Figure 7.

Polyplexes Induce Little to No Hemolysis and Mediate Reporter Gene Expression in Mouse Brains

(A) Whole human blood was centrifuged at 2,000 rpm. Pelleted red blood cells were mixed in saline at a 1:6 ratio and incubated at 37°C with polyplexes (2:1 w/w ratio) for 3 hr. PBS (1X) and deionized water were used as negative and positive controls, respectively. Post-incubation, supernatants were analyzed to measure % hemolysis compared to controls. Data shown represent mean ± SEM from three donors with a minimum of triplicate determinations/donor (***p < 0.001). (B) Athymic nude mice were injected intravenously with S2 A₅P₁₀:pLuc polyplexes (100 μg polymer and 50 μg pDNA/dose) for 3 days. Brains and livers were cut into small pieces and incubated with in vivo glo-d-luciferin in well plates. Luciferase activity was assessed in tissue isolates harvested at 24, 48, and 72 hr after last injection. Data shown represent mean ± SEM for 3 to 4 mice per condition (***p < 0.001). (C1–E4) In a parallel experiment, brains harvested at 24 and 48 hr were fixed with 4% paraformaldehyde, and 40 μM frozen coronal sections were cut immunostained for GFAP (green) and luc (red). (C) Control, (D) 24 hr, (E) 48 hr. Arrowheads represent cells co-stained for luciferase and GFAP. n = 3 or 4 mice per condition. Each image was captured as 8–15 Z sections using a confocal microscope. Representative images from the cortical regions of brain are shown. Original magnification ×100.