Pitfalls in analyzing release from chitosan/tripolyphosphate micro- and nanoparticles

Submicron particles prepared by complexing chitosan with tripolyphosphate (TPP) attract widespread interest as potential drug, gene and vaccine delivery vehicles, and many published studies examine their release properties. Despite these sustained efforts, however, literature on the release performance of chitosan/TPP micro- and nanoparticles is filled with conflicting results, with some reporting nearly instantaneous release, while others showing the release to be sustained for up to multiple days. To resolve these opposing findings, we recently postulated that the in vitro release profiles obtained from chitosan/TPP particles by the standard sample and separate or solvent replacement method (where the solvent was periodically replaced with fresh buffer and analyzed for the released bioactive molecule content) may have been subject to strong experimental artifacts and not have reflected their true release behavior. To explore this possibility, here we examine several experimental artifacts that may arise during such in vitro experiments and show that conflicting findings on release from chitosan/TPP particles can arise from: (1) incomplete particle separation from the release media upon centrifugation; (2) irreversible particle coagulation; and (3) failure to maintain sink conditions. Moreover, we show that some of the longer-lasting release profiles may reflect the use of physiologically irrelevant (low-ionic strength) release media. By analyzing and discussing these effects, this article provides guidelines for obtaining more reliable release profiles for chitosan/TPP micro- and nanoparticles and other/related colloidal carriers.