Design and biological characterization of novel cell-penetrating peptides preferentially targeting cell nuclei and subnuclear regions

Within this study, we report about the design and biological characterization of novel cell-penetrating peptides (CPPs) with selective suborganelle-targeting properties. The nuclear localization sequence N50, as well as the nucleoli-targeting sequence NrTP, respectively, were fused to a shortened version of the cell-penetrating peptide sC18. We examined cellular uptake, subcellular fate and cytotoxicity of these novel peptides, N50-sC18* and NrTP-sC18*, and found that they are nontoxic up to a concentration of 50 or 100 µM depending on the cell lines used. Moreover, detailed cellular uptake studies revealed that both peptides enter cells via energy-independent uptake, although endocytotic processes cannot completely excluded. However, initial drug delivery studies demonstrated the high versatility of these new peptides as efficient transport vectors targeting specifically nuclei and nucleoli. In future, they could be further explored as parts of newly created peptide–drug conjugates.

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Coupling the Antimalarial Cell Penetrating Peptide TP10 to Classical Antimalarial Drugs Primaquine and Chloroquine Produces Strongly Hemolytic Conjugates

Molecules ◽

10.3390/molecules24244559 ◽

2019 ◽

Vol 24 (24) ◽

pp. 4559 ◽

Cited By ~ 4

Author(s):

Luísa Aguiar ◽

Arnau Biosca ◽

Elena Lantero ◽

Jiri Gut ◽

Nuno Vale ◽

...

Keyword(s):

Cell Penetrating Peptides ◽

Antiplasmodial Activity ◽

Erythrocyte Membranes ◽

Cell Penetrating Peptide ◽

Peptide Conjugates ◽

Drug Conjugates ◽

Cell Penetrating ◽

A Cell ◽

Peptide Drug Conjugates ◽

The Cost

Recently, we disclosed primaquine cell penetrating peptide conjugates that were more potent than parent primaquine against liver stage Plasmodium parasites and non-toxic to hepatocytes. The same strategy was now applied to the blood-stage antimalarial chloroquine, using a wide set of peptides, including TP10, a cell penetrating peptide with intrinsic antiplasmodial activity. Chloroquine-TP10 conjugates displaying higher antiplasmodial activity than the parent TP10 peptide were identified, at the cost of an increased hemolytic activity, which was further confirmed for their primaquine analogues. Fluorescence microscopy and flow cytometry suggest that these drug-peptide conjugates strongly bind, and likely destroy, erythrocyte membranes. Taken together, the results herein reported put forward that coupling antimalarial aminoquinolines to cell penetrating peptides delivers hemolytic conjugates. Hence, despite their widely reported advantages as carriers for many different types of cargo, from small drugs to biomacromolecules, cell penetrating peptides seem unsuitable for safe intracellular delivery of antimalarial aminoquinolines due to hemolysis issues. This highlights the relevance of paying attention to hemolytic effects of cell penetrating peptide-drug conjugates.

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siRNA versus pharmacological inhibition of endocytic pathways for studying cellular uptake of cell penetrating peptides and other drug delivery vectors

Drug Discovery Today ◽

10.1016/j.drudis.2010.09.411 ◽

2010 ◽

Vol 15 (23-24) ◽

pp. 1103-1103

Author(s):

Monerah H. Al-Soraj ◽

Catherine L. Watkins ◽

Dries Vercauteren ◽

Stefaan De Smedt ◽

Kevin Braeckmans ◽

...

Keyword(s):

Drug Delivery ◽

Cellular Uptake ◽

Cell Penetrating Peptides ◽

Pharmacological Inhibition ◽

Cell Penetrating ◽

Endocytic Pathways

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Cellular Uptake of Arginine-Rich Cell-Penetrating Peptides and the Contribution of Membrane-Associated Proteoglycans

Trends in Glycoscience and Glycotechnology ◽

10.4052/tigg.1420.1 ◽

2015 ◽

Vol 27 (155) ◽

pp. 81-88 ◽

Cited By ~ 3

Author(s):

Ikuhiko Nakase ◽

Yoshimasa Kawaguchi ◽

Motoyoshi Nomizu ◽

Shiroh Futaki

Keyword(s):

Cellular Uptake ◽

Cell Penetrating Peptides ◽

Cell Penetrating

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β-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides

Biomolecules ◽

10.3390/biom8030051 ◽

2018 ◽

Vol 8 (3) ◽

pp. 51 ◽

Cited By ~ 5

Author(s):

Shane Stone ◽

Tatjana Heinrich ◽

Suzy Juraja ◽

Jiulia Satiaputra ◽

Clinton Hall ◽

...

