Cyclization of a cell‐penetrating peptide via click‐chemistry increases proteolytic resistance and improves drug delivery

In this work we report synthesis and biological evaluation of a cell‐penetrating peptide (CPP), that is partly cyclized via a triazole bridge. Recently, beneficious properties have been reported for cyclized peptides concerning their metabolic stability and intracellular uptake. A CPP based on human calcitonin was used in this study, and side chain cyclization was achieved via copper catalyzed alkyne‐azide click reaction. Cell viability studies in several cell‐lines revealed no cytotoxic effects. Furthermore, efficient uptake in breast cancer MCF‐7 cells could be determined. Moreover, preliminary studies using this novel peptide as drug transporter for daunorubicin were performed. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

A biological transporter for the delivery of peptide nucleic acids (PNAs) to the nuclear compartment of living cells

To facilitate nuclear delivery of biomolecules we describe the synthesis of a modular transporter bearing a cellular membrane transport peptide (pAntp) and, as a cargo, a 16-mer peptide nucleic acid (PNA) covalently linked to a nuclear localisation signal (NLS[SV40-T]). Transport peptide and PNA are connected via N-terminal activated cysteine to form cleavable disulphide bonds. Internalization and subsequent delivery of PNA to the nucleus was verified in living and fixed cells by confocal laser scanning microscopy (CLSM) and fluorescence correlation spectroscopy (FCS). Double-labelling experiments indicate the cytoplasmic cleavage of the two modules and the effective nuclear import of the chromophore-tagged cargo. A non-degradable linker between transport module and cargo as well as a construct without NLS did not enable nuclear PNA import under the described experimental conditions. FCS-measurements revealed that most of the PNAs delivered into the cytoplasm by the modular transporter are anchored or encapsulated, indicating that intracellular transport of these compounds is not governed by molecular diffusion. Our results clearly demonstrate efficient compartment-directed transport using a synthetic, non-toxic modular transporter in living cells.     

Structural requirements for cellular uptake of α‐helical amphipathic peptides

The structure of the cell‐permeable α‐helical amphipathic model peptide FLUOS‐KLALKLALKALKAALKLA‐NH₂ ( I ) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 μm . These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non‐amphipathic 18‐mer peptides with different primary structure, net charge and helix parameters from I . The amphipathic counterparts were internalized into the cells to a comparable extent as I , whereas no cellular uptake could be detected for the non‐amphipathic analogues. The mode of uptake remains unclear and involves both temperature‐sensitive and ‐insensitive processes, indicating non‐endocytic contributions. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Studies on the cellular uptake of substance P and lysine-rich, KLA-derived model peptides

In the last decade many peptides have been shown to be internalized into various cell types by different, poorly characterized mechanisms. This review focuses on uptake studies with substance P (SP) aimed at unravelling the mechanism of peptide-induced mast cell degranulation, and on the characterization of the cellular uptake of designed KLA-derived model peptides.Studies on structure-activity relationships and receptor autoradiography failed to detect specific peptide receptors for the undecapeptide SP on mast cells. In view of these findings, a direct interaction of cationic peptides with heterotrimeric G proteins without the participation of a receptor has been proposed. Such a process would require insertion into and translocation of peptides across the plasma membrane.In order to clarify whether a transport of cationic peptides into rat peritoneal mast cells is possible, transport studies were performed by confocal laser scanning microscopy (CLSM) using fluorescence-labeled Arg(3),Orn(7)-SP and its D-amino acid analog, all-D-Arg(3),Orn(7)-SP, as well as by electron microscopic autoradiography using (3)H-labelled SP and (125)I-labelled all-D-SP. The results obtained by CLSM directly showed translocation of SP peptides into pertussis toxin-treated cells. Kinetic experiments indicated that the translocation process was rapid, occurring within a few seconds. Mast cell degranulation induced by analog of magainin 2 amide, neuropeptide Y and the model peptide acetyl-KLALKLALKALKAALKLA-amide was also found to be very fast, pointing to an extensive translocation of the peptides. In order to learn more about structural requirements for the cellular uptake of peptides, the translocation behavior of a set of systematically modified KLA-based model peptides has been studied in detail. By two different protocols for determining the amount of internalized peptide, evidence was found that the structure of the peptides only marginally affects their uptake, whereas the efflux of cationic, amphipathic peptides is strikingly diminished, thus allowing their enrichment within the cells. Although the mechanism of cellular uptake, consisting of energy-dependent and -independent contributions, is not well understood, KLA-derived peptides have been shown to deliver various cargos (PNAs, peptides) into cells. The results obtained with SP- and KLA-derived peptides are discussed in the context of the current literature.     

