2.6. DNA Release From the Nanoparticles DNA-Cy5 nanoparticles were resuspended in phosphate buffer pH 7.4. The nanoparticles were left in a shaker at 60rpm

and 37°C. Aliquots were taken at different time intervals and spun down at 2,000g and 4°C for 10min. The supernatant was used to determine the fluorescence of released DNA-Cy5. After 24 hours, Inhibitors,research,lifescience,medical the particles were spun down and resuspended in phosphate buffer pH 5 to test the effect of pH on DNA release from the nanoparticles. 2.7. Transfection of DNA with Nanoparticles HCT116 cells were plated at ~50% density in a 24-well culture plate and allowed to attach overnight. Cells were then treated with nanoparticles encapsulating 50 to 100ng of labeled or unlabeled DNA for 30 minutes, 1, 2, 3, or 4 hours in the presence Inhibitors,research,lifescience,medical of regular media with 10% serum. The media was then replaced with 500μL of fresh media in each well after washing to remove excess nanoparticles. For DNA-Cy5 analysis, the cells were immediately analyzed by fluorescence microscopy (Nikon and NIS Elements software) and flow cytometry Inhibitors,research,lifescience,medical (Accuri C6) by detecting fluorescence in the far red spectrum (670nm). To analyze GFP

expression, the cells were treated with nanoparticles for 4 hours,then the media was replaced and incubated for 48 hours. The cells were subsequently analyzed by fluorescence microscopy or flow cytometry (Accuri C6) to detect green fluorescence. For Inhibitors,research,lifescience,medical microscopy analysis, cells were placed in wells containing glass coverslips. For flow cytometry, cells were first trypsinized for 5 minutes followed by two washes with PBS and analyzed immediately. To test the requirement for low R406 nmr endosomal pH, cells were treated with Bafilomycin A1 at a final concentration Inhibitors,research,lifescience,medical of 300nM prior to adding nanoparticles.

The cells were then incubated for 4 hours, followed by replacement of media and incubation for 48 hours. 3. Results and Discussion 3.1. DNA Encapsulation Adenylyl cyclase and Stability Study Considering the obstacles to gene delivery, including DNA packaging, transport across the membrane, endosomal escape and transport into the nucleus, we aimed to demonstrate the effectiveness of our dual pH-responsive nanoparticles to meet these challenges. We first determined the stability and effectiveness of DNA encapsulation in the dual pH-responsive nanoparticles. The dual pH-responsive nanoparticles containing plasmid DNA were prepared with poly-β-aminoester ketal-2 using a double-emulsion method. The supernatant and washes of the preparations were kept and analyzed to estimate the percent of nonencapsulated DNA. The encapsulation efficiency was estimated to be approximately 100% since no DNA was detectable in these fractions (Figure 2(a)).