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...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-05-01
|
| Series: | International Journal of Molecular Sciences |
| Subjects: | |
| Online Access: | https://www.mdpi.com/1422-0067/23/10/5839 |
| _version_ | 1797499125156020224 |
|---|---|
| author | Manuel Fernández-Delgado Luis Sendra María José Herrero Gladys G. Olivera-Pasquini Alexander Batista-Duharte Salvador F. Aliño |
| author_facet | Manuel Fernández-Delgado Luis Sendra María José Herrero Gladys G. Olivera-Pasquini Alexander Batista-Duharte Salvador F. Aliño |
| author_sort | Manuel Fernández-Delgado |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T03:42:58Z |
| format | Article |
| id | doaj.art-e5e867a8e34349a196d20988917b99b5 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T03:42:58Z |
| publishDate | 2022-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-e5e867a8e34349a196d20988917b99b52023-11-23T11:29:20ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-05-012310583910.3390/ijms23105839Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear CellsManuel Fernández-Delgado0Luis Sendra1María José Herrero2Gladys G. Olivera-Pasquini3Alexander Batista-Duharte4Salvador F. Aliño5Service of Hematology and Hemotherapy, Hospital General Universitario de Castellón, 12004 Castelló de la Plana, SpainFarmacogenetics and Gene Therapy Group, Instituto de Investigación Sanitaria La Fe, Av. Fernando Abril Martorell, 106, 46026 Valencia, SpainFarmacogenetics and Gene Therapy Group, Instituto de Investigación Sanitaria La Fe, Av. Fernando Abril Martorell, 106, 46026 Valencia, SpainFarmacogenetics and Gene Therapy Group, Instituto de Investigación Sanitaria La Fe, Av. Fernando Abril Martorell, 106, 46026 Valencia, SpainGC01 Immunology and Allergy Group, Maimonides Biomedical Research Institute of Cordoba (IMIBIC), Av. Menéndez Pidal, s/n, 14004 Córdoba, SpainFarmacogenetics and Gene Therapy Group, Instituto de Investigación Sanitaria La Fe, Av. Fernando Abril Martorell, 106, 46026 Valencia, SpainTherapeutic 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.https://www.mdpi.com/1422-0067/23/10/5839flow cytometryoligonucleotidesTregcell uptakequenchingfluorescent labeling |
| spellingShingle | Manuel Fernández-Delgado Luis Sendra María José Herrero Gladys G. Olivera-Pasquini Alexander Batista-Duharte Salvador F. Aliño Study of Oligonucleotides Access and Distribution in Human Peripheral Blood Mononuclear Cells International Journal of Molecular Sciences flow cytometry oligonucleotides Treg cell uptake quenching fluorescent labeling |
| 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 | flow cytometry oligonucleotides Treg cell uptake quenching fluorescent labeling |
| url | https://www.mdpi.com/1422-0067/23/10/5839 |
| work_keys_str_mv | AT manuelfernandezdelgado studyofoligonucleotidesaccessanddistributioninhumanperipheralbloodmononuclearcells AT luissendra studyofoligonucleotidesaccessanddistributioninhumanperipheralbloodmononuclearcells AT mariajoseherrero studyofoligonucleotidesaccessanddistributioninhumanperipheralbloodmononuclearcells AT gladysgoliverapasquini studyofoligonucleotidesaccessanddistributioninhumanperipheralbloodmononuclearcells AT alexanderbatistaduharte studyofoligonucleotidesaccessanddistributioninhumanperipheralbloodmononuclearcells AT salvadorfalino studyofoligonucleotidesaccessanddistributioninhumanperipheralbloodmononuclearcells |