Structural requirements for cellular uptake and antisense activity of peptide nucleic acids conjugated with various peptides

Peptide nucleic acids (PNAs) have shown great promise as potential antisense drugs; however, poor cellular delivery limits their applications. Improved delivery into mammalian cells and enhanced biological activity of PNAs have been achieved by coupling to cell-penetrating peptides (CPPs). Structural requirements for the shuttling ability of these peptides as well as structural properties of the conjugates such as the linker type and peptide position remained controversial, so far. In the present study an 18mer PNA targeted to the cryptic splice site of a mutated beta-globin intron 2, which had been inserted into a luciferase reporter gene coding sequence, was coupled to various peptides. As the peptide lead we used the cell-penetrating alpha-helical amphipathic peptide KLAL KLAL KAL KAAL KLA-NH2 [model amphipathic peptide (MAP)] which was varied with respect to charge and structure-forming properties. Furthermore, the linkage and the localization of the attached peptide (C- vs N-terminal) were modified. Positive charge as well as helicity and amphipathicity of the KLA peptide was all required for efficient dose-dependent correction of aberrant splicing. The highest antisense effect was reached within 4 h without any transfection agent. Stably linked conjugates were also efficient in correction of aberrant splicing, suggesting that a cleavable disulfide bond between CPP and PNA is clearly not essential. Moreover, the placement of the attached peptide turned out to be crucial for attaining antisense activity. Coadministration of endosome disrupting agents such as chloroquine or Ca2+ significantly increased the splicing correction efficiency of some conjugates, indicating the predominant portion to be sequestered in vesicular compartments.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Summary Genomic sequencing makes it possible to identify all the genes of an organism, now including Homo sapiens. Yet measurement of the expression of each gene of interest still presents a daunting prospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately, no reliable method has been available to measure levels of specific mRNAs in vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless, we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cells are taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAs in human cancer xenografts in a mouse model. The oncogenes cyclin D1, ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experiments provide a proof-of-principle for noninvasive detection of oncogene expression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Summary Genomic sequencing makes it possible to identify all the genes of an organism, now includingHomo sapiens. Yet measurement of the expression of each gene of interest still presents a daunting prospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately, no reliable method has been available to measure levels of specific mRNAsin vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless, we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cells are taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAs in human cancer xenografts in a mouse model. The oncogenes cyclin D1,ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experiments provide a proof-of-principle for noninvasive detection of oncogene expression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

Receptor-specific targeting with complementary peptide nucleic acids conjugated to peptide analogs and radionuclides

Genomic sequencing makes it possible to identify all the genes of an organism, now including Homo sapiens. Yet measurement of the expression of each gene of interest still presents a dauntingprospect. Northern blots, RNase protection assays, as well as microarrays and related technologies permit measurement of gene expression in total RNA extracted from cultured cells or tissue samples. It would be most valuable, however, to quantitate gene expression noninvasively in living cells and tissues. Unfortunately,no reliable method has been available to measure levels of specificmRNAs in vivo. Peptide nucleic acids (PNAs) display superior ruggedness and hybridization properties as a diagnostic tool for gene expression, and could be used for this purpose. On the down side, they are negligibly internalized by normal or malignant cells in the absence of conjugated ligands. Nevertheless,we have observed that Tc-99m-peptides can delineate tumors, and PNA-peptides designed to bind to IGF-1 receptors on malignant cellsare taken up specifically and concentrated in nuclei. We have postulated that antisense Tc-99m-PNA-peptides will be taken up by human cancer cells, will hybridize to complementary mRNA targets, and will permit scintigraphic imaging of oncogene mRNAsin human cancer xenografts in a mouse model. The oncogenes cyclinD1, ERBB2, c-MYC, K-RAS, and tumor suppressor p53 are being probed initially. These experimentsprovide a proof-of-principle for noninvasive detection of oncogeneexpression in living cells and tissues. This scintigraphic imaging technique should be applicable to any particular gene of interest in a cell or tissue type with characteristic receptors.     

