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Bladder tissue distribution of paclitaxel and docetaxel from polymeric nanoparticles Tsallas, Antonia Theodora 2010

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BLADDER TISSUE DISTRIBUTION OF PACLITAXEL AND DOCETAXEL FROM POLYMERIC NANOPARTICLES  by ANTONIA THEODORA TSALLAS B.Sc., The University of Toronto, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  May 2010  © Antonia Theodora Tsallas, 2010  ABSTRACT Taxane based drugs are commonly used second‐line adjuvant therapies for the treatment of superficial bladder cancer. However, the effectiveness of these drugs following intravesical instillation is limited, because of a short drug residence time and poor drug penetration into the bladder wall. The goals of this project were to use PTX and DTX loaded nanoparticulate formulations and investigate their bladder mucosal permeability and distribution characteristics in ex vivo isolated porcine bladders. PTX and DTX were loaded into nanoparticles of methoxy poly(ethylene glycol)‐block‐ poly(D,L‐lactic acid) (MePEG‐PDLLA), methoxy poly(ethylene glycol)‐block‐ poly(caprolactone) (MePEG‐PCL) and hydrophobically derivatized hyperbranched polyglycerol, HPG‐C10‐MePEG. In vitro drug release profiles of PTX and DTX loaded MePEG‐ PDLLA micelles (approx. 20 nm in size) demonstrated controlled and complete release of the drugs over 7 days. The penetration and distribution characteristics of PTX and DTX from nanoparticulate formulations (spiked with tritium labelled drugs) into freshly excised porcine bladder tissue were evaluated using Franz diffusion cells. Nanoparticle dispersions were instilled onto bladder tissue for 2 h. The tissues were frozen, sectioned and drugs quantified using liquid scintillation counting. The drug tissue levels were highest in the urothelium and then decreased exponentially with increasing tissue depth. Micellar formulations of MePEG‐ PDLLA showed higher bladder tissue uptake levels of PTX and DTX compared to control commercial formulations. Drug uptake levels did not differ significantly between concentrations of 0.5 and 1 mg/mL. However, DTX was present in bladder tissue at significantly higher concentrations than PTX. Micellar formulations of MePEG‐PDLLA showed higher bladder tissue uptake levels of PTX compared to both MePEG‐PCL copolymer formulations. The rapid and high levels of PTX and DTX penetration observed in bladder tissue using the MePEG‐PDLLA micellar formulation of these drugs was likely due to the use of high concentrations of the drugs with these polymeric micelles, greater free drug released from the MePEG‐PDLLA micelles and the ability to increase the contact of the formulation at the bladder wall surface allowing for improved partitioning of the drug into the tissues.  ii  TABLE OF CONTENTS ABSTRACT ...............................................................................................................................................ii TABLE OF CONTENTS ........................................................................................................................ iii LIST OF TABLES ....................................................................................................................................vi LIST OF FIGURES .................................................................................................................................vii LIST OF ABBREVIATIONS ............................................................................................................... xiii ACKNOWLEDGEMENTS ....................................................................................................................xiv DEDICATION ......................................................................................................................................... xv 1 PROJECT OVERVIEW ..................................................................................................................... 1 2 BACKGROUND ................................................................................................................................. 4 2.1 Urinary Bladder Anatomy ....................................................................................................... 4 2.1.1 Bladder structure .............................................................................................................................4 2.1.1.1 Superficial umbrella cells.......................................................................................................... 9 2.1.1.2 Mucin glycoproteins ................................................................................................................. 11 2.1.2 Bladder permeability...................................................................................................................12 2.1.3 Bladder filling and voiding ........................................................................................................12 2.2 Superficial Bladder Cancer .................................................................................................. 13 2.2.1 Etiology and pathogenesis.........................................................................................................13 2.2.2 Classification of the disease ......................................................................................................14 2.2.3 Diagnosis and disease management .....................................................................................16 2.2.3.1 Intravesical immunotherapy................................................................................................ 16 2.2.3.2 Intravesical chemotherapy ................................................................................................... 17 2.3 Paclitaxel and Docetaxel....................................................................................................... 19 2.3.1 Chemistry..........................................................................................................................................19 2.3.2 Commercial formulations ..........................................................................................................22 2.3.3 Pharmacology and clinical indications ................................................................................22 2.3.4 Toxicology ........................................................................................................................................23 2.3.5 Pharmacokinetics..........................................................................................................................25 2.3.6 Clinical use of intravesical taxanes ........................................................................................26 2.4 Polymeric Drug Delivery Systems ..................................................................................... 26 2.4.1 Polymer structure and morphology......................................................................................27 2.4.2 Polymeric nanoparticles ............................................................................................................28 2.4.2.1 Micelles and nanospheres of amphiphilic diblock copolymers.............................. 28 2.4.2.2 Nanoparticles of MePEG­PDLLA......................................................................................... 31 2.4.2.3 Nanoparticles of MePEG­PCL ............................................................................................... 32 2.4.2.4 Hydrophobically derivatized hyperbranched polyglycerols................................... 33 2.4.3 Drug release from polymeric nanoparticles......................................................................35 2.5 Intravesical Drug Delivery ................................................................................................... 36 2.5.1 Drug transport into the bladder wall....................................................................................36 2.5.2 Intravesical administration of anticancer drugs .............................................................39 2.5.3 Intravesicular mucoadhesive drug delivery......................................................................40 2.6 Thesis Goals, Hypothesis and Objectives ........................................................................ 42 3 EXPERIMENTAL ........................................................................................................................... 43 iii  3.1  3.2  3.3 3.4 3.5  3.6  3.7 3.8  3.9  Materials .................................................................................................................................... 43 3.1.1 Chemicals and solvents...............................................................................................................43 3.1.2 Tyrode buffer ..................................................................................................................................43 3.1.3 Paclitaxel and docetaxel .............................................................................................................43 3.1.4 Polymers ...........................................................................................................................................44 3.1.5 Porcine bladder tissue.................................................................................................................44 Equipment ................................................................................................................................. 45 3.2.1 Liquid scintillation counter.......................................................................................................45 3.2.2 Cryotome...........................................................................................................................................45 3.2.3 Dynamic light scattering ............................................................................................................45 3.2.4 UV‐Vis spectrophotometer........................................................................................................45 3.2.5 High performance liquid chromatography ........................................................................46 3.2.6 Glassware..........................................................................................................................................46 3.2.7 General equipment and supplies............................................................................................46 Preparation of PTX and DTX Loaded MePEG­PDLLA Micelles ................................. 48 Preparation of Control Commercial Formulations of PTX and DTX ...................... 48 Micelle Characterization....................................................................................................... 48 3.5.1 Particle size analysis ....................................................................................................................48 3.5.2 Preparation and loading of MePEG‐PCL19 micelles ........................................................49 3.5.3 Preparation and loading of MePEG‐PCL104 nanospheres.............................................49 3.5.4 In vitro drug release of PTX and DTX from MePEG‐PDLLA micelles ......................50 3.5.5 Evaluation of mucoadhesive properties of MePEG‐PDLLA micelles.......................50 Bladder Tissue Distribution ................................................................................................ 51 3.6.1 Preparation of PTX and DTX loaded nanoparticulate formulations .......................51 3.6.2 Tissue preparation........................................................................................................................52 3.6.3 Cryotome sectioning of tissue..................................................................................................54 3.6.4 Quantification of drug in tissue...............................................................................................54 3.6.5 Analysis of tissue level‐depth profiles .................................................................................54 3.6.6 Drug recovery .................................................................................................................................55 3.6.7 PTX metabolites in tissue and stability of tritium label................................................55 Tissue Viability ........................................................................................................................ 55 3.7.1 Lactate dehydrogenase assay ..................................................................................................55 3.7.2 Mannitol paracellular permeability assay..........................................................................56 Toxicity of Nanoparticulate formulations on Bladder Tissue.................................. 56 3.8.1 Preparation of formulations .....................................................................................................56 3.8.2 Lactate dehydrogenase assay ..................................................................................................57 3.8.3 Mannitol paracellular permeability assay..........................................................................57 Statistical Analysis.................................................................................................................. 58  4 RESULTS......................................................................................................................................... 59 4.1 Characterization of Nanoparticulate formulations ..................................................... 59 4.1.1 Particle size and release profiles for MePEG‐PDLLA micelles...................................59 4.1.2 PTX loading in MePEG‐PCL nanoparticles..........................................................................62 4.1.3 Mucoadhesive properties of polymeric nanoparticles..................................................64 4.2 Bladder Tissue Distribution of PTX and DTX Loaded Nanoparticles .................... 67 4.2.1 PTX and DTX analysis by liquid scintillation counting .................................................67 4.2.2 Effect of concentration of drug on bladder tissue uptake ...........................................68 4.2.3 Effect of incubation time on bladder tissue uptake........................................................77 4.2.4 Effect of formulation on bladder tissue uptake................................................................80 iv  4.2.5 Presence of PTX metabolites by HPLC and stability of tritium label ......................85 Bladder Tissue Viability........................................................................................................ 88 4.3.1 Lactate dehydrogenase assay ..................................................................................................88 4.3.2 Mannitol paracellular permeability assay..........................................................................89 4.4 Bladder Tissue Toxicity of Nanoparticulates ................................................................ 90 4.4.1 Lactate dehydrogenase assay ..................................................................................................90 4.4.2 Mannitol paracellular permeability assay..........................................................................91 4.3  5 DISCUSSION................................................................................................................................... 93 6 SUGGESTIONS FOR FUTURE WORK.....................................................................................101 REFERENCES......................................................................................................................................102 APPENDIX I. UBC BIOHAZARD APPROVAL CERTIFICATE...................................................119  v  LIST OF TABLES Table 1 Probabilities of recurrence and progression of bladder tumors in patients undergoing controlled adjuvant intravesical chemotherapy following TUR (excerpted with permission from (Hendricksen and Witjes, 2007), who previously adapted from Oosterlinck, 2002 #291;Sylvester, 2006 #351}).............18 Table 2 Common adverse side effects when using PTX and DTX for the treatment of many cancers (Verweij et al., 1994; Piccart et al., 1995; van Oosterom et al., 1995) including urothelial cancer (Vaughn et al., 2002; Barlow et al., 2009) ......................24 Table 3 List of General Equipment and Supplies..................................................................................47  vi  LIST OF FIGURES Figure 1 Anatomy of the human urinary bladder (Adapted from Human Anatomy (Marieb and Mallant, 1997))............................................................................................................................6 Figure 2 Layers of the bladder wall represented by a view of the human urinary bladder. 