Because polyplexes cause toxicity and are relatively unstable, nano- and micro-particles provide an alternative method for delivery. Nanoparticles provide superior protection from circulating nuclease activity and offer an array of possible targeting advantages when combined with specific peptides. Nanoparticles composed of synthetic polymers such as poly(lactic-co-glycolic acid) (PLGA) are safe and attractive methods Inhibitors,research,lifescience,medical for DNA delivery applications and have been

used in several studies [17]. Encapsulation of DNA with PLGA protects it from nuclease degradation, but the DNA is released slowly over time as PLGA degrades through ester hydrolysis [18, 19]. An additional limitation of using PLGA nanoparticles is their negative charge that must be modified to reduce this barrier to DNA encapsulation and delivery [20]. In this paper, we investigated a novel gene delivery system using Logic Gate Nanoparticles developed with a dual pH-responsive random copolymer (poly-β-aminoester ketal-2, Figure 1) [21]. Inhibitors,research,lifescience,medical Current pH-responsive polymers have been demonstrated and are promising gene delivery systems [22]. However, our random copolymer is unique because it remains

hydrophobic at physiological pH (pH 7.4) but undergoes a switch from hydrophobic to hydrophilic at low endosomal pH, which Inhibitors,research,lifescience,medical initiates rapid fragmentation into small molecules. The amine moieties in the backbone undergo a sharp hydrophobic-hydrophilic switch. This leads to an increase in water

uptake (bulk dissolution) and hence an increase in ketal hydrolysis (degradation) [23]. The nanoparticle Inhibitors,research,lifescience,medical formulations are stable for 24 hours in physiological pH [21], as TEM revealed well-formed particles, and upon PLX4032 in vivo reducing the pH to endosomal levels, pH 5, these dual responsive nanoparticles undergo a rapid and dramatic fragmentation followed by concomitant release of their payloads (Figure 1). We hypothesized that Inhibitors,research,lifescience,medical these nanoparticles would be suitable for gene delivery and efficient gene expression. In this study, we demonstrate that nanoparticles composed of the dual pH-responsive polymer offer effective endosomal release and expression of encapsulated DNA due to its ability to undergo rapid fragmentation. Figure 1 Schematic representation of the dual pH-responsive nanoparticles used for gene transfection. 2. Materials and Methods 2.1. Materials Dichloromethane (DCM, methylene chloride) and trehalose were purchased from Fisher Scientific (Hampton, NH, USA). Poly (vinyl Adenosine alcohol) (PVA) (MW 30–70k) and bafilomycin A1 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). PLGA (Resomer RG 502H) was purchased from Boehringer Ingelheim (Germany). Cy5 labeling kit was obtained from Mirus Bio (Madison, WI, USA). Cell culture media was purchased from Life Technologies (Carlsbad, CA, USA). All reagents were purchased from commercial sources and were used without further purification unless otherwise stated. 2.2.