scholarly journals Breaking in and busting out: cell-penetrating peptides and the endosomal escape problem

2017 ◽  
Vol 8 (3-4) ◽  
pp. 131-141 ◽  
Author(s):  
Julia C. LeCher ◽  
Scott J. Nowak ◽  
Jonathan L. McMurry
Keyword(s):  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Great Promise ◽  
Delivery Methods ◽  
Oligomeric Proteins ◽  
Cell Penetrating ◽  
History Of ◽  
A Cell ◽  
Escape Problem ◽  
Primary Focus

AbstractCell-penetrating peptides (CPPs) have long held great promise for the manipulation of living cells for therapeutic and research purposes. They allow a wide array of biomolecules from large, oligomeric proteins to nucleic acids and small molecules to rapidly and efficiently traverse cytoplasmic membranes. With few exceptions, if a molecule can be associated with a CPP, it can be delivered into a cell. However, a growing realization in the field is that CPP-cargo fusions largely remain trapped in endosomes and are eventually targeted for degradation or recycling rather than released into the cytoplasm or trafficked to a desired subcellular destination. This ‘endosomal escape problem’ has confounded efforts to develop CPP-based delivery methods for drugs, enzymes, plasmids, etc. This review provides a brief history of CPP research and discusses current issues in the field with a primary focus on the endosomal escape problem, for which several promising potential solutions have been developed. Are we on the verge of developing technologies to deliver therapeutics such as siRNA, CRISPR/Cas complexes and others that are currently failing because of an inability to get into cells, or are we just chasing after another promising but unworkable technology? We make the case for optimism.

scholarly journals A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos

2021 ◽  
Author(s):  
Jonathan L. McMurry ◽  
Schuyler B. Gentry ◽  
Scott J. Nowak ◽  
Xuelei Ni ◽  
Stephanie A. Hill ◽  
...  
Keyword(s):  
Real Time ◽  
Cultured Cells ◽  
Flow Chamber ◽  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Calmodulin Binding ◽  
Distance Analysis ◽  
Cell Penetrating ◽  
Broad Array ◽  
Escape Problem

Cell-penetrating peptides (CPPs) are capable of transporting molecules to which they are tethered across cellular membranes. Unsurprisingly, CPPs have attracted attention for their potential drug delivery applications, but several technical hurdles remain to be overcome. Chief among them is the so-called ‘endosomal escape problem,’ i.e. the propensity of CPP-cargo molecules to be endocytosed but remain entrapped in endosomes rather than reaching the cytosol. Previously, a CPP fused to calmodulin that bound calmodulin binding site-containing cargos was shown to efficiently deliver cargos to the cytoplasm, effectively overcoming the endosomal escape problem. The CPP-adaptor, “TAT-CaM,” evinces delivery at nM concentrations and more rapidly than we had previously been able to measure. To better understand the kinetics and mechanism of CPP-adaptor-mediated cargo delivery, a real-time cell penetrating assay was developed in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for observation ab initio . Also examined in this study was an improved CPP-adaptor that utilizes naked mole rat ( Heterocephalus glaber ) calmodulin in place of human and results in superior internalization, likely due to its lesser net negative charge. Adaptor-cargo complexes were delivered into the flow chamber and fluorescence intensity in the midpoint of baby hamster kidney cells was measured as a function of time. Delivery of 400 nM cargo was observed within seven minutes and fluorescence continued to increase linearly as a function of time. Cargo-only control experiments showed that the minimal uptake which occurred independently of the CPP-adaptor resulted in punctate localization consistent with endosomal entrapment. A distance analysis was performed for cell-penetration experiments in which CPP-adaptor-delivered cargo showing wider dispersions throughout cells as compared to an analogous covalently-bound CPP-cargo. Small molecule endocytosis inhibitors did not have significant effects upon delivery. The real-time assay is an improvement upon static endpoint assays and should be informative in a broad array of applications.

scholarly journals A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0254468
Author(s):  
Schuyler B. Gentry ◽  
Scott J. Nowak ◽  
Xuelei Ni ◽  
Stephanie A. Hill ◽  
Lydia R. Wade ◽  
...  
Keyword(s):  
Real Time ◽  
Cultured Cells ◽  
Flow Chamber ◽  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Calmodulin Binding ◽  
Distance Analysis ◽  
Cell Penetrating ◽  
Broad Array ◽  
Escape Problem

