Phosphodiester and phosphorothioate oligonucleotide condensation and preparation of antisense nanoparticles

Protamine is a cationic peptide with a molecular mass of approx. 4000 Da th at is able to condense DNA. In the present study it was used to complex ant isense oligonucleotides (ODNs) and to form solid particles with initial dia meters of 90-150 nm. The reaction was very rapid and occurred by simple mix ing of diluted solutions of the polycation with the oligonucleotide. The ag gregation was dependent on the oligonucleotide chain length and the protami ne/ODN mass ratio. Particle formation required a minimal chain length of ni ne nucleotides and a mass ratio of 0.5:1. The particle surface charge and t he mumber of particles depended on the mass ratio. With increasing amounts of the peptide, the number of particles and the zeta potential increased. B oth negatively and positively charged particles improved the stability of o ligonucleotides against DNase I digestion. Above a mass ratio of 2.5:1 no d egradation was found. The uptake of unbound rhodamine-labelled ODNs and its complexes with protamine was determined with Vero cells under in vitro cel l culture conditions at 37 degreesC and 4 degreesC. At 37 degreesC the cell ular uptake increased with increasing mass ratio. The internalized oligonuc leotides were localized in the cytoplasm and in the nucleus of the cells. W hen Vero cells were treated with these samples at 4 degreesC for 4 h, no fl uorescence could be detected inside the cells. Therefore, our data indicate an energy dependent endocytotic uptake mechanism. In contrast, spermine an d spermidine, which are also known condensation agents, did not aggregate w ith oligonucleotides into nanoparticles under the same conditions. (C) 2001 Elsevier Science B.V. All rights reserved.