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Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells

Therapeutic oligonucleotides have achieved great clinical interest since their approval as drug agents by regulatory agencies but their access and distribution in blood cells are not completely known. We evaluated by flow cytometry the ability of short fluorescent scramble oligonucleotides (ON*) to...

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Autores principales: Fernández-Delgado, Manuel, Sendra, Luis, Herrero, María José, Olivera-Pasquini, Gladys G., Batista-Duharte, Alexander, Aliño, Salvador F.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9143973/
https://www.ncbi.nlm.nih.gov/pubmed/35628649
http://dx.doi.org/10.3390/ijms23105839
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author Fernández-Delgado, Manuel
Sendra, Luis
Herrero, María José
Olivera-Pasquini, Gladys G.
Batista-Duharte, Alexander
Aliño, Salvador F.
author_facet Fernández-Delgado, Manuel
Sendra, Luis
Herrero, María José
Olivera-Pasquini, Gladys G.
Batista-Duharte, Alexander
Aliño, Salvador F.
author_sort Fernández-Delgado, Manuel
collection PubMed
description Therapeutic oligonucleotides have achieved great clinical interest since their approval as drug agents by regulatory agencies but their access and distribution in blood cells are not completely known. We evaluated by flow cytometry the ability of short fluorescent scramble oligonucleotides (ON*) to access human peripheral blood mononuclear cells (PBMC) after incubating with ON* during 1 h and 7 days of culture follow-up ‘in vitro’. Blood samples were treated with chemically modified oligonucleotides (phosphorothioate backbone and 2′ O-Me ends) to resist nuclease digestion under culture conditions. The ON* internalization was determined after discarding the membrane-associated fluorescence by trypan blue quenching. Whereas the oligonucleotide accessed neutrophils and monocytes rapidly, achieving their maximum in 1 h and 24 h, respectively, lymphocytes required 7 days to achieve the maximum (80% of cells) transfection. The ON*ability to access lymphocyte types (T, B, and NK) and T cell subtypes (CD4+, CD8+, and CD4-CD8-) were similar, with T cells being more accessible. Regulatory CD4+ and CD8+ T cells were classified in low and high Foxp3 expressers, whose expression proved not to alter the ON* internalization during the first hour, achieving 53% of CD4+Foxp3+ and 40% of CD8+Foxp3+ cells. Our results contribute to understanding and improving the management of therapeutic ONs.
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spelling pubmed-91439732022-05-29 Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells Fernández-Delgado, Manuel Sendra, Luis Herrero, María José Olivera-Pasquini, Gladys G. Batista-Duharte, Alexander Aliño, Salvador F. Int J Mol Sci Article Therapeutic oligonucleotides have achieved great clinical interest since their approval as drug agents by regulatory agencies but their access and distribution in blood cells are not completely known. We evaluated by flow cytometry the ability of short fluorescent scramble oligonucleotides (ON*) to access human peripheral blood mononuclear cells (PBMC) after incubating with ON* during 1 h and 7 days of culture follow-up ‘in vitro’. Blood samples were treated with chemically modified oligonucleotides (phosphorothioate backbone and 2′ O-Me ends) to resist nuclease digestion under culture conditions. The ON* internalization was determined after discarding the membrane-associated fluorescence by trypan blue quenching. Whereas the oligonucleotide accessed neutrophils and monocytes rapidly, achieving their maximum in 1 h and 24 h, respectively, lymphocytes required 7 days to achieve the maximum (80% of cells) transfection. The ON*ability to access lymphocyte types (T, B, and NK) and T cell subtypes (CD4+, CD8+, and CD4-CD8-) were similar, with T cells being more accessible. Regulatory CD4+ and CD8+ T cells were classified in low and high Foxp3 expressers, whose expression proved not to alter the ON* internalization during the first hour, achieving 53% of CD4+Foxp3+ and 40% of CD8+Foxp3+ cells. Our results contribute to understanding and improving the management of therapeutic ONs. MDPI 2022-05-23 /pmc/articles/PMC9143973/ /pubmed/35628649 http://dx.doi.org/10.3390/ijms23105839 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Fernández-Delgado, Manuel
Sendra, Luis
Herrero, María José
Olivera-Pasquini, Gladys G.
Batista-Duharte, Alexander
Aliño, Salvador F.
Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells
title Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells
title_full Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells
title_fullStr Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells
title_full_unstemmed Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells
title_short Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells
title_sort study of oligonucleotides access and distribution in human peripheral blood mononuclear cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9143973/
https://www.ncbi.nlm.nih.gov/pubmed/35628649
http://dx.doi.org/10.3390/ijms23105839
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