Figure 4.

Polyethylenimine-(5) myristic acid/polyethylene glycol oleic acid/cholesterol (PEI₆₀₀-MA₅/PEG-OA/Cho) nanoparticles deliver cytomegalovirus (CMV)-GFP (green fluorescent protein) reporter plasmid to pulmonary endothelial cells. (A and B) GFP fluorescence is detected in pulmonary endothelial cells after treatment with PEI₆₀₀-MA₅/PEG-OA/Cho nanoparticles. CMV-GFP plasmid was encapsulated into nanoparticles, and the nanoparticle–DNA complexes were injected at Postnatal Day 2 (P2). Lungs were harvested at P5 and P7 and used for fluorescence-activated cell sorter (FACS) analysis. Histograms in A show GFP fluorescence in different cell types. GFP (green area) is detected in endothelial cells (CD31⁺CD45⁻CD326⁻). Autofluorescence is shown as the black area. Data were quantitated in B by comparing GFP mean fluorescence intensity (MFI) with autofluorescence in each cell type (n = 3–4 mice per group). Error bars are mean ± SE. **P < 0.01. (C) Confocal images show that GFP is present in perinuclear regions of endothelial cells stained with endomucin (arrowheads). Lungs of mice treated with nanoparticles containing CMV–empty plasmid were used as controls. Scale bars: top panels, 10 μm; bottom panels, 2 μm. (D) High-magnification confocal images show the presence of GFP (green) and DyLight 650 quantum dots (purple) in cytoplasm of microvascular endothelial cells stained with endomucin. Cell nuclei were stained with DAPI (blue). Cell surface endomucin was removed using a deconvolution option in Imaris software (imaris.oxinst.com) (bottom image), indicating the presence of GFP and DyLight 650 inside the cell. Scale bars, 2 μm. (E) RT-PCR shows the presence of exogenous, plasmid-derived Foxf1 (forkhead box F1) mRNA in endothelial cells (CD31⁺CD45⁻CD326⁻) and myofibroblasts (CD140a⁺CD31⁻CD45⁻CD326⁻) that were FACS sorted from P4 lungs. β-Actin was used as a loading control. endo = endothelial cells; hema = hematopoietic cells; HO = hyperoxia; n.s. = not significant; RA = room air.