pH-sensitive nanogels with ortho ester linkages were conveniently prepared through reaction of thiol-ene click chemistry. Through adjusting feed reactant ratios and concentrations of ortho ester diacrylamide (OEAM), pentaerythritol tetra(3-mercaptopropionate) (PT), and methoxyl poly(ethyleneglycol) acrylate (mPEGA), the size of the nanogels could be controlled at 100-200 nm with relatively narrow size distributions. The nanogels with size of 149.1 +/- 17.7 nm (designed as NG) were verified by proton nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), dynamic laser scattering (DLS) and transmission electron microscopy (TEM). Doxorubicin (DOX) was loaded into NG with high drug loading efficiency up to 73.7%. In vitro drug release studies showed that up to 75.9% DOX from NG was released in 24 hat pH 5.0 because of hydrolysis of ortho ester. Cellular uptake studies confirmed that DOX-loaded NG (NG/DOX) could be readily internalized by two-dimensional cells, resulting in efficient antitumor efficiency of cancer cells. Three-dimensional (3D) multicellular tumor spheroids (MCTS) as in vitro tumor model was used to further evaluate the antitumor effect of NG/DOX. The results demonstrated that NG/DOX showed a significantly enhanced penetration and growth inhibition in 3D multicellular tumor spheroids (MCTS), compared to free DOX. (C) 2017 Elsevier Inc. All rights reserved.