7 Figure 3 Composition of the bladder wall. Several layers comprise the wall (from top to bottom) an adhering glycosaminoglycan layer, the urothelium, the lamina propria, the muscularis and the serosa. ....................................................................................8 Figure 4 A. Structure of superficial umbrella cell. Apical membrane is covered with scalloped‐shaped membrane plaques separated by hinges. Each umbrella cell is joined together by tight junctions in the laternal membrane. The cell cytoplasm is full of cytoplasmic vesicles held together by cytoskeletal fibrils. These fibrils connect at the tight junctions and desmosomes in the basal membrane (Adapted from (Lewis, 2000)). B. Structure of a contracted superficial umbrella cell. During the expansion‐contraction cycle, the urothelium accommodates changes in bladder volume by stretching/relaxing the umbrella cells to eliminate/form microscopic folds and expand/contract the apical surface area during bladder filling and voiding (Adapted from (Minsky and Chlapowski, 1978))........................10 Figure 5 Stage of superficial tumors. Tis (carcinoma in situ) are high grade tumors, flat and restricted to the mucosa, Ta tumors are papillary or solid and confined to the urothelium and T1 tumors are invasive to the lamina propria but do not invade the muscle. ...........................................................................................................................15 Figure 6 Chemical structure of A. PTX B. DTX (Adapted from (Kingston, 1994))..................21 Figure 7 Structure of A. micelles in dynamic equilibrium with unimers comprised of a hydrophilic block (blue) and a hydrophobic block (black). Micelles form at concentrations above the critical micelle concentration (CMC). B. nanospheres containing copolymer unimers in a frozen state................................................................30 Figure 8 Structure of methoxy poly(ethylene glycol)‐block‐poly(D,L‐lactic acid) (MePEG‐ PDLLA)..................................................................................................................................................31 Figure 9 Structure of methoxy poly(ethylene glycol)‐block‐polycaprolactone (MePEG‐ PCL)........................................................................................................................................................32  vii  Figure 10 Structure of hyperbranched polyglycerol, HPG‐C10‐MePEG (Adapted from (Kainthan et al., 2008b)). ...........................................................................................................34 Figure 11 A proposed model for vesicle dynamics in umbrella cells during bladder filling and voiding. “Filling stimulates both exocytosis and endocytosis of vesicles. The net rates of these processes are such that membrane is added to the apical surface. Endocytosed membraneis delivered to lysosomes where contents are degraded. Upon voiding the added membrane isinternalized and reestablishment of the vesicle pool may result from both endocytosis and de novo synthesis along the biosynthetic pathway.” [Figure and text excerpted from: (Apodaca, 2004)] ..............................................................................................................38 Figure 12  Diffusion cell apparatus. Fresh bladder tissue mounted between donor and receptor chambers with luminal side of the bladder wall exposed to the drug solution. .............................................................................................................................................53  Figure 13 A representative size distribution plot of A. 5 mg/mL MePEG‐PDLLA micelles and B. 0.5 mg/mL PTX in 5 mg/mL MePEG‐PDLLA micelles. Measurements by dynamic light scattering at 25 °C in tyrode buffer..........................................................60 Figure 14  Release of A. PTX and B. DTX from polymeric micelles of MePEG‐PDLLA at 0.5 mg/mL (▲) and 1 mg/mL (▼) into pH 7.4 tyrode buffer at 37 °C. The movement of free drug (■) out of the dialysis bag is also shown. Data are mean ± SD (n = 4). ....................................................................................................................................61  Figure 15 Solubilization of PTX by MePEG‐PCL19 micelles formed by nanoprecipitation and dialysis of copolymer and drug in DMF solutions. Micelles were formed in PBS (pH 7.4) with final copolymer concentrations of 5 % w/v. [PTX] solubilized (▲), Loading efficiency (☐)......................................................................................................62 Figure 16 Solubilization of PTX by MePEG‐PCL104 micelles formed by nanoprecipitation and dialysis of copolymer and drug in DMF solutions. Micelles were formed in PBS (pH 7.4) with final copolymer concentrations of 1.2 % w/v. [PTX] solubilized (▲), Loading efficiency (☐). .............................................................................63 Figure 17 Turbidity measurements for varying volume ratios of 0.1% w/v polymer and 1% w/v mucin dispersions in acetate buffer at pH 4.4. Data are mean turbidity ± SD (n = 3).......................................................................................................................................65  viii  Figure 18 Changes in particle size of 1% w/v mucin when mixed with 0.1% w/v polymer solutions. Values are means ± SD (n = 3). * p < 0.05 .......................................................66 Figure 19 Standard curves for 0.5 mg/mL PTX and DTX prepared from MePEG‐PDLLA (PTX and DTX), Cremophor EL + EtOH (PTX) or Tween 80 (DTX) based formulations. Each curve was repeated three times using freshly made formulations and values are means ± SD (n = 3).............................................................67 Figure 20 Tissue level‐depth profiles of PTX in bladder tissue following exposure to A. 0.5 mg/ml PTX in MePEG‐PDLLA micelles (●) and 0.5mg/ml PTX in Cremophor EL and EtOH (▲) and B. 1mg/ml PTX in MePEG‐PDLLA micelles (♦) and 1 mg/ml PTX in Cremophor EL and EtOH (■). Tissues were incubated for 2 h and sectioned at 60 μm thickness. Uro, Lam and Mus refer to the urothelium, lamina propria and superficial muscle layers. Values are means ± SD (n = 18). ..............70 Figure 21 Average tissue levels of PTX in various layers of the bladder wall (60‐240 μm: urothelium, 240‐1260 μm: the lamina propria, 1260‐2160 μm: the superficial muscle layer and 60‐2160 μm: the whole tissue) following incubation with PTX at either 0.5 mg/ml or 1 mg/ml from either MePEG‐PDLLA or Cremophor EL and EtOH formulations for 2 hours. The average tissue levels were determined as the total amount of drug found in the tissue layer divided by the total tissue weight for that layer. Data are means ± SD (n = 18). *** p < 0.001, 0.5 mg/mL MePEG‐PDLLA PTX vs. 0.5 mg/mL PTX + Cremophor EL + EtOH and 1 mg/mL MePEG‐PDLLA PTX vs. 1 mg/mL PTX + Cremophor EL + EtOH ...............................71 Figure 22 AUCs of PTX in various tissue layers of the bladder wall (0‐240 μm: urothelium, 240‐1260 μm: the lamina propria, 1260‐2160 μm: the superficial muscle layer and 0‐2160 μm: the whole tissue) following incubation with PTX at either 0.5 mg/ml or 1 mg/ml from either MePEG‐PDLLA or Cremophor EL and EtOH formulations for 2 hours. The AUCs were calculated using the linear trapezoid rule. Data are means ± SD (n = 18). *** p < 0.001, 0.5 mg/mL MePEG‐PDLLA PTX vs. 0.5 mg/mL PTX + Cremophor EL + EtOH and 1 mg/mL MePEG‐PDLLA PTX vs. 1 mg/mL PTX + Cremophor EL + EtOH.........................................................................72 Figure 23 Tissue level‐depth profiles of DTX in bladder tissue following exposure to A. 0.5 mg/ml DTX in MePEG‐PDLLA micelles (●) and 0.5mg/ml DTX in Tween 80 (▲) and B. 1mg/ml DTX in MePEG‐PDLLA micelles (♦) and 1 mg/ml DTX in Tween ix  80 (■). Tissues were incubated for 2 h and sectioned at 60 μm thickness. Uro, Lam and Mus refer to the urothelium, lamina propria and superficial muscle layers. Values are means ± SD (n = 18)................................................................................73 Figure 24 Average tissue levels of DTX in various layers of the bladder wall (60‐240 μm: urothelium, 240‐1260 μm: the lamina propria, 1260‐2160 μm: the superficial muscle layer and 60‐2160 μm: the whole tissue) following incubation with DTX at either 0.5 mg/ml or 1 mg/ml from either MePEG‐PDLLA or Tween 80 formulations for 2 hours. The average tissue levels were determined as the total amount of drug found in the tissue layer divided by the total tissue weight for that layer. Data are means ± SD (n = 18). ** p < 0.01, 1 mg/mL MePEG‐PDLLA DTX vs 1 mg/mL DTX + Tween 80 and *** p < 0.001, 0.5 mg/mL MePEG‐PDLLA DTX vs 0.5 mg/mL DTX + Tween 80 .....................................................................................74 Figure 25 AUCs of DTX in various tissue layers of the bladder wall (0‐240 μm: urothelium, 240‐1260 μm: the lamina propria, 1260‐2160 μm: the superficial muscle layer and 0‐2160 μm: the whole tissue) following incubation with DTX at either 0.5 mg/ml or 1 mg/ml from either MePEG‐PDLLA or Tween 80 formulations for 2 hours. The AUCs were calculated using the linear trapezoid rule. Data are means ± SD (n = 18). ** p < 0.01, 1 mg/mL MePEG‐PDLLA DTX vs 1 mg/mL DTX + Tween 80 and *** p < 0.001, 0.5 mg/mL MePEG‐PDLLA DTX vs 0.5 mg/mL DTX + Tween 80.............................................................................................................................75 Figure 26 Mass balance analysis of drug recovered from donor, washes, receptor and tissue fractions following a 2 h incubation with PTX or DTX at A. 0.5 mg/mL and B. 1 mg/mL PTX and DTX following a 2 h incubation. All portions of the diffusion cell including washes and tissue were analyzed for drug content. Data are means ± SD (n = 18). ............................................................................................................76 Figure 27 Tissue level‐depth profile of PTX in bladder tissue from MePEG‐PDLLA micelle formulations (1 mg/ml) following incubation for 30 min (■), 60 min (▲), 120 min (●) and 180 min (◆) at 37°C. Uro, Lam and Mus refer to the urothelium, lamina propria and superficial muscle layers. Inset: The time course of drug uptake into bladder tissue for PTX at 1 mg/mL from MePEG‐PDLLA micelles. Points show average tissue levels of drug recovered from all bladder tissue. The average tissue levels were determined as the total amount of drug found in the x  tissue layer divided by the total tissue weight for that layer.Data are expressed as µg of drug per g of tissue and the means ± SD (n = 4). * p < 0.05, PTX 180 min vs. 30 min..........................................................................................................................................78 Figure 28 Tissue level‐depth profile of DTX in bladder tissue from MePEG‐PDLLA micelle formulations (1 mg/ml) following incubation for 30 min (■), 60 min (▲), 120 min (●) and 180 min (◆) at 37°C. Uro, Lam and Mus refer to the urothelium, lamina propria and superficial muscle layers. Inset: The time course of drug uptake into bladder tissue for DTX at 1 mg/mL from MePEG‐PDLLA micelles. Points show average tissue levels of drug recovered from all bladder tissue. The average tissue levels were determined as the total amount of drug found in the tissue layer divided by the total tissue weight for that layer.Data are expressed as µg of drug per g of tissue and the means ± SD (n = 4). * p < 0.05, DTX 120 min vs. 60 min. ** p < 0.01, DTX 120 min vs. 30 min................................................................79 Figure 29 Tissue level‐depth profiles of PTX in bladder tissue following exposure to 0.5 mg/mL PTX in MePEG‐PDLLA micelles (▲), Cremophor EL + EtOH (●), MePEG‐ PCL19 micelles (■) and MePEG‐PCL104 nanospheres (♦).Tissues were incubated for 2 h and sectioned at 60 μm thickness. Uro, Lam and Mus refer to the urothelium, lamina propria and superficial muscle layers. Values are means ± SD (n = 6). .........................................................................................................................................81 Figure 30 Average tissue levels of 0.5 mg/mL PTX in whole bladder tissue (60‐3060 μm) following a 2 h incubation. The average tissue levels were determined as the total amount of PTX found in the tissue divided by the total tissue weight. Data are means ± SD (n = 6). ** p < 0.01, MePEG‐PDLLA vs. MePEG‐PCL19 and *** p < 0.001, MePEG‐PDLLA vs. MePEG‐PCL104 .............................................................................82 Figure 31 Tissue level‐depth profiles of DTX in bladder tissue following exposure to 1 mg/mL DTX in MePEG‐PDLLA micelles (▲), Tween 80 (●) and HPG‐C10‐MePEG (■). Tissues were incubated for 2 h and sectioned at 60 μm thickness. Uro, Lam and Mus refer to the urothelium, lamina propria and superficial muscle layers. Values are means ± SD (n = 4). ................................................................................................83 Figure 32 Average tissue levels of 1 mg/mL DTX in whole bladder tissue (60‐3060 μm) following a 2 h incubation. The average tissue levels were determined as the  xi  total amount of PTX found in the tissue divided by the total tissue weight. Data are means ± SD (n = 4). ...............................................................................................................84 Figure 33 Chromatogram of PTX extracted from bladder tissue in ACN by HPLC. Mobile phase: 58:37:5 ACN:H2O:methanol, flow rate: 1 mL/min, injection volume 20 μL, detection at 232 nm. .............................................................................................................86 Figure 34 Eluted samples of 3H PTX from HLPC measured by liquid scintillation counting. ...........................................................................................................................................................87 Figure 35 Bladder tissue viability following a 0.5, 1, 3, 5, 8 and 24 h incubated at 37°C in tyrode buffer. Values were measured by UV‐Vis spectroscopy and are expressed as the change in absorbance with time, which represent the amount of LDH released into supernatant. Data are means ± SD (n = 9). *p<0.05, 8 h vs. 0 h, **p<0.01, 24 h vs 0, 0.5, 1, 3 and 5 h and ***p<0.001 vs. all groups...................88 Figure 36 Tissue level‐depth profile of 3H mannitol in bladder tissue following a 3 and 24 h incubation. Amount of 3H is expressed as CPM divided by the weight of tissue slices in mg. Values are n = 1....................................................................................................89 Figure 37 Tissue toxicity of nanoparticulate formulations (no drug) following a 2 h incubation. Values were measured by UV‐Vis spectroscopy and are expressed as the change in absorbance with time, which represent the amount of LDH released into supernatant. Values are means ± SD (n = 3‐9). ***p<0.001 vs. all groups.................................................................................................................................................90 Figure 38 Tissue level‐depth profile of 14C mannitol in bladder tissue following a 2 h incubation. Mannitol delivered to bladder in tyrode buffer containing nanoparticulate vehicles. Data are means ± SD (n = 3). ...............................................92  xii  LIST OF ABBREVIATIONS °C ACN BCG CO2 CMC CPM DCM DLS DMF DMSO DTX GPC HPG HPLC IV LDH LSC MePEG Mn MMC MW Mw nm O2 PCL PDLLA PTX PVA SD SEM TB TCC Tis Tg Tm TUR UV‐Vis w/v w/w  Degrees celsius Acetonitrile Bacillus Calmette‐Guerin Carbon dioxide Critical micelle concentration Counts per minute Dichloromethane Dynamic light scattering Dimethyl formamide Dimethyl sulfoxide Docetaxel Gel permeation chromatography Hyperbranched polyglycerol High performance liquid chromatography Intravenous Lactate dehydrogenase Liquid scintillation counter Methoxy polyethylene glycol Number average molecular weight Mitomycin C Molecular weight Weight average molecular weight Nanometer Oxygen Polycaprolactone Poly(D,L‐lactic acid) Paclitaxel Poly(vinyl alcohol) Standard deviation Standard error of the mean Tyrode buffer Transitional cell carcinoma Carcinoma in situ Glass transition Melting transition Transurethral resection Ultraviolet‐visible Weight per volume Weight per weight  xiii  ACKNOWLEDGEMENTS I would first like to thank my supervisor, Dr. Helen Burt, for her support over the course of my M.Sc studies. Thank you for giving me the opportunity to grow and the freedom to be independent. Your patience and help during this time has been much appreciated. I thank my committee members, Drs. Kishor Wasan, Wayne Riggs, Don Brooks and Frank Abbott for their guidance and direction on my research project. I would also like to thank my colleagues in the lab for their help and encouragement: Michelle Chakraborti, Lucy Ye, Melanie ter Borg, Dr. Chiming Yang, Leon Wan, Clement Mugabe, Sam Gilchrist and Dr. Kevin Letchford. Thank you to John Jackson for the advice and direction with my work, especially during the difficult times. Thanks to Elissa Aeng for the extra helping hands. A special thanks to Sam Gilchrist for the illustrations in Figures 2 and 5 of the background. Thank you to Barb Conway, Suzi Topic and Rita Zhao for their friendship. A special thanks to Wesley Wong, Jamal Kurtu, Mike Parr and Raina Tamakawa for driving to Langley to pick up porcine bladders. I would like to thank Irina Chafeeva in Dr. Brooks’ lab and Dr. Kevin Letchford in Dr. Burt’s lab for providing samples of diblock copolymers and HPGs. I am grateful for the financial support offered by Merck Frosst Inc. and the Canadian Institute of Health Research. Thank you to my sister for always being there. Thank you to Erich for being patient and understanding with me. Thank you for your unconditional love.  xiv  DEDICATION I would like to dedicate this work to my parents, Tina and George Tsallas. Thank you for believing in me, this work could not have been done without you.  xv  1  PROJECT OVERVIEW Bladder cancer is a disease that affects over 1 million worldwide and is three to four  times more prevalent in men than in women (Parkin, 2008; Ploeg et al., 2009) At initial diagnosis, the majority of bladder cancer patients present with superficial tumors and surgical removal by transurethral resection (TUR) is the effective method of treatment (Clark, 2007). Despite TUR and adjuvant chemotherapy, superficial bladder cancer continues to be a difficult disease to treat due to tumor recurrence and progression and limitations in current drug delivery methods. Systemic treatment provides little therapeutic benefit to patients with tumors located at the bladder surface urothelium, as the layer is non‐vascularized (Wientjes et al., 1991). Consequently, intravesical chemotherapy becomes the treatment of choice offering high drug concentrations to be delivered directly to the tumor site with minimal systemic uptake (Dalton et al., 1991; Chai et al., 1994; Song et al., 1997). The current first line therapies for superficial bladder cancer treatment include mitomycin C (MMC) and Bacillus Calmette‐Guerin (BCG) (Pashos et al., 2002). While MMC may be effective in treating patients after TUR with stage T1 and high‐grade Ta tumors, response rates have been highly variable, which may be due to the poor tissue penetration and rapid‐wash out of the water‐soluble drug (Song et al., 1997; Au et al., 2002; Chade et al., 2009). BCG is typically reserved as the most effective immunotherapeutic regimen for high‐risk tumors, such as carcinoma in situ, and bladder cancer prophylaxis (Ratliff, 1991; Pashos et al., 2002), but it is associated with frequent local and systemic side effects (Chade et al., 2009). As a result, the chemotherapeutic agents paclitaxel (PTX) and docetaxel (DTX) are being investigated as second‐line therapies for the treatment of superficial tumors. PTX has emerged as an attractive candidate for intravesical treatment because of its high lipophilicity and tissue penetration characteristics. PTX has been shown to penetrate the bladder tissue 20 times greater than water‐soluble drugs like MMC or doxorubicin (Song et al., 1997). In vivo, PTX has demonstrated good activity against metastatic bladder cancer with response rates of 50% in previously untreated patients (Bokemeyer et al., 1998). DTX has gained importance because it possesses greater potency than PTX against tumor cell lines and increased affinity for microtubules (Gueritte‐Voegelein et al., 1991). Like PTX, DTX has shown promising efficacy as a second‐line treatment for patients with metastatic bladder cancer, generating response rates of 31% (de Wit et al., 1998; Galsky, 1  2005). In fact, in a recent phase I trial, intravesical DTX exhibited minimal toxicity and several disease free responses 4 weeks post‐treatment in patients treated for recurrent superficial bladder cancer (McKiernan et al., 2006). However, the effectiveness of both PTX and DTX following intravesical administration is limited by a 2 h dwell time within the bladder, inadequate drug exposure to the bladder wall and minimal drug uptake due to dilution by urine (Au et al., 2002). New methods for optimizing intravesical chemotherapy have been proposed to maximize the exposure of therapeutic agents to tumors located in the lining of the bladder wall. Nano‐sized drug carriers with mucoadhesive properties may increase the residence time of drugs at the bladder mucosa and enhance drug uptake. While only a limited number of groups have used mucoadhesive delivery systems to localize drug at the bladder wall, even fewer groups have looked at the permeability of PTX from such nanoparticulate formulations and no studies have examined the penetration and distribution of DTX in normal bladder tissue. Using ex vivo bladder tissue, the penetration of various drugs such as PTX, moxifloxacin, MMC and doxorubicin within the bladder wall has been studied by a number of groups (Wientjes et al., 1993; Wientjes et al., 1996; Song et al., 1997; Knemeyer et al., 1999; Chen et al., 2003; Kerec et al., 2005). For instance, Wientjes et al. (1993) examined the penetration of intravesical MMC in bladder tissue and determined a profile of the concentration of drug in bladder tissue as a function of tissue depth. Drug concentrations declined linearly and then exponentially with tissue depth, which the group described in terms of drug diffusion and drug distribution processes (Wientjes et al., 1993). Kerec et al. (2005) investigated the effect of chitosan, the mucoadhesive polymer, on the permeation of moxifloxacin into isolated pig bladder. Chitosan was found to significantly increase the permeation of moxifloxacin into the bladder wall compared to moxifloxacin alone (Kerec et al., 2005). Knemeyer et al. (1999) found that much higher bladder tissue levels of PTX were achieved after intravesical administration when the drug was delivered in water than when solubilized in the commercial vehicle, Cremophor EL/ethanol. It was suggested that Cremophor reduced PTX penetration into the bladder wall of dogs by sequestration of the drug within the micelle core (Knemeyer et al., 1999). We have previously described the effective encapsulation of PTX in polymeric micelles composed of methoxy poly(ethylene glycol)‐block‐poly(D,L‐lactic acid) (MePEG‐PDLLA) 2  and methoxy poly(ethylene glycol)‐block‐poly(caprolactone) (MePEG‐PCL) for intravenous administration (Zhang et al., 1996; Burt et al., 1999; Letchford et al., 2008; Letchford et al., 2009). These biocompatible and biodegradable micellar systems effectively solubilize PTX and DTX allowing for the use of concentrated drug solutions. In our previous work, PTX loaded MePEG‐PDLLA micelles have shown significant cytotoxicity against bladder cancer cell lines and significant inhibition of tumor growth following intravesical administration in a mouse model of superficial bladder cancer (Hadaschik et al., 2008). Furthermore, after a 2 hour instillation of PTX loaded MePEG‐PDLLA micelles into healthy mice bladders, significant bladder tissue levels were achieved (Hadaschik et al., 2008). The goal of this work was to investigate the penetration and distribution characteristics of PTX and DTX from nanoparticulate formulations into freshly excised porcine bladder tissue in diffusion cells. The objectives were, firstly, to prepare and characterize PTX and DTX loaded micelles of MePEG‐PDLLA and MePEG‐PCL and hyperbranched polyglycerols. Secondly, tissue levels of PTX and DTX in the porcine bladder wall as a function of tissue depth were determined and factors influencing the tissue distribution were investigated. Finally, bladder tissue viability over the time course of the studies and tissue toxicity of nanoparticulate formulations were evaluated.  3  2  BACKGROUND  2.1 Urinary Bladder Anatomy 2.1.1 Bladder structure The urinary bladder (Figure 1) is a hollow muscular organ lined with a mucous membrane and covered by a visceral peritoneum also referred to as serosa. The interior mucous membrane contains folds called rugae when the bladder is empty and appears smooth when the bladder is filled (Gray and Lewis, 1918). Outside the serosa, a layer of perivesical fat surrounds the organ. Located on the floor of the pelvic cavity, the bladder has two ureters that extend posterior from the bladder to the kidneys and a urethra that leads urine to the exterior of the body. The bladder is designed to store urine produced by the kidneys until release. The bladder wall (Figure 2) consists of several layers, the urothelium, the connective lamina propria, the underlying muscularis and the serosa. The urothelium is a transitional epithelial layer (3‐7 cell layers) composed of three morphologically distinct cell types of superficial umbrella cells, intermediate and basal urothelial cells (Figure 3) (Congiu et al., 2004). The deep basal cell layer is germinal in nature with cells of 5‐10 µm in diameter. Intermediate cells are 20 µm and superficial cells are hexagonal and range from 50‐120 µm across, depending on the degree of bladder stretch (Lewis, 2000). The superficial umbrella cells are replaced by the gradual turnover of the underlying intermediate and basal cells (Martin, 1972). The rate of turnover is slow, due to the low mitotic division of basal cells and a long life span of superficial cells (Khandelwal et al., 2009; Kreft et al., 2009). The estimated turnover rate of the urothelium is approximately 3‐6 months (Hicks, 1975; Jost, 1989). However, when injured, the urothelium shows enormous regenerative capability and can rapidly restore itself within days of significant damage (Kreft et al., 2005; Veranic et al., 2009). Beneath the urothelium is the lamina propria, which is a subepithelial connective tissue containing blood vessels and nerves. Within this layer is a thin, discontinuous layer of smooth muscle called the muscularis mucosa that connects the true muscular layer of the bladder wall to the urothelium (Gray and Lewis, 1918). The muscularis is a thick wall of smooth muscle composed of three layers of muscular fibers. An internal and external layer of fibers arrange longitudinally within the bladder wall, while a middle layer of fibers arrange in a circular configuration (Davis, 1913; Gray et al., 1999). The outer surface of the bladder is covered by the serosa. This membrane is derived from 4  the peritoneum of the abdominal cavity and consists of mesothelium and elastic fibrous connective tissue (Gray and Lewis, 1918). Blood vessels collect in a complicated venous plexus on the serosal surface of the bladder wall and drain into the vesical veins (Wientjes et al., 1991).  5  0&%+%& !"#$%&'() *%&"+,-%./ 1%+&.#,&) /.#$(% 0&%+%&'()) ,*%-"-2#  ;.2'%),<)/.$,#'  3('44%&)-%$5 6-+%&-'().&%+7&'()#*7"-$+%&  89+%&-'().&%+7&'()#*7"-$+%& 0&,2%-"+'()4"'*7&'2/ 0&%+7&'  89+%&-'().&%+7&'(),&":$%  Figure 1  Anatomy of the human urinary bladder (Adapted from Human Anatomy (Marieb and Mallant, 1997)).  6  Perivesical Fat Serosa Muscularis Lamina Propria Urothelium  Figure 2  Layers of the bladder wall represented by a view of the human urinary bladder.  7  Mucin Glycoproteins Apical Membrane  Urothelium  Lamina Propria Muscularis Serosa Perivesical Fat  Figure 3  Composition of the bladder wall. Several layers comprise the wall (from top to bottom) an adhering glycosaminoglycan layer, the urothelium, the lamina propria, the muscularis and the serosa.  8  2.1.1.1 Superficial umbrella cells The superficial layer provides a barrier between urine and blood, protecting the underlying layers from high concentrations of urea, ammonia and toxins in the urine (Congiu et al., 2004). The apical surface of superficial cells has thickened membrane plaques that are separated by plasma membrane domains called “hinges” (Figure 4A). These polygonal‐shaped plaques occupy 70‐90% of the apical surface area with a diameter of 0.5 µm and a thickness of 12 nm (Staehelin et al., 1972; Lewis, 2000). The hinge membrane surrounds each individual plaque of 1000 subunits occupying 10‐30% of the remaining apical surface area (Walz et al., 1995). The subunits are composed of 4 transmembrane domain proteins (UPIa and UPIb) and single transmembrane domain proteins (UPII and UPIII) called uroplakins (Yu et al., 1994; Lewis, 2000). Uroplakins Ia and Ib belong to the family of tetraspanins, which play various roles in cell migration, cell signaling, viral infections, and membrane architecture (Hemler, 2003). Uroplakin IIIa has a 50‐amino acid‐long cytoplasmic domain and may be involved in mediating the interaction between urothelial plaques and the underlying cytoskeleton (Wu and Sun, 1993; Lin et al., 1994). Umbrella cells are joined together by tight junctions composed of 4 to 6 interconnecting strands beneath the apical membrane (Peter, 1978). The thickened membranes and the tight junctions contribute to the permeability barrier formed by the cell layer. The cell cytoplasm of the umbrella cells has a high density of cytoplasmic vesicles joined together by cytoskeletal fibrils (Figure 4A) (Hicks, 1965; Staehelin et al., 1972). These vesicles are composed of two opposing plaques connected by the hinge membrane. The fibrils attach at the tight junctions and the desmosomes in the basal membrane (Minsky and Chlapowski, 1978). Upon bladder expansion these vesicles extend to the surface of the bladder wall increasing the surface to volume ratio of the bladder.  9  A  Expansion  Contraction  B  Figure 4  A. Structure of superficial umbrella cell. Apical membrane is covered with scalloped‐shaped membrane plaques separated by hinges. Each umbrella cell is joined together by tight junctions in the laternal membrane. The cell cytoplasm is full of cytoplasmic vesicles held together by cytoskeletal fibrils. These fibrils connect at the tight junctions and desmosomes in the basal membrane (Adapted from (Lewis, 2000)). B. Structure of a contracted superficial umbrella cell. During the expansion‐contraction cycle, the urothelium accommodates changes in bladder volume by stretching/relaxing the umbrella cells to eliminate/form microscopic folds and expand/contract the apical surface area during bladder filling and voiding (Adapted from (Minsky and Chlapowski, 1978)) 10  2.1.1.2 Mucin glycoproteins A mucus layer of membrane bound O‐linked glycoproteins called mucins cover the apical membrane of the urothelium and protect the underlying urothelium from changes in pH, bacterial invasion, tumor implantation and noxious agents (Rofeim et al., 2001; Okegawa et al., 2003). The thickness of this layer varies on different mucosal surfaces, but has been reported as approximately 13.6 μm in porcine bladder (N'Dow et al., 2005). N’Dow et al. (2005) collected 2.7 mg of mucin in normal urine over 24 hours, which represented less than 0.6% of the total bladder mucin (N'Dow et al., 2005). Hurst (1994) estimated the total turnover of urothelial cells to be on the order of 104‐105 per day, assuming an average urinary output of 1000 mL/day with 10‐100 cells/mL contained in normal male urine. Performing a simple calculation, Hurst (1994) estimated that the turnover of urothelial cells was extremely small, somewhere on the order of 0.01%‐0.1% per day (Hurst, 1994). Mucins are large macromolecules, with high molecular weights ranging from 10 to 50 x 106 g/mol (Lindh et al., 2002; Scholfield et al., 2003; Dawson et al., 2004). A protein backbone composed of mostly amino acids, threonine, serine and proline, accounts for 10‐ 30 wt% of the overall glycoprotein, while 70‐80% of the remaining weight constitutes the carbohydrate chains (Khanvilkar et al., 2001; Chayed and Winnik, 2007). Carbohydrate chains are attached to the backbone by O‐glycosidic links between N‐acetyl galactosamine and serine or threonine (Peppas et al., 2000). The sugar residues include fucose, galactose, sialic acid, N‐acetyl glucosamine and N‐acetyl galactosamine (N'Dow et al., 2004). The carbohydrate chains vary from 2 to 20 sugars in length and may occur both as linear and branched structures (Peppas and Huang, 2004). The oligosaccharide side chains of mucin are long and negatively charged due to sialic acid and sulfated sugar residues (Bogataj et al., 1999; Peppas and Huang, 2004). Numerous human MUC genes have been identified, each aiding in the lubrication of epithelial surfaces and providing protection against dehydration and infection (Scholfield et al., 2003). MUCs 1, 3 and 4 are predominately expressed in the normal urothelium and form a membrane bound barrier at the cell surface (N'Dow et al., 2000). Some mucins, however, are secreted and in larger quantities in bladder cancer cell lines and in transitional cell carcinomas (Retz et al., 1998). For example, MUC 7 is over expressed in bladders of patients with bladder cancer, but not in normal bladder mucosa (Okegawa et 11  al., 2003). 2.1.2 Bladder permeability Apical membrane glycoproteins, umbrella cells, plaques and tight junctions composing the urothelium provide a permeability barrier to the movement of substances between urine and blood (Kreft et al., 2005). The barrier is primarily associated with umbrella cells, which have a transepithelial electric resistance from 20,000 Ω • cm2 to > 75,000 Ω • cm2 (Lewis and Diamond, 1976; Khandelwal et al., 2009). Although the urothelium demonstrates an impermeable barrier to all substances present in the urine or blood, the bladder has a small, but finite passive permeability to most substances (Hicks et al., 1974; Negrete et al., 1996). The specialized lipid molecules and uroplakin proteins comprising the umbrella cells reduce the permeability of the urothelium to small molecules such as water, urea and protons whereas the tight junctions reduce the movement of ions and solutes (Birder, 2005). However, in the presence of neoplastic cells having an impaired production of membrane plaques, tight junctions and surface glycoproteins, the permeability of this barrier has been shown to increase (Wientjes et al., 1993; Highley et al., 1999). 2.1.3 Bladder filling and voiding In 24 hours, the mammalian urinary bladder undergoes a series of slow fillings and rapid emptying of urine (Lewis and de Moura, 1984). The epithelium accommodates increases in bladder volume by stretching the cells to eliminate microscopic folds and expand the apical surface area (Lewis and de Moura, 1984). In the contracted bladder, the cells are goblet shaped with an irregularly folded apical membrane, a smooth lateral membrane and folded basal membrane (Figure 4). During expansion, the apical membrane is smoothed as the surface cells change from a goblet shape to a flattened/squamous shape (Porter et al., 1967; Lewis and Diamond, 1976; Minsky and Chlapowski, 1978). The basal membrane becomes unfolded and the lateral membrane becomes folded. With further cell stretching, by bladder filling, an even greater expansion of the apical membrane surface area results from insertion of cytoplasmic vesicles, termed apical membrane endosomes (Minsky and Chlapowski, 1978). On emptying, the excess apical membrane area appears to be taken up into these vesicles (Chang et al., 1994). Movement of vesicles into and out of the apical membrane may be associated with intracellular filaments and is controlled by 12  expansion of the bladder during filling and collapse of the bladder during voiding (Lewis and de Moura, 1982; Lewis, 2000). Morphometric analysis has shown that umbrella cells from full bladders contained significantly fewer vesicles than those from contracted bladders, supporting the theory of vesical insertion (Minsky and Chlapowski, 1978). 2.2 Superficial Bladder Cancer Superficial bladder carcinoma is a type of transitional cell carcinoma (TCC) limited to growth in the urothelium and does not invade beyond the lamina propria of the bladder wall. Superficial tumors are heterogeneous and include those that are high grade, flat and restricted to the mucosa (Tis) (carcinoma in situ), papillary or solid and confined to the urothelium (Ta) and invasive to the lamina propria (T1) (Figure 5) (Kurth, 1997; Borden et al., 2005). 2.2.1 Etiology and pathogenesis Both smoking and occupational exposure to carcinogens have been recognized as leading risk factors in bladder cancer. Chemicals such as acrolein, 4‐amino‐biphenyl, arylamine and oxygen free radicals contained in cigarette smoke have been recognized as urothelial carcinogens (Lower, 1982; Cohen et al., 1992; Yu et al., 2002). Occupational exposure to aromatic amines such as benzidine used in the rubber industry may account for up to one fourth of all urothelial cancers (Golka et al., 2004). In an attempt for a better understanding of the development of bladder cancer, genetic alterations have also been studied (Miyao et al., 1993; Wu, 2005, 2009). These genetic alterations have resulted in activation of oncogenes or inactivation of tumor‐ suppressor genes, causing uncontrolled cell proliferation (Dalbagni and Fair, 1997). In superficial bladder cancer, most tumors show a loss of heterozygosity of chromosome 9 (Wu, 2005). Deletions to 9p and 9q have led to the development of urothelial hyperplasia and low‐grade papillary Ta tumors (Spruck et al., 1994; Kitamura and Tsukamoto, 2006). In fact, low‐grade papillary tumors exhibit a constitutive activation of the receptor tyrosine kinase‐ras pathway, activating mutations in the Harvey rat sarcoma virus oncogene and fibroblast growth factor receptor 3 gene (Mitra et al., 2006).  13  2.2.2 Classification of the disease Carcinoma in situ (Tis) is characterized by flat, disordered proliferation of epithelial cells with polymorphous nuclei replacing the superficial umbrella cells (Jung and Jakse, 1995; Amling, 2001; Pasin et al., 2008). The cells may appear by cystoscopy as a reddish, inflammatory patch of urothelium due to increased lymphoid infiltration of the lamina propria and significant neoangiogenesis (Jung and Jakse, 1995). Although Tis is non invasive, it has been recognized as a likely precursor lesion of solid or papillary TCC (Koss et al., 1965; Koss et al., 1969). Stage Ta lesions account for approximately 70% of superficial carcinoma and are typically low grade (Amling, 2001). These papillary tumors are anchored to the bladder wall and extend out into the lumen of the bladder. The tumors are composed of branching fibrovascular cores with greater than eight cell layers (Pasin et al., 2008). Unlike Tis, the superficial umbrella layer remains intact, but no mitotic activity exists (Pasin et al., 2008). Stage T1 bladder carcinoma invades the lamina propria. Progression is significant in these high‐grade tumors. The malignant potential of these lesions separate them from Ta and Tis lesions (Amling, 2001). Further stage subdivision has been proposed for T1 tumors (Younes et al., 1990; Angulo et al., 1995). For instance, T1a is used to designate those tumors not involving the muscularis mucosa, whereas T1b to T1c represents deeper invasion of the lamina propria to the level of the muscularis mucosa or beyond (Amling, 2001).  14  Tis  Ta  T1  Figure 5  Stage of superficial tumors. Tis (carcinoma in situ) are high grade tumors, flat and restricted to the mucosa, Ta tumors are papillary or solid and confined to the urothelium and T1 tumors are invasive to the lamina propria but do not invade the muscle.  