Cellular uptake experiments

Cells were plated at a density of 150,000 cells/mL in complete medium with 100 μL of media per well in 96 well plates. PBAE particles labeled with Cy5 were formed with RR-CDG at a 500 w/w ratio, then were added to cells at a dose of 20 μg polymer/well and incubated for 1h at 37°C after which cells were washed with PBS twice and analyzed via flow cytometry. A BD Accuri C6 (BD Biosciences) flow cytometer with two lasers (488 and 633 nm) with four channels corresponding to green, yellow, red, and far-red fluorescence (FL1 at 530 ± 15 nm, FL2 at 565 ± 10nm, FL3 a t610 ± 10 nm and FL4 at 675 ± 12.5 nm, respectively) was used for all flow cytometry experiments in combination with a HyperCyt autosampler (IntelliCyt). Cell counts per well and nanoparticle concentrations in media were equal between cell types. Statistical significance was assessed between the uptake levels of the treated cells by one-way ANOVA with multiple comparisons corrected for by Tukey’s method.

For confocal microscopy experiments, PBMCs transfected in suspension in 96 well round bottom plates as described above were stained with Hoechst 33342 (H3570; Thermo Fisher) to label nuclei. Cells were resuspended in live cell imaging solution and transferred to 8 well Nunc Lab-Tek chambered borosilicate coverglass well plates (155411; Thermo Fisher) at 37,500 cells/well for imaging. Images were acquired using a Zeiss LSM 780 microscope with Zen software and 63 oil immersion lens. Specific laser channels used were 405 nm diode, 488 nm argon, 561 nm solid-state, and 639 nm diode lasers.