Phosphodiester and phosphorothioate oligonucleotide condensation and preparation of antisense nanoparticles.

Protamine is a cationic peptide with a molecular mass of approx. 4000 Da that is able to condense DNA. In the present study it was used to complex antisense oligonucleotides (ODNs) and to form solid particles with initial diameters of 90-150 nm. The reaction was very rapid and occurred by simple mixing of diluted solutions of the polycation with the oligonucleotide. The aggregation was dependent on the oligonucleotide chain length and the protamine/ODN mass ratio. Particle formation required a minimal chain length of nine nucleotides and a mass ratio of 0.5:1. The particle surface charge and the number of particles depended on the mass ratio. With increasing amounts of the peptide, the number of particles and the zeta potential increased. Both negatively and positively charged particles improved the stability of oligonucleotides against DNase I digestion. Above a mass ratio of 2.5:1 no degradation was found. The uptake of unbound rhodamine-labelled ODNs and its complexes with protamine was determined with Vero cells under in vitro cell culture conditions at 37 degrees C and 4 degrees C. At 37 degrees C the cellular uptake increased with increasing mass ratio. The internalized oligonucleotides were localized in the cytoplasm and in the nucleus of the cells. When Vero cells were treated with these samples at 4 degrees C for 4 h, no fluorescence could be detected inside the cells. Therefore, our data indicate an energy dependent endocytotic uptake mechanism. In contrast, spermine and spermidine, which are also known condensation agents, did not aggregate with oligonucleotides into nanoparticles under the same conditions.

Enhanced antisense efficacy of oligonucleotides adsorbed to monomethylaminoethylmethacrylate methylmethacrylate copolymer nanoparticles.

The purpose of this study was the investigation of cationic nanoparticles as drug delivery systems for antisense oligonucleotides. Cationic monomethylaminoethylmethacrylate (MMAEMA) copolymer nanoparticles were prepared from N-monomethylaminoethylmethacrylate hydrochloride and methylmethacrylate. Oligonucleotides were adsorbed onto MMAEMA nanoparticles. Cell penetration was investigated in vitro with fluorescently labeled oligonucleotides and nanoparticles. Antisense effects of oligonucleotides adsorbed to MMAEMA nanoparticles were evaluated by sequence specific inhibition of ecto-5'-nucleotidase expression. The amount of enzyme expressed in PC12 cells was detected and quantified by immunocytochemistry using fluorescein isothiocyanate-labeled antibodies. Oligonucleotides were adsorbed to MMAEMA nanoparticles by the formation of ion-pairs between the positively charged secondary amino groups located on the particle surface and the anionic phosphodiester or phosphorothioate backbones of the oligonucleotides. Adsorption to nanoparticles led to an increased cellular uptake of oligonucleotides and to a significantly enhanced antisense efficacy of unmodified phosphodiester oligonucleotides as well as phosphorothioates. The results of the cell penetration and the antisense assay demonstrated that MMAEMA nanoparticles are promising carriers for oligonucleotide administration.

Antisense delivery using protamine-oligonucleotide particles.

Protamine, a polycationic peptide (mol. wt 4000-4500), was evaluated as a potential penetration enhancer for phosphodiester antisense oligonucleotides (ODNs). Unique complexes in the form of nanoparticles were spontaneously formed, which we call 'proticles'. The stability of the particles and the ODNs bound into the proticles was examined in foetal calf serum and cell culture medium. FITC-labelled ODNs bound to protamine showed an increased cellular uptake into human histiocytic lymphoma U 937 cells compared to free ODNs. Proticles significantly decreased cellular growth in a cell proliferation assay using ODNs against the c- myc proto-oncogene.