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

• Autor(a) principal: Fernández Delgado, Manuel
• Data de Publicação: 2022
• Outros Autores: Sendra, Luis, Herrero, María José, Olivera-Pasquini, Gladys G., Duharte, Alexander Batista [UNESP], Aliño, Salvador F.
• Tipo de documento: Artigo
• Idioma: eng
• Título da fonte: Repositório Institucional da UNESP
• Texto Completo: http://dx.doi.org/10.3390/ijms23105839; http://hdl.handle.net/11449/241020
• topic: CD8+ Treg; cell uptake; flow cytometry; fluorescent labeling; FOXP3; oligonucleotides; PBMC; quenching; Treg

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 neutro-phils 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 lym-phocyte 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.