Pro-aCD25-NDs exhibit tunable release kinetics of both prostratin and aCD25. (a) and (b) Pro-aCD25-NDs were incubated in PBS at 37 °C. Cumulative release of (a) prostratin and (b) aCD25 into the solution was analyzed at the listed time periods by UV–Vis spectroscopy and BCA protein assay, respectively, (c) Pro-aCD25-NDs were incubated in PBS at 37 °C for 24 h. At indicated timepoints, release of aCD25 from NDs into the solution was measured by BCA assay. Fresh PBS was used for replacement at each timepoint in the replacement group (shaded circles). No fresh PBS was added to the no replacement group (empty circles), (d) and (e) After synthesis, Pro-aCD25-NDs were incubated at 4 °C. After 24 h, Pro-aCD25-NDs were incubated in PBS at 37 °C for six days. At each listed timepoint, an aliquot was centrifuged, and released (d) prostratin and (e) aCD25 was measured by UV-Vis spectroscopy and BCA assay, respectively. Fresh PBS was replaced at every timepoint measured (i.e. 24, 48, and 72 h). Values represent means ± standard deviation (n = 3/group).