Preparation and characterization of nanoparticle/miRNA complexes

PLGA nanoparticles were prepared using a water-in-oil-in-water solvent evaporation technique as described previously with minor modification.24 Briefly, 100 mg of PLGA was dissolved in 1 mL of methylene chloride to form a 10% (w/v) polymer solution. Then, 3 mL of 7% (w/v) poly(vinyl alcohol) aqueous solution was added to the above polymer solution and emulsified by sonification for 120 seconds (Bandelin Electronic, Berlin, Germany). The above emulsion was further added into 50 mL of 1% (w/v) PVA aqueous solution. The resulting mixture was sonicated for 180 seconds and then stirred for 24 hours at room temperature to evaporate the residual methylene chloride. Subsequently, the nanoparticles suspension was washed twice with ddH₂O by centrifugation at 13,000 rpm for 5 minutes and then resuspended in the ddH₂O. A total of 100 μL of the nanoparticles aqueous solution (10 μg/μL) was mixed with 2 μL of polyethylenimine (PEI; Mw =~25 kDa) polymer aqueous solution (100 μg/μL) to form PEI-modified nanoparticles. A gel retardation assay was used to detect the most appropriate ratio of nanoparticles and miRNA (N/P). Then, PEI-modified nanoparticles suspension was added to the miRNA solution at the 6:1 ratio between nitrogen of polymer and phosphate of RNA (N/P), mixed gently, and then incubated for 15 minutes at room temperature to form nanoparticle/miRNA complexes. The morphology of the nanoparticle/miRNA complexes was characterized by scanning electron microscopy (SEM; S-3400N; Hitachi Ltd., Tokyo, Japan). The mean hydrodynamic diameter measurements of nanoparticle/miRNA complexes were performed by dynamic light scattering (Brookhaven Instruments, Holtsville, NY, USA). The zeta potential (surface charge) of the complexes was determined at 25°C with a scattering angle of 90° using Zetaplus (Brookhaven Instruments).