A methodology for quantitation and characterization of oligonucleotides in albumin microspheres

Abstract

A fluorescence assay was developed to quantify oligonucleotides (ODNs) encapsulated in bovine serum albumin (BSA) microspheres using antisense to Nuclear Factor-kappaB (NF-kappaB) as a model ODN and employing Oligreen as the fluorescent dye. Methodologies were optimized for the suspension of the microspheres as well as release of the encapsulated ODN using protease digestion. This was followed by the detection and quantitation of the ODN using the Oligreen dye. The Oligreen fluorescence assay gave a concentration-dependent fluorescent interaction with the ODN. Further characterization of the ODN with respect to their structural integrity in non-irradiated and gamma-irradiated antisense encapsulated in BSA microspheres was performed using HPLC, infrared spectroscopy and polyacrylamide gel electrophoresis. Results showed no structural modification of antisense in the BSA microspheres as determined by HPLC retention times for the pure antisense and microsphere-encapsulated ODN. The migration pattern of the antisense in polyacrylamide gels confirmed the absence of significant alterations as a result of the encapsulation process or due to gamma-irradiation. The infrared spectra of non-irradiated and gamma-irradiated antisense to NF-kappaB microspheres also displayed peaks characteristic of the functional groups. The fluorescence assay could also detect NF-kappaB antisense in the serum of rats administered with encapsulated antisense by oral and intravenous routes. This methodology should be valuable for the analysis of BSA-encapsulated antisense ODN and for pharmacokinetic studies during antisense therapy.