(A) Design of CD4-AsiC, containing a CD4 aptamer and a CCR5 siRNA. (B and C) CD4-AsiCs or PSMA-AsiCs targeting CCR5 were Cy3 labeled at the 3′ end of the antisense siRNA strand and incubated with primary CD4⁺ T lymphocytes from a healthy donor. Uptake was assessed 24 hours later by flow cytometry (B) and fluorescence microscopy (C; original magnification, ×60). Data are representative of 3 independent experiments. MFI of each peak is shown (mock, blue; treated, red). Transfection controls used nucleofection. (D) Specific siRNA delivery to CD4⁺ cells in a mixed population of resting PBMCs was assessed by flow cytometry 24 hours after incubation with 4 μM Cy3-labeled AsiCs. In the absence of oligofectamine (OF), Cy3-labeled CD4-AsiCs were preferentially taken up by CD3⁺CD4⁺ T cells and CD4⁺CD14⁺ monocytes, whereas PSMA-AsiCs only transfected monocytes with OF. CD3⁺CD8⁺ T cells remained relatively label free. Representative dot plots of 3 experiments with different donors are shown. (E and F) To test for gene silencing, CD4⁺ T lymphocytes were treated with CD4-AsiCs or PSMA-AsiCs targeting CCR5, with or without transfection. Chimeras bearing scrambled siRNAs (Scr) and CD4 aptamers served as controls. Shown are mean ± SEM MFI normalized to the mock-treated sample (E; n = 4; *P < 0.005, **P < 0.0005, ***P < 0.00005, 2-tailed t test) and representative histograms (F; mock, blue; treated, red). In the absence of nucleofection, CCR5 was knocked down only by CCR5 CD4-AsiCs.