In vitro microtubule polymerization assay

CT26 cells were grown in glass dishes and cultured for 24 h. Then, the culture medium was discarded, and the fresh medium containing Taxol or prodrug nanoassemblies (100 nmol/L equivalent to PTX) was added, and further cultured for 48 h. Then, the cells were washed by PBS for three times, and fixed with 4% formaldehyde. After that, the cells were treated according to the procedure suggested in Microtubule Tracker Red detection kit. Subsequently, the cells were washed again, and stained with Hoechst 33342. Finally, the samples were observed using confocal laser scanning microscopy (CLSM, TCS SP2/AOBS, LEICA, Germany). Images were analyzed using Image J software.