Effect of polyisobutylcyanoacrylate nanoparticles and lipofectin loaded with oligonucleotides on cell viability and PKC alpha neosynthesis in HepG2 cells.

The aim of the present study was to evaluate the inhibitory effect on protein kinase C alpha (PKC alpha) neosynthesis of antisense oligonucleotides delivered by two types of carriers. First, PKC alpha antisense oligonucleotides were associated with polyisobutylcyanoacrylate (PIBCA) nanoparticles pre-coated with cetyltrimethyl ammonium bromide (CTAB), a hydrophobic cation. Adsorption of oligonucleotides onto PIBCA nanoparticles was shown to be a saturating process. From these studies, it was possible to identify two types of particles: positively and negatively charged. Secondly, Lipofectin was used as another carrier system. These systems were incubated with HepG2 cells. Toxicity was evaluated by the MTT assay, and PKC alpha neosynthesis was determined by Western blots in conditions where nanoparticles and Lipofectin were not inducing cytotoxicity. It was observed that both mismatch and antisense oligonucleotides induced an inhibition of PKC alpha neosynthesis when loaded onto cationic or anionic nanoparticles as well as when complexed to cationic liposomes (Lipofectin). This non-specific effect was only observed in the phase of PKC alpha neosynthesis when the cells were first depleted in PKC alpha by phorbol 12-myristate beta-acetate (12-PMA) and in the absence of serum. These results strongly suggest that delivery systems, PIBCA nanoparticles or Lipofectin, containing a positively charged component (CTAB or cationic lipids), are able to induce a perturbation in the intracellular metabolic activity. In conclusion, it was shown that the commonly used strategy of oligonucleotides targeting with cationic non-viral vectors may display non-specific effects which can lead to artifactual results.