Microscopy analysis confirmed that Cy5-labeled pEGFP DNA was delivered inside cells and reached #selleck chem randurls[1|1|,|CHEM1|]# the periphery of the nucleus (Figure 3(b)). The uptake efficiency of the dual selleck catalog pH-responsive nanoparticles by HCT116 cells was very similar to PLGA nanoparticles (Supplementary Figure3). Figure 3 Cy5-labeled DNA delivery into cells via pH-responsive nanoparticles. HCT116 cells were incubated with

pH-responsive nanoparticles containing Cy5-DNA for 0–4 hours and analyzed by flow cytometry (a) and microscopy Inhibitors,research,lifescience,medical (b) after 4 hours. Delivery of labeled plasmid into the cells via nanoparticles does not necessarily mean that EGFP will be expressed. To test expression and transfection efficiency, we Inhibitors,research,lifescience,medical incubated HCT116 cells with DNA-containing nanoparticles for 4 hours before washing to remove excess nanoparticles and incubating cells in fresh media for 48 hours. Cells treated with the dual pH-responsive nanoparticles produced intense green fluorescence when analyzed by microscopy. In contrast, cells treated with PLGA nanoparticles containing similar amounts of pEGFP DNA produced relatively low fluorescence (Figure 4(a)). Flow cytometry analysis revealed that approximately 6% of

Inhibitors,research,lifescience,medical the cell population treated with the dual pH-responsive nanoparticles had fluorescence intensity higher than that of nontreated cells (Figure 4(b)), compared to 2% of cells treated with PLGA-DNA nanoparticles. The percentage of EGFP positive cells correlates well with the number of Cy5-positive cells, indicating that expression efficiency is high for cells that take up the nanoparticles. Additionally, comparing the intensity of EGFP-positive cells transfected with our nanoparticles Inhibitors,research,lifescience,medical Inhibitors,research,lifescience,medical or with an equal amount of DNA complexed with PEI, we show that our nanoparticles produce similar levels of

EGFP expression within transfected cells (Supplementary Figure 4). Figure 4 Expression of EGFP DNA with pH-responsive nanoparticles (NPs) compared to PLGA NPs: HCT116 cells were incubated with NPs for Carfilzomib 3 hours, washed, and then incubated in media for 48 hours followed by (a) microscopy and (b) flow cytometry analysis. Immediate burst degradation and release of DNA from the dual pH-responsive nanoparticles only occurs in an environment of low pH, similar to that present in endosomes. This low pH offers the appropriate stimulus to solubilize the polymer, resulting in an accelerated degradation via ketal hydrolysis. Bafilomycin A1, an inhibitor of V-ATPase, blocks the acidification of endosomes and has been previously used to characterize the mechanism of release of pH-dependent polyplexes and nanoparticles [26]. To verify that DNA release is pH-independent, the transfection efficiency of our dual pH-responsive nanoparticles was examined in the presence of 300nM bafilomycin A1.