In vitro assay for measuring real time topotecan release from liposomes: release kinetics and cellular internalization.

Topotecan is a drug that is under investigation for the treatment of neuroblastoma and has been encapsulated into liposomes to improve its therapeutic efficacy. However, liposomal formulations still need to be optimized for drug retention and new techniques to measure drug release are required to better understand this process. Here, a novel in vitro method based on fluorescence de-quenching and an automated microscopy imaging platform were developed for monitoring, in real time, the release of topotecan from a liposomal formulation. Drug release from liposomes was monitored for up to 15 h under different conditions including topotecan concentrations, fetal bovine serum amounts (0-20%), and temperatures (25 and 37 °C). A cell-based assay was used to assess liposome association with cells in culture and to quantify amounts of topotecan internalized into cells after release from liposomes. Our results show that the liposomal topotecan concentration had an influence on drug release kinetics: there was a reduction in release rate as a function of increasing concentration. Our data also show that topotecan release from the liposomal formulation was dependent on serum concentration where faster release was observed at higher serum concentrations, and on temperature where faster release was found at 37 °C. This real-time liposomal drug release assay allows for better understanding of the factors important in governing release of topotecan. The assay will be essential towards designing liposomal formulations of topotecan (and potentially of other camptothecin derivatives such as irinotecan) with optimized retention times and better therapeutic efficacy for testing in the clinic.