Magnetofection--a highly efficient tool for antisense oligonucleotide delivery in vitro and in vivo.
Delivery of antisense oligodesoxynucleotides (ODN) into primary cells is a specific strategy for research with therapeutic perspectives but transfection-associated difficulties. We established the technique of magnetofection to enhance ODN delivery at low toxicity and procedure time in vitro and in vivo. In vitro, target knockout was assessed at protein and mRNA levels and by measuring superoxide generation after antisense magnetofection against the p22(phox) subunit of endothelial NAD(P)H-oxidase. Under magnetic field guidance, low-dose magnetic particle-bound ODN were transfected to 84% human umbilical vein endothelial cells within 15 min followed by nuclear accumulation within 2 h, which required 24 h using standard methods. Antisense magnetofection against p22(phox) significantly decreased basal and prevented stimulated superoxide release due to loss of NAD(P)H-oxidase activity by mRNA knockout as assessed after 24 h. Knockout of endothelial phosphatase SHP-1 and connexin 37 proteins confirmed the method's efficiency. Transfection-associated toxicity was minimal. Twenty-four hours after injection of fluorescence-labeled ODN into femoral arteries of male mice, there was specific ODN uptake only into cremaster vessels exposed to magnetic fields during injection. Magnetofection is an ideal tool for delivery of functionally active ODN to difficult-to-transfect cells to study gene/protein function and a promising strategy for targeted ODN delivery in vivo.
Krötz
Florian
F
Institute of Physiology, Munich, Germany. fkroetz@lmu.de
de Wit
Cor
C
Sohn
Hae Young
HY
Zahler
Stefan
S
Gloe
Torsten
T
Pohl
Ulrich
U
Plank
Christian
C
D000818:Animals; D002470:Cell Survival / Q000528:radiation effects; D045304:Cytochromes c / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods*; D004574:Electromagnetic Fields; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D026901:Membrane Transport Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D009097:Multienzyme Complexes / Q000378:metabolism; D009132:Muscles / Q000166:cytology / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D019255:NADPH Oxidases; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage*; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014471:Umbilical Veins / Q000378:metabolism
D026901:Membrane Transport Proteins; D009097:Multienzyme Complexes; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D013481:Superoxides; D045304:Cytochromes c; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase