Degradable PEG-folate coated poly(DMAEA-co-BA)phosphazene-based polyplexes exhibit receptor-specific gene expression

A new cationic biodegradable polyphosphazene was developed, bearing both pendant primary and tertiary amine side groups, poly(2-dimethylaminoethylamine-co-diaminobutane)phosphazene (poly(DMAEA-co-BA)phosphazene). PEG and PEG-folate were coupled to polyplexes based on this poly(DMAEA-co-BA)phosphazene, leading to small (size 100 and 120nm, respectively) and almost neutral particles. In vitro tissue culture experiments showed a low cytotoxicity of both uncoated and coated polyplexes. However, the PEG coated polyplexes showed a 2-fold lower transfection activity in OVCAR 3 cells as compared to the uncoated polyplexes. On the other hand, the PEG-folate coated polyplexes had a 3-fold higher transfection than the PEGylated polyplexes. When free folate was added to the transfection medium, only the transfection activity of the targeted polyplexes was reduced, indicating internalization of the targeted PEG polyplexes via the folate receptor. Confocal laser scanning microscopy confirmed a lower binding and uptake of the PEGylated polyplexes by OVCAR-3 cells when compared to uncoated and folate-PEGylated polyplexes. While uncoated polyplexes induced aggregation of erythrocytes at polymer concentrations of 0.09microg/mL, the PEGylated systems could be incubated at ten times higher concentration before aggregation occurred indicating excellent shielding of the surface charge of the polyplexes by grafting of PEG. In conclusion, the targeted delivery of poly(DMAEA-co-BA)phosphazene bases polyplexes and their improved compatibility with erythrocytes makes them interesting for in vivo applications.