In vitro transfection efficiency of ACU-4429-PEG-HZ-ECO/pDNA nanoparticles. (A) Confocal fluorescence images (B) Flow cytometry measurements of the cytosolic delivery of ACU-PEG-HZ-ECO/Cy3-pDNA and PEG-ECO/Cy3-pDNA nanoparticles at an N/P ratio of 10 in ARPE-19 cells. DNA plasmid was labeled with Cy3 (red), nuclei with Hoechst 33342 (blue), and Late endosomes with LysoTracker Green (green). Flow cytometry recorded mean fluorescence intensities of Cy3 positive ARPE-19 cells at each time point. (C) qRT-PCR of ABCA4 mRNA expression at 48 h in ARPE-19 cells transfected with ACU-PEG-HZ-ECO/pCMV-ABCA4 and PEG-ECO/pCMV-ABCA4 nanoparticles (n = 3, error bars = ± SD, **, ##P < 0.05. Scale bars represent 20 μm).