| DNA nanoparticles are in vivo gene transfer vectors in development for treating cystic fibrosis. We previously discovered that they deliver transgenes efficiently to the mouse airway without inducing significant inflammatory cells and cytokines. Using rhodamine-labeled DNA nanoparticle, we found that they accumulate in the nucleoli in well-differentiated airway epithelial cells, colocalizing with nucleolin. We employed surface plasmon resonance (SPR) technology to demonstrate direct binding of nucleolin to DNA nanoparticles with K D = 25.9 nM. Nucleolin is expressed on the surface of HeLa cells and human tracheal 16HBEo-cells. Cell surface nucleolin shares a similar intracellular trafficking pattern with rhodamine-nanoparticles. Manipulations of nucleolin indicate that it is essential for the transfection of the nanoparticles. Moreover, purified nucleolin significantly blocks transfection, supporting the hypothesis that nucleolin is a critical receptor for the nanoparticles. We then studied the regulation of surface expression of nucleolin and found that cyclin dependent kinase 1 (Cdk1), rather than casein kinase 2 (CK2) phosphorylation, promotes its surface appearance. The N-terminus of nucleolin including the 8 consecutive Cdk sites is required for efficient surface expression. Inhibition of Cdk1 blocks the increase of surface nucleolin at G2/M phase transition. Mutations of the Cdk sites to glutamate increase its surface expression. We also found that nucleolin exists in lipid rafts on the membrane, and DNA nanoparticles can be recovered from lipid raft fractions following cellular uptake. Moreover, transfection of the nanoparticles is blocked by cholesterol depletion using drugs such as filipin and methyl-beta-cyclodextrin. Nucleolin directly associates with flotillin-1, an integral lipid raft protein regulating raft-mediated endocytosis; and the association is not disrupted by cholesterol depletion. Taken together, DNA nanoparticles enter cells via lipid rafts and cell surface nucleolin, which is positively regulated by cyclin dependent kinase Cdk1. |
