© 2018 by the authors. Licensee MDPI, Basel, Switzerland.Amphiphilic cyclodextrins are biocompatible oligosaccharides that can be used for drug delivery especially for the delivery of drugs with solubility problems thanks to their unique molecular structures. In this paper, Paclitaxel was used as a model anticancer drug to determine the inclusion complex properties of amphiphilic cyclodextrins with different surface charge. Paclitaxel-loaded cyclodextrin nanoparticles were characterized in terms of mean particle diameter, zeta potential, encapsulation efficacy, drug release profile and cell culture studies. It was determined that the nanoparticles prepared from the inclusion complex according to characterization studies have a longer release profile than the conventionally prepared nanoparticles. In order to mimic the tumor microenvironment, breast cancer cells and healthy fibroblast cells were used in 3-dimensional (3D) cell culture studies. It was determined that the activities of nanoparticles prepared by conventional methods behave differently in 2-dimensional (2D) and 3D cell cultures. In addition, it was observed that the nanoparticles prepared from the inclusion complex have a stronger anti-tumoral activity in the 3D multicellular tumor model than the drug solution. Furthermore, polycationic amphiphilic cyclodextrin nanoparticles can diffuse and penetrate through multilayer cells in a 3D tumor model, which is crucial for an eventual antitumor effect.