Triggered rapid degradation of nanoparticles for gene delivery

Abstract

Effective gene delivery tools offer the possibility of addressing multiple diseases; current strategies rely on viruses or polyplexes. Encapsulation of DNA within nanoparticles is an attractive alternative method for gene delivery. We investigated the use of our recently developed Logic Gate Nanoparticle for gene delivery. The nanoparticles, composed of a dual pH response random copolymer (poly-β-aminoester ketal-2), can undergo a two-step "in series" response to endosomal pH. The first sep is a hydrophobic-hydrophilic switch, which is followed immediately by rapid degradation. Rapid fragmentation is known to increase cytoplasmic delivery from nanoparticles. Therefore, we hypothesized that our Logic Gate Nanoparticles would enable increased gene delivery and expression relative to nanoparticles that degrade more slowly such as PLGA-based nanoparticles. Passive nanoparticle entry into cells was demonstrated by delivering Cy5-labeled pDNA encoding EGFP into HCT116, a colon carcinoma cell line. Flow cytometry analysis showed that cells are positive for Cy5-DNA-nanoparticles and produced EGFP expression superior to PLGA nanoparticles. Inhibition of V-ATPases using bafilomycin A1 demonstrates that expression of EGFP is dependent on low endosomal pH. The advanced Logic Gate Nanoparticles offer new therapeutic possibilities in gene delivery and other applications where rapid release is important.

Figure 1

Schematic representation of the dual pH-responsive nanoparticles used for gene transfection.

Figure 2

DNA encapsulation efficiency and release study. (a) DNA encapsulation efficiency was analyzed by comparing band intensity of control DNA (lane 1) to that of nonencapsulated DNA collected during the tangential flow filtration process (lane 2). (b) Cy5 fluorescence of released DNA from nanoparticles in buffer pH 7.4 over 24 hours followed by addition of pH 5 buffer. The pH changes to 5 at 24 hours.

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 (b) after 4 hours.

Figure 4

Expression of EGFP DNA with pH-responsive nanoparticles (NPs) compared to PLGA NPs: HCT116 cells were incubated with NPs for 3 hours, washed, and then incubated in media for 48 hours followed by (a) microscopy and (b) flow cytometry analysis.

Figure 5

Dependence on endosomal low pH was analyzed by comparing transfection of nanoparticles in the presence or absence of 300 nM bafilomycin A1, a V-ATPase inhibitor.