15  2.2.3 Diagnosis and disease management The diagnosis of superficial bladder cancer can be difficult as many presenting symptoms mimic other common urologic conditions such as a urinary tract infection and general lower urinary tract symptoms including bladder spasms, frequent urination and pain or burning during urination (Pasin et al., 2008). The predominant sign of superficial bladder cancer is painless hematuria, detected in urine (Cummings et al., 1992). Urine cytology is a simple technique, most commonly used for detecting cancerous cells in urine. However, due to low sensitivity in detecting low‐grade tumors, cystoscopy remains the primary mode for diagnosing superficial cancer (Barocas and Clark, 2008). Visualization of the urothelium not only leads to the detection of lesions, but it also provides useful information, including tumor number, size, and distribution (Cummings et al., 1992). When urine cytology reads positive, but no grossly visible tumor is apparent by cystoscopy, bladder biopsies are performed (Pasin et al., 2008). Furthermore, various imaging tests may be performed including IV pyelography, a standard radiological method in which a contrasting agent is administered through the vein and x‐rays are taken as the dye moves through the urinary tract detecting any possible tumors (Amling, 2001). At diagnosis, the standard method of treatment for superficial carcinoma is surgical removal of tumors by transurethral resection (TUR). This effective technique establishes the diagnosis and allows pathologic analysis of the resected tumor specimen for tumor grade and depth of bladder invasion (Amling, 2001). However, within 5 years of having TUR, tumor recurrence will develop in 70% of patients, with 25% showing progression to muscle‐invading tumors (Shen et al., 2008). Problems managing bladder tumors may be due to incomplete removal of the tumor and/or implantation of cancer cells in normal or resected areas of the mucosa during or after TUR (Nakamura et al., 2002; Gasion and Cruz, 2006). Therefore, in an attempt to reduce tumor recurrence and progression, intravesical immunotherapy and chemotherapy are used as adjuvant therapies to TUR. 2.2.3.1 Intravesical immunotherapy Intravesical therapy has been used for patients with tumors in the superficial lining of the bladder wall. Prior to treatment, the bladder is emptied with a urethral catheter, and drug is instilled into the bladder. After instillation of the therapeutic agent, the urethral catheter is removed, and the drug is left for a period of 1‐2 hours. The rationale for 16  intravesical therapy is to expose tumors directly to high concentrations of drug while minimizing systemic toxicities, thereby eradicating the existing disease and preventing tumor recurrence and progression (Au et al., 2002; Malmstrom, 2003). Intravesical immunotherapy uses Bacillus Calmette‐Guerin (BCG) as the standard immunotherapeutic agent for the treatment and prophylaxis of superficial bladder cancer. It is the first line therapy for Tis and high‐risk superficial tumors. In 1976, Morales et al. (1976) first reported the use of BCG for the treatment of superficial bladder cancer (Morales et al., 1976). BCG is a live attenuated tuberculosis vaccine that stimulates an immune response to tumors. However, its exact mechanism of action is still unknown. BCG is thought to induce inflammation of the bladder with a broad range of cell types, such as macrophages, T and B lymphocytes and killer cells (Patard et al., 2003; Borden et al., 2005). BCG immunotherapy also results in cytokine production, enhancing killer cell activity and activating tumor necrosis factor alpha and interleukins‐1 responsible in tumor killing. After intravesical instillation, BCG may also be internalized by tumor cells, leaving cell surface glycoproteins that serve as antigens, to mediate an immune response (Morales and Nickel, 1986). However, the success of BCG is limited by side effects such as cystitis, hematuria, malaise and nausea (Pashos et al., 2002). 2.2.3.2 Intravesical chemotherapy Intravesical chemotherapy has played an important role in the management of superficial bladder cancer. The duration of benefits for intravesical chemotherapeutic agents is relatively short term (~2 yrs) (Table 1). Studies have demonstrated only limited efficacy as treatments fail to impact disease progression or survival (Oosterlinck et al., 1993; Bouffioux et al., 1995; Tolley et al., 1996; Kamat and Lamm, 2000). Intravesical chemotherapy is generally used for low and intermediate risk superficial carcinomas because the incidence of local and systemic adverse effects is significantly lower than with BCG (Gasion and Cruz, 2006). A single dose of intravesical chemotherapy immediately (within 6 hours) after tumor resection has been shown to significantly decrease the rate of tumor recurrence likely due to its effect against residual tumor cells and prevention of reimplantation of exfoliated tumor cells (Okuno et al., 1997; Kamat and Lamm, 2000). However, the impact of timing of immediate bladder instillation following TUR is still unknown (Oosterlinck et al., 2002). In a meta‐analysis of seven trials comparing 17  TUR alone to TUR plus one immediate instillation, Sylvester et al. (2004) could not find significant differences in the efficacy when the instillation was given within 24 h (Sylvester et al., 2004). Furthermore, Oosterlinck et al. (1993) instilled either 80 mg epirubicin or sterile water in superficial bladder cancer patients within 6 hours of TUR and found a significantly lower overall recurrence rate for epirubicin treated patients (17% vs 32%; mean follow up 2 years) (Oosterlinck et al., 1993). Some chemotherapeutic agents used to treat superficial bladder cancer include mitomycin (MMC), gemcitabine and doxorubicin. These agents have been evaluated in patients with superficial tumors and were shown to reduce recurrence rates by 46.5%, 25.9% and 16%, respectively (Kamat and Lamm, 2000; Hendricksen and Witjes, 2007). More recently, the taxanes, paclitaxel and docetaxel have been introduced as a new line of therapy for patients who have failed standard intravesical BCG or MMC treatment.  Table 1  Probabilities of recurrence and progression of bladder tumors in patients undergoing controlled adjuvant intravesical chemotherapy following TUR (excerpted with permission from (Hendricksen and Witjes, 2007), who previously adapted from Oosterlinck, 2002 #291;Sylvester, 2006 #351})  Risk group  Low (Ta tumors, ≤3cm in diameter)  Probability of progression  Probability of recurrence  (%)  (%)  1 year  5 years  1 year  5 years  15‐24  31‐46  ≤1  ≤ 1‐6  24‐38  46‐62  ≤ 1‐5  ≤ 1‐17  24‐61  46‐78  1‐17  6‐45  Intermediate (Ta‐T1 tumors, >3cm in diameter) High (T1 & Tis tumors, highly recurrent)  18  2.3 Paclitaxel and Docetaxel Paclitaxel (PTX) was isolated in the early 1960s from the bark of the Pacific Yew tree, Taxus brevifolia (Wani et al., 1971). Its significant cytotoxic effects against many tumors were later reported in the early 1970s (Wani et al., 1971). In 1986, docetaxel (DTX) was discovered from the collaborative effort between Rhône‐Poulenc and the Institut de Chimie des Substances Naturelles (France) (Lavelle et al., 1993). DTX was extracted from the needles of the European Yew tree, Taxus baccata (Sparreboom et al., 1998). The physicochemical properties, pharmacology and toxicology and pharmacokinetics of these drugs are discussed below. 2.3.1 Chemistry PTX is 5β, 20‐epoxy‐1,2α,4,7β,10β,13α‐hexahydroxytax‐11‐en‐9‐one 4,10‐diacetate 2‐benzoate 13‐ester with (2R,3S)‐N‐bezoyl‐3‐phenylisoserine (Pazdur et al., 1993). Its structure is shown in Figure 6A. PTX has a chemical formula of C47H51NO14 and a molecular weight of 853.92 g/mol (Singla et al., 2002). PTX occurs as a white to off‐white crystalline powder and melts at around 216‐217 °C (Singla et al., 2002). PTX is poorly water soluble and highly lipophilic. Various values for the aqueous solubility of PTX at 37 °C have been reported as 0.7 μg/mL (Mathew et al., 1992), 1 μg/mL (Liggins et al., 1997), 6 μg/mL (Tarr and Yalkowsky, 1987), 11 μg/mL (Lundberg, 1997) and 30 μg/mL (Swindell et al., 1991). PTX can be dissolved in organic solvents. The solubility of PTX was reported to be approximately 46 mM in ethanol, 20 mM in acetonitrile and 14 mM in isopropanol (Adams et al., 1993). DTX (Figure 6B) is 4‐acetoxy‐2α‐benzoyloxy‐5β, 20‐epoxy‐l, 7β, 10β‐trihydroxy‐9‐ oxotax‐11‐ene‐13α‐yl‐(2R,3S)‐3‐tert‐butoxycarbonylamino‐2‐hydroxy‐3‐ phenylpropionate (Pazdur et al., 1993). It has a molecular formula of C43H53NO14, a molecular weight of 807.89 g/mol (Verweij et al., 1994) and is available as a trihydrate form. DTX is a semisynthetic derivative of PTX prepared from 10‐deacetylbaccatin III (Huizing et al., 1995a). DTX is a white to off‐white powder that melts at approximately 232°C (Gennaro and Remington, 1995). DTX is poorly water soluble, but has a slightly higher aqueous solubility than PTX. Values for the aqueous solubility of DTX of 5‐6 μg/mL (Ali et al., 1995) and 6‐7 μg/mL at 23°C (Vyas et al., 1993) have been reported. The drug is also soluble in 0.1 N hydrochloric acid, chloroform, dimethylformamide, 95%‐96% v/v 19  ethanol, 0.1 N sodium hydroxide and methanol (Pazdur et al., 1993). PTX and DTX consist of a complex taxane ring system with a four membered oxetane ring at positions C4 and C5 and an ester side chain at position 13 of the taxane ring (Figure 6) (Pazdur et al., 1993; Verweij et al., 1994; Sparreboom et al., 1998). The C‐13 side chain has been recognized as an essential part of the cytotoxic activity of the taxanes (Kingston, 1994). DTX differs structurally from PTX by the presence of a hydroxyl group rather than an acetyl group on position 10 of the baccatin III ring and a trimethylmethoxy moiety on the tertiary position of the side chain at position 13 of the taxane ring instead of a benzamide phenyl group (Bissery et al., 1995). Dordunoo and Burt (1996) determined the degradation of PTX in aqueous buffers. The hydrolytic degradation of PTX in aqueous buffers displayed a pseudo first order kinetic behaviour (Dordunoo and Burt, 1996). The degradation of PTX was pH dependent and subject to general acid‐base catalysis. The major degradation products were baccatin III, 10‐deacetylbaccatin III and baccatin V and the maximum stability of PTX was reported between pH 3‐5 (Dordunoo and Burt, 1996). Epimerization of the C‐7 hydroxyl group has been responsible for a slight decrease in cytotoxic activity of PTX in KB cells (Lataste et al., 1984). The transformation, which results from intramolecular hydrogen bonding between the 7α‐hydroxyl and the carbonyl oxygen of the 4α‐acetoxy group, was discovered to be reversible. In solution, both PTX and 7‐epi‐PTX were in equilibrium with each other and with longer storage, the ester side chains were hydrolyzed forming 10‐deacetylbaccatin III and 10‐deacetylbaccatin V (McLaughlin et al., 1981). Kumar et al. (2007) isolated and characterized the degradation impurities in DTX by performing forced degradation studies using heat, acid, base and peroxide. In the acid and thermal stressed conditions, one major product was observed, 7‐epi‐docetaxel. The group did not observe DTX degradation in the peroxide stressed conditions, but under alkaline conditions, five major degradation products were observed including, 10‐deacetyl baccatin III, 7‐epi‐10‐deacetyl baccatin III, 7‐epi‐10‐oxo‐10‐deacetyl baccatin III, 7‐epi‐docetaxel and 7‐epi‐10‐oxo‐docetaxel (Kumar et al., 2007).  20  O  A Paclitaxel  R. Panchagnula / International Journal of Pharmaceutics 172 (1998) 1–15  O  B Docetaxel  H  Fig. 3. Structure – activity relationships of paclitaxel (from Kingston, 1994, with permission).  in various cellular functions such as move 1994). In addition, it was shown that ingestion of food, ce of an accessible hydroxyl group at O controlling the shape of sensory transduction and spindle formation ! of the ester side-chain enhances the ing cell division (Rowinsky et al., 1990, Ho activity of the drug (Guenard et al., 1992). Paclitaxel has a unique mechanism dification of the side-chain has resulted tion and differs from that of other currently potent analog, docetaxel (Fabre et al., se structure – activity relationships are able anticancer agents (Kuhn, 1994). It rtant in the development of paclitaxel polymerization of tubulin dimers to form Figure 6 Chemical structure of A. PTX B. DTX (Adapted from (Kingston, 1994)). ig. 3). Paclitaxel undergoes epimerizatubules, even in the absence of factors th ure media and forms 7-epitaxol, which normally required for microtubule assembl ive as paclitaxel (Ringel and Horwitz, guanine triphosphate, GTP) (Fig. 4), and importance of structure – activity relastabilizes the microtubules by preventing d Fig. 3. Structure – activity relationships of paclitaxel (from Kingston, 1994, with1979, permission). and conformational studies of derivamerization (Schiff et al., Schiff and Ho 21 microtu clitaxel have been reviewed (Hepperle 1980). Paclitaxel mainly binds to e,1994). 1994).In addition, it was shown that rather than cellular to tubulin dimers such (Parness and in various functions as mov  2.3.2 Commercial formulations PTX is currently formulated as Taxol®, an injection concentrate in a 1:1 mixture of Cremophor EL (polyoxyethylated castor oil) and dehydrated ethanol intended for dilution with a parenteral fluid prior to intravenous administration. DTX is commercially available as Taxotere®, an injection concentrate in polysorbate 80 with an accompanying diluent of 13% w/v ethanol in water for injection. Taxotere® is intended for dilution with a parenteral fluid prior to intravenous administration. 2.3.3 Pharmacology and clinical indications PTX and DTX have a unique mechanism of action. Unlike other microtubule agents such as vinca alkaloids, the taxanes promote the polymerization of tubulin by premature stabilization of the microtubule assembly (Singla et al., 2002). PTX binds to the beta‐ tubulin subunit of the tubulin polymers (Rao et al., 1994). This causes inhibition of the normal dynamic equilibrium of the microtubule network between tubulin dimers and microtubules essential for interphase and mitotic cellular function, thereby freezing the cell in late G2 and M phase (Manfredi and Horwitz, 1984). Disruption of microtubule bundles causes abnormal mitotic spindle aster production and microtubule bundling (Rose, 1995). DTX exerts its therapeutic effect essentially in the same way as PTX, but exhibits 1.9‐fold greater binding affinity for microtubules than PTX (Gaucher et al., 2009). DTX has been shown to induce assembly of microtubule bundles, but without altering the number of protofilaments in the microtubules (Ringel and Horwitz, 1991; Pazdur et al., 1993). In vitro uptake experiments revealed that a 3‐fold higher intracellular concentration of DTX was obtained in P388 leukemia cells compared with PTX for the same initial extracellular concentration of 0.1 μM (Riou et al., 1994). Efflux studies revealed that the half‐time efflux of DTX from P388 leukemia cells was at least three times slower than that of PTX (150 min vs 45 min, respectively) (Riou et al., 1994). The cytotoxicity of DTX in murine and human tumor cell lines was found to be 1.3 to 12 fold more potent than PTX (Riou et al., 1992). IC50 values of DTX ranged from 4 to 35 ng/mL and the cytotoxic effects were greater on proliferating cells than on non‐proliferating cells (Riou et al., 1992). PTX is an extremely effective drug in the management of several common malignancies. It is the first line treatment for breast, lung and ovarian cancer (Rosenberg et 22  al., 2005; Gaucher et al., 2009). In a phase II trial, PTX given as a 24 h intravenous (IV) infusion of 250 mg/m2 weekly for three weeks produced a 42% partial response rate and a 27% complete response rate in advanced and metastatic bladder cancer (Roth, 1995). The usual dosage range for PTX is 200‐250 mg/m2 given as 3 and 24 h IV infusions (Singla et al., 2002). In combination chemotherapy in the treatment of advanced bladder cancer, 100 to 200 mg/m2 of PTX has been employed (Bokemeyer et al., 1998). PTX given at 175 mg/m2 as a 24 h continuous infusion was combined with cisplatin given at 75 mg/m2 in 20 patients with advanced bladder cancer and a response rate of 75% was observed (Bokemeyer et al., 1998). DTX is used for the treatment of breast, ovarian, lung, prostate, and head and neck cancer (Pronk et al., 1995). Although several administration schedules have been studied for phase I development of DTX, DTX has been primarily evaluated at a dose of 100 mg/m2 as a 1h IV infusion every three weeks (van Oosterom et al., 1995). In a phase II study, De Wit et al. (1998) observed an overall response rate of 31% using IV DTX at 100 mg/m2 every 3 weeks among 30 patients with metastatic urothelial cancer (de Wit et al., 1998). DTX also showed activity in patients with TCC who failed to respond to prior cisplatin‐ based therapy (McCaffrey et al., 1997). The combination of DTX and cisplatin both at 75 mg/m2 administered intravenously every 3 weeks was evaluated in 38 previously untreated patients with advanced TCC. An overall response rate of 58% with a 19% complete response and a 39% partial response was observed (Garcia del Muro et al., 2002). 2.3.4 Toxicology When administered intravenously, the major dose limiting side effect of PTX and DTX is neutropenia (Verweij et al., 1994; Aapro and Bruno, 1995). Other dose limiting side effects such as hypersensitivity reactions, mucositis, neurotoxicity and myelosuppression have been reported (Table 2) (Huizing et al., 1995a; Piccart et al., 1995; van Oosterom et al., 1995Vaughn, 2002 #293; Bajorin et al., 1998; Di Lorenzo et al., 2009). Stomatitis has been more frequent with DTX than with PTX, but unlike PTX, no cardiac effects have been reported with DTX (Huizing et al., 1995a). Other toxic effects include anemia, granulocytopenia, alopecia, diarrhea, nausea, vomiting, thrombocytopenia, and myalgia (Pazdur et al., 1993; Verweij et al., 1994; Ajani et al., 1995; Rowinsky and Donehower, 1995; Rowinsky, 1997). 23  Table 2  Side effect  Common adverse side effects when using PTX and DTX for the treatment of many cancers (Verweij et al., 1994; Piccart et al., 1995; van Oosterom et al., 1995) including urothelial cancer (Vaughn et al., 2002; Barlow et al., 2009) PTXa 80 mg/m2 (N=30) (%)  PTX 175 mg/m2 (N=260) (#)  PTX 250 mg/m2 (N=270) (#)  DTX 100 mg/m2 (N=496) (#)  Neutropenia 13 Thrombocytopenia 17 Anemia 37 Asthemia 37 Musculoskeletal 13 Nail disorder 3 Anorexia 10 Alopecia 17 89 90 Neuropathy 43 51 59 Diarrhea 7 25 40 Hypersensitivity 42 40 Myalgia 53 51 Nausea/Vomiting 45 47 Skin reactions 2 23 Cardiac 2 14 Fever 20 30 Leukocytopenia 45 52 Mucositis 23 25 Stomatitis Frequency to urinate Urgency to urinate Dysuria Hematuria Facial flushing Rash Urinary track infection Premature voiding aAdverse effects for IV treatment of urothelial cancer bAdverse effects for IV treatment of ovarian cancer cAdverse effects for intravesicular treatment of urothelial cancer  81 5 40 25 27 45 53 0 17 55 34  DTXb 100 mg/m2 (N=90) (%)  DTXc 75 mg (N=33) (%)  43 43 29 13 55/46 58  55 15 3 18 18 12 3 9 3  24  2.3.5 Pharmacokinetics After a 6 and 24 hour IV infusion of PTX at 275 mg/m2 maximum plasma concentrations were 10 μmol/L and 5 μmol/L (Wiernik et al., 1987a; Wiernik et al., 1987b). The drug is widely distributed into body fluids and tissues with an apparent volume of distribution at steady state ranging from 227‐688 L/m2 (Kuhn, 1994; Rowinsky and Donehower, 1995). In blood, 95 to 98% of PTX is protein bound with a plasma clearance in the range of 8 – 26 L/h/m2 (Rowinsky et al., 1992; Rowinsky and Donehower, 1995). The average distribution half‐life is 0.27 to 0.34 hours and the average elimination half‐life is 2.33 to 5.8 hours depending on infusion time (Rowinsky et al., 1992). PTX is metabolized in the liver to the major metabolite 6α‐hydroxypaclitaxel and eliminated primarily in feces (see Scheme 1) (Kumar et al., 1994). Urinary excretion of PTX accounts for less than 10% of an administered dose (Rowinsky et al., 1992). Extra et al. (1993) reported the IV pharmacokinetics of DTX at 20 ‐ 115 mg/m2 in 23 patients with a variety of tumor types. The plasma pharmacokinetics of DTX were described as biphasic for doses between 20 mg/m2 and 70 mg/m2 and triphasic for doses between 85 mg/m2 and 115 mg/m2 (Extra et al., 1993; Huizing et al., 1995a). After a 1 – 2 h infusion of a 20 – 70 mg/m2 dose of DTX the maximum concentration in plasma was 0.42 – 3.8 μg/mL. At a 85 – 115 mg/m2 dose given as a 3 h infusion, the maximum plasma concentration was 2.41‐2.68 μg/mL. The volume of distribution at steady state ranged from 12 – 93 L/m2 depending on the administered dose (Extra et al., 1993). In blood, more than 90% of DTX is protein bound (Pronk et al., 1995), with a plasma clearance ranging between 17.0 and 39.9 L/h/m2 (Extra et al., 1993). DTX is metabolized by hepatic cytochrome P‐450 mixed function enzymes and undergoes oxidative metabolism of the tert‐butyl ester group (Marre et al., 1996). Fecal excretion of DTX accounts for 70%–80% of the total dose (De Valeriola et al., 1993). Renal excretion of DTX is low, with 2‐6% of the administered dose recovered in urine (Huizing et al., 1995a).  