Preparation of solid DNA nanoparticles for use in gene therapy

Background: Non-viral gene therapy, based on nanosized particles, is a potential therapeutic option in various diseases. The success is mainly dependent on an efficient gene delivery vector. Aerosol synthesis can provide pure solid DNA particles with substantial high dose of DNA per particle and thereby increase the amount of DNA delivered into cells as compared to commonly used DNA polymer complexes. The purpose of this study was to test the suitability of plasmid DNA alone or in complex with cationic polymers for the preparation of solid DNA nanoparticles by an aerosol flow reactor method. Methods: The sample solutions contained either plasmid DNA (pDNA) alone or complexed at a ratio of 1:1 (w/w) with branched or linear polyethylenimine (PEI) with the molecular weight of 25 kDa. The additive agents, l-leucine and mannitol, were added to PEI/DNA complexes at a ratio of 1:8 (w/w). The aerosol flow reactor method [1] involved atomization of sample solutions to nanosized droplets, which were immediately dried in a heated flow reactor tube by the evaporation of the solvent. The dried nanoparticles were collected with a low-pressure impactor and the size distribution was determined by a differential mobility analyzer. The surface morphology was analyzed using field emission scanning electron microscopy and the structural integrity of pDNA was evaluated by agarose gel electrophoresis. Results: The produced pure pDNA nanoparticles were spherical and had a mean diameter of 125 nm. However, pDNA in such nanoparticles did not preserve its supercoiled structure due to the shearing stresses caused by the atomization process. The complexation of pDNA with PEIs before atomization allowed the maintanence of pDNA integrity. The further addition of either l-leucine or mannitol to initial sample solution, stabilized nanoparticles structure and prevented them from water uptake and subsequent deformation. The resulting solid nanoparticles had a mean size between 65 and 125 nm and the loading content of pDNA in a single nanoparticle was approximately 10% (w/w). Conclusions: The aerosol flow reactor method provides an effective way of producing solid DNA nanoparticles with a size optimal for cell uptake and for potential use in non-viral gene delivery.