PLGA microspheres containing plasmid DNA: Preservation of supercoiled DNA via cryopreparation and carbohydrate stabilization

Biodegradable microspheres containing plasmid DNA have potential uses as me diators of transfection in cells, particularly phagocytic cells such as mac rophages. 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 ini tially 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 microenc apsulation procedure nicked (degraded) the supercoiled DNA, and (3) lyophil ization of the microsphere also nicked the DNA. We have therefore designed a new microsphere preparation method (called cryopreparation) to specifical ly address these concerns. Using the cryopreparation method, the aqueous ph ase of the primary emulsion containing the plasmid DNA is frozen and then s ubjected to homogenization. Because there is no shear stress inside a solid , we hypothesized that freezing the aqueous phase of the primary emulsion w ould help to preserve the supercoiled plasmid DNA during formation of the s econdary emulsion. We also hypothesized that the formation of crystals from buffers within the primary emulsion was a causative factor for nicking dur ing freezing or lyophilization, and that disruption of the crystal formatio n by the addition of saccharides into the primary emulsion would improve th e supercoiled-DNA content of the spheres; Our results support the two hypot heses. 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 %.