Cationic transfectors increase accumulation in cultured tumor cells of radiolabeled antisense DNAs without entrapment.
While cellular accumulations in culture of oligomers, such as interfering RNA and antisense DNA, are reported to benefit from the addition of transmembrane transfectors (TFs), the extent to which individual TFs improve cellular delivery is usually inferred and rarely measured. The goal of this investigation was to use radioactivity to measure in cells in culture the degree to which accumulations of DNA increased when complexed with TFs and without DNA entrapment in vesicles. The antisense (AS) DNA targeting mdr1 mRNA coding for P-glycoprotein (Pgp) and its sense (S) complement DNA were radiolabeled with 99mTc and mixed with jetPEI, Chariot, or Neophectin over a range of TF/DNA ratios. Thereafter,the radiolabeled DNAs with and without TFs were incubated with KB-G2 (mdr1(+/+)) and KB-31 (mdr(+/-))cells at 37 degrees C in serum or serum-free media for 20-24 hours at a fixed DNA concentration of 13 nM. Cellular accumulations were increased under most incubation conditions and by as much as threefold with jetPEI and eightfold with Neophectin. As evidence against entrapment, the accumulations of AS DNAs were higher than S DNAs in virtually all measurements and higher in virtually all accumulations in the mdr1(+/+) cells, compared to the mdr1(+/-) cells. In conclusion, by using radiolabeled DNAs, definitive evidence was obtained showing that the addition of Neophectin and jet PEI increased cellular accumulations of both AS and S DNA without evidence of vesicle entrapment.
Nakamura
Kayoko
K
Department of Radiology, Keio University School of Medicine, Tokyo, Japan. kumako@sc.itc.keio.ac.jp
Wang
Yi
Y
Liu
Xinrong
X
Kubo
Atsushi
A
Hnatowich
Donald J
DJ
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D002308:Cardiolipins / Q000737:chemistry; D002412:Cations / Q000737:chemistry; D045744:Cell Line, Tumor; D003543:Cysteamine / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D007097:Imines / Q000737:chemistry; D007553:Isotope Labeling; D008081:Liposomes / Q000737:chemistry; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011073:Polyamines / Q000737:chemistry*; D000071228:Polyelectrolytes; D011095:Polyethylenes / Q000737:chemistry; D013670:Sodium Pertechnetate Tc 99m / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry; D014162:Transfection / Q000379:methods*
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002308:Cardiolipins; D002412:Cations; D003543:Cysteamine; D007097:Imines; D008081:Liposomes; D009838:Oligodeoxyribonucleotides; D020319:Oligodeoxyribonucleotides, Antisense; D009842:Oligopeptides; D010455:Peptides; D054735:Phosphorothioate Oligonucleotides; D011073:Polyamines; D000071228:Polyelectrolytes; D011095:Polyethylenes; D013670:Sodium Pertechnetate Tc 99m; D013388:Succinimides; C513055:ABCB1 protein, human; C503924:Pep-1 peptide; C118517:S-acetylmercaptoacetyltriglycine N-hydroxysuccinimide; C505405:poly(ethylene imine)