Mirus label it tracker intracellular nucleic acid localization kit

The pBAL was produced by Copernicus Therapeutics Inc. (Cleveland, OH) and pEGFP plasmid was purchased by Clontech Laboratories Inc. (Mountain View, CA). The plasmids were expanded and purified as previously described.²⁶ Plasmid was fluorescently labeled with Cy5 or Cy3 using a Mirus Label IT® Tracker™ Intracellular Nucleic Acid Localization Kit (Mirus Bio, Madison, WI). The gene vector complexation was achieved by the drop-wise addition (1 mL min⁻¹) of 10 volumes of plasmid DNA at a concentration of 0.1 mg mL⁻¹ to 1 volume of a swirling polymer solution. Various polymer solutions were prepared with one or more dendrimer conjugates at a nitrogen-to-phosphate (N/P) ratio of 5 unless otherwise specified. For the formulation of gene vectors with varying amounts of TA, blends of BiD-TA and BiD with a varying percentage of amine groups contributed by the BiD-TA polymer (100%, 75%, 50%, 25%, 10% and 5%) were used. For the formulation of PEGylated gene vectors, blends of BiD-TA and D-NH₂-PEG with a varying percentage of amine groups contributed by D-NH₂-PEG (75%, 50% and 25%) were used. The plasmid-polymer solutions were incubated for 30 min at room temperature to allow for the formation of gene vectors. PEI and PEG-poly-L-lysine (PEG-PLL) gene vectors were formulated as previously described²⁶ and used as controls in the following experiments.