Detection of early induction of apoptosis was done using JC-1 dye by flow cytometry.

Briefly, 1×10⁵ cells/ml were treated with native nutlin-3a, Nut-NPs, Fol-Nut-NPs (1.5 µg/ml), curcumin, Cur-NPs, Fol-Cur-NPs (2 µg/ml) and Nutlin+Curcumin, Nut-Cur-NPs, Fol-Nut-Cur-NPs (1.5 nutlin+2 curcumin µg/ml) for 48 hrs. Cells treated with only medium was used as controls. After completion of incubation period, cells were treated with JC1 staining solution (1 µg/ml in warm DPBS) and incubated at 37°C for 20 min in a CO2 incubator. After incubation period, cells were examined by flow cytometer. Data represented as mean±s.e.m., (n = 3). Folate targeted dual drug loaded NPs showed higher loss of MMP in Y79 cells compared native drugs (single or in combination) and unconjugated single or dual drug loaded NPs.