Cell-penetrating peptides (CPPs) are capable of transporting molecules to which they are tethered across cellular membranes. Unsurprisingly, CPPs have attracted attention for their potential drug delivery applications, but several technical hurdles remain to be overcome. Chief among them is the so-called ‘endosomal escape problem,’ i.e. the propensity of CPP-cargo molecules to be endocytosed but remain entrapped in endosomes rather than reaching the cytosol. Previously, a CPP fused to calmodulin that bound calmodulin binding site-containing cargos was shown to efficiently deliver cargos to the cytoplasm, effectively overcoming the endosomal escape problem. The CPP-adaptor, “TAT-CaM,” evinces delivery at nM concentrations and more rapidly than we had previously been able to measure. To better understand the kinetics and mechanism of CPP-adaptor-mediated cargo delivery, a real-time cell penetrating assay was developed in which a flow chamber containing cultured cells was installed on the stage of a confocal microscope to allow for observation ab initio. Also examined in this study was an improved CPP-adaptor that utilizes naked mole rat (Heterocephalus glaber) calmodulin in place of human and results in superior internalization, likely due to its lesser net negative charge. Adaptor-cargo complexes were delivered into the flow chamber and fluorescence intensity in the midpoint of baby hamster kidney cells was measured as a function of time. Delivery of 400 nM cargo was observed within seven minutes and fluorescence continued to increase linearly as a function of time. Cargo-only control experiments showed that the minimal uptake which occurred independently of the CPP-adaptor resulted in punctate localization consistent with endosomal entrapment. A distance analysis was performed for cell-penetration experiments in which CPP-adaptor-delivered cargo showing wider dispersions throughout cells as compared to an analogous covalently-bound CPP-cargo. Small molecule endocytosis inhibitors did not have significant effects upon delivery. The real-time assay is an improvement upon static endpoint assays and should be informative in a broad array of applications.

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

RSC Advances ◽  
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.

scholarly journals Recent Advances in Cell Penetrating Peptide-Based Anticancer Therapies

Molecules ◽  
2019 ◽  
Vol 24 (5) ◽  
pp. 927 ◽  
Author(s):  
Justine Habault ◽  
Jean-Luc Poyet
Keyword(s):  
Tissue Specificity ◽  
Chemical Compounds ◽  
Drug Carriers ◽  
Cell Penetrating Peptides ◽  
Amino Acid Sequences ◽  
Recent Advances ◽  
Cell Penetrating ◽  
A Cell ◽  
Low Toxicity ◽  
Applied Biology

Cell-penetrating-peptides (CPPs) are small amino-acid sequences characterized by their ability to cross cellular membranes. They can transport various bioactive cargos inside cells including nucleic acids, large proteins, and other chemical compounds. Since 1988, natural and synthetic CPPs have been developed for applications ranging from fundamental to applied biology (cell imaging, gene editing, therapeutics delivery). In recent years, a great number of studies reported the potential of CPPs as carriers for the treatment of various diseases. Apart from a good efficacy due to a rapid and potent delivery, a crucial advantage of CPP-based therapies is the peptides low toxicity compared to most drug carriers. On the other hand, they are quite unstable and lack specificity. Higher specificity can be obtained using a cell-specific CPP to transport the therapeutic agent or using a non-specific CPP to transport a cargo with a targeted activity. CPP-cargo complexes can also be conjugated to another moiety that brings cell- or tissue-specificity. Studies based on all these approaches are showing promising results. Here, we focus on recent advances in the potential usage of CPPs in the context of cancer therapy, with a particular interest in CPP-mediated delivery of anti-tumoral proteins.

Decoding the Entry of Two Novel Cell-Penetrating Peptides in HeLa Cells:  Lipid Raft-Mediated Endocytosis and Endosomal Escape†

Biochemistry ◽  
2005 ◽  
Vol 44 (1) ◽  
pp. 72-81 ◽  
Author(s):  
Christina Foerg ◽  
Urs Ziegler ◽  
Jimena Fernandez-Carneado ◽  
Ernest Giralt ◽  
Robert Rennert ◽  
...  
Keyword(s):  
Lipid Raft ◽  
Hela Cells ◽  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Cell Penetrating

scholarly journals Cell-Penetrating Peptide and Transferrin Co-Modified Liposomes for Targeted Therapy of Glioma

Molecules ◽  
2019 ◽  
Vol 24 (19) ◽  
pp. 3540 ◽  
Author(s):  
Xi Wang ◽  
Yarong Zhao ◽  
Shiyan Dong ◽  
Robert J. Lee ◽  
Dongsheng Yang ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Therapeutic Agent ◽  
Spherical Shape ◽  
Cell Penetrating Peptides ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Great Promise ◽  
Uniform Size ◽  
Cell Penetrating ◽  
Low Toxicity

