Original article Paclitaxel-hyaluronan hydrosoluble bioconjugate: Mechanism of action in human bladder cancer cell lines  Isabella Monia Montagner, M.S. a,1 , Alessandra Banzato, Ph.D. b,1 , Gaia Zuccolotto, M.S. c , Davide Renier, M.S. d , Monica Campisi, Ph.D. d , PierFrancesco Bassi, M.D. e , Paola Zanovello, M.S. a,c , Antonio Rosato, M.D., Ph.D. a,c, * a Istituto Oncologico Veneto IRCCS, Padova, Italy b Department of Cardiac, Thoracic and Vascular Sciences, University of Padova, Padova, Italy c Department of Oncology and Surgical Sciences, University of Padova, Padova, Italy d Fidia Farmaceutici, Abano Terme, Italy e Department of Urology, Catholic University Medical School, Rome, Italy Received 25 March 2011; received in revised form 6 January 2012; accepted 7 January 2012 Abstract Objectives: A previously described hydrosoluble paclitaxel-hyaluronan bioconjugate appears particularly well suited for treatment of superficial bladder cancer because of its in vitro cytotoxic profile against urothelial carcinoma (UC) cell lines and in vivo biocompatibility. The aim of this work was to assess the mechanism of action of the bioconjugate in UC cells. Materials and methods: Expression of CD44 and RHAMM hyaluronan-binding receptors in RT-4 and RT-112/84 UC cell lines, interaction of fluorochrome-labeled bioconjugate with tumor cells, CD44 modulation upon incubation with the compound or free hyaluronan, and caspase activation were assessed by flow cytometry. Cytotoxicity was studied by the MTT assay. Analysis of bioconjugate intracellular localization and effects on -tubulin organization was carried out by confocal microscopy. Results: The paclitaxel-hyaluronan bioconjugate bound to UC tumor cells entered intracellular compartments through a saturable and energy-dependent mechanism that involved CD44, as assessed by blocking with specific antibody. Upon internalization, the bioconjugate accumulated into lysosomes where the esteric bond between paclitaxel and the hyaluronan moiety was cleaved, leading to cytoplasmic diffusion of the free drug, caspase activation, and disruption of the -tubulin microtubular mesh with subsequent cell death. Conclusions: Conjugation of paclitaxel to hyaluronan results in a new chemical entity, characterized by selective targeting to polymer receptors on plasma membrane and cell entry through receptor-mediated endocytosis, followed by lysosomal accumulation. Ultimately, the active molecule is released, fully preserving the cytotoxic potential and profile of clinically used free paclitaxel. © 2012 Elsevier Inc. All rights reserved. Keywords: Bladder cancer; Paclitaxel; Hyaluronan; Bioconjugate 1. Introduction Bladder cancer represents a leading cause of malignancy in the world, with urothelial carcinoma (UC) being the most common histotype, as it represents more than 90% of cases [1]. Among UC, about 70% are diagnosed as superficial tumors with a high likelihood of recurrence (50% to 70%); thus, the real prevalence of UC exceeds its primary inci- dence significantly [2,3]. Management of superficial bladder cancer usually relies on transurethral resection (TUR) of neoplastic foci, fol- lowed by adjuvant intravesical immunotherapy or chemo- therapy to reduce recurrence and/or progression [2– 4]. Bladder instillation of chemotherapeutics has a potential targeting advantage due to a selective delivery of high concentration of drugs to the urothelial cancer tissues, while minimizing systemic exposure.  This work was partly supported by the Italian Association for Can- cer Research (AIRC) and the Italian Ministry of Health. * Corresponding author. Tel.: +39-49-8215800/8215858; fax: +39- 49-8072854. E-mail address: antonio.rosato@unipd.it (A. Rosato). 1 These authors contributed equally to this work. Urologic Oncology: Seminars and Original Investigations xx (2012) xxx 1078-1439/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.urolonc.2012.01.005