In this study, paclitaxel was chemically conjugated to a poly(poly(ethylene glycol) methyl ether acrylate)-b-poly(carboxyethyl acrylate) (PPEGMEA-b-PCEA) block copolymer to assess the effect of core-crosslinking on nanoparticle efficacy. The nanoparticles were used to treatment human prostate carcinoma LNCaP cells and the effects were tested using a multicellular tumor spheroids (MCTS) model. Conjugation of paclitaxel was completed via an esterification reaction using solid phase peptide synthesis reagents to afford a final conjugated compound containing 22 wt% paclitaxel. The core was also irreversibly crosslinked with a diamino compound to produce crosslinked micelles. The sizes of the resultant uncrosslinked and crosslinked micelles were 178 nm and 182 nm, respectively. The cytotoxicity of both micelles were then compared to the activity of free paclitaxel using both 2D and 3D cell culture models. The incorporation of a 3D cell culture model and the subsequent comparison of its results to the 2D model are due to the enhanced similarity between the 3D model and in vivo tumours 1. In particular, 3D models better mimic cell-cell interactions, an aspect which is mostly missing from the monolayer cell culture model. Thus, 3D models have the potential to better predict the effect of nanoparticle-drug formulations as well effective dosages. Cytotoxicity tests using monolayer cell culture showed that crosslinked micelles were more effective than its uncrosslinked counterpart with IC50 values of 193.2 nM and 270.9 nM, respectively. Both micelle variants had significantly lower toxicity than free paclitaxel, which had an IC 50 value of 15.2nM. Cytotoxicity tests using a 3D MCTS model, however, indicated that the performances of the micelle variants were extremely similar to each other. In addition, spheroids treated by both micelles demonstrated comparable growth inhibition as that shown by spheroids treated with free paclitaxel. DNA content was used to quantify cytotoxic effect on the MCTS and it was shown that after treatment, the DNA content of the MCTS incubated with free paclitaxel and the micelles were very similar to the DNA content of the spheroids harvested on Day 0. That is, there was a substantial and similar inhibition of spheroid growth across all three treatment groups, a result that was not expected based upon the monolayer cytotoxicity results. Despite the 12-18 fold lower IC50 value exhibited by the micelle variants in 2D cultured LNCaP cells, the micelles performed similarly to free paclitaxel when tested in a 3D in vitro model. Hence, the use of polymer-paclitaxel conjugate micelles allows …