Glioma is one of the most aggressive and common malignant brain tumors. Due to the presence of the blood-brain barrier (BBB), the effectiveness of therapeutics is greatly affected. In this work, to develop an efficient anti-glioma drug with targeting and which was able to cross the BBB, cell-penetrating peptides (R8) and transferrin co-modified doxorubicin (DOX)-loaded liposomes (Tf-LPs) were prepared. Tf-LPs possessed a spherical shape and uniform size with 128.64 nm and their ξ-potential was 6.81 mV. Tf-LPs were found to be stable in serum within 48 h. Uptake of Tf-LPs in both U87 and GL261 cells was analyzed by confocal laser scanning microscopy and by flow cytometry. Tf-LPs were efficiently taken up by both U87 and GL261 cells. Moreover, Tf-LPs exhibited sustained-release. The cumulative release of DOX from Tf-LPs reached ~50.0% and showed excellent anti-glioma efficacy. Histology of major organs, including brain, heart, liver, spleen, lungs and kidney, and the bodyweight of mice, all indicated low toxicity of Tf-LPs. In conclusion, Tf-LPs showed great promise as an anti-glioma therapeutic agent.

Cellular siRNA delivery using cell-penetrating peptides modified for endosomal escape

2009 ◽  
Vol 61 (9) ◽  
pp. 704-709 ◽  
Author(s):  
Tamaki Endoh ◽  
Takashi Ohtsuki
Keyword(s):  
Sirna Delivery ◽  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Cell Penetrating

scholarly journals Cell-Penetrating Peptides: a Useful Tool for the Delivery of Various Cargoes Into Cells

2018 ◽  
pp. S267-S279 ◽  
Author(s):  
E. BÖHMOVÁ ◽  
D. MACHOVÁ ◽  
M. PECHAR ◽  
R. POLA ◽  
K. VENCLÍKOVÁ ◽  
...  
Keyword(s):  
Cellular Uptake ◽  
Cellular Membrane ◽  
Cell Penetrating Peptides ◽  
Cell Entry ◽  
Biologically Active ◽  
Cell Internalization ◽  
Structural Types ◽  
Cell Penetrating ◽  
History Of ◽  
Preclinical And Clinical Studies

Cell-penetrating compounds are substances that enhance the cellular uptake of various molecular cargoes that do not easily cross the cellular membrane. The majority of cell-penetrating compounds described in the literature are cell-penetrating peptides (CPPs). This review summarizes the various structural types of cell-penetrating compounds, with the main focus on CPPs. The authors present a brief overview of the history of CPPs, discuss the various types of conjugation of CPPs to biologically active cargoes intended for cell internalization, examine the cell-entry mechanisms of CPPs, and report on the applications of CPPs in research and in preclinical and clinical studies.

scholarly journals Conjugation of oligo-His peptides to magnetic γ-Fe2O3@SiO2 core-shell nanoparticles promotes their access to the cytosol

2021 ◽  
Author(s):  
Mathilde Le Jeune ◽  
Emilie Secret ◽  
Michaël Trichet ◽  
Aude Michel ◽  
Delphine Ravault ◽  
...  
Keyword(s):  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Core Shell ◽  
Shell Nanoparticles ◽  
Heating Efficiency ◽  
Multiple Substrates ◽  
Transmission Electron ◽  
Cell Penetrating ◽  
Calcein Leakage ◽  
Core Shell Nanoparticles

The endosomal entrapment of functional nanoparticles is a severe limitation to their use for biomedical applications. In the case of magnetic nanoparticles (MNPs), this entrapment leads to poor heating efficiency for magnetic hyperthermia and suppresses the possibility to manipulate them in the cytosol. Current strategies to limit their entrapment are based on their functionalization with cell-penetrating peptides in order to promote their translocation directly across the cell membrane or their endosomal escape. However, these strategies suffer from potential release of free peptides in cell and to the best of our knowledge there is currently a lack of effective methods for the cytosolic delivery of MNPs after incubation with cells. Herein, we report the conjugation of fluorescently labelled cationic peptides to γ-Fe2O3@SiO2 core-shell nanoparticles by click chemistry to improve MNP access to the cytosol. We compare the effect of Arg9 and His4 peptides. On one hand, Arg9 is a classical cell-penetrating peptide, able to enter cells by direct translocation and on the other hand, it has been demonstrated that sequences rich in histidine residues promote endosomal escape, most probably by the proton sponge effect. The methodology developed allows to have a high co-localization of the peptides and core-shell nanoparticles in cells and to attest that the grafting onto nanoparticles of peptides rich in histidine promotes NP access to the cytosol. The endosomal escape was confirmed by a calcein leakage assay and by ultrastructural analysis in transmission electron microscopy. No toxicity of the nanoparticles functionalized with peptides was found. We show that our conjugation strategy is compatible with the addition of multiple substrates and can thus be used for the delivery of cytoplasm-targeted therapeutics.

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