25  Paclitaxel CYP3A4  CYP2C8  3’p‐hydroxypaclitaxel 6α‐hydroxypaclitaxel  CYP2C8  CYP3A4  6α,3’p‐dihydroxypaclitaxel Scheme 1  Major metabolic pathways of PTX (Adapted from (Malingre et al., 2000)).  2.3.6 Clinical use of intravesical taxanes During the last decade, PTX and DTX have emerged as important drugs for intravesical chemotherapy. Currently in phase I clinical trials, intravesical DTX given weekly for 6 weeks demonstrated excellent tolerability at doses up to 0.75 mg/mL (diluted in normal saline) with no evidence of systemic absorption or dose‐limiting toxicities (McKiernan et al., 2006). In the 18 patients treated with the 0.75 mg/mL dose, a complete response rate of 67% was observed (Barlow et al., 2009). Localized dose limiting toxicities of intravesical DTX have included, hematuria, dysuria, urinary retention, urinary frequency/urgency, and bladder spasms (McKiernan et al., 2006). Although intravesical PTX has been studied by a number of groups either in vivo or using ex vivo bladders of dogs, rabbits and mice (Song et al., 1997; Hadaschik et al., 2008; Tringali et al., 2008), the drug has not yet been investigated for clinical use in humans. 2.4 Polymeric Drug Delivery Systems A range of different biomaterials have been explored as carriers of drugs and fabricated as nanoparticles, including lipids, polymers, metals and ceramics. Synthetic biodegradable polymers constitute the most important type of biomaterials for use as nanoparticulate drug delivery systems.  26  2.4.1 Polymer structure and morphology Polymers are macromolecules composed of repeat units called monomers (Odian, 1991). If the polymer is prepared from more than one monomer the product is referred to as a copolymer. More specifically, a polymer composed of two homopolymers linked by covalent bonds is a diblock copolymer. The molecular weight of a synthetic polymer is not well defined since the polymer is always a mixture of molecules with different chain lengths and is therefore a distribution of molecular weights (Allcock and Lampe, 1981). Polymer molecular weights may therefore be defined in terms of number (Mn) and weight (Mw) averages of molecular weight. Mn is defined as:  Mn =  W = N  ∑ niMi ∑ ni  Equation 1  Where W is the total weight of the polymer sample, N, the total number of moles of the polymer and ni, the number of moles of the polymer molecules of molecular weight Mi. Mw is defined as:  ∑ wiMi = ∑ niMi Mw = ∑ wi ∑ niMi  2  Equation 2  Where wi is the weight of the polymer molecules of molecular weight Mi Mw is always larger than Mn. When the ratio of Mw to Mn is calculated, the resulting value is called the polydispersity index, which defines the distribution of the polymer molecular weights. For instance, a polymer with a narrow distribution of chain lengths would have a polydispersity index close to 1. Gel permeation chromatography can be used to determine the molecular weight of the polymer by separation of polymer chains according to their molecular size. The morphology of a polymer can be described as the arrangement of polymer chains with respect to long‐range order (Rosen, 1993). Crystallinity refers to the degree of order within a polymer matrix. Many polymers are semicrystalline and contain both ordered 27  crystalline regions and disordered amorphous regions within the polymer matrix (Rosen, 1993). The degree of crystallinity of a polymer can affect drug release, thermal behaviour, degradation rates and permeability. 2.4.2 Polymeric nanoparticles Nanoparticles are submicron colloidal carriers in the size range of 10 to 1000 nm and include liposomes, nanospheres, polymersomes, micelles and hyperbranched polyglycerols or “unimolecular micelles” (Sakuma et al., 2001). Micelles, nanospheres and hyperbranched polyglycerols are nanoparticulate drug delivery systems used in this project. Their features and properties will be discussed below. 2.4.2.1 Micelles and nanospheres of amphiphilic diblock copolymers Polymeric micelles (Figure 7A) are spherical molecular aggregates formed by the self‐assembly of amphiphilic block copolymers. These structures are characterized by a core‐shell architecture with diameters ranging from 10‐100 nm and a fluid‐like core. The hydrophilic block, typically composed of poly(ethylene glycol) (PEG) or methoxy poly(ethylene glycol) (MePEG), hydrates in water forming a dense, water bound corona. MePEG restricts protein adsorption and subsequent clearance by the reticuloendothelial system (RES) and prolongs circulation within the body (Letchford et al., 2008). MePEG chains provide stealth‐like properties and may even intercalate with other membrane bound chains like mucin and enhance mucoadhesion of the polymer material. The hydrophobic blocks, forming the core are composed of biocompatible, biodegradable polyesters such as poly(lactic‐co‐glycolic acid) (PLGA), poly(L‐lactic acid) (PLLA), poly(caprolactone) (PCL) and have proven to be very effective in solubilizing hydrophobic drugs such as PTX and indomethacin (Zhang et al., 1996; Kim et al., 1998). The formation of micelles is dependent on the solubility of the polymer. For instance, if the copolymer is relatively water soluble, micelles may be formed by direct dissolution or film casting methods. In the first method, the copolymer is added to aqueous media at a concentration above the critical micelle concentration (CMC) and drug partitions into the core of the micelles (Kabanov and Alakhov, 2002). In the second method, the copolymer is dissolved together with a drug in a volatile solvent, which is then evaporated leaving a film or matrix to be reconstituted with buffer or water for the formation of micelles (Zhang et al., 1996). A disadvantage of micelles, is their instability following dilution below the CMC due to the 28  dissociation of micelles into the free polymer chains or unimers (Kainthan et al., 2008b). Nanospheres (Figure 7B) have a core‐shell architecture, but are typically larger (100‐200 nm) than micelles and possess a solid‐like matrix core, exhibiting generally greater stability than micelles (Gref et al., 1994; Letchford et al., 2009). Drugs may be dissolved, entrapped, encapsulated, chemically bound or absorbed to the polymer matrix (Letchford and Burt, 2007). The copolymer unimers of the micelle are in a frozen state and the structure is considered phase separated (Gref et al., 1994; Letchford et al., 2009). Several methods have been proposed for the preparation of nanospheres (Galindo‐ Rodriguez et al., 2004; Sinha et al., 2004), however the most common method is nanoprecipitation (Pinto Reis et al., 2006). In this method, the polymer is dissolved in an organic, water‐miscible solvent and added to aqueous solution where the organic solvent diffuses out, leading the polymer to precipitate and nanoparticles to form (Vauthier and Bouchemal, 2009).  29  A Micelle  Unimers  CMC  B Nanosphere  Figure 7  Structure of A. micelles in dynamic equilibrium with unimers comprised of a hydrophilic block (blue) and a hydrophobic block (black). Micelles form at concentrations above the critical micelle concentration (CMC). B. nanospheres containing copolymer unimers in a frozen state  30  2.4.2.2 Nanoparticles of MePEG­PDLLA One of the diblock copolymers (Figure 8) used in this work was composed of a block of PDLLA approximately 9 repeat units long, linked to a block of MePEG, approximately 45 repeat units long and previously characterized by Zhang et al. (1996). The copolymer was synthesized from D,L‐lactide and MePEG by ring opening bulk polymerization in the presence of stannous octoate (Zhang et al., 1996). The ratio of the two polymer components was 40% PDLLA and 60% MePEG with a MePEG molecular weight of 2000 g/mol. The theoretical polymer molecular weight was 3333 g/mol, but the observed molecular weight was reported to be MGPC = 5240 g/mol (Zhang et al., 1996). Micelles were formed above the CMC and the aqueous solubility of PTX was increased up to 5000‐fold (5 mg/mL) (Burt et al., 1999). CMC values measured at 25 °C ranged between 23‐90 μM depending on the measurement technique (Zhang et al., 1996; Burt et al., 1999).  CH" O CH" O  CH! CH! O  CH n  Figure 8  C  O m  Structure of methoxy poly(ethylene glycol)‐block‐poly(D,L‐lactic acid) (MePEG‐PDLLA)  31  2.4.2.3 Nanoparticles of MePEG­PCL Another diblock copolymer, MePEG‐PCL (Figure 9), used in this work was composed of a block of PCL of 19 or 104 repeat units long, coupled to a block of MePEG approximately 114 repeat units long. The copolymer was previously characterized by Letchford et al. (2009) and synthesized from ε‐caprolactone and MePEG by ring opening polymerization in the presence of stannous octoate (Letchford et al., 2009). Our group uses the following nomenclature for nanoparticles of MePEG‐PCL. The short PCL block length nanoparticles (MePEG114‐PCL19) are referred to as micelles since they were shown to possess a fluid‐like core and may be formed by self‐assembly above the CMC (Letchford et al., 2009). The long PCL block length nanoparticles (MePEG114‐PCL104) are referred to as nanospheres due to their phase separated, solid‐like core (Letchford et al., 2009). The ratios of the two polymer components were 30:70 and 70:30 MePEG:PCL with a MePEG molecular weight of 5000 g/mol for nanospheres and micelles, respectively. The hydrodynamic diameters of these nanoparticles were 80 nm and 40 nm for nanospheres and micelles, respectively. The theoretical polymer molecular weights were 16,651 and 7143 g/mol, but the observed molecular weights were reported to be MGPC = 12,238 and 7044 g/mol for MePEG114‐PCL104 and MePEG114‐PCL19, respectively (Letchford et al., 2009).  O CH" O  CH  CH! CH! O  C O  2  5  n Figure 9  m  Structure of methoxy poly(ethylene glycol)‐block‐polycaprolactone (MePEG‐PCL)  32  2.4.2.4 Hydrophobically derivatized hyperbranched polyglycerols Hyperbranched polyglycerols (HPGs) (Figure 10), often referred to as “unimolecular micelles” possess a dendrimer like architecture. The core may be derivatized with alkyl chains by sequential addition of 1,2‐epoxyoctadecane to hydroxyl end groups formed from the anionic ring opening multibranching polymerization of glycidol (Kainthan et al., 2006b; Kainthan et al., 2008b). MePEG chains can be conjugated to the surface of HPGs to enhance water solubility. The excellent stability and capacity to encapsulate hydrophobic drugs makes them good candidates for drug delivery (Liu et al., 2000; Kainthan and Brooks, 2007). These systems are small in size (< 10 nm) and display similar core‐shell architectures to micelles, however the hydrophobic core and hydrophilic shell can be found within the single molecule connected by covalent bonds (Newkome et al., 1991; Kainthan and Brooks, 2007). HPGs can be synthesized in a single, yet controlled synthetic procedure based on ring opening polymerization of epoxides with predetermined molecular weights and narrow polydispersities (Sunder et al., 1999; Kainthan et al., 2008b). Biocompatibility evaluations of these water‐soluble polymers conducted both in vitro and in vivo have shown good compatibility with blood and no evident animal toxicity (Kainthan et al., 2006a; Kainthan et al., 2006b; Kainthan and Brooks, 2007; Kainthan et al., 2007).  33  O 7  O 7  O O  HO  HO  O O  O 7  O  O  R  O  O  O HO  7  O  O  O  OH O  O  RO  OH  OH O O  O O  O  O  O 7  OH  O  O  O  7  )O 7  R  O  O O  ( O  O  O  O  HO  OH OH  O  O  R  O  O O H  O  O  O  O  O  HO HO  O  O  O  O  O  O O  HO  OH  O  HO O  OH  O  O  O O 7  O  OH  OH  O  O 7  O  OH  O  HO  HO  O  R O  7  OH  O  O 7 O O  7  alkyl chain chain) R= C10(C10 alkyl  Figure 10  Structure of hyperbranched polyglycerol, HPG‐C10‐MePEG (Adapted from (Kainthan et al., 2008b)).  34  2.4.3 Drug release from polymeric nanoparticles The mechanism of drug release from nanoparticles primarily involves, diffusion and copolymer degradation (Kim et al., 2001). Polymer degradation may govern the rate of drug release if the interaction between the polymer and the drug is strong and the rate of degradation is fast (Lee et al., 2007). However, in vitro, the rate of drug release from micelles can exceed the rate of copolymer degradation and so polymer degradation is often ruled out as one of the main mechanisms of drug release (Lee et al., 2007). Thus, diffusion becomes the principle mechanism of drug release from nanoparticles. Three main factors influence the release of drug from a polymeric micelle. These include the characteristics of the drug, the properties of the core‐forming block and the degree of polymer‐drug compatibility. The amount of drug present in a micelle can influence the drug release kinetics such that an increase in the drug loading results in an increase in the rate of drug release (Liu et al., 2004; Lim Soo et al., 2005). An increase in the molecular volume and molecular weight of the drug leads to a lower diffusion coefficient and decreased release rate. The rate of diffusion can be greatly influenced by the properties of the micelle core. The hydrophobicity of the core can affect the permeability of the core to aqueous media. Micelles with a highly hydrophobic core will likely have a slow rate of drug release due to poor water penetration into the core in comparison to micelles that have a more hydrophilic core. The physical state of the core can also affect the diffusion of the drug from the micelles. Polymers that are more crystalline or have a high Tg or bulky groups present on their backbone have limited flexibility since the ordered alignment of the polymer chains and bulky side groups lower the free volume. As a result, these polymers will form micelle cores with high microviscosity, thus preventing diffusion of the drug and water molecules through the polymer core because of limited movement (Kwon et al., 1994). This is in contrast to polymers that are amorphous or more rubbery or gel‐like that will tend to form cores that have lower microviscosity and a higher rate of diffusion (Lee et al., 2007). Finally, the degree of polymer‐drug compatibility can greatly influence the rate of drug release from the micelles. Drugs that are miscible with the core‐forming block possess good polymer‐drug compatibility. However, an increase in the extent of interaction between a drug and a polymer can result in a slower release (Saltzman, 2001).  35  2.5 Intravesical Drug Delivery 2.5.1 Drug transport into the bladder wall Penetration of an agent through bladder tissue involves passage through the urothelium and the underlying tissue (Badalament and Farah, 1997; Malmstrom, 2003). The human urothelium is approximately 200 μm thick, does not have capillaries and is covered by a mucous layer of mucin glycoproteins (Dalton et al., 1991; Badalament and Farah, 1997). Drug transport across the urothelium occurs by passive diffusion via the transcellular or paracellular pathways. It is more likely that drug molecules diffuse transcellularly through umbrella cells, as the tight junctions in this layer offer a barrier to the paracellular movement of substances. Intermediate and basal cells of the urothelium do not express tight junctions and thus do not offer a significant barrier to paracellular transport through the urothelium (Lewis, 2000). The decline in drug concentration across the urothelium has been reported to be linear, due to the homogeneity of the barrier and the lack of vasculature (Gao et al., 1998). However, beneath the urothelium, the lamina propria (200 – 1000 μm) and the underlying muscularis (1000 – 10,000 μm) are well vascularized (Au et al., 2002; Malmstrom, 2003) allowing for rapid penetration of an agent into the blood. The cell packing in the deeper tissue is less dense and diffusion can occur more readily through the extracellular space. Wientjes and coworkers (1991 and 1993) determined the bladder tissue pharmacokinetics of mitomycin C (MMC) in humans and dogs. An intravesical dose of 20 mg and 40 mg MMC in water was instilled into human and dog bladders and maintained for 120 min. The bladders were removed and tissues were sectioned in layers parallel to the urothelium and analyzed for MMC concentration. Within the urothelium, it was shown that the decline in drug concentration was linear with depth and dependent on the concentration gradient across the urothelium and the initial drug concentration. The average MMC tissue concentrations with the 40 mg dose in dogs and human bladders were reported twice that achieved with the 20 mg dose, suggesting that the tissue uptake of MMC was linearly related to its concentration in urine. Beyond the urothelium, the MMC concentrations in the bladder wall declined exponentially with tissue depth since the capillary density increased as the distance increased. The authors concluded that the tissue MMC concentration‐depth profiles were the result of a combined drug diffusion process and a drug removal process by tissue blood flow (Wientjes et al., 1991; Wientjes et al., 36  1993). Enhancements to the delivery of agents across the urothelium have been demonstrated by the use of permeability enhancers, such as dimethyl sulfoxide (DMSO) and chitosan (Chen et al., 2003; Grabnar et al., 2003). DMSO can penetrate living tissue and has been used as a vehicle to enhance the bladder absorption of various chemotherapeutic agents, such as cisplatin and doxorubicin (Schoenfeld et al., 1983; Hashimoto et al., 1992). Approved by the U.S Food and Drug Administration for the treatment of interstitial cystitis, DMSO has been safely instilled, up to 50% (v/v), in the bladders of patients (Schoenfeld et al., 1983). In dog bladders, DMSO has been shown to destabilize Cremophor EL micelles encapsulating PTX, promoting the penetration of PTX across the urothelium (Chen et al., 2003). Chitosan has been reported to influence bladder permeability by desquamation of the urothelium, resulting in the removal of diffusion barriers (Kerec et al., 2005) and increasing drug transport across the urothelium. In response to bladder filling, the apical plasma membrane coordinates changes in size and function with the processes of endocytosis and exocytosis (Figure 11) (Khandelwal et al., 2009). Exocytosis is involved in the cell surface delivery and secretion of newly synthesized and recycled molecules, whereas endocytosis is required for the internalization and recovery of exocytosed membranes (Truschel et al., 2002). In superficial cells of the urinary bladder urothelium, endocytosis represents a mechanism for the removal of the apical membrane and is involved in the forming of different cytoplasmic vesicles (Romih and Jezernik, 1994). Studies have shown that when fluid phase markers, such as horseradish peroxidase and colloidal gold were added to the bladder lumen, the markers were detected in vesicles after voiding (Hicks, 1966; Porter et al., 1967; Chang et al., 1994). Observations of markers within the cytoplasm of umbrella cells may suggest a possible mechanism for the internalization of drugs into superficial tumor cells.  