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

Abstract

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.

Keywords: CD8+ Treg; FOXP3; PBMC; Treg; cell uptake; flow cytometry; fluorescent labeling; oligonucleotides; quenching.

Figure 1

Dose-response fluorescent Cy5-ON (ON*) and size. Kinetics of Cy5 labeled oligonucleotide (ON*) on PBMC. Labeled 13-, 17- or 20-mer ONs were incubated at concentrations of 0.2, 0.5, 1, 2, 4 and 10 µM at 37 °C during 10, 20, 40, and 90 min. At each time point, the leukocytes (CD45+) were analyzed by flow cytometry to measure the ON* uptake and the emitted fluorescence, expressed as fluorescence intensity per cell (FI/cell). Lines through experimental points fit a sigmoidal dose-response model and were drawn with Graphpad Prism software.

Figure 2

Comparison between RPMI and DMEM culture media in leukocyte viability for 7 days. 2.5 mL of blood samples were centrifuged and plasma was substituted with either DMEM or RPMI containing 25% of its own plasma and antibiotics. The viability of neutrophils, monocytes, and lymphocytes was determined by the 7AAD test in flow cytometry.

Figure 3

Location of fluorescent ON* in peripheral blood leukocytes treated with FITC-labeled ON* and incubated for 0.5 h at 0.5 µM. Then, the oligonucleotide was removed and PBMC in phosphate-buffered saline (PBS) solution was extended on a slide and dried. Cells were marked with DAPI and analyzed using a fluorescence microscope to observe the uptake of ON* by neutrophils, monocytes, and lymphocytes.

Figure 4

Fluorescent leukocytes (%) after 20-mer ON* treatment at 4 µM with DMEM incubation before and after Quenching. The bar graphics represent the percentage of viable cells positive for green fluorescence provided by ON*. Percentage of viable cells with fluorescence before and after TB membrane fluorescence quenching were compared. Different leukocyte cells (lymphocytes, neutrophils, and monocytes), lymphocyte types (T cells, B cells, and NK cells), and T cells subtypes (CD4+, CD8+, and CD4-CD8-) are depicted.

Figure 5

Fluorescence intensity per cell in blood cells treated with fluorescent 20-mer ON leukocytes at 4 µM and DMEM incubation + Quenching. Monocytes vs neutrophils p < 0.05; monocytes vs lymphocytes p < 0.001; neutrophils vs lymphocytes p < 0.01.

Figure 6

The panel shows representative cytometry plot of fluorescence intensity of ON:FITC vs Foxp3:PE of CD3+CD4+CD25+Foxp3+ and CD3+CD8+Foxp3+ cells population. Cells were divided into ON-/ON+ depending on their FITC fluorescence (ON- < 1E3; ON+ > 1E3) and classified them upon their Foxp3 expression (low: fluorescence < 1E3; high: fluorescence > 1E3).

Figure 7

The figure shows representative plot of cell distribution according to their level of Foxp3 expression. CD3+CD4+CD25+Foxp3+ (upper panel) and CD3+CD8+Foxp3+ (lower panel) cells are represented. In each case, two different populations can be clearly differentiated upon their Foxp3 expression (low: fluorescence < 1E3; high: fluorescence > 1E3).