Improved transfection efficiency of chitosan-DNA complexes employing reverse transfection

摘要

The present study was designed to systematically compare the conventional and reverse transfection methodologies for chitosan/DNA complexes using a low molecular weight (MW) chitosan. The hydrodynamic diameter of the complexes, measured by Dynamic Light Scattering (DLS) was found to be ～ 216 nm and TEM investigations showed spherical and compact complexes with an average size of 200 nm. The transfection efficiency of chitosan using the two methodologies was assessed by employing reporter gene coding for green fluorescent protein (GFP) and luciferase. More than 50% of HEK 293 cells were transfected when transfection done using reverse transfection strategy at pH 6.5 with 10% serum for 24 h followed by media replenishment with pH 7.4 with 10% serum for an additional 24 h period. Also, the cytotoxicity of chitosan/DNA complexes was also considerably lower than the commercially available transfection reagent lipofectamine. Our investigation concludes that maximal transgene expression levels could be achieved using reverse transfection where the chitosan/DNA complexes are pre-incubated on the plate surface followed by plating of cells at pH 6.5 with 10% serum for 24h and media resupplemented with pH 7.4 with 10% serum for an additional 24 h period.