Figure 3: In vitro assays.

(a) Binding assay with BC cells. MCF-7, BT474 and MDA-MB 468 cells were incubated at 37 °C for 1 h with 13 and 65 nM FITC-labelled NPs functionalized with 5NP-1Tz and 5NP-2Tz, and then processed with flow cytometry, in which labelling of cells with NPs was detected. FITC-labelled NPs functionalized with generic rabbit IgG (IgG) or only with PEG were used as specificity controls. Untreated cells were used to create viable cells and singlet gates, and to set the positive region assigned to labelled cells. (b) Viability of cells treated with NPs. MCF-7 cells were treated with 13 and 65 nM of NPs or Tz for up to 72 h. Viability was tested by measuring the conversion of MTT into formazan. Reported values are the mean of six replicates±s.e., normalized on cell proliferation of untreated MCF-7 cells (control), respectively. *P<0.01, 5NP-1Tz or 5NP-2Tz versus control; ^§P<0.01, 5NP-1Tz versus 5NP-2Tz antibodies (Student’s t-test). (c) Cell death assay on incubation with NPs. MCF-7 cells were treated with 13 and 65 nM of NPs for 24, 48 and 72 h. Untreated cells and Tz-treated cells (13 and 65 nM) were used as control. Cell death was assessed based on the exposure of Annexin V, evaluated by flow cytometry. Live cells were used to set the region of positivity. Reported values are the mean of three replicates±s.e. *P<0.01, 5NP-1Tz or 5NP-2Tz versus control; ^§P<0.01; ^§§P<0.0005, 5NP-1Tz versus 5NP-2Tz (Student’s t-test). CTRL, control.