Figure 2. Development of G7-FLT3L dendrimers and their selective targeting to FLT3-overexpressing AML cells.

(A) G7-NH₂ PAMAM dendrimers were first conjugated with the near-infrared dye Cy5.5 using an N-hydroxysuccinimide ester linker sulfo-SMCC. Next, the fluorescently-tagged G7-NH₂ (G7-Cy5.5) was conjugated to human recombinant FLT3L (i.e., the soluble FLT3L form with 155 amino acids; ProSpec-Tany Technogene Ltd., East Brunswick, NJ) at a 1:2 ratio using the heterofunctional linker sulfosuccinimidyl 4-((N-maleimidomethyl) cyclohexane-1-carboxylate) (sulfo-SMCC), resulting in the G7-FLT3L conjugates. (B) MONOMAC-6 cells were treated with Cy5.5-conjugated G7-FLT3L or G7-H2B nanoparticles for 24 hrs at the indicated doses. The proportion of Cy5.5⁺ cells were detected through flow cytometry analysis. (C) MONOMAC-6 and U937 cells were treated with 50 nM Cy5.5-conjugated G7-FLT3L or G7-H2B nanoparticles for 1 hr. The proportion of Cy5.5⁺ cells were detected through flow cytometry analysis. (D) MONOMAC-6 cells transfected with MSCV-PIG-miR-150 (miR-150-PIG) or MSCV-PIG (PIG) were treated with 50 nM Cy5.5-conjugated G7-FLT3L or G7-H2B nanoparticles for 1 hr. The proportion of Cy5.5⁺ cells were detected through flow cytometry analysis. Each experiment was repeated independently for at least three times. Average levels of at least three replicates are shown. (E) Cell viability (left panel) and apoptosis (right panel) of MONOMAC-6 cells treated with PBS (Ctrl), 50 nM G7-FLT3L or G7-H2B nanoparticles for 48 hr. *, P<0.05; **, P<0.01.