Critical evaluation of quantification methods for oligonucleotides formulated in lipid nanoparticles

There is a very large variety in the types of nanoparticulate lipid formulations for oligonucleotides, and remarkably, also a very large heterogeneity in the methods that are used for analyzing oligonucleotide load, encapsulation efficiency and oligonucleotide release. Furthermore, a literature survey showed that the extent to which these procedures are reported in scientific literature varies greatly, with some of them not even reporting any quantification at all. This greatly hampers the reproducibility of nanoparticle preparation between different researchers and between different laboratories, which slows down the clinical translation of such nanomedicines. In this work, a standardized extraction method from liposomes is proposed, in which potential contaminants from the carrier are removed by a simple extraction of the oligonucleotides. These extracts were then analyzed with seven commonly used methods for oligonucleotide quantification, including several absorbance based methods and the most commonly applied dye binding assay. Strikingly, differences in absolute values up to fourfold were found when the same sample was analyzed using different methods which should be taken into consideration when reports using different methods are compared. Furthermore, these results indicate that the most commonly applied method, the dye binding assay, may -without adaptations- not be suitable for short oligonucleotides like siRNAs. The found differences in quantification methods as described here underscore the need for proper documentation of methods to correctly interpret published results, which -with regard to oligonucleotide analysis- is currently lacking in many reports.