Self-assembled amphiphilic phosphopyridoxyl-polyethylenimine polymers exhibit high cell viability and gene transfection efficiency in vitro and in vivo

Branched polyethylenimine (bPEI) was conjugated with hydrophobic pyridoxal phosphate (PLP) in the side chain via reaction with primary amines to obtain amphiphilic phosphopyridoxyl-polyethylenimine (PPyP) polymers. These polymeric amphiphiles with a defined degree of hydrophobicity self-assembled into nanostructures, which were characterized by DLS and evaluated for their capability to condense nucleic acids and carry them into cells. Further condensation of pDNA compacted the size of the self-assembled nanostructures from 421-559 nm to 134-210 nm with zeta potentials from +20-32 mV to +18-28 mV. Conjugation of PLP with bPEI not only reduced the density of the primary amines (i.e. charge density) but also improved the cell viability of the modified polymers considerably and weakened the binding of pDNA with these polymers. Efficient unpackaging of the pDNA complexes inside the cells led to a several fold enhancement in the transfection efficiency with one of the formulations, PPyP-3/pDNA complex, among the series, exhibiting similar to 4.9 to 8.2 folds higher gene delivery activity than pDNA complexes of bPEI and Lipofectamine (TM) in HeLa and MCF-7 cells. Flow cytometry analysis revealed a very high percentage of transfected cells by PPyP/pDNA complexes compared to pDNA complexes of bPEI and Lipofectamine (TM). Further, GFP-specific siRNA delivery using PPyP-3 as a vector resulted in similar to 84% knockdown of the target gene expression (cf. similar to 54% by Lipofectamine (TM)/pDNA/siRNA complex). Moreover, the PPyP-3/pDNA complex displayed similar to 6.7 fold higher transfection efficiency than the bPEI/pDNA complex in human peripheral blood dendritic cells. Intravenous administration of PPyP-3/pGL3 complex showed the highest gene expression in spleen tissue, advocating the potential of these vectors in future gene delivery applications.