Advanced fluorescence microscopy methods illuminate the transfection pathway of nucleic acid nanoparticles

A great deal of attention in biopharmacy and pharmaceutical technology is going to the development of nanoscopic particles to efficiently deliver nucleic acids to target cells. Despite the great potential of nucleic acids for treatment of various diseases, progress in the field is fairly slow. One of the causes is that development of suitable nanoscopic delivery vehicles is hampered by insufficient knowledge of their physicochemical and biophysical properties during the various phases of the transfection process. To address this issue, in the past decade we have developed and applied advanced fluorescence microscopy techniques that can provide a better insight in the transport and stability of nanoparticles in various biological media. This mini-review discusses the basic principles of fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT), and gives an overview of studies in which we have employed these techniques to characterize the transport and stability of nucleic acid containing nanoparticles in extracellular media and in living cells.