397. Compacted DNA Nanoparticles Transfect Cells by Binding to Cell Surface Nucleolin
Authors: Xuguang Chen, Pamela B. Davis
Journal: Molecular Therapy (2006)

DNA nanoparticles formulated with polyethylene glycol (PEG)- substituted polylysine peptides have minimal toxicity, are non- immunogenic, and can transfect non-dividing cells in vivo at remarkably high efficiencies. We previously reported that compacted DNA nanoparticles labeled with rhodamine are only identified intracellularly in cells that express cell surface nucleolin. Nucleolin is a ubiquitous protein participating in ribosomal biogenesis, and mainly localizes in the nucleolus and cytoplasm. In some cell types, nucleolin is also present on the cell surface, and is the target of multiple extracellular ligands, which include lipoproteins, lipopolysaccharides, viruses, bacterial adhesins, and small peptides. In this report, we test the hypothesis that cell surface nucleolin acts as a receptor for DNA nanoparticles and is essential for their uptake into the cell. Initial experiments tested directly whether purified nucleolin binds to DNA nanoparticles by Surface Plasma Resonance (SPR). We observed tight binding when we immobilized either the naonoparticles or nucleolin on the sensorchip. The calculated KD for a single site is about 1.9 nM. The sensogram also suggested multiple binding sites on DNA nanoparticles for nucleolin. Three nanoparticle preparations manufactured by Copernicus Therapeutics using PEG-substituted polylysine from different vendors were evaluated by SPR for their affinity to nucleolin. Their KD values had the same rank order as the mean luciferase activity detected in mouse lung two days following in vivo administration of the three preparations. No binding was detected between nanoparticles and purified green fluorescence protein. We confirmed the cell surface expression of nucleolin in HeLa cells by both surface biotinylation and immunofluorescence. Cell surface nucleolin was biotinylated and pulled down by avidin-agarose beads, then identified by Western blot. We also labeled cell surface nucleolin with FITC conjugated primary antibody and observed colocalization of nucleolin with a cell membrane marker, concanavalin A. To test the importance of nucleolin in the uptake of DNA nanoparticles, we manipulated expression of cell surface nucleolin and then studied activity of the reporter gene luciferase from DNA nanoparticles. When we treated HeLa cells for 12 hours with serum free media, which is reported to reduce cell surface nucleolin, luciferase expression decreased by 70%. When we used siRNA to reduce surface expression of nucleolin in HeLa cells by 50%, we observed a 50% decrease in reporter gene expression. In addition, we tested whether exogenous nucleolin could prevent uptake and expression of DNA nanoparticles. When we premixed DNA nanoparticles with purified nucleolin protein, then applied these mixtures to HeLa cells, the transfection efficiency decreased by 70%- 90%. So purified nucleolin competes with cell surface nucleolin to bind to DNA nanoparticles, thereby inhibiting their uptake and expression. These data indicate that nucleolin is a cell surface target of compacted DNA nanoparticles and plays an essential role in their cellular uptake. Dr. Davis' patents are licensed by Copernicus Therapeutics and she holds equity in this company, which is developing DNA nanoparticles for therapeutic purposes.