Abstract
We have reported that ternary complexes of plasmid DNA with conventional linear polyethylenimine (l-PEI) and certain polyanions were very stably dispersed, and, with no cryoprotectant, they could be freeze-dried and re-hydrated without the loss of transfection ability. These properties enabled the preparation of a concentrated suspension of very small pDNA complex, by preparing the complexes at highly diluted conditions, followed by condensation via lyophilization-and-rehydration procedure. Recently, a high potency linear polyethylenimine having no residual protective groups, i.e., Polyethylenimine “Max” (PEI “Max”), is available, which has been reported to induce much higher gene expression than conventional l-PEI. We tried to prepare the small DNA/PEI “Max”/polyanion complexes by a similar freeze-drying method. Small complex particles could be obtained without apparent aggregation, but transfection activity of the rehydrated complexes was severely reduced. Complex-preparation conditions were investigated in details to achieve the freeze-dried DNA/PEI “Max”/polyanion small ternary complexes with high transfection efficiency. DNA/PEI “Max”/polyanion complexes containing cytokine-coding plasmids were then prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice.
Highlights
Considerable efforts have been invested in developing non-viral vector systems as safer alternatives to live viruses [1]
We mixed plasmid DNA (pDNA) with hyaluronic acid, and linear polyethylenimine (l-PEI), in the given order, as pre-addition of the polyanion had been found to afford complexes of smaller size than those obtained by post-addition of the polyanion to the pre-formed pDNA/polycation complexes [5]
We explored the optimum conditions for preparation of the freeze-dried pDNA/(PEI “Max”)/polyanion ternary complexes, which could be re-hydrated into fine suspensions having high activity for in vivo applications
Summary
Considerable efforts have been invested in developing non-viral vector systems as safer alternatives to live viruses [1]. We mixed pDNA with hyaluronic acid, and linear polyethylenimine (l-PEI), in the given order, as pre-addition of the polyanion had been found to afford complexes of smaller size than those obtained by post-addition of the polyanion to the pre-formed pDNA/polycation complexes [5] Preparation of these complexes was performed under very dilute conditions ([pDNA] = 6 μg/mL). Mixing of pDNA, HA, and PEI “Max” in this order at [pDNA] = 6 μg/mL generated fairly small stable complex particles They showed significantly higher (70-fold) gene expression than achieved by conventional l-PEI in cultured cells, as was expected. We explored the optimum conditions for preparation of the freeze-dried pDNA/(PEI “Max”)/polyanion ternary complexes, which could be re-hydrated into fine suspensions having high activity for in vivo applications. Effective pDNA/(PEI “Max”)/polyanion complexes containing cytokine-coding plasmid were prepared, and their anti-tumor therapeutic efficacy was examined in tumor-bearing mice
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