930. Magnetically Responsive Polylactide-Based Nanoparticles Potently Enhance Non-Viral Gene Transfer

Abstract

Top of pageAbstract Clinical use of non-viral gene delivery vectors is compromised by their comparatively low efficacy. To address this, we explored the possibility of enhancing DNA expression using magnetically responsive biodegradable polylactide (PLA)-based nanoparticles (NP). Magnetite-loaded NP of varying size (190 nm |[ndash]| 320 nm) have been formulated with polyethyleneimine (PEI 25K) surface coating using modified emulsification-solvent evaporation. Preparation procedure resulted in high yields of magnetite incorporation in the polymer matrix (95-100%) and PEI-NP association (up to 50%). NP demonstrated complete DNA binding in the theoretical charge ratio range 5-15 +/|[minus]| as shown by PicoGreen displacement. BODIPY 650/665-X-labeled NP and GFP reporter were used to determine the gene transfer efficacy and NP uptake in rat aortic smooth muscle cells (A10) and bovine aortic endothelial cells (BAEC). Plasmid DNA was associated with increasing amounts of magnetic particles and diluted in cell medium containing 10% fetal bovine serum. NP were applied to cells grown on 96-well plates (0.25 |[mu]|g DNA/well) for 15 min with or without magnetic field (500 G) using PEI and non-magnetic NP as a control. The gene expression was dependent on the NP size, with the largest-sized NP (320 nm) exhibiting the highest efficacy. The percent of GFP-expressing cells treated with 320 nm sized magnetic NP was NP dose dependent and amounted to 22|[plusmn]|3% and 14|[plusmn]|6% at 15 +/|[minus]| for BAEC and A10 cells, respectively, in the presence of magnetic field, whereas in the absence of magnetic exposure it was below 0.2% in both cell types. Similarly, the transfection mediated by non-magnetic NP and by free PEI was negligible with or without the magnetic exposure; this indicates that the effect was specific to the magnetic formulation and not due to the cell membrane permeability affected by the magnetic field. This was also supported by the observation that the magnetic NP uptake under magnetic force was substantially higher than that of magnetic NP in the absence of the magnetic field or that of the non-magnetic NP exposed to the magnet (A10: 12- and 7-fold increase, respectively, BAEC: 12- and 5-fold increase, respectively). Cell viability measured on day 3 post transfection using the AlamarBlue assay was comparable to that of free PEI (85-105% in both A10 and BAEC) and was unaffected by the magnetic field exposure shown to facilitate the internalization of the magnetic NP. In conclusion, PEI-coated magnetically responsive NP have been shown to mediate high levels of gene transfer into smooth muscle cells and endothelial cells upon 15 min incubation in a serum containing medium in the presence of a magnetic field. The efficacy of this strategy demonstrated under challenging conditions, together with the small submicronial size of the carrier and its minor effect on the cell viability, makes PEI-coated magnetically responsive biodegradable NP an important candidate DNA delivery system for in vivo applications.