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A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos
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-c...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8412273/ https://www.ncbi.nlm.nih.gov/pubmed/34473728 http://dx.doi.org/10.1371/journal.pone.0254468 |
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author | Gentry, Schuyler B. Nowak, Scott J. Ni, Xuelei Hill, Stephanie A. Wade, Lydia R. Clark, William R. Keelaghan, Aidan P. Morris, Daniel P. McMurry, Jonathan L. |
author_facet | Gentry, Schuyler B. Nowak, Scott J. Ni, Xuelei Hill, Stephanie A. Wade, Lydia R. Clark, William R. Keelaghan, Aidan P. Morris, Daniel P. McMurry, Jonathan L. |
author_sort | Gentry, Schuyler B. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-8412273 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-84122732021-09-03 A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos Gentry, Schuyler B. Nowak, Scott J. Ni, Xuelei Hill, Stephanie A. Wade, Lydia R. Clark, William R. Keelaghan, Aidan P. Morris, Daniel P. McMurry, Jonathan L. PLoS One Research Article 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. Public Library of Science 2021-09-02 /pmc/articles/PMC8412273/ /pubmed/34473728 http://dx.doi.org/10.1371/journal.pone.0254468 Text en © 2021 Gentry et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Gentry, Schuyler B. Nowak, Scott J. Ni, Xuelei Hill, Stephanie A. Wade, Lydia R. Clark, William R. Keelaghan, Aidan P. Morris, Daniel P. McMurry, Jonathan L. A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos |
title | A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos |
title_full | A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos |
title_fullStr | A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos |
title_full_unstemmed | A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos |
title_short | A real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos |
title_sort | real-time assay for cell-penetrating peptide-mediated delivery of molecular cargos |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8412273/ https://www.ncbi.nlm.nih.gov/pubmed/34473728 http://dx.doi.org/10.1371/journal.pone.0254468 |
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