Figure 6. Role of cholesterol in the induction of apoptotic cell death by α_vβ₃ integrin–targeted melittin-loaded nanoparticles.

(A) C32 melanoma cells were treated with 2 concentrations of methyl-β-cyclodextrin (MBCD) to remove cholesterol as described in Methods and in Results. These cells were washed and exposed to the α_vβ₃ integrin–targeted nanoparticles. After incubation in culture, cell proliferation was determined by MTT assay as described in Methods. Data are represented as mean ± SD. **P < 0.01. (B–G) Cells treated as described in A were stained with annexin V–FITC and 7-AAD before analysis by cell sorting after treatment with the nanoparticles: (B) nanoparticles; (C) 2.5 μM α_vβ₃ integrin-targeted melittin-loaded nanoparticles; (D) 0.25 mM MBCD; (E) 0.5 mM MBCD; (F) 0.25 mM MBCD and 2.5 μM α_vβ₃ integrin–targeted melittin-loaded nanoparticles; and (G) 0.5 mM MBCD and 2.5 μM α_vβ₃ integrin–targeted melittin-loaded nanoparticles. Cells stained with annexin V–FITC were considered early in apoptosis, and those stained with 7-AAD also were late apoptotic cells. Numbers show the percentage of cells in the respective quadrants. (H) Schematic depiction of interaction of C32 melanoma cells with α_vβ₃ integrin–targeted melittin-loaded nanoparticles.