Spectrophotometric determination of polyethylenimine in the presence of an oligonucleotide for the characterization of controlled release formulations.

摘要

Polyethylenimine (PEI) is a cationic polymer that can be associated to oligonuclotides to promote their transfection both in vitro and in vivo. The controlled release of oligonucleotide/polyethylenimine complexes from biodegradable systems can result in an increased cellular internalisation of the oligonucleotide and a reduced cytotoxicity of the complex. This effect strongly depends on the amount of PEI loaded in and released from the delivery system. In this work we describe a rapid, sensitive and reproducible spectrophotometric method for the quantitative analysis of PEI by itself or in the presence of an associated oligonucleotide. PEI does not possess chromophores, hence the determination by ordinary spectrophotometry is not possible. However, upon addition of copper (II) ions, PEI forms a dark blue cuprammonium complex that can be detected by UV-vis spectrophotometry. The optimum conditions in terms of optical parameters, copper (II) concentration required for a quantitative PEI complexation, andthe most suitable medium for the reaction were ascertained. A linear relationship (r(2)=0.9997) between absorbance and amounts of PEI was found at lambda(max) of 285 nm over the concentration range 5.0-50.0 microg ml(-1). The detection limit (QOD) was 4.0 microg ml(-1). The method was validated for the quantitation of PEI in the presence of an oligonucleotide, which absorbs at 285 nm as well.