siILs can specifically target DCs in vitro. (A) siRNA encapsulation efficiency within liposomes. The entrapment efficiency of siRNA within neutral and positively charged liposomes was determined using an initial 250 μg amount of Cy3-labeled siRNA. Mass spectrofluorometry (excitation/emission 550/570) was used to measure the encapsulation efficiency of fluorescent siRNA within liposomes formulated using increasing amounts of the positive lipid DDAB. (B) Specific binding of siILs to dendritic cells. BMDCs were incubated for 30 minutes at 4°C with 1 mol/dL positive, DC-specific siILs containing Cy3-labeled CD40 siRNA. Cells were washed and imaged. Cy3-CD40 siRNA was complexed with GP, a commercial transfection reagent, and used as a positive control treatment. Isotype IgG conjugated siILs, or siILs lacking Cy3-CD40 siRNA (Empty siILs), or the no DC-targeting mAb (siRNA liposomes) were used as negative controls. A cell line (L929) that does not express DEC-205 was also used as a negative control. The specificity of siIL binding was determined by epifluorescence microscopy. (C) Confocal imaging of siIL binding to dendritic cells. BMDCs were fixed, permeabilized, stained with Alexa 488–phalloidin, and incubated with empty or Cy3-CD40 siILs for 15 minutes at room temperature. Unbound siILs were washed off using PBS, and cells were imaged by confocal microscopy with a 40× objective.