37  Figure 11  A proposed model for vesicle dynamics in umbrella cells during bladder filling and voiding. “Filling stimulates both exocytosis and endocytosis of vesicles. The net rates of these processes are such that membrane is added to the apical surface. Endocytosed membraneis delivered to lysosomes where contents are degraded. Upon voiding the added membrane isinternalized and reestablishment of the vesicle pool may result from both endocytosis and de novo synthesis along the biosynthetic pathway.” [Figure and text excerpted from: (Apodaca, 2004)]  38  2.5.2 Intravesical administration of anticancer drugs Hadaschik et al. (2008) instilled PTX loaded MePEG‐PDLLA micelles intravesically in an orthotopic mouse model of superficial bladder cancer. The group reported maximum serum concentrations of PTX of approximately 1 μg/mL and 5.5 μg/mL at 60 min from administered doses of 500 μg and 1000 μg, respectively. Bladder tissue concentrations ranged from 72 μg/g to 241 μg/g following a 2 h intravesical instillation (Hadaschik et al., 2008). Song et al. (1997) instilled a non‐micellar solution of PTX at 25 μg/mL into the bladder of dogs and reported an initial 13% decrease in the concentration of PTX in urine within the first 5 min followed by a slower decrease for the remaining 115 min. Plasma concentrations of PTX were found to increase with time, reaching a plateau of 0.3 ng/mL at 30 min, but remained several orders of magnitude lower than the concentrations in urine and bladder tissue. Concentrations of PTX in bladder tissue were also found to be 1.8 μg/g with the highest concentration found in the urothelium (7.5 μg/g) (Song et al., 1997). Tringali et al. (2008) administered Oncofid‐P® (PTX conjugated to hyaluronic acid) at a PTX concentration of 1 mg/mL and 3 mg/mL into the bladders of rabbits. The group obtained similar results for both concentrations with 0.68 % of the administered drug recovered within the bladder wall. Oncofid‐P® was also found uniformly distributed in the bladder mucosa as no significant differences in PTX levels were observed over four different areas of the bladder wall (Tringali et al., 2008). Gao, Wientjes and coworkers (1998, 1993 and 1991) examined the kinetics of mitomycin C (MMC) in the bladder of dogs and humans after intravesical instillation of a 20 mg and 40 mg dose (Wientjes et al., 1991; Wientjes et al., 1993; Gao et al., 1998). A comparison of the doses showed the average tissue concentration in dog and human bladders attained with the 40 mg dose (8.77 μg/g and 7.55 μg/g, respectively) to be about twice the concentration achieved with the 20 mg dose (4.33 μg/g and 3.91 μg/g, respectively) (Gao et al., 1998). Plasma concentrations obtained at the 40 mg dose in dogs were comparable to the levels obtained at the 20 mg dose and ranged from 2.2 to 8.5 ng/mL, at a concentration 80,000 fold lower than concentrations in urine. Furthermore following a 40 mg dose, the average MMC concentration in human tumor bearing bladder tissue was reported to be approximately 40% higher than the concentration in adjacent normal bladder tissue (Gao et al., 1998). 39  HPGs have been synthesized by members of the Brooks’ Lab and investigated as drug delivery systems for the treatment of bladder cancer (Mugabe et al., 2009). Mugabe et al. (2009) instilled nude mice bearing orthotopic KU7‐tumors with intravesical Taxol® and PTX loaded hydrophobically derivatized HPG at 1 mg/mL. The PTX loaded HPG formulation showed significantly greater tumor inhibition compared to Taxol®. In addition, the group reported no evidence of systemic toxicity, hematuria or weight loss in PTX loaded HPG treated mice (Mugabe et al., 2009). 2.5.3 Intravesicular mucoadhesive drug delivery Mucoadhesion is the adhesive attachment of a material to mucus or a mucous membrane by interfacial forces (Smart, 2005). Mucoadhesion is controlled by ionic, covalent, hydrogen, Van der Waals or hydrophobic bonds between the chemical functional groups comprising the mucin and the delivery system (Smart, 2005). The mucin molecules have both glycosylated hydrophilic sugar blocks and nonglycosylated hydrophobic peptide blocks allowing for strong affinity to both hydrophilic and hydrophobic moieties (Peppas and Huang, 2004). Mucins contain hydroxyl groups on the branched sugars chains, amide groups in the backbone chains and carboxylic or sulphate groups in the terminal segments of the branched chains (Peppas and Huang, 2004). The negatively charged sialic acid and sulfated sugars of mucin at pH > 3 increase the strength of hydrogen‐bonding formation ability with polymers (MacAdam, 1993). For example, positively charged polymers, such as chitosan have been shown to form polyelectrolyte complexes between amine groups and the negatively charged mucin exhibiting strong mucoadhesion (Genta et al., 1998). Drug exposure to bladder tissue has been successfully increased using the approach of bioadhesion. Binding to a mucosal surface can provide a high concentration of drug for extended periods while encouraging drug uptake and reduced wash out. The coupling of bioadhesion characteristics to carrier particles can provide additional advantages to intravesical drug delivery systems, including a much more intimate mucosal contact, enhanced drug absorption and improved bioavailability (Tyagi et al., 2006). Lu and coworkers (2004) developed PTX loaded gelatin nanoparticles for use in intravesical therapy of superficial bladder cancer. The nanoparticles rapidly released most of the loaded PTX within 2 hours in dog urine. In order to determine whether the gelatin nanoparticles provided enhanced drug delivery to bladder tissues, the authors compared 40  the data obtained with two earlier studies (Song et al., 1997; Chen et al., 2003). Bladder tissue concentrations of PTX delivered from loaded nanoparticles were higher than for micellar PTX in Cremophor (Lu et al., 2004). Lee et al. (2005) instilled o/w emulsions of PTX in glyceryl monooleate/tricaprylin into the bladders of rabbits. The PTX concentrations in the urothelium were significantly higher for the PTX emulsions compared to the Cremophor control, which was attributed to the emulsions being more bioadhesive (Lee et al., 2005). Using a mouse bladder cancer model, Le Visage et al. (2004) instilled bioadhesive microspheres of PTX made from poly(methylidene malonate) that adhered to the urothelium of mice for up to 2 days. Scanning electron microscopy showed most superficial urothelial cells decorated with multiple bioadhesive microspheres. There was a significantly higher survival rate for the mice with microspheres compared to similar doses of PTX in Tween 80 (Le Visage et al., 2004). Although literature evidence suggests that higher drug concentrations maintained at the bladder urothelium should increase tissue levels, the effect of loading drugs at high concentrations in nanoparticulate delivery systems on bladder tissue levels is not clear. Furthermore, whether mucoadhesion of the drug carrier to the bladder wall plays a role in enhancing bladder tissue levels is not well understood.  41  2.6 Thesis Goals, Hypothesis and Objectives The overall goal of this research was to investigate, ex vivo, the bladder mucosal permeability and distribution of PTX and DTX from nanoparticulate formulations. We hypothesized that the intravesical administration of mucoadhesive nanoparticulate formulations of PTX and DTX would bind to the bladder mucosa and allow for increased drug uptake into the bladder wall compared to commercial formulations of both drugs.  The specific objectives were to: 1. Determine the release profiles of drugs from PTX and DTX loaded MePEG‐PDLLA micelles. 2. Investigate tissue levels of PTX and DTX in different layers of ex vivo porcine bladder wall exposed to PTX and DTX loaded nanoparticulate delivery systems. 3. Determine the effects of incubation time, drug concentration and nanoparticulate composition on tissue levels of PTX and DTX in ex vivo porcine bladder wall. 4. Evaluate the bladder tissue viability over the time course of the studies and the tissue toxicity of nanoparticulate formulations.  42  3  EXPERIMENTAL  3.1 Materials 3.1.1 Chemicals and solvents HPLC‐grade acetonitrile, dichloromethane, ethanol, methanol and spectranalyzed N,N‐dimethylformamide were obtained from Fisher Scientific (Fairlawn, NJ). ACS‐grade sodium chloride, magnesium chloride, calcium chloride, sodium dihydrogen orthophosphate, sodium bicarbonate, glacial acetic acid and anhydrous sodium acetate were purchased from Fisher Scientific (Fairlawn, NJ) for use in buffer solutions. ACS‐grade potassium chloride was acquired from Sigma‐Aldrich (St. Louis, MO), sodium phosphate dibasic from EMD chemicals Inc. (Gibbstown, NJ) and α‐D(+)‐Glucose from Acros Organics (Geel, Belgium). Triton X‐100, and Cremophor EL were obtained from Fluka Biochemika (Buchs, Switzerland) and Tween 80 (polyoxyethylene‐sorbitan monoleate) from Sigma‐ Aldrich (St. Louis, MO). Mucin from porcine stomach was purchased from Sigma‐Aldrich (St. Louis, MO). Liquid scintillation fluid, CytoScintTMES, was purchased from MP Biomedicals (Irvine, CA). LDH cytotoxicity detection kit was obtained from Takara Bio Inc. (Shiga, Japan). [14C] and [3H] D‐Mannitol, 250 μCi in 9:1 ethanol:H2O were purchased from Moravek Biochemicals (Brea, CA) with a 55 mCi/mmol specific activity. 3.1.2 Tyrode buffer Tyrode salts were purchased from Sigma‐Aldrich (St. Louis, MO). One litre of tyrode buffer prepared from ACS‐grade salts as described above contained 8 g sodium chloride, 0.2 g potassium chloride, 0.1 g magnesium chloride, 0.2 g calcium chloride, 0.05 g sodium dihydrogen orthophosphate, 1 g sodium bicarbonate and 1 g glucose in deionised water adjusted to pH 7.4. Tyrode buffer is a solution that resembles physiological fluid used to irrigate the peritoneal cavity (Tyrode, 1910), which is a space between the parietal peritoneum that lines the abdominal wall and the visceral peritoneum (serosa) that covers the bladder and other abdominal organs in the abdominal cavity. 3.1.3 Paclitaxel and docetaxel Paclitaxel (MW 853.92 g/mol) was purchased from Polymed Therapeutics Inc. (Houston,TX). Docetaxel (anhydrous, MW 807.89 g/mol) was obtained from Natural Pharmaceuticals (Beverly, MA). Commercial Taxol® 6 mg/mL (Biolyse Pharma, St 43  Catharines, ON) and Taxotere® 20 mg/0.5 mL (Sanofi Aventis, Laval, QC) were purchased from the BC Cancer Agency at the Vancouver General Hospital. Tritium labeled PTX and DTX in ethanol were purchased from Moravek Biochemicals (Brea, CA) with specific activities of 19.7 Ci/mmol and 23.2 Ci/mmol, respectively. 3.1.4 Polymers Diblock copolymer, methoxy polyethylene glycol‐block‐poly(D,L‐lactic acid) (MePEG‐ PDLLA) was provided by Angiotech Pharmaceuticals (Vancouver, BC). The copolymer was manufactured by ring‐opening polymerization in the presence of stannous octoate with a weight ratio of 60:40 (MePEG:PDLLA), using a MePEG molecular weight of 2000 g/mol. Diblock copolymers of methoxy polyethylene glycol‐block‐polycaprolactone, MePEG114‐PCL19 and MePEG114‐PCL104, were previously synthesized by Dr. Kevin Letchford in weight ratios of 70:30 and 30:70 with a MePEG molecular weight of 5000 g/mol, using ring‐opening polymerization in the presence of stannous octoate (Letchford et al., 2008; Letchford et al., 2009). Hydrophobically derivatized hyperbranched polyglycerols, HPG‐C10‐MePEG were previously synthesized by Dr. Don Brooks and coworkers using anionic ring‐opening polymerization of epoxides. The polymer was prepared from multibranching polymerization of glycidol in the presence of trimethyloyl propane. Some of the hydroxyl groups within the core of the HPGs were modified with C10 alkyl chains. Short MePEG chains of number average molecular weight 350 g/mol were added to surface hydroxyls by addition of MePEG‐epoxide to improve water solubility (Kainthan et al., 2008a; Kainthan et al., 2008b). Water‐soluble chitosan, Protasan UP CL 213, was purchased from FMC Biopolymer (Drammen, Norway). Poly(vinyl alcohol) (MW 125,000) was purchased from Polysciences Inc. (Warrington, PA). 3.1.5 Porcine bladder tissue Porcine bladders were purchased from Britco Inc. (Langley, BC). Freshly excised urinary bladders were removed on‐site from 6‐10 month old male pigs weighing between 90‐113 kg. Pigs arrived from various farms and were executed in accordance with the National Farm Animal Care Council. 44  3.2 Equipment 3.2.1 Liquid scintillation counter The amount of tritium present in dialysis bags after in vitro drug release and porcine bladder tissue studies was determined using a LS6500 Multi‐Purpose Scintillation Counter (Beckman Coulter, Fullerton, CA) featuring a Motorola 68000 Series microprocessor, a digital signal processor and a 32,768 channel multichannel analyzer. Measurements were made using an automatic count‐operating mode. 3.2.2 Cryotome Frozen bladder tissue was mounted with Shandon CryomatrixTM (Themo Scientific, Pittsburgh, PA) onto a cryotome object holder. Bladder tissue was sectioned with Shandon MB35 Premier Low Grade Microtome Blades (Themo Scientific, Pittsburgh, PA) at ‐20 °C on a Shandon Cryotome Electronic (Thermo Electron Corporation, Cheshire, England) with a R404A refrigeration system. 3.2.3 Dynamic light scattering Particle size analysis of micelles was conducted on a Malvern Zetasizer Nano ZS (Malvern Instrument Ltd, UK) with a 4 mW Helium‐Neon laser of 633 nm and 173° backscatter detecting optics. Samples were measured at 25 °C with 12.5 x 12.5 x 45 mm acrylic cuvettes. Data were analyzed using Zetasizer v6.01 software. Particle size analysis of mucin was measured on a Malvern 3000HS Zetasizer (Malvern Instrument Ltd, UK) with a nominal 5 mW Helium‐Neon laser of 633 nm and 90° collecting optics. Samples were measured at 25 °C with 10 x 10 x 45 mm acrylic cuvettes. Data were analyzed using CONTIN algorithms with photon correlation spectroscopy (PCS) software. 3.2.4 UV­Vis spectrophotometer Spectroscopic analysis of lactate dehydrogenase (LDH) and mucin mixed with various polymer solutions was performed on a Multiskan Ascent spectrophotometer (Labsystems, Helsinki, Finland) equipped with a 96 well microplate reader, interference filters (405, 450, 492, 540 and 620 nm), a quartz‐halogen lamp source and silicon  45  photodetector, amplifier and lens optics with Ascent software. Single and two‐point measurements were made. 3.2.5 High performance liquid chromatography Chromatographic analysis of PTX was performed on a Waters HPLC system (Milford, MA) consisting of a 717+ autosampler, a 600‐controller pump and a 486 tunable absorbance detector with Waters Millennium v4.0 software. The analytical column was a Novo‐Pak C18 column with dimensions 3.9 x 150 mm (Millipore Corporation). 3.2.6 Glassware For in vitro drug release studies, 500 mL I‐Chem brand (Rockwood, TN) wide mouth glass jars were used with Teflon® lined screw capped lids. Beakers, bottles and graduated cylinders were Pyrex® brand (Lowell, MA). Nanoparticles and drug solutions were stored in 20 mL glass scintillation vials with polypropylene lined screw capped lids. All glassware was obtained from Fisher Scientific (Toronto, ON). 3.2.7 General equipment and supplies General equipment and supplies used for this project are listed in Table 3.  46  Table 3  List of General Equipment and Supplies  Equipment and Supplies  Supplier  Isotemp 210 water bath  Fisher Scientific (Fairlawn, NJ)  Innova 4000 incubator shaker  New Brunswick Scientific (Edison, NJ)  Mettler AE 163 and AJ100 balance  Mettler Instruments (Zurich, Switzerland)  Beckman GS‐6 and microfuge 18 centrifuge  Beckman Coulter (Palo Alto, CA)  Shel‐Lab oven  Sheldon Manufacturing (Portland, OR)  Thelco model 16 oven  Precision Scientific (Chicago, IL)  Accumet model 230 and 610A pH meter  Fisher Scientific (Fairlawn, NJ)  Model 370 magnetic stirrer/heater  VWR Scientific (Missaussauga, ON)  Franz Diffusion cells  PermeGear (Hellertown, PA)  Vanlab vortex mixer  VWR Scientific (Missaussauga, ON)  PolyScience dual action shaker  PolyScience (Niles, IL)  Sonic Dismembrator model 100  Fisher Scientific (Fairlawn, NJ)  PowerGen model 125 homogenizer  Fisher Scientific (Ottawa, ON)  Surgical Tools  Fine Science Tools (North Vancoucer, BC)  1.5 mL and 2.0 mL micro eppendorf tubes  Sarstedt (Montreal, QC)  Pipetman of variable volume  Gilson Company (Middleton, WI)  Nalgene 500 mL containers  Fisher Scientific (Fairlawn, NJ)  7 mL plastic scintillation vials  Fisher Scientific (Fairlawn, NJ)  3500 MWCO Spectra/Por® dialysis membrane and dialysis clips  Spectrum Laboratories Inc (Rancho Dominguez, CA)  1 mL clear HPLC vials with plug (8 x 40 mm)  Canadian Life Science (Peterborough, ON)  47  3.3 Preparation of PTX and DTX Loaded MePEG­PDLLA Micelles Micelles loaded with PTX and DTX were prepared via the solvent evaporation technique. Briefly, 7 mg of PTX or DTX and 70 mg of MePEG‐PDLLA copolymer (10% w/w) were dissolved in 500 μL of acetonitrile (ACN) and dried with nitrogen gas. Polymer and drug amounts were adjusted appropriately depending on the final concentration and number of samples needed. When necessary, prior to drying, the stock polymer/drug solution was spiked with 100 μL of 3H PTX or 3H DTX. The resulting polymer/drug matrix was reconstituted with 60 °C tyrode buffer (pH 7.4) and vortexed for 2 min such that the final concentration of drug was either 0.5 or 1 mg/mL. 3.4 Preparation of Control Commercial Formulations of PTX and DTX PTX was prepared in Cremophor EL and ethanol (EtOH) by diluting Taxol® (containing 6 mg of PTX, 527 mg Cremophor EL and 49.7% (v/v) ethanol per mL) with tyrode buffer to yield a final concentration of either 0.5 or 1 mg/mL PTX. Solutions were doped with 100 µL of 3H PTX prior to dilution. Figure legends refer to commercial formulations as PTX + Cremophor EL + EtOH. However, in the text, the diluted commercial formulation may also be referred to as the control commercial formulation of PTX. DTX was prepared in Tween 80 by diluting Taxotere® concentrated solution (containing 40 mg of DTX and 1040 mg of Tween 80 per mL) with tyrode buffer to yield a final concentration of either 0.5 or 1 mg/mL DTX. The ethanol diluent was not used in these studies. The control formulation was the same as used in a recent phase I trial with intravesical DTX, where the drug was reconstituted in polysorbate 80 (without the use of ethanol) then diluted with 0.9% saline (McKiernan et al., 2006). Solutions were doped with 100 µL of 3H DTX prior to dilution. In figures, Taxotere® is referred to as DTX + Tween 80 and in the text, may also be referred to as control commercial formulation of DTX. 3.5 Micelle Characterization 3.5.1 Particle size analysis The particle size of the copolymer micelles was analyzed by light scattering measurements on a Malvern Zetasizer Nano ZS. Measurements were performed at 25°C on copolymer samples of 5 and 10 mg/mL, with and without drug, in pH 7.4 tyrode buffer. MePEG‐PDLLA micelles without drug were prepared by the direct dissolution method due 48  to the relative solubility of the copolymer. Micelles with loaded drug were prepared by the solvent evaporation method as described above. Samples were run in triplicate. 