coli cells and purified using maxi prep kits (Life Technologies). The DNA was Cy5-labeled using the Label IT Tracker Intracellular Nucleic Acid Localization Kit (Mirus, USA). In brief, the DNA plasmid was incubated with Label IT tracker reagent in the labeling reaction at 37°C for 1hr. Then, labeled DNA was separated from free dye using Micro Bio-Spin 30 chromatography columns (BioRad, Hercules, CA, USA). 2.3. Synthesis and Characterization of Poly-β-Aminoester Inhibitors,research,lifescience,medical ketal-2 Following the literature procedures and in agreement with previously described polymer characterization [21], the polymer was prepared by Michael addition of the corresponding diacrylates with trimethyl dipiperidine. Molecular weight was

estimated by size exclusion chromatography against polystyrene standards Inhibitors,research,lifescience,medical in DMF/0.01% LiBr with a VWD (variable wavelength detector) at 250nm. Mw = 6300, Mn = 2880, and PDI = 2.18. 2.4. Preparation of Nanoparticles The nanoparticles were prepared

using PLGA or the pH-responsive polymer using W/O/W method. Inhibitors,research,lifescience,medical In a vial, 10mg of the polymer was dissolved in 300μL of DCM. Subsequently, 30μL DNA solution prepared in Tris-HCl buffer pH 8 was added. The two phases were sonicated for 30s at 6W (amplitude of 2, Misonix S-4000, 5.5′′ cup horn, USA). Then, an aqueous solution of 3mL 1% PVA in Tris-HCl buffer pH 8 was added and sonicated for two 30s cycles at 7W (amplitude of 5) using the same cup horn. The nanoparticle suspension was stirred

at 500rpm under vacuum using a magnetic stirrer to evaporate DCM. A concentrated mode tangential Inhibitors,research,lifescience,medical flow filtration system using 500kDa MicroKros modules (Spectrum Labs) was used to remove the PVA and free DNA [24]. The nanoparticle suspension was concentrated and washed two times. Finally, the suspension was lyophilized after 3 MA adding Inhibitors,research,lifescience,medical 5% trehalose. The nanoparticle characterization and properties were in agreement with the previously described literature [21]. In brief, dynamic light scattering (DLS, Malvern Zetasizer) revealed that pH-responsive particles had Z-average diameters of 300nm (PDI = 0.3, zeta-potential = −0.562mV in pH 8 PB), and PLGA particles were 340nm (PDI = 0.37). 2.5. Nanoparticles Encapsulation Efficiency and DNA Integrity To test the integrity and amount of encapsulated DNA in PLGA nanoparticles, 0.2mL nanoparticles dispersion in 10mM Tris-HCl (pH Mephenoxalone 8) was extracted with 0.2mL phenol:chloroform: isoamyl alcohol (25:24:1) and spun down at 12,100g for 20min. Then, 50μL of the aqueous layer was diluted with 250μL 10mM Tris-HCl (pH 8) and extracted with 300μL CHCl3. The aqueous layer was separated by spinning down and analyzed by gel electrophoresis for DNA. To test the integrity of encapsulated DNA in the pH-responsive nanoparticles, nanoparticles (0.2mL) in 10mM Tris-HCl (pH 8) with heparin (1:100 DNA to heparin), were extracted and analyzed as previously described with PLGA nanoparticles.