pDNA bioparticles: comparative heterogeneity, surface, binding, and activity analyses
Section snippets
Materials and methods
Reagents. All model compounds were obtained from Sigma/Aldrich (St Louis, MO). 1:1 wt:wt DOTAP/cholesterol and rhodamine labeled plasmid DNA were generous gifts from Megabios (Burlingame, CA). Luciferase and green fluorescent protein plasmid were obtained from Aldevron (Fargo, ND). Gold microparticles were obtained from PowderJect Vaccines (Madison, WI).
Controlled complex formation. Compounds were added to plasmid at approximately 1 ml/min while the plasmid was stirring within a 12 ml glass vial
Model compounds
Chemical features of the model compounds, lipid or tributyrin controls are shown in Table 1. All compounds’ transfection and toxicity in combination with DNA had been previously described [3], [4], [5], [6], [7], [8]. All agents contained cationic–NH-groups permitting stabilizing charge–charge interactions with the anionic phosphodiester backbone on the DNA, except tributyrin whose charge was mediated through combination with RGDS peptide at the vesicles’ surface. In addition, intercalation of
Discussion
In brief summary we have characterized the DNA-binding, charge and size heterogeneity, surface, sedimentation, protein-binding, transfection activity, and immunotoxicity of a range of non-lipid biomaterials which produce micro/nanoparticles when complexed to plasmid DNA. Unlike viral or liposomal DNA particles [1], [2], our results especially with chitosan and protamine materials suggest that these materials will be less limited from the standpoint of aggregation, sedimentation, and
Acknowledgements
We gratefully acknowledge J. Koe, G. Koe, and S. Venkatesh (Megabios Corp., Burlingame, CA) for the provision of liposome, rhodamine labeled plasmid DNA, and assistance with the sedimentation assays, B. Lemos (Geron Corp.) for technical assistance with mouse intra-peritoneal assays, S. Ghivizzani and T. Oligino (University of Pittsburg Center for Biotechnology and Bioengineering) for assistance with the rabbit knee model white blood cell assays, and J. Haynes (PowderJect Vaccines, Madison, WI)
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