and cause huge losses to the global swine industry. The CD2v protein is a transmembrane protein encoded by the ASFV's EP402R gene, which can effectively inhibit the bystander lymphocyte proliferation in response to mitogens and mediate the absorption of red blood cells to ASFV-infected cells. The CD2v protein contains repetitive amino acid sequences ([KPCPPP] 3 labeled as RAAS), which is reported as a genetic marker and an epitope. However, the specific biological function of the RAAS is unknown. Here, we have found that the truncated CD2v protein with RAAS can enter Chinese hamster ovary cells, but the truncated CD2v protein without RAAS cannot enter the cells. Also, the RAAS can carry the macromolecular protein EGFP to enter various cells through multiple endocytic processes that are dependent on time, concentration, and location. Besides, the RAAS enter the cells via the macropinocytosis or the clathrin-mediated endocytosis. These results indicate that the RAAS can function as a cell-penetrating peptide that provides a new insight for ASFV research and has potential application value as a tool for drug delivery.

The toolbox for genetic engineering has quickly evolved from CRISPR/Cas9 to a myriad of different gene editors, each with promising properties and enormous clinical potential. However, a major challenge remains: delivering the CRISPR machinery to the nucleus of recipient cells in a nontoxic and efficient manner. In this article, we repurpose an RNA-delivering cell-penetrating peptide, PepFect14 (PF14), to deliver Cas9 ribonucleoprotein (RNP). The RNP-CPP complex achieved high editing rates, e.g., up to 80% in HEK293T cells, while being active at low nanomolar ranges without any apparent signs of toxicity. The editing efficiency was similar to or better compared to the commercially available reagents RNAiMAX and CRISPRMax. The efficiency was thoroughly evaluated in reporter cells and wild-type cells by restriction enzyme digest and next-generation sequencing. Furthermore, the CPP-Cas9-RNP complexes were demonstrated to withstand storage at different conditions, including freeze-thaw cycles and freeze-drying, without a loss in editing efficiency. This CPP-based delivery strategy complements existing technologies and further opens up new opportunities for Cas9 RNP delivery, which can likely be extended to other gene editors in the future. [ABSTRACT FROM AUTHOR]

A specific series of peptides, called a cell-penetrating peptide (CPP), is known to be free to directly permeate through cell membranes into the cytosol (cytolysis); hence, this CPP would be a potent carrier for a drug delivery system (DDS). Previously, we proposed the mechanism of cytolysis as a temporal and local phase transfer of membrane lipid caused by positive membrane curvature generation. Moreover, we showed how to control the CPP cytolysis. Here, we investigate the phospholipid vesicle's size effect on CPP cytolysis because this is the most straightforward way to control membrane curvature. Contrary to our expectation, we found that the smaller the vesicle diameter (meaning a higher membrane curvature), the more cytolysis was suppressed. Such controversial findings led us to seek the reason for the unexpected results, and we ended up finding out that the mobility of membrane lipids as a liquid crystal is the key to cytolysis. As a result, we could explain the cause of cytolysis suppression by reducing the vesicle size (because of the restriction of lipid mobility); osmotic pressure reduction to enhance positive curvature generation works as long as the membrane is mobile enough to modulate the local structure. Taking all the revealed vital factors and their effects as a tool, we will further explore how to control CPP cytolysis for developing a DDS system combined with appropriate cargo selection to be tagged with CPPs. [ABSTRACT FROM AUTHOR]

Cell-penetrating peptide (CPP) can directly penetrate the cytosol (cytolysis) and is expected to be a potent vector for a drug delivery system (DDS). Although there is general agreement that CPP cytolysis is related to dynamic membrane deformation, a distinctive process has yet to be established. Here, we report the key process and factors controlling CPP cytolysis. To elucidate the task, we have introduced trypsin digestion of adsorbed CPP onto giant unilamellar vesicle (GUV) to quantify the adsorption and internalization (cytolysis) separately. Also, the time-course analysis was introduced for the geometric calculation of adsorption and internalization amount per lipid molecule consisting of GUV. As a result, we found that adsorption and internalization assumed to occur successively by CPP molecule come into contact with membrane lipid. Adsorption is quick to saturate within 10 min, while cytolysis of each CPP on the membrane follows successively. After adsorption is saturated, cytolysis proceeds further linearly by time with a different rate constant that is dependent on the osmotic pressure. We also found that temperature and lipid composition influence cytolysis by modulating lipid mobility. The electrolyte in the outer media is also affected as a chemical mediator to control CPP cytolysis by following the Hoffmeister effect for membrane hydration. These results confirmed the mechanism of cytolysis as temporal and local phase transfer of membrane lipid from Lα to Mesh1, which has punctured bilayer morphologies. [ABSTRACT FROM AUTHOR]

