Cell Surface Nucleolin Serves as Receptor for DNA Nanoparticles Composed of Pegylated Polylysine and DNA

Abstract

Compacted DNA nanoparticles deliver transgenes efficiently to the lung following intrapulmonary dosing. Here we show that nucleolin, a protein known to shuttle between the nucleus, cytoplasm, and cell surface, is a receptor for DNA nanoparticles at the cell surface. By using surface plasmon resonance (SPR), we demonstrate that nucleolin binds to DNA nanoparticles directly. The presence of nucleolin on the surface of HeLa and 16HBEo- cells was confirmed by surface biotinylation assay and immunofluorescence. Rhodamine-labeled DNA nanoparticles colocalize with nucleolin on the cell surface, as well as in the cytoplasm and nucleus, but not with transferrin or markers of early endosome or lysosome following cellular uptake. Reducing nucleolin on the cell surface by serum-free medium or siRNA against nucleolin treatment leads to significant reduction in luciferase reporter gene activity, while overexpressing nucleolin has the opposite effect. Competition for binding to DNA nanoparticles with exogenous purified nucleolin decreases the transfection efficiency by 60-90% in a dose-dependent manner. Therefore, the data strongly suggest that cell surface nucleolin serves as a receptor for DNA nanoparticles, and that nucleolin is essential for internalization and/or transport of the nanoparticles from cell surface to the nucleus.

Pre-clinical evaluation of three non-viral gene transfer agents for cystic fibrosis after aerosol delivery to the ovine lung

“…Although the plasmid dose received by the NP group was only B20% less, it has been argued that the markedly reduced inflammation in response to instillation of NP in the mouse lung is due to the pDNA within the NP formulation being masked from the toll-like receptor 9 in the endosome, 18 as a consequence of a nucleolin-mediated route of entry to the cell and into the nucleus. 29 Despite this, a modest BAL neutrophil response has been reported for a 100 mg dose administered to the lungs of Balb/c mice, 18 which could be consistent with the trend towards increased BAL neutrophils observed after lung delivery in the sheep.…”

Kruppel-like Factor 4 Is a Novel Mediator of Kallistatin in Inhibiting Endothelial Inflammation via Increased Endothelial Nitric-oxide Synthase Expression

“…However, it is also possible that KLF4 may be migrated to cell surface under overexpression conditions. Recent studies showed that nucleolin, a nuclear protein, was present on the surface of various cell types, despite lacking a transmembrane domain or a signal sequence (24,25). Therefore, nucleolin acts as a receptor on plasma membrane and shuttles the ligand from the plasma membrane to the cytoplasm and nucleus (24,26).…”

Channel Replacement Therapy for Cystic Fibrosis

“…Cleveland-based Copernicus Therapeutics, Inc. developed a compacted DNA/DNA nanoparticle therapy (PLASmin ® ) that decreases the volume of the complex by up to 1000-fold, to a single molecule small enough to permeate the membrane and nuclei of target cells. The complex contains only one copy of the DNA to be delivered, increasing the stability and efficacy of the treatment (Chen et al, 2007). In phase 1a trials, plasmid DNA nanoparticles with the gene responsible for encoding CFTR were applied intra-nasally with lysine peptides substituted with polyethylene glycol.…”