Preparation of radioiodinated peptide nucleic acids with high specific activity

Peptide nucleic acids (PNAs) have stronger affinity and greater specificity than do oligonucleotides for binding to DNA and RNA and, as such, have potential utility as probes in molecular biology applications. In this study, a novel approach for labeling the PNA with radioiodine that avoided solubility issues and poor labeling encountered when trying to radioiodinate PNAs directly in solution was developed. For this approach, a purpose-designed prosthetic group that incorporated both a radioiodinatable tyrosine and a triphenylphosphonium (TPP) moiety was synthesized. The latter is an organic cation that combines the properties of good solubility in both aqueous and organic solvents with a strong retention by reverse phase HPLC. Following radioiodination of the TPP-based prosthetic group in phosphate buffer, the prosthetic group was purified and coupled to the terminal amine of 15-mer PNA on the solid phase resin. After cleavage and deprotection of the PNA from the resin, the presence of the TPP group resulted in a clean separation of radioiodinated PNA from unlabeled PNA, yielding a high-specific activity probe in a single HPLC run. As an example of a potential molecular biology application of the resultant (125)I-labeled PNA probe, it was used to detect mRNA for the Lcn2 gene in Northern blotting.     

Peptide nucleic acids: Cellular delivery and recognition of DNA and RNA targets

Peptide nucleic acids (PNAs) can be conveniently delivered into cells in complex with DNA and cationic lipid. This advance enables researchers to test the hypothesis that PNAs offer advantages for recognition of DNA or RNA targets within cells. In this review, I describe the intracellular delivery of PNAs as DNA-PNA-cationic lipid complexes and discuss recognition of three classes of nucleic acid target: duplex DNA, single-stranded mRNA, and the ribonucleoprotein telomerase. These targets differ dramatically in their potential for base-paired structure, offering distinct challenges for hybridization by PNAs. It is apparent that PNAs can exert sequence-specific effects within cells, and their full potential has only begun to be explored.     

Peptide nucleic acids: Cellular delivery and recognition of DNA and RNA targets

Summary Peptide nucleic acids (PNAs) can be conveniently delivered into cells in complex with DNA and cationic lipid. This advance enables researchers to test the hypothesis that PNAs offer advantages for recognition of DNA or RNA targets within cells. In this review, I describe the intracellular delivery of PNAs as DNA-PNA-cationic lipid complexes and discuss recognition of three classes of nucleic acid target: duplex DNA, single-stranded mRNA, and the ribonucleoprotein telomerase. These targets differ dramatically in their potential for base-paired structure, offering distinct challenges for hybridization by PNAs. It is apparent that PNAs can exert sequence-specific effects within cells, and their full potential has only begun to be explored.     

Kinetic uptake profiles of cell penetrating peptides in lymphocytes and monocytes

Background

Nucleolar targeting peptides (NrTPs), resulting from structural minimization of the rattlesnake toxin crotamine, are a novel family of cell-penetrating peptides (CPPs) shown to internalize and deliver cargos into different cell types.

Methods

In this study, we address NrTP kinetics of translocation into primary cells. We used flow cytometry to measure the intracellular uptake of rhodamine B-labeled NrTPs in peripheral blood mononucleated cells (PBMCs).

Results

The kinetic profiles for each peptide are concentration-independent but significantly different among NrTPs, pointing out for the amino acid sequence importance. Arginine-containing peptides (NrTP7 and Tat_48–60, used for comparison) were found to be more toxic than lysine-containing ones, as expected. On the other hand, one same peptide behaves differently in each of the lymphocyte and monocyte cell populations, suggesting differences in entry mechanism that in turn reflect diversity in cell functionality. Uptake results obtained at 4 °C or using chemical endocytosis inhibitors support the importance of non-endocytic mechanisms in the cellular internalization of NrTP1 and NrTP5, while confirming endocytosis as the main mechanism of NrTPs entry.

