530. The Effects DNA Molecular Weights and Conformations on Cell Uptake and the Binding Interaction with Cationic Polymers Used for Non-Viral Gene Delivery

Synthetic polymers are used in non-viral gene therapy as vectors to facilitate the transfer of DNA into the pertinent cells. These polymers compact the DNA into a dense particle, mediate entry across the plasma membrane, and protect the genes from degradation by the host defense mechanism. While it has been established previously that a strong binding of polymer to DNA does not correlate directly with gene expression3, no study thus far has determined whether such correlation also exist for the cell uptake of DNA. Further, as DNA molecules can exist in different conformations and molecular weights, the accessibility of ionic groups for polymers to interact may also vary between structurally distinct DNA molecules. Whether such differences translate in subsequent transfection events remain to be seen. In this study, we compared the binding affinity between PLL (25 kDa), PLL (25 kDa) modified with palmitic acid (PLL-PA), Linear PEI (2.5 kDa and 25 kDa), branched PEI (25 kDa), and Lipofectamine2000 to three DNA molecules: a 4.2 kb circular plasmid DNA extracted and purified from Escherichia coli, a 4.2 kb linear plasmid DNA prepared by single digestion of the circular plasmid DNA and a 2.1 kb linear PCR product amplified using the circular plasmid DNA as template. A known quantity of DNA were mixed with polymer at polymer-to-DNA ratios (w/w) of 0, 0.02, 0.05, 0.1, 0.2, 0.5, and 1 in 0.1M HEPES buffer (pH7.2) for 30 minutes at room temperature. Polyplex solutions were then loaded onto a 0.6% TBE agarose gel stained with EtBr and applied with 130 volts for 60 minutes. For the uptake studies, DNA was labeled with Cy5.5 probe (Amershaim), complexed with polymer and added to rat bone marrow stromal cells (BMSC) cultured in a basic medium then incubated for 24 hours in 37°C with 5% CO2 after which cells are processed for flow cytometry. Polymer binding to DNA neutralizes anionic charges on the phosphate backbone, rendering the complex immobile under electric field. Binding was generally poor for the linear PEIs as most of the DNA migrated through the agarose matrix. Lipofectamine only showed a preferential binding for linear DNAs. PLL-PA and PLL binds equally well to all three types of DNA. Branched PEI showed a significant selectivity for both linear plasmid DNA and linear PCR product over the circular plasmid. In short, a correlation between binding affinity and DNA physical properties cannot be generalized for all polymers. Almost all of the BMSC transfected with branched PEI as polymer exhibited DNA uptake. PLL, PLL-PA and Lipofectamine2000 all induced an elevated level of DNA uptake, but at a lower level compared to branched PEI. Linear PEI was least effective as delivery vector, consistent with very low binding data. No apparent correlation between DNA uptake and the differences in molecular weight and conformation can be drawn. Polymers also did not show a clear pattern about the relative efficiency towards different DNA molecules. In conclusion, non-viral polymer vectors are capable of transferring different sizes of DNA into the cell with no effect of being either of linear or circular configuration.

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