494. Development of a pH Sensor to Probe Endosomal Buffering of Polymeric Nanoparticles Effective for Gene Delivery

Abstract

Introduction: Polymeric gene delivery suffers from low efficacy compared to viral gene delivery with one of the primary barriers to successful transfection being efficient endosomal escape. Cationic polymers have been hypothesized to facilitate endosomal escape via the proton sponge mechanism by buffering hydrogen ions in the endosomal compartment. Here we have created a nucleic acid based pH sensor and applied it using flow cytometry and confocal microscopy to investigate endosomal buffering of synthetic biodegradable cationic polymers for gene delivery, correlating the pH of delivered DNA with transfection. Methods: We created a nucleic acid pH sensor by conjugating pH sensitive (FITC, OG) and insensitive (Cy5) fluorophores to plasmid DNA. The fluorescence ratio of the sensor was calibrated to pH using flow cytometry and confocal microscopy following electroporation into cells. Cells were transfected with the plasmid pH sensor complexed with cationic polymers including poly(beta-amino ester)s (PBAEs) of variable transfection efficacy to investigate endosomal buffering. Additionally, confocal microscopy was used to assess colocalization of the plasmid pH sensor with a lysosomal dye. Results: PBAEs were demonstrated to effectively buffer endosomes and avoid lysosomal fate, whereas the negative controls poly-L-lysine (PLL) and polyethylenimine (PEI) were shown to accumulate in lysosomes by 24h post-transfection. The polymer molecular weight and weight-weight ratio to plasmid DNA was shown to have an effect on endosomal buffering as well as transfection efficacy in the case of PBAEs. From confocal microscopy analysis of endosomes, local pH was observed to be higher at the endosomal membrane than in the center, which may be indicative of polymer enrichment along the membrane due to excess soluble polymer. Conclusions: The constructed DNA sensor gave a linear relationship with intracellular pH. When investigating PBAE-induced buffering, results were consistent with the hypothesized proton sponge mechanism. These cationic polymers were demonstrated to affect endosomal pH and DNA lysosomal fate and lead to successful transfection.Fig. 1Confocal microscopy was used to assess the compartmental pH of endosomes containing nanoparticles formed with the pH sensor. Lysosome colocalization is shown in merged thresholded images with lysosomes (blue), DNA non-colocalized with lysosomes (pink) and DNA colocalized with lysosomes (yellow). Scattergrams show DNA fluorescence (horizontal) and lysosome fluorescence (vertical). Scale bar 10 µm.View Large Image | Download PowerPoint SlideFig. 2Confocal microscopy images were analyzed at (A) 1 hour and (B) 24 hours post-transfection using Pearson's correlation coefficient M1 to assess the fraction of DNA colocalized with lysosomal stain. When delivered via PBAE 447, the fraction of DNA colocalized with lysosomes was significantly different than both bPEI and PLL at both time points.View Large Image | Download PowerPoint Slide