Seven conjugates composed of well-known fluoroquinolone antibacterial agents, ciprofloxacin (CIP) or levofloxacin (LVX), and a cell-penetrating peptide transportan 10 (TP10-NH2) were synthesised. The drugs were covalently bound to the peptide via an amide bond, methylenecarbonyl moiety, or a disulfide bridge. Conjugation of fluoroquinolones to TP10-NH2 resulted in congeners demonstrating antifungal in vitro activity against human pathogenic yeasts of the Candida genus (MICs in the 6.25–100 µM range), whereas the components were poorly active. The antibacterial in vitro activity of most of the conjugates was lower than the activity of CIP or LVX, but the antibacterial effect of CIP-S-S-TP10-NH2 was similar to the mother fluoroquinolone. Additionally, for two representative CIP and LVX conjugates, a rapid bactericidal effect was shown. Compared to fluoroquinolones, TP10-NH2 and the majority of its conjugates generated a relatively low level of reactive oxygen species (ROS) in human embryonic kidney cells (HEK293) and human myeloid leukemia cells (HL-60). The conjugates exhibited cytotoxicity against three cell lines, HEK293, HepG2 (human liver cancer cell line), and LLC-PK1 (old male pig kidney cells), with IC50 values in the 10–100 µM range and hemolytic activity. The mammalian toxicity was due to the intrinsic cytoplasmic membrane disruption activity of TP10-NH2 since fluoroquinolones themselves were not cytotoxic. Nevertheless, the selectivity index values of the conjugates, both for the bacteria and human pathogenic yeasts, remained favourable. [ABSTRACT FROM AUTHOR]

Branched polymers as drug delivery carriers have been widely attempted due to their outstanding drug loading capability and complex stability like branched polyethyleneimine (B-PEI). However, branched polymers without biodegradability may cause toxicity as they can accumulate in the body. Herein, we report branched modified nona-arginine (B-mR9) composed of redox-cleavable disulfide bonds to form stable complexes with methotrexate (MTX) as an anticancer agent, which is further coated with hyaluronic acid (HA). The HA-coated nanoparticles provide targetability for the CD44 cell surface receptor. The B-mR9-MTX/HA can effectively aid in intracellular MTX delivery to CD44 overexpressing cancer cells being degradable by the reducing environments of the cancer cells. The B-mR9-MTX/HA exhibits not only a glutathione-triggered degradability but also an outstanding CD44-mediated MTX delivery efficacy. In addition, its superior tumor inhibition capability was confirmed through an in vivo study. The results suggest that the HA-coated B-mR9 nanoparticle can be used as a drug delivery platform.

The cell membrane is a protective layer that strictly controls the passage of molecules restricting the delivery of biomolecules such as drugs, oligonucleotides, peptides, and siRNA into the cells. This shortcoming has been overcome by the discovery of Cell-Penetrating Peptides (CPPs) that has undergone 30 years of evolution. To date, CPPs are largely modified to improve its efficacy and to suit the different delivery applications. The modes of CPPs penetration are still an unresolved mystery and requires further investigations to increase its effectiveness and to diversify its use. Despite having huge potential as a biomolecule carrier, CPPs also have some drawbacks. In this review, the natural and synthetic CPPs, the modifications that have been conducted on CPPs to improve its efficacy, its extended applications, modes of penetration and limitation as well as challenges will be discussed., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Disulfide-rich plant peptides with molecular masses of 2-6 kDa represent an expanding class of peptidyl-type natural products with diverse functions. They are structurally compact, hyperstable, and underexplored as cell-penetrating agents that inhibit intracellular functions. Here, we report the discovery of an anionic, 34-residue peptide, the disulfide-rich roseltide rT7 from Hibiscus sabdariffa (of the Malvaceae family) that penetrates cells and inhibits their proteasomal activities. Combined proteomics and NMR spectroscopy revealed that roseltide rT7 is a cystine-knotted, six-cysteine hevein-like cysteine-rich peptide. A pair-wise comparison indicated that roseltide rT7 is >100-fold more stable against protease degradation than its S -alkylated analog. Confocal microscopy studies and cell-based assays disclosed that after roseltide rT7 penetrates cells, it causes accumulation of ubiquitinated proteins, inhibits human 20S proteasomes, reduces tumor necrosis factor-induced IκBα degradation, and decreases expression levels of intercellular adhesion molecule-1. Structure-activity studies revealed that roseltide rT7 uses a canonical substrate-binding mechanism for proteasomal inhibition enabled by an IIML motif embedded in its proline-rich and exceptionally long intercysteine loop 4. Taken together, our results provide mechanistic insights into a novel disulfide-rich, anionic, and cell-penetrating peptide, representing a potential lead for further development as a proteasomal inhibitor in anti-cancer or anti-inflammatory therapies., (© 2019 Kam et al.)

The absorption of drugs is limited by the epithelial barriers of the gastrointestinal tract. One of the strategies to improve drug delivery is the modulation of barrier function by the targeted opening of epithelial tight junctions. In our previous study the 18-mer amphiphilic PN159 peptide was found to be an effective tight junction modulator on intestinal epithelial and blood–brain barrier models. PN159, also known as KLAL or MAP, was described to interact with biological membranes as a cell-penetrating peptide. In the present work we demonstrated that the PN159 peptide as a penetration enhancer has a dual action on intestinal epithelial cells. The peptide safely and reversibly enhanced the permeability of Caco-2 monolayers by opening the intercellular junctions. The penetration of dextran molecules with different size and four efflux pump substrate drugs was increased several folds. We identified claudin-4 and -7 junctional proteins by docking studies as potential binding partners and targets of PN159 in the opening of the paracellular pathway. In addition to the tight junction modulator action, the peptide showed cell membrane permeabilizing and antimicrobial effects. This dual action is not general for cell-penetrating peptides (CPPs), since the other three CPPs tested did not show barrier opening effects. [ABSTRACT FROM AUTHOR]