Conclusion

Overall, both direct translocation and endocytosis mechanisms play a role in NrTP entry. Yet, there is predominance of endocytosis-mediated mechanisms. NrTPs (especially NrTP6) are an excellent intracellular delivery tool, with efficient internalization and no toxicity.

General significance

This work validates NrTPs as potential therapeutic tools for, e.g., cancer or inhibition of viral replication and establishes a new comparative and quantitative method to test CPP efficiency.     

Transduction of peptides and proteins into live cells by cell penetrating peptides

Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines.     

Temporal switching and cell‐to‐cell variability in Ca2+ release activity in mammalian cells

Genetically identical cells in a uniform external environment can exhibit different phenotypes, which are often masked by conventional measurements that average over cell populations. Although most studies on this topic have used microorganisms, differentiated mammalian cells have rarely been explored. Here, we report that only approximately 40% of clonal human embryonic kidney 293 cells respond with an intracellular Ca²⁺ increase when ryanodine receptor Ca²⁺ release channels in the endoplasmic reticulum are maximally activated by caffeine. On the other hand, the expression levels of ryanodine receptor showed a unimodal distribution. We showed that the difference in the caffeine sensitivity depends on a critical balance between Ca²⁺ release and Ca²⁺ uptake activities, which is amplified by the regenerative nature of the Ca²⁺ release mechanism. Furthermore, individual cells switched between the caffeine‐sensitive and caffeine‐insensitive states with an average transition time of approximately 65 h, suggestive of temporal fluctuation in endogenous protein expression levels associated with caffeine response. These results suggest the significance of regenerative mechanisms that amplify protein expression noise and induce cell‐to‐cell phenotypic variation in mammalian cells.     

A stable meta‐carborane enables the generation of boron‐rich peptide agonists targeting the ghrelin receptor

Boron neutron capture therapy (BNCT) is a binary cancer therapy, which combines the biochemical targeting of a boron‐containing drug with the regional localization of radiation treatment. Although the concept of BNCT has been known for decades, the selective delivery of boron into tumor cells remains challenging. G protein‐coupled receptors that are overexpressed on cancer cells in combination with peptidic ligands can be potentially used as shuttle system for a tumor‐directed boron uptake. In this study, we present the generation of short, boron‐rich peptide conjugates that target the ghrelin receptor. Expression of the ghrelin receptor on various cancer cells makes it a viable target for BNCT. We designed a novel hexapeptide super‐agonist that was modified with different specifically synthesized carborane monoclusters and tested for ghrelin receptor activation. A meta‐carborane building block with a mercaptoacetic acid linker was found to be optimal for peptide modification, owing to its chemical stability and a suitable activation efficacy of the conjugate. The versatility of this carborane for the development of peptidic boron delivery agents was further demonstrated by the generation of highly potent, boron‐loaded conjugates using the backbone of the known ghrelin receptor ligands growth hormone releasing peptide 6 and Ipamorelin.     

Identification of insect cell lines and cell‐line cross‐contaminations by nuclear ribosomal ITS sequences

This report shows how the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (rDNA) can be used to determine the species identity of insect cell lines and to distinguish between cell lines derived from closely related insect species. A PCR‐RFLP method with the endonucleases HincII and PstI produces restriction fragment profiles that could distinguish between insect cell lines at the species level. Another PCR‐based method used three species‐specific primer sets, Ly‐ITS1/Ly‐ITS2, ITS1‐1/Ld‐ITS1 and Sf9‐F2/ITS4, to identify the cell lines from Lymantria xylina, L. dispar and Spodoptera frugiperda, respectively. This method also detected cell‐line cross‐contaminations (CLCC) with contamination levels as low as 1% (10 cells in a population of 1000 cells) even when the contaminating cells were from a closely related species. Compared with conventional methods used for cell‐line identification and CLCC detection, the methods presented here are fast and sensitive and could easily be applied to other cell culture laboratories.