Biodegradable microspheres containing plasmid DNA have potential uses as mediators of transfection in cells, particularly phagocytic cells such as macrophages. However, the hydrophilic nature and the structural instability of supercoiled DNA preclude its facile encapsulation in polymer matrixes such as poly(d,l-lactic-co-glycolic acid) (PLGA) by traditional methods. We initially studied the microencapsulation of plasmid DNA using the established water-in-oil-in-water double-emulsion solvent-evaporation method and found that (1) the encapsulation efficiency was low (about 20%), (2) the microencapsulation procedure nicked (degraded) the supercoiled DNA, and (3) lyophilization of the microsphere also nicked the DNA. We have therefore designed a new microsphere preparation method (called cryopreparation) to specifically address these concerns. Using the cryopreparation method, the aqueous phase of the primary emulsion containing the plasmid DNA is frozen and then subjected to homogenization. Because there is no shear stress inside a solid, we hypothesized that freezing the aqueous phase of the primary emulsion would help to preserve the supercoiled plasmid DNA during formation of the secondary emulsion. We also hypothesized that the formation of crystals from buffers within the primary emulsion was a causative factor for nicking during freezing or lyophilization, and that disruption of the crystal formation by the addition of saccharides into the primary emulsion would improve the supercoiled-DNA content of the spheres; Our results support the two hypotheses. Not only was the supercoiled-DNA content increased from 39% to over 85%, but the encapsulation efficiency was also elevated from 23% to over 85%.
