The influence of cationic lipid type on in-vitro release kinetic profiles of antisense oligonucleotide from cationic nanoemulsions.

Novel formulations of cationic nanoemulsions based on three different lipids were developed to strengthen the attraction of the polyanionic oligonucleotide (ODN) macromolecules to the cationic moieties on the oil nanodroplets. These formulations were developed to prolong the release of the ODN from the nanoemulsion under appropriate physiological dilutions as encountered in the eye following topical application. Increasing the concentration of the new cationic lipid exhibiting two cationic amine groups (AOA) in the emulsion from 0.05% to 0.4% did not alter markedly the particle size or zeta potential value of the blank cationic nanoemulsion. The extent of ODN association did not vary significantly when the initial concentration of ODN remained constant at 10 microM irrespective of the cationic lipid nature. However, the zeta potential value dropped consistently with the low concentrations of 0.05% and 0.1% of AOA in the emulsions suggesting that an electrostatic attraction occurred between the cationic lipids and the polyanionic ODN molecules at the o/w interface. Only the nanoemulsion prepared with N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts (DOTAP) remained physically stable over time. DOTAP cationic lipid nanoemulsion was the most efficient formulation capable of retaining the ODN despite the high dilution of 1:100 with simulated tear solution (STS). Less than 10% of the ODN was exchanged in contrast to 40-50% with the other cationic nanoemulsions. The in-vitro release kinetic behavior of ODN exchange with physiological anions present in the STS appears to be complex and difficult to characterize using mathematical fitting model equations. Further pharmacokinetic studies are needed to verify our kinetic assumptions and confirm the in-vitro ODN release profile from DOTAP cationic nanoemulsions.

Topical and intravitreous administration of cationic nanoemulsions to deliver antisense oligonucleotides directed towards VEGF KDR receptors to the eye.

Antisense oligonucleotides (ODNs) specific for VEGFR-2-(17 MER) and inhibiting HUVEC proliferation in-vitro were screened. One efficient sequence was selected and incorporated in different types of nanoemulsions the potential toxicity of which was evaluated on HUVEC and ARPE19 cells. Our results showed that below 10 microl/ml, a 2.5% mid-chain triglycerides cationic DOTAP nanoemulsion was non-toxic on HUVEC and retinal cells. This formulation was therefore chosen for further experiments. In-vitro transfection of FITC ODNs in ARPE cells using DOTAP nanoemulsions showed that nanodroplets do penetrate into the cells. Furthermore, ODNs are released from the nanoemulsion after 48 h and accumulate into the cell nuclei. In both ex-vivo and in-vivo ODN stability experiments in rabbit vitreous, it was noted that the nanoemulsion protected at least partially the ODN from degradation over 72 h. The kinetic results of fluorescent ODN (Hex) distribution in DOTAP nanoemulsion following intravitreal injection in the rat showed that the nanoemulsion penetrates all retinal cells. Pharmacokinetic and ocular tissue distribution of radioactive ODN following intravitreal injection in rabbits showed that the DOTAP nanoemulsion apparently enhanced the intraretinal penetration of the ODNs up to the inner nuclear layer (INL) and might yield potential therapeutic levels of ODN in the retina over 72 h post injection.