Down-regulation of EGFR by different concentrations of siRNA-loaded nanogels. A. A dose curve was established for EGFR expression by immunoblotting of Hey cell lysates. Nanogels were loaded with siRNA at a concentration of 16.6 μg siRNA/mg of nanogels, and 1000 μg/mL of the loaded particles was added to 5 × 105 cells. For other concentrations, nanogels were diluted serially by 10-fold (1000 μg/mL, 100 μg/mL, 10 μg/mL, and 1 μg/mL). All cells were harvested at 48 hours after the addition of the siRNA loaded nanogels. A significant decrease (*P < 0.01) in EGFR expression was observed at the highest nanogel concentration when compared to EGFR expression in untreated (UT) cells using the averaged values from three experiments. YSA peptide alone (YSA) was included as an additional control. B. An immunoblot from one of three experiments demonstrating EGFR expression in Hey cells after treatment with different concentrations of siRNA-loaded nanogels is shown. Note in the study shown that complete reduction in EGFR expression was observed when the concentration of nanogels used was as little as 10 μg/mL. C. Hey cells were treated with a non-specific siRNA, siGLO at a concentration of 1000 μg/mL of siGLO-loaded nanogels. Cells were harvested after 48 hours, and the levels of EGFR were examined by immunoblot. Differences in EGFR expression were not observed in siGLO-nanogel treated cells when compared with YSA peptide alone (YSA), nanogels (Ng), or untreated (UT) controls.