Nanoparticles of compacted DNA transfect postmitotic cells.
Charge-neutral DNA nanoparticles have been developed in which single molecules of DNA are compacted to their minimal possible size. We speculated that the small size of these DNA nanoparticles may facilitate gene transfer in postmitotic cells, permitting nuclear uptake across the 25-nm nuclear membrane pore. To determine whether DNA nanoparticles can transfect nondividing cells, growth-arrested neuroblastoma and hepatoma cells were transfected with DNA/liposome mixtures encoding luciferase. In both models, growth-arrested cells were robustly transfected by compacted DNA (6,900-360-fold more than naked DNA). To evaluate mechanisms responsible for enhanced transfection, HuH-7 cells were microinjected with naked or compacted plasmids encoding enhanced green fluorescent protein. Cytoplasmic microinjection of DNA nanoparticles generated a approximately 10-fold improvement in transgene expression as compared with naked DNA; this enhancement was reversed by the nuclear pore inhibitor, wheat germ agglutinin. To determine the upper size limit for gene transfer, DNA nanoparticles of various sizes were microinjected into the cytoplasm. A marked decrease in transgene expression was observed as the minor ellipsoidal diameter approached 25 nm. In summary, suitably sized DNA nanoparticles productively transfect growth arrested cells by traversing the nuclear membrane pore.
Liu
Ge
G
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Li
DeShan
D
Pasumarthy
Murali K
MK
Kowalczyk
Tomasz H
TH
Gedeon
Christopher R
CR
Hyatt
Susannah L
SL
Payne
Jennifer M
JM
Miller
Timothy J
TJ
Brunovskis
Peter
P
Fink
Tamara L
TL
Muhammad
Osman
O
Moen
Robert C
RC
Hanson
Richard W
RW
Cooper
Mark J
MJ
D021581:Active Transport, Cell Nucleus; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008156:Luciferases / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008854:Microscopy, Electron; D008938:Mitosis; D036103:Nanotechnology / Q000379:methods; D009447:Neuroblastoma / Q000378:metabolism; D022022:Nuclear Pore / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D012542:Scattering, Radiation; D013997:Time Factors; D014162:Transfection; D019076:Transgenes; D014407:Tumor Cells, Cultured
D008164:Luminescent Proteins; D010455:Peptides; D010714:Phosphatidylethanolamines; D049452:Green Fluorescent Proteins; C020888:1,2-dielaidoylphosphatidylethanolamine; D004247:DNA; D008156:Luciferases; D008239:Lysine