Novel pH-sensitive protamine sulfate (PS) hollow microspheres with controllable structure were fabricated and can be used as carriers for drug delivery and medical diagnostics. The carboxyl group-functionalized polystyrene microspheres prepared by soap-free emulsion polymerization were used as the templates. The self-assembled PS microspheres were prepared via electrostatic attraction between PS and carboxyl group-functionalized polystyrene. The single-layer PS was self-assembled and subsequently crosslinked with glutaraldehyde (GA). Then, the PS hollow microspheres (PSHMs) were obtained after the templates were removed. It was found that the pH of the external environment played an important role on the particle size of the PSHMs. To estimate the feasibility as novel carriers, an antitumor model drug 5 fluorouracil (5 Fu) and gold nanoclusters (Au NCs) were incorporated into hollow microspheres. The antitumor activity of the 5Fu/Au NCs-loaded PSHMs against cancer HepG2 was evaluated by measuring the body weight change and tumor volume of tumor bearing mice. The gold nanoclusters kept their fluorescent stability during the whole study. The 5 Fu/Au NCs-loaded PSHMs showed comparable anticancer efficacy with the free drug. (c) 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013