Spectroscopic methods for characterization of nonviral gene delivery systems from a pharmaceutical point of view

Introduction of nucleic acids into mammalian and plant cells has become, in recent years, a well established procedure in basic and applied research, and the methods involved therein are now useful for the development of gene-based medicines as a new class of pharmaceuticals. The incorporation of the gene of interest into a plasmid construct by molecular biology techniques, the complexation and condensation of nucleic acids by means of cationic agents, is the first step for enabling DNA to get into the cells of interest in a biologically active state. Monitoring this condensation procedure for scientific and quality control purposes is mostly performed by optical spectroscopy methods. A fluorimetric, high-throughput-assay has been developed for the characterization of non-viral gene delivery systems. Protamine sulfate and cationic DOTAP liposomes were used to condense the plasmid DNA. The DNA was labeled with nucleic acid stains, and the quenching of fluorescence by the addition of condensing agents was measured. PicoGreen(TM) was compared to a number of common nucleic acid stains (ethidium bromide, ethidium-homodimer-2, acridine orange, BOBO-1, POPO-1, TOTO-1 YOYO-I, BOBO-3, POPO-3, TOTO-3, YOYO-3, BO-PRO-1, PO-PRO-1, TO-PRO-1, YO-PRO-1, BO-PRO-3, PO-PRO-3, TO-PRO-3, YO-PRO-3), and was found to be an advantageous nucleic acid stain for the fluorimetric characterization of condensation. Stability of nucleic acid-dye complex, reproducibility and the signal-to-noise ratio make PicoGreen(TM) a dye of choice for condensation experiments. Condensation was quantified by the fitting of condensation curves to the "Hill equation". Relative fluorescence intensity, Hill slope and kinetics of DNA-condensation provide the calculated values for advanced characterization of DNA-condensation. Fluorescence spectroscopy are compared to agarose gel electrophoresis as another approach for biophysical characterization of DNA-condensation. Advantages and disadvantages of the two methods are discussed.