3.5.2 Preparation and loading of MePEG­PCL19 micelles MePEG‐PCL19 micelles were prepared by Dr. Kevin Letchford using the nanoprecipitation and dialysis technique. The final copolymer concentration of 5% w/v was chosen to match the concentration of diblock, MePEG‐PDLLA, used in our previous efficacy studies with the orthotopic mouse model (Hadaschik et al., 2008). Stock solutions of copolymer and PTX were prepared in N,N‐dimethylformamide (DMF) at 300 mg/mL and 12 mg/mL. Several 0.5 mL solutions of copolymer at 150 mg/mL in DMF were prepared with increasing amounts of PTX. The copolymer/drug solutions of 0.5 mL were added drop‐wise to 1 mL of rapidly stirring phosphate buffered saline (PBS) (pH 7.4). The DMF was removed from the solution by dialysis in PBS overnight using 3500 MWCO Spectra/Por® dialysis membranes. After dialysis, an aliquot of 50 µL of dialysate was dried under a stream of nitrogen gas and reconstituted in 1 mL 60:40 ACN:H2O. Each solution was analyzed for PTX content by HPLC. Standards of 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μg/mL PTX in 60:40 ACN:H2O were prepared by serial dilution. Standards were analyzed by HPLC and the amount of PTX in each sample was quantified. The loading efficiency of the micelles was calculated as:  Loading efficiency (%) =  [ PTX ]so lub ilized × 100 [ PTX ]added  Equation 3  3.5.3 Preparation and loading of MePEG­PCL104 nanospheres Nanospheres of MePEG‐PCL104 were prepared by Dr. Kevin Letchford using the nanoprecipitation and dialysis technique as described in his previous work (Letchford et al., 2009). The final copolymer concentration was 1.2% w/v. Stock solutions of copolymer and PTX were prepared in DMF at 120 mg/mL and 4 mg/mL. Several 0.5 mL solutions of copolymer at 36 mg/mL in DMF were prepared with increasing amounts of PTX. The copolymer/drug solutions of 0.5 mL were added drop‐wise to 1 mL of rapidly stirring PBS (pH 7.4). The DMF was removed from the solution by dialysis in PBS overnight using 3500 MWCO Spectra/Por® dialysis membranes. After dialysis, an aliquot of 50 µL of dialysate 49  was dried under a stream of nitrogen gas and reconstituted in 1 mL 60:40 ACN:H2O. Each solution was analyzed for PTX content by HPLC. Standards of 100, 50, 25, 12.5, 6.25, 3.125, and 1.56 μg/mL PTX in 60:40 ACN:H2O were prepared by serial dilution. Standards were run by HPLC and the amount of PTX in each sample was quantified. The loading efficiency of the nanospheres was determined using equation 3 as previously described in section 3.5.2. 3.5.4 In vitro drug release of PTX and DTX from MePEG­PDLLA micelles MePEG‐PDLLA micelles were loaded with PTX and DTX at 0.5 and 1 mg/mL drug concentrations as described above and where appropriate, a 20 μL aliquot of 3H PTX or 3H DTX was added to the stock solutions. A control of cold PTX or DTX in ethanol, adjusted to 1 μg/mL in tyrode buffer, a concentration equivalent to the aqueous solubility of PTX, was also prepared and spiked with 20 μL of 3H PTX and 3H DTX. Solutions (2 mL) were added to 3500 MWCO Spectra/Por® dialysis bags and transferred into bottles containing 500 mL of tyrode buffer (pH 7.4) at 37°C shaking at 75 rpm. At time points 0, 1, 2, 4, 8, 12, 24, 48, 96 and 168 hours, a 10 μL aliquot was taken from each dialysis bag and measured for remaining 3H PTX or 3H DTX by liquid scintillation counting. At each time point the release media was exchanged with fresh tyrode buffer. The data were presented as cumulative percentage of drug released as a function of time. The cumulative percent drug release was calculated as:  Cumulative Drug Release (%) =  Druginitial − Drugremaining Druginitail  × 100  Equation 4  Where Druginitial is the amount of drug in the dialysis bag at the beginning of the experiment and Drugremaining is the amount of drug remaining in the dialysis bag at given sampling times.  3.5.5 Evaluation of mucoadhesive properties of MePEG­PDLLA micelles To evaluate the mucoadhesive properties of MePEG‐PDLLA copolymers, a series of turbidity and particle size measurements were performed on mucin mixed with varying amounts of polymer solution by adapting previously published methods (Sogias et al., 50  2008; Thongborisute and Takeuchi, 2008). The turbidity of the solution was measured in order to assess the interaction between the polymer and mucin. A mucin suspension was prepared at a concentration of 1% w/v by stirring mucin particles in 100 mM acetate buffer at pH 4.4, overnight. The suspension was sonicated at 0.4 watts for 10 min to fully disperse the particles and then centrifuged at 18,000 xg for 15 min. The supernatant was filtered twice through a 0.22 μm filter and collected for experimental use. A 0.1 % w/v solution of MePEG‐PDLLA was prepared by dissolving 10 mg of polymer in 10 mL of 100 mM acetate buffer. Solutions of 0.1% w/v solutions of MePEG‐PCL19 and MePEG‐PCL104 were prepared by first dissolving 300 mg of MePEG‐PCL19 in 2 mL of DMF and 240 mg of MePEG‐PCL104 in 2mL of DMF. Then, 20 μL of MePEG‐PCL19 and 25 μL of MePEG‐PCL104 in DMF were added drop wise to 2.98 mL and 2.975 mL of rapidly stirring 100 mM acetate buffer. The DMF was removed from the solution by dialysis in acetate buffer overnight using 3500 MWCO Spectra/Por® dialysis membranes. Positive and negative control solutions of chitosan and poly(vinyl alcohol) (PVA) were prepared at 0.1% w/v in 100 mM acetate buffer. In a 96 well plate, mucin solution was mixed with polymer solutions at various volume ratios of 100/0, 90/10, 80/20, 70/30, 60/40 and 50/50 (mucin/polymer). Changes in absorbance were measured at 540 nm. Changes in particle size due to aggregation of mucin with adhesive polymers were measured by dynamic light scattering on a Malvern 3000HS Zetasizer. Mucin solution (as described above) was mixed with polymer solutions of chitosan, PVA, MePEG‐PDLLA, MePEG‐PCL19 and MePEG‐PCL104 and HPG‐C10‐MePEG at a volume ratio of 70/30 (mucin/polymer) in 100 mM of pH 4.4 acetate buffer. Studies were performed in triplicate and chitosan and PVA were used as positive and negative control polymers. 3.6 Bladder Tissue Distribution 3.6.1 Preparation of PTX and DTX loaded nanoparticulate formulations MePEG‐PDLLA micelles loaded with 0.5 and 1 mg/mL PTX and DTX were prepared via the solvent evaporation technique as described above, except that 100 μL of either 3H PTX or 3H DTX were added prior to setting of the drug/copolymer matrix. The resulting polymer/drug matrix was reconstituted so that the final copolymer concentration was 5 and 10 mg/mL. In bladder tissue distribution studies comparing MePEG‐PCL copolymers to MePEG‐PDLLA copolymers, MePEG‐PDLLA was prepared at 1% w/v instead of 0.5% w/v 51  with 0.5 mg/mL PTX such that the copolymer concentration was closer to the copolymer concentrations of 5% w/v and 1.2% w/v used to prepare MePEG‐PCL19 and MePEG‐PCL104 nanoparticles. Nanoparticles of MePEG‐PCL were prepared by Dr. Kevin Letchford using the nanoprecipitation and dialysis technique. Briefly, four 0.5 mL solutions each of MePEG‐ PCL19 and MePEG‐PCL104 were prepared at concentrations of 20% w/v and 5% w/v in DMF with PTX at 2 mg/mL and spiked with 25 μL 3H PTX. The 0.5 mL copolymer/drug solutions were added drop‐wise to 0.75 mL of rapidly stirring tyrode buffer (pH 7.4) in each of four glass vials. The contents of each vial was dialyzed in tyrode buffer overnight using 3500 MWCO Spectra/Por® dialysis membranes. The dialysates from each of the four samples were pooled and diluted with tyrode buffer such that the final copolymer concentration was 5% w/v and 1.2% w/v and the final drug concentration was 0.5 mg/mL. Derivatized hyperbranched polyglycerols were synthesized by Irina Chafeeva from Dr. Brooks’ lab. HPG‐C10‐MePEG loaded with DTX was prepared at 1 mg/mL via the solvent evaporation technique. Briefly, 500 mg of HPG‐C10‐MePEG and 5 mg of DTX, were dissolved in 1 mL ACN, spiked with 100 μL of 3H DTX and dried in an oven at 60°C. The resulting matrix was hydrated with 5 mL of 60°C tyrode buffer (pH 7.4) and vortexed for 2 min. 3.6.2 Tissue preparation Freshly excised porcine urinary pig bladders were obtained from the slaughterhouse (Britco Inc., Langley, BC) and transported to the laboratory in tyrode buffer at 4 °C. Excess adipose tissue on the exterior wall of the bladders was removed and opened longitudinally into left and right lateral sides and cut into pieces approximately 2 cm x 2 cm in a shallow bath of 37 °C tyrode buffer bubbled with carbogen (95% O2 / 5% CO2). Studies were performed within 5 h after sacrifice. Bladder pieces were mounted onto a Franz diffusion cell apparatus (Figure 12), such that the luminal side of the bladder wall was exposed to the drug solution. Receptor chambers were filled with 10 mL of 37 °C tyrode buffer (pH 7.4). Excess tissue was trimmed around the perimeter of the diffusion cell. The donor chamber of the diffusion cell was filled with 1 mL of either 0.5 or 1 mg/mL drug formulations and the tissue exposure area was 0.64 cm2. Each diffusion cell was set into a shallow water bath and incubated at 37 °C for 2 hours, with the exception of time dependent studies where tissues were incubated for 0.5, 1, 2 and 3 hours. Following 52  incubation, the donor and receptor solutions were collected and stored at –20 °C for further analysis. Tissue samples were washed three times with tyrode buffer to remove all unbound drug and wash solutions were collected and stored at ‐20 °C. Tissue samples were trimmed to remove area of tissue compressed from clamping by the diffusion cell and rapidly frozen on metal plates with liquid nitrogen on a bed of dry ice.  Figure 12  Diffusion cell apparatus. Fresh bladder tissue mounted between donor and receptor chambers with luminal side of the bladder wall exposed to the drug solution.  53  3.6.3 Cryotome sectioning of tissue Frozen tissue samples were sectioned by cryostat. The frozen tissue was glued onto the cryotome object holder with Shandon CryomatrixTM, such that the outer surface of the serosa was anchored onto the cryotome object holder and the urothelial surface was exposed for sectioning. Tissues were sectioned into 60 μm thickness. Tissues between 60 and 240 µm (urothelium) were collected individually for analysis. Two tissue sections between 240 and 1260 μm (lamina propria) were collected and pooled for analysis. Three tissue sections between 1260 and 2160 or 3060 μm (muscle layer) were collected and pooled for analysis. Tissue sections were placed in pre‐weighed 1.5 mL eppendorf tubes and stored frozen at ‐20°C. 3.6.4 Quantification of drug in tissue To the weighed tissue slices, 200 µL of ACN was added for drug extraction. Samples were vortexed until all tissue slices were freely submerged in ACN and shaken for 24 h at room temperature to ensure complete extraction of drug. The samples (including tissue slices) were transferred to scintillation vials and 5 mL of scintillation fluid was added. Counts of 3H PTX and 3H DTX were measured by liquid scintillation counting and quantitated using calibration curves from the original stock solution. 3.6.5 Analysis of tissue level­depth profiles The tissue level‐depth profiles were analyzed for average PTX and DTX concentrations in the urothelium, lamina propria, muscularis and whole bladder tissue. The average tissue levels were determined as the total amount of drug found in the tissue layer divided by the total tissue weight for that layer. The area under the tissue‐level depth profile was calculated using the linear trapezoid rule, as follows: (ti +1 − ti ) µ gi µ m × (C1 + Ci +1 ) = 2 g i=0  n −1  AUC0t = ∑  Where, t is tissue depth in μm and C is concentration in μg/g An estimation by extrapolation of the drug concentration (μg/g) to 0 μm was performed in order to calculate the AUC from 0‐2160 μm. 54  3.6.6 Drug recovery The amounts of drug in the donor, receptor and tissue wash solutions were also determined by liquid scintillation counting. Receptor solutions (10 mL) collected from diffusion cells were treated with 1 mL of dichoromethane (DCM), shaken and centrifuged for 10 min at 250 xg. The supernatant was aspirated and the organic phase containing drug was dried under nitrogen gas and reconstituted in 1 mL 60:40 ACN:H2O (v/v) and counted using a Beckman Scintillation Counter. Aliquots of 20 μL of donor solution and 100 – 200 μL of tissue wash solutions were also analyzed using scintillation counting. Total drug recovery was accounted for by summing the amounts of drug found in the tissue, donor, receptor and tissue wash fractions. 3.6.7 PTX metabolites in tissue and stability of tritium label Frozen tissue samples from previous studies where bladders were treated with 3H PTX were sectioned into 60 μm sections by cryostat. Tissue slices were collected and pooled (35‐50 slices) into 2 mL eppendorf tubes. To each tube 750 μL of ACN was added and the tissues were homogenized for 2 min. The blade was washed with 500 μL of ACN and collected for further analysis. Each tube was centrifuged at 18,000 xg for 15 min. Supernatants were collected and pooled into a 20 mL glass scintillation vial and dried with nitrogen gas to 500 μL. Standards of 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.75 and 0.43 μg/mL PTX in ACN were prepared by serial dilution. Standards and sample solutions were run by HPLC. The tissue‐extracted samples were run for 60 min. As sample solutions eluted from the column, 1 mL fractions were collected every minute and analyzed by liquid scintillation counting. 3.7 Tissue Viability 3.7.1 Lactate dehydrogenase assay Freshly excised porcine urinary pig bladders obtained from the slaughterhouse (Britco Inc., Langley, BC) were cut into pieces approximately 2 cm x 2 cm in a shallow bath of 37°C tyrode buffer bubbled with carbogen (95% O2 / 5% CO2) and mounted onto a Franz diffusion cell apparatus such that the luminal surface was exposed to 1 mL of tyrode buffer or 2% Triton X‐100. Receptor chambers were filled with 10 mL of 37 °C tyrode buffer to 55  keep the tissue hydrated. Diffusion cells were incubated at 37°C for 0, 0.5, 1, 3, 5, 8 and 24 hours. At each time point, the donor chamber solution was collected for analysis. Donor solutions were centrifuged at 18,000 xg for 10 min. The supernatants were collected and diluted 10x with tyrode buffer. In a 96‐well plate, 100 μL of the diluted supernatant was mixed with 100 μL of the diaphorase catalyst and tetrazolium salt reaction mixture from the assay kit. LDH release was measured by UV‐Vis spectroscopy at 492 nm together with a reference wavelength at 620 nm. 3.7.2 Mannitol paracellular permeability assay A 12 μL aliquot of 3H mannitol was added to a 1 mL sample of tyrode buffer. As a positive control, 0.2% Triton X‐100 solution was prepared and 6 μL of 3H mannitol was added. The luminal surfaces of freshly excised bladder pieces were exposed to 500 μL of tyrode buffer and 0.2% Triton X‐100 solution at 37°C for 3 and 24 hours on a Franz diffusion cell apparatus. Tissue samples were trimmed as described in section 3.6.2 and rapidly frozen on metal plates with liquid nitrogen. Tissues were sectioned by cryostat into 60 μm thickness. 3H mannitol was extracted from tissue slices with 100 μL ethanol in 1.5 mL eppendorf tubes for 24 h and measured by liquid scintillation counting. 3.8 Toxicity of Nanoparticulate formulations on Bladder Tissue 3.8.1 Preparation of formulations MePEG‐PDLLA micelles were prepared by dissolving copolymer in tyrode buffer to concentrations of 5 and 10 mg/mL, equivalent to concentrations of copolymer used to prepared micelles loaded with 0.5 and 1 mg/mL PTX and DTX. Cremophor EL micelles were prepared by direct dissolution as a 50:50 mixture of Cremophor EL and ethanol in tyrode buffer. Samples contained either 4% w/v or 8% w/v Cremophor EL and ethanol. Tween 80 micelles were also prepared by direct dissolution in tyrode buffer, but at concentrations of 1.25 and 2.5% w/v. MePEG‐PCL19 and MePEG‐PCL104 nanoparticles were prepared by the nanoprecipitation and dialysis technique because of the relatively poor solubility of the polymers. Copolymer solutions were prepared in DMF at concentrations of 150 mg/mL and 120 mg/mL. Briefly, 1 mL and 0.3 mL of each solution was added drop‐ wise to 2 mL and 2.7 mL of rapidly stirring tyrode buffer (pH 7.4). The DMF was removed from the solution by dialysis in tyrode buffer overnight using 3500 MWCO Spectra/Por® dialysis membranes. The final copolymer concentrations of MePEG‐PCL19 and MePEG‐ 56  PCL104 were 5% w/v and 1.2% w/v. HPG‐C10‐MePEG of 100 mg/mL was prepared by dissolving the polymer directly in tyrode buffer (pH 7.4). 3.8.2 Lactate dehydrogenase assay Fresh bladder pieces approximately 2 cm x 2 cm were mounted onto a Franz diffusion cell apparatus. The luminal surface of the bladder tissue was exposed to solutions of MePEG‐PDLLA, Cremophor EL and ethanol, Tween 80, MePEG‐PCL19 and MePEG‐PCL104 and HPG‐C10‐MePEG of various concentrations equivalent to those used in drug loaded formulations. The diffusion cells were incubated at 37°C for 2 hours. Donor solutions were collected and centrifuged at 18,000 xg for 10 min. The supernatant of the donor solutions were diluted 20x with tyrode buffer. In a 96‐well plate, 100 μL of diluted supernatant was mixed with 100 μL of the diaphorase catalyst and tetrazolium salt reaction mixture from the assay kit. LDH release was measured by UV‐Vis spectroscopy at 492 nm together with a reference wavelength at 620 nm. 3.8.3 Mannitol paracellular permeability assay The penetration of 14C mannitol into bladder tissue was determined in the presence of Cremophor EL, Tween 80 and MePEG‐PDLLA micelles. MePEG‐PDLLA micelles were prepared by direct dissolution of the copolymer in tyrode buffer to a final concentration of 10 mg/mL and 35 μL of 14C mannitol was added. Cremophor EL and Tween 80 micelles were prepared by dissolving 8% w/v Cremophor EL and ethanol and 2.5% w/v Tween 80 in tyrode buffer and adding 35 μL of 14C mannitol. A 30 μL aliquot of 14C mannitol was added to a 3 mL sample of tyrode buffer (negative control). As a positive control, 0.2% Triton X‐100 solution was prepared and 30 μL of 14C mannitol was added. The luminal surfaces of freshly excised bladder pieces were exposed to 1 mL of formulations or 0.2% Triton X‐100 solution (with mannitol) at 37°C for 2 hours in a Franz diffusion cell apparatus. Tissue samples were trimmed as described in section 3.6.2 and rapidly frozen on metal plates with liquid nitrogen. Tissues were sectioned by cryostat into 60 μm thickness. 14C mannitol was extracted from tissue slices with 100 μL ethanol in eppendorf tubes shaking for 24 h and measured by liquid scintillation counting.  57  3.9 Statistical Analysis Data collected are reported as the mean ± standard deviation, where n represents the size of the sample. For example, for tissue level versus depth profile experiments showing n = 18, each replicate for each treatment group used a piece of tissue from 18 bladders. The first trial was performed by mounting a piece of tissue from 6 bladders onto one of the 6 diffusion cells for each treatment group, followed by two addition trials each using 6 new freshly excised bladders. Statistical analysis was performed using GraphPad Prism version 4.0b (GraphPad Software, La Jolla, CA) with a significance level of p<0.05. The two‐tailed unpaired t‐test was used for comparisons between two groups with a significance level of α < 0.05 considered to be statistically significant and marked with an asterisk (*). For comparisons between three or more groups, the results were analyzed for statistical significance using a one‐way ANOVA. Differences were considered significant at p < 0.05. A Bonferroni or a Tukey‐Kramer post hoc test was performed when a difference was detected.  