Keyword(s):

Functional Characterization ◽

Cell Penetrating Peptides ◽

Stable Cell Line ◽

Intracellular Space ◽

Biologically Relevant ◽

Cell Internalization ◽

Cytosolic Delivery ◽

Cell Penetrating ◽

Split Protein

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 β-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length β-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of β-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split β-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 µM. Together, the β-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.

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A comparison of acyl-moieties for noncovalent functionalization of PLGA and PEG-PLGA nanoparticles with a cell-penetrating peptide

RSC Advances ◽

10.1039/d1ra05871a ◽

2021 ◽

Vol 11 (57) ◽

pp. 36116-36124

Author(s):

Omar Paulino da Silva Filho ◽

Muhanad Ali ◽

Rike Nabbefeld ◽

Daniel Primavessy ◽

Petra H. Bovee-Geurts ◽

...

Keyword(s):

Cellular Uptake ◽

Plga Nanoparticles ◽

Cell Penetrating Peptides ◽

Cell Penetrating Peptide ◽

Noncovalent Functionalization ◽

Cell Penetrating ◽

A Cell

Noncovalent functionalization with acylated cell-penetrating peptides achieves an efficient cellular uptake of PLGA and PEG-PLGA nanoparticles.

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Backbone rigidity and static presentation of guanidinium groups increases cellular uptake of arginine-rich cell-penetrating peptides

Nature Communications ◽

10.1038/ncomms1459 ◽

2011 ◽

Vol 2 (1) ◽

Cited By ~ 166

Author(s):

Gisela Lättig-Tünnemann ◽

Manuel Prinz ◽

Daniel Hoffmann ◽

Joachim Behlke ◽

Caroline Palm-Apergi ◽

...

Keyword(s):

Cellular Uptake ◽

Cell Penetrating Peptides ◽

Cell Penetrating

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Internalization mechanisms of cell-penetrating peptides

Beilstein Journal of Nanotechnology ◽

10.3762/bjnano.11.10 ◽

2020 ◽

Vol 11 ◽

pp. 101-123 ◽

Cited By ~ 20

Author(s):

Ivana Ruseska ◽

Andreas Zimmer

Keyword(s):

Cellular Uptake ◽

Cell Types ◽

Cell Penetrating Peptides ◽

Uptake Mechanism ◽

Black And White ◽

Cell Penetrating ◽

Basic Peptides ◽

Different Cell Types ◽

Immense Potential

In today’s modern era of medicine, macromolecular compounds such as proteins, peptides and nucleic acids are dethroning small molecules as leading therapeutics. Given their immense potential, they are highly sought after. However, their application is limited mostly due to their poor in vivo stability, limited cellular uptake and insufficient target specificity. Cell-penetrating peptides (CPPs) represent a major breakthrough for the transport of macromolecules. They have been shown to successfully deliver proteins, peptides, siRNAs and pDNA in different cell types. In general, CPPs are basic peptides with a positive charge at physiological pH. They are able to translocate membranes and gain entry to the cell interior. Nevertheless, the mechanism they use to enter cells still remains an unsolved piece of the puzzle. Endocytosis and direct penetration have been suggested as the two major mechanisms used for internalization, however, it is not all black and white in the nanoworld. Studies have shown that several CPPs are able to induce and shift between different uptake mechanisms depending on their concentration, cargo or the cell line used. This review will focus on the major internalization pathways CPPs exploit, their characteristics and regulation, as well as some of the factors that influence the cellular uptake mechanism.

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