Specific uptake of anti-GD₂-nanoparticles exclusively by GD₂ expressing cells.

<p>Effects on growth and caspase activity by anti-GD₂ conjugated nanoparticles containing miR-34a. (A) FACS analysis, using an anti-GD₂ primary antibody and goat anti-mouse IgG2a-PE secondary antibody indicated little, if any, GD₂ surface antigen on HEK293 cells. Conversely NB1691 (B) and SK-N-AS (C) cells showed significant GD₂ reactivity. (D) Varying concentrations of FITC-anti-GD₂-NPs were added to cell culture media of NB1691 or HEK293 (1×10⁶ cells). A concentration of 6.8×10⁹ particles/ml (40 µg/ml) was the optimal dosage tested <i>in vitro</i> for specific delivery of encapsulated FITC fluorophore to NB1691 neuroblastoma cells with minimal incorporation into HEK293 cells. (E) Anti-GD2-miR34a or control-anti-GD2-nanoparticles were added to adhered neuroblastoma SK-N-AS and NB1691, and to HEK293 cells for 4 hours in standard media. Media was removed, the cells washed and media was replaced. RNA was isolated from these cells after 24 hours and assessed for miR34a levels. Anti-GD₂-miR34a-NPs treatment (40 µg/ml) led to ∼2 fold increase in miR-34a levels in NB1691 cells, ∼30 fold increase in SK-N-AS cells (*p<0.05, n=3), with no significant change in miR-34a transcript levels in HEK293 cells. (F) NB1691 and SK-N-AS cells showed a significant increase in caspase 3/7 activity relative to anti-GD₂-miRneg-NP-treated cells after 72 hrs (*p<0.05, n=3) while HEK293 cells showed no significant increase in caspase activity under these conditions. Acid phosphatase assays indicated a significant reduction in viable cell numbers for (G) NB1691 and (H<b>)</b> SK-N-AS cells treated with anti-GD₂-miR34a-NPs, over a 96 hour period (***p<0.001) relative to anti-GD₂-miRneg-NP-treated controls. (I) HEK293 cells showed no significant reduction of viable cells following treatment with anti-GD₂-miR34a-NP (p>0.05, n=3).