Effects of Microfluidic Shear on the Plasmid DNA Structure: Implications for Polymeric Gene Delivery Vectors

Microfluidic manufacturing of advanced gene deliveryvectors necessitatesconsideration of the effects of microfluidic shear forces on the structuralintegrity of plasmid DNA (pDNA). In this paper, we expose pDNA tovariable shear forces in a two-phase, gas-liquid microfluidicreactor and apply gel electrophoresis to analyze the products of on-chipshear-induced degradation. The effects of shear rate, solvent environment,pDNA size, and copolymer complexation on shear-induced degradationare investigated. We find that small naked pDNA (pUC18, 2.7 kb) exhibitsshear rate-dependent shear degradation in the microfluidic channelsin a mixed organic solvent (dioxane/water/acetic acid; 90/10/<0.1w/w/w), with the extents of both supercoil isoform relaxation andcomplete fragmentation increasing as the maximum shear rates increasefrom 4 x 10(5) to 2 x 10(6) s(-1). However, over the same range of shear rates, the same pDNA sampleshows no evidence of microfluidic shear-induced degradation in a pureaqueous environment. Quiescent control experiments in the same mixedorganic solvent prove that a combination of solvent and shear forcesis involved in the observed shear-induced degradation. Furthermore,we show that shear degradation effects in mixed organic solvents canbe significantly attenuated by complexation of pDNA with the blockcopolymer polycaprolactone-block-poly(2-vinylpyridine)prior to exposure to microfluidic shear. Finally, we demonstrate thatmedium (pDSK519, 8.1 kb) and large (pRK290, 20 kb) naked pDNA aremore sensitive to shear-induced microfluidic degradation in the mixedorganic solvent environment than small pDNA, with both plasmids showingcomplete fragmentation even at the lowest shear rate, although wefound no evidence of shear-induced damage in water for the largestinvestigated naked pDNA even at the highest flow rate. The resultingunderstanding of the interplay of the solvent and shear effects duringmicrofluidic processing should inform microfluidic manufacturing routesto new gene therapy formulations.