58  4  RESULTS  4.1 Characterization of Nanoparticulate formulations 4.1.1 Particle size and release profiles for MePEG­PDLLA micelles MePEG‐PDLLA micelles are routinely prepared in our lab and the physicochemical properties are well established (Zhang et al., 1996; Burt et al., 1999; Liggins and Burt, 2002). PTX can be loaded up to a maximum of 25 % w/w in MePEG‐PDLLA. Micelles of this diblock copolymer have demonstrated low critical micelle concentrations and good biocompatibility with no evident animal toxicity. The aqueous solubility of PTX can be increased up to 5000‐fold (5 mg/mL) and remain stable in solution up to 24 h (Zhang et al., 1996; Burt et al., 1999). In this work, other than particle size analysis and in vitro release studies no further characterization was performed. The hydrodynamic diameters of MePEG‐PDLLA micelles at both 5 and 10 mg/mL, as determined by dynamic light scattering, were 20.1 ± 0.3 nm and 19.1 ± 0.2 nm, respectively. These micelles had monodispersed distributions (Figure 13). Upon loading of PTX into the micelles, the hydrodynamic diameter of the copolymers did not change. Similarly, the hydrodynamic diameter of both 5 and 10 mg/mL MePEG‐PDLLA micelles did not change upon DTX loading and were 18.0 ± 0.2 nm and 17.9 ± 0.3 nm, respectively. The in vitro release profiles of PTX and DTX from MePEG‐PDLLA micelles using the dialysis method are shown in Figures 14A and 14B. Free PTX and DTX were released to completion by 24 hours, demonstrating the lack of interference of the dialysis membrane on the release of the drugs. In general, DTX was released more rapidly from MePEG‐PDLLA micelles than PTX. Release profiles of PTX from micelles were characterized by an initial burst for the first 12 hours followed by a slower release for the remaining 6.5 days. After 2 hours, MePEG‐PDLLA micelles of 0.5 and 1 mg/mL PTX or DTX released approximately 10% of the drug. PTX loaded micelles of both 0.5 and 1 mg/mL released all of the drug by 7 days and DTX loaded micelles released all loaded drug by 4 days.  59  A  B  Figure 13  A representative size distribution plot of A. 5 mg/mL MePEG‐PDLLA micelles and B. 0.5 mg/mL PTX in 5 mg/mL MePEG‐PDLLA micelles. Measurements by dynamic light scattering at 25 °C in tyrode buffer. 60  A 110 100  Free PTX  90  0.5mg/mL MePEG-PDLLA PTX  80  1mg/mL MePEG-PDLLA PTX  70 60 50 40 30 20 10 0 0  1  2  3  4  5  6  7  8  Time (d) B 110 100  Free DTX  90  0.5mg/mL MePEG-PDLLA DTX  80  1mg/mL MePEG-PDLLA DTX  70 60 50 40 30 20 10 0 0  1  2  3  4  5  6  7  8  Time (d)  Figure 14  Release of A. PTX and B. DTX from polymeric micelles of MePEG‐PDLLA at 0.5 mg/mL (▲) and 1 mg/mL (▼) into pH 7.4 tyrode buffer at 37 °C. The movement of free drug (■) out of the dialysis bag is also shown. Data are mean ± SD (n = 4). 61  4.1.2 PTX loading in MePEG­PCL nanoparticles The MePEG‐PCL19 micelles solubilized PTX up to 1.0 mg/mL with approximately an 87% loading efficiency (Figure 15). However after 48 h, there was evidence of physical instability as the formulation became cloudy due to PTX precipitation. MePEG‐PCL104 loaded nanospheres could only be prepared by the nanoprecipitation and dialysis technique because of the poor aqueous solubility of the polymer. The MePEG‐ PCL104 nanospheres were able to solubilize PTX up to approximately 0.5 mg/mL with an 72% loading efficiency with much less polymer (1.2 % w/v) than MePEG‐PCL19 micelles (5 % w/v) (Figure 16).  1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.00  100 80 60 40 20  0.25  0.50  0.75  1.00  1.25  0 1.50  [PTX]added mg/mL Loading Efficiency (%) Figure 15  [PTX]solubilized mg/mL  Solubilization of PTX by MePEG‐PCL19 micelles formed by nanoprecipitation and dialysis of copolymer and drug in DMF solutions. Micelles were formed in PBS (pH 7.4) with final copolymer concentrations of 5 % w/v. [PTX] solubilized (▲), Loading efficiency (☐).  62  1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.00  100 80 60 40 20  0.25  0.50  0.75  0 1.00  [PTX]added mg/mL Loading Efficiency (%) Figure 16  [PTX]solubilized mg/mL  Solubilization of PTX by MePEG‐PCL104 micelles formed by nanoprecipitation and dialysis of copolymer and drug in DMF solutions. Micelles were formed in PBS (pH 7.4) with final copolymer concentrations of 1.2 % w/v. [PTX] solubilized (▲), Loading efficiency (☐).  63  4.1.3 Mucoadhesive properties of polymeric nanoparticles The mucoadhesive interaction between polymer and mucin was examined by changes in turbidity and particle size, which are related to the aggregation of mucin particles in the presence of small portions of polymer. These changes were measured by absorbance (Figure 17) and light scattering particle size determinations (Figure 18). Chitosan is well known to be a mucoadhesive polymer and serves as a positive control in these studies. The addition of chitosan to mucin resulted in an increase in solution turbidity until mucin/polymer volume ratios greater than 70/30 when the turbidity decreased with further polymer addition. No change in the absorbance of MePEG‐PDLLA, HPG‐C10‐MePEG, MePEG‐PCL19 and MePEG‐PCL104 was observed in the presence of mucin and absorbance values were the same as solutions of PVA. The particle size of mucin remained constant after mixing with MePEG‐PDLLA, HPG‐C10‐MePEG, MePEG‐PCL19 or MePEG‐PCL104. However, in the presence of chitosan, mucin particles formed larger aggregates whose size exceeded 1200 nm.  64  0.6  Mucin PVA Chitosan MePEG‐PCL19 MePEG‐PCL104 HPG‐C10‐MePEG MePEG‐PDLLA  Absorbance (540 nm)  0.5  0.4  0.3  0.2  0.1  0 100/0  90/10  80/20  70/30  60/40  50/50  Mucin/Polymer vol. ratios  Figure 17  Turbidity measurements for varying volume ratios of 0.1% w/v polymer and 1% w/v mucin dispersions in acetate buffer at pH 4.4. Data are mean turbidity ± SD (n = 3).  65  1800  *  1600  Particle Size (nm)  1400 1200 1000 800 600 400 200 0  Figure 18  Changes in particle size of 1% w/v mucin when mixed with 0.1% w/v polymer solutions. Values are means ± SD (n = 3). * p < 0.05  66  4.2 Bladder Tissue Distribution of PTX and DTX Loaded Nanoparticles 4.2.1 PTX and DTX analysis by liquid scintillation counting In order to quantify the amount of drug in the bladder tissue, a series of standards (0.025 ‐ 5 μg/mL) were prepared from stock solutions of 0.5 and 1 mg/mL of drug. A fresh set of standards were prepared for each experiment. A linear relationship between the concentration of drug and the radioactivity (CPM) was established. Figure 19 shows representative standard curves used to quantify the amount of drug in bladder tissue. Using linear regression analysis, the coefficient of determination (R2) values were typically >0.99.  25000  PTX + Cremophor EL + EtOH MePEG‐PDLLA PTX DTX + Tween 80 MePEG‐PDLLA DTX  20000  y = 3427x ‐ 361.79 R² = 0.99827 y = 3245.1x ‐ 296.66 R² = 0.99689  15000 ³H CPM  y = 2513.8x ‐ 240.68 R² = 0.99707 y = 2316.3x ‐ 311.2 R² = 0.9931  10000  5000  0 0  1  2  3  4  5  6  Concentration (μg/mL)  Figure 19  Standard curves for 0.5 mg/mL PTX and DTX prepared from MePEG‐PDLLA (PTX and DTX), Cremophor EL + EtOH (PTX) or Tween 80 (DTX) based formulations. Each curve was repeated three times using freshly made formulations and values are means ± SD (n = 3).  67  4.2.2 Effect of concentration of drug on bladder tissue uptake Both drugs were used at concentrations of 0.5 and 1 mg/mL because previously completed in vivo efficacy studies using PTX and DTX in a mouse model of bladder cancer were performed with these concentrations (Mugabe et al., 2009). Figures 20A and 20B show the concentration of PTX in bladder tissue as a function of tissue depth following exposure to 0.5 and 1 mg/mL micellar solutions. Concentrations of PTX decreased with increasing tissue depth. PTX loaded into MePEG‐PDLLA micelles showed greater bladder tissue uptake compared to PTX in Cremophor and EtOH. However, there was no difference in tissue uptake levels observed between the 0.5 and 1 mg/mL PTX formulations. Figure 21 shows the average tissue levels of PTX within each layer of the bladder wall including the entire tissue (60‐2160 μm). The average concentration of PTX taken up into the bladder wall from the MePEG‐PDLLA micellar formulation was significantly greater than for the Cremophor EL and EtOH formulation. In the different tissue sections the values for tissue levels following exposure to 0.5 and 1 mg/mL diblock micellar PTX, respectively, were as follows: 44.1 and 48.3 μg/g (urothelium), 26.4 and 29.7 μg/g (lamina propria) and 17.2 and 17.4 μg/g (superficial muscle). Much lower values were obtained for the same layers after exposure to 0.5 and 1 mg/mL Cremophor EL and EtOH solutions: 16.3 and 16.2 μg/g (urothelium), 10.1 and 10.0 μg/g (lamina propria) and 5.0 and 5.3 μg/g (superficial muscle). Figure 22 shows the AUCs for PTX calculated using the linear trapezoid rule for the urothelium (0‐240 μm), the lamina propria (240‐1260 μm) and the muscle tissue (1260‐2160 μm). The AUC for the whole tissue is designated 0‐2160 μm. AUCs give a measure of drug “exposure” and it is clear that much greater PTX exposure is achieved using MePEG‐PDLLA micelles (Figure 22). Tissue levels of DTX within each layer of the bladder wall following incubation with DTX in MePEG‐PDLLA micelles or Tween 80 (at either 0.5 or 1 mg/mL) are shown in Figures 23A and 23B. For both formulations, the penetration of DTX decreased with increasing tissue depth. Although DTX formulated in MePEG‐PDLLA micelles penetrated tissue in significantly higher concentrations than for the drug in Tween 80 solutions, there was no significant difference observed between DTX concentrations of 0.5 and 1 mg/mL. In Figure 24, the average tissue levels in the different tissue layers following exposure to 0.5 and 1 mg/mL DTX respectively were as follows: 81.9 and 68.5 μg/g (urothelium), 48.9 and 42.6 μg/g (lamina propria) and 29.1 and 28.6 μg/g (superficial muscle). Much lower 68  values were obtained for the same layers after exposure to 0.5 mg/mL and 1 mg/mL DTX in Tween 80 solutions: 44.1 and 36.3 μg/g (urothelium), 26.4 and 23.5 μg/g (lamina propria) and 17.2 and 15.8 μg/g (superficial muscle). In Figure 25, AUCs are shown for DTX calculated for each tissue layer as described above. Figures 21 and 24 show the average tissue levels of the two drugs in the urothelium, lamina propria and superficial muscle layers. At both 0.5 mg/mL and 1 mg/mL incubation concentrations, DTX was taken up into the bladder wall in greater amounts than PTX as shown by comparing tissue levels in Figures 21 and 24. All components of the diffusion cell apparatus including donor, receptor, washes and tissue were analyzed for drug content. Mass balance analysis showed that the drugs in both the 0.5 and 1 mg/mL PTX and DTX diffusion experiments could be almost fully recovered (Figure 26A and 26B). The majority of drug was recovered in the donor chamber, less than 3% in the washes and less than 0.04% in the receptor solutions. Between 0.3 ‐ 0.5 % of the initial dose was recovered in bladder tissue treated with 0.5 and 1 mg/mL PTX loaded MePEG‐PDLLA micelles, whereas between 0.1 – 0.2 % of the initial dose was recovered in bladder tissue treated with 0.5 and 1 mg/mL PTX in Cremophor EL and EtOH. Between 0.5 ‐ 1% of the initial dose was recovered in bladder tissue treated with 0.5 and 1 mg/mL DTX loaded MePEG‐PDLLA micelles, whereas between 0.3 ‐ 0.4 % of the initial dose was recovered in bladder tissue treated with 0.5 and 1 mg/mL DTX in Tween 80.  69  A Tissue levels (μg of PTX/g of tissue)  Mus  Lam  Uro 90  0.5mg/mL MePEG‐PDLLA PTX  80  0.5mg/mL PTX + Cremophor EL + EtOH  70 60 50 40 30 20 10 0 0  200  400  600  800 1000 1200 1400 1600 1800 2000 2200 Tissue depth (μm)  Tissue levels (μg of PTX/g of tissue)  B  Mus  Lam  Uro 90  1mg/mL MePEG‐PDLLA PTX  80  1mg/mL PTX + Cremophor EL + EtOH  70 60 50 40 30 20 10 0 0  200  400  600  800 1000 1200 1400 1600 1800 2000 2200 Tissue depth (μm)  Figure 20  Tissue level‐depth profiles of PTX in bladder tissue following exposure to A. 0.5 mg/ml PTX in MePEG‐PDLLA micelles (●) and 0.5mg/ml PTX in Cremophor EL and EtOH (▲) and B. 1mg/ml PTX in MePEG‐PDLLA micelles (♦) and 1 mg/ml PTX in Cremophor EL and EtOH (■). Tissues were incubated for 2 h and sectioned at 60 μm thickness. Uro, Lam and Mus refer to the urothelium, lamina propria and superficial muscle layers. Values are means ± SD (n = 18). 70  A  Average tissue levels (μg of PTX/g of tissue)  80 70  1mg/mL MePEG‐PDLLA PTX  ***  0.5mg/mL MePEG‐PDLLA PTX 1mg/mL PTX + Cremophor + EtOH  ***  0.5mg/mL PTX + Cremophor EL + EtOH  60 50  *** ***  40  ***  ***  *** ***  30 20 10 0 60‐240 μm  Figure 21  240‐1260 μm  1260‐2160 μm  60‐2160 μm  Average tissue levels of PTX in various layers of the bladder wall (60‐240 μm: urothelium, 240‐1260 μm: the lamina propria, 1260‐2160 μm: the superficial muscle layer and 60‐2160 μm: the whole tissue) following incubation with PTX at either 0.5 mg/ml or 1 mg/ml from either MePEG‐PDLLA or Cremophor EL and EtOH formulations for 2 hours. The average tissue levels were determined as the total amount of drug found in the tissue layer divided by the total tissue weight for that layer. Data are means ± SD (n = 18). *** p < 0.001, 0.5 mg/mL MePEG‐PDLLA PTX vs. 0.5 mg/mL PTX + Cremophor EL + EtOH and 1 mg/mL MePEG‐PDLLA PTX vs. 1 mg/mL PTX + Cremophor EL + EtOH  71  B ***  80000  1mg/mL MePEG‐PDLLA PTX  ***  0.5mg/mL MePEG‐PDLLA PTX  70000  1mg/mL PTX + Cremophor EL + EtOH  AUC (μg•μm/g)  60000  0.5mg/mL PTX + Cremophor EL + EtOH  ***  50000  *** 40000 30000 20000  ***  *** *** ***  10000 0 0‐240 μm  Figure 22  240‐1260 μm  1260‐2160 μm  0‐2160 μm  AUCs of PTX in various tissue layers of the bladder wall (0‐240 μm: urothelium, 240‐1260 μm: the lamina propria, 1260‐2160 μm: the superficial muscle layer and 0‐2160 μm: the whole tissue) following incubation with PTX at either 0.5 mg/ml or 1 mg/ml from either MePEG‐PDLLA or Cremophor EL and EtOH formulations for 2 hours. The AUCs were calculated using the linear trapezoid rule. Data are means ± SD (n = 18). *** p < 0.001, 0.5 mg/mL MePEG‐PDLLA PTX vs. 0.5 mg/mL PTX + Cremophor EL + EtOH and 1 mg/mL MePEG‐PDLLA PTX vs. 1 mg/mL PTX + Cremophor EL + EtOH  72  Tissue levels (μg of DTX/g of tissue)  A  Mus  Lam  Uro 200 180  0.5mg/mL MePEG‐PDLLA DTX  160  0.5mg/mL DTX + Tween 80  140 120 100 80 60 40 20 0 0  200  400  600  800 1000 1200 1400 1600 1800 2000 2200 Tissue depth (μm)  Tissue levels (μg of DTX/g of tissue)  B  Uro  Mus  Lam  160 1mg/mL DTX + Tween 80  140  1mg/mL MePEG‐PDLLA DTX  120 100 80 60 40 20 0 0  200  400  600  800 1000 1200 1400 1600 1800 2000 2200 Tissue depth (μm)  Figure 23  Tissue level‐depth profiles of DTX in bladder tissue following exposure to A. 0.5 mg/ml DTX in MePEG‐PDLLA micelles (●) and 0.5mg/ml DTX in Tween 80 (▲) and B. 1mg/ml DTX in MePEG‐PDLLA micelles (♦) and 1 mg/ml DTX in Tween 80 (■). Tissues were incubated for 2 h and sectioned at 60 μm thickness. Uro, Lam and Mus refer to the urothelium, lamina propria and superficial muscle layers. Values are means ± SD (n = 18). 73  Average tissue levels (μg of DTX/g of tissue)  140 1mg/mL MePEG‐PDLLA DTX  ***  120  0.5mg/mL MePEG‐PDLLA DTX 1mg/mL DTX + Tween 80  ** 100  0.5mg/mL DTX + Tween 80  80  ** *** 60  ** *** **  40  ***  20 0 60‐240 μm  Figure 24  240‐1260 μm  1260‐2160 μm  60‐2160 μm  Average tissue levels of DTX in various layers of the bladder wall (60‐240 μm: urothelium, 240‐1260 μm: the lamina propria, 1260‐2160 μm: the superficial muscle layer and 60‐2160 μm: the whole tissue) following incubation with DTX at either 0.5 mg/ml or 1 mg/ml from either MePEG‐ PDLLA or Tween 80 formulations for 2 hours. The average tissue levels were determined as the total amount of drug found in the tissue layer divided by the total tissue weight for that layer. Data are means ± SD (n = 18). ** p < 0.01, 1 mg/mL MePEG‐PDLLA DTX vs 1 mg/mL DTX + Tween 80 and *** p < 0.001, 0.5 mg/mL MePEG‐PDLLA DTX vs 0.5 mg/mL DTX + Tween 80  74  1mg/mL MePEG‐PDLLA DTX 0.5mg/mL MePEG‐PDLLA DTX 1mg/mL DTX + Tween 80 0.5mg/mL DTX + Tween 80  140000 120000  ** ***  AUC (μg•μm/g)  100000 80000  ** ***  60000  **  40000  **  ***  ***  20000 0 0‐240 μm  Figure 25  240‐1260 μm  1260‐2160 μm  0‐2160 μm  AUCs of DTX in various tissue layers of the bladder wall (0‐240 μm: urothelium, 240‐1260 μm: the lamina propria, 1260‐2160 μm: the superficial muscle layer and 0‐2160 μm: the whole tissue) following incubation with DTX at either 0.5 mg/ml or 1 mg/ml from either MePEG‐ PDLLA or Tween 80 formulations for 2 hours. The AUCs were calculated using the linear trapezoid rule. Data are means ± SD (n = 18). ** p < 0.01, 1 mg/mL MePEG‐PDLLA DTX vs 1 mg/mL DTX + Tween 80 and *** p < 0.001, 0.5 mg/mL MePEG‐PDLLA DTX vs 0.5 mg/mL DTX + Tween 80  75  A  120  0.5mg/mL MePEG-PDLLA PTX 0.5mg/mL MePEG-PDLLA DTX 0.5mg/mL DTX + Tween 80  95  0.5mg/mLPTX+ Cremophor EL+EtOH  70  2.5 2.0 1.5 1.0 0.5 0.0  Donor  Washes  Receptor  Tissue  B 120  1mg/mL MePEG-PDLLA PTX 1mg/mL MePEG-PDLLA DTX 1mg/mL DTX + Tween 80  95  1mg/mL PTX + Cremophor EL+EtOH  70 4 3 2 1 0  Figure 26  Donor  Washes  Receptor  Tissue  Mass balance analysis of drug recovered from donor, washes, receptor and tissue fractions following a 2 h incubation with PTX or DTX at A. 0.5 mg/mL and B. 1 mg/mL PTX and DTX following a 2 h incubation. All portions of the diffusion cell including washes and tissue were analyzed for drug content. Data are means ± SD (n = 18). 76  4.2.3 Effect of incubation time on bladder tissue uptake The effect of incubation times between 30 to 180 min on PTX and DTX uptake into bladder tissues using MePEG‐PDLLA solutions at 1 mg/ml are shown in Figures 27 and 28 respectively. All the uptake profiles determined at each incubation time were similar. Tissue levels decreased with increasing tissue depth and DTX uptake was greater than PTX. For both PTX and DTX, high levels of drug were found in the urothelium layers at 30 minutes with smaller increases after that time. The average concentrations of PTX or DTX that penetrated the bladder wall are shown in the insets of Figures 27 and 28. Tissue concentrations of PTX differed significantly between 180 min and 30 min incubation times. Tissue concentrations of DTX were significantly higher following a 120 min incubation than a 30 and 60 min incubation.  77  Uro  Lam  Mus  140 Average tissue levels (μg of PTX/g of tissue)  Tissue levels (μg of PTX/g of tissue)  130 120 110 100 90 80 70 60  40 35 30 25 20 15 10 5 0  *  0  50  30 60 90 120 150 180 210 Time (min)  40 30 20 10 0 0  250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 Tissue depth (μm)  Figure 27  Tissue level‐depth profile of PTX in bladder tissue from MePEG‐PDLLA micelle formulations (1 mg/ml) following incubation for 30 min (■), 60 min (▲), 120 min (●) and 180 min (◆) at 37°C. Uro, Lam and Mus refer to the urothelium, lamina propria and superficial muscle layers. Inset: The time course of drug uptake into bladder tissue for PTX at 1 mg/mL from MePEG‐ PDLLA micelles. Points show average tissue levels of drug recovered from all bladder tissue. The average tissue levels were determined as the total amount of drug found in the tissue layer divided by the total tissue weight for that layer.Data are expressed as µg of drug per g of tissue and the means ± SD (n = 4). * p < 0.05, PTX 180 min vs. 30 min.  78  Uro  Lam  Mus  240 Average tissue levels (μg of DTX/g of tissue)  220 Tissue levels (μg of DTX/g of tissue)  200 180 160 140 120 100 80  *, **  60 50 40 30 20 10 0 0  30 60 90 120 150 180 210 Time (min)  60 40 20 0 0  250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 Tissue Depth (μm)  Figure 28  Tissue level‐depth profile of DTX in bladder tissue from MePEG‐PDLLA micelle formulations (1 mg/ml) following incubation for 30 min (■), 60 min (▲), 120 min (●) and 180 min (◆) at 37°C. Uro, Lam and Mus refer to the urothelium, lamina propria and superficial muscle layers. Inset: The time course of drug uptake into bladder tissue for DTX at 1 mg/mL from MePEG‐ PDLLA micelles. Points show average tissue levels of drug recovered from all bladder tissue. The average tissue levels were determined as the total amount of drug found in the tissue layer divided by the total tissue weight for that layer.Data are expressed as µg of drug per g of tissue and the means ± SD (n = 4). * p < 0.05, DTX 120 min vs. 60 min. ** p < 0.01, DTX 120 min vs. 30 min.  79  4.2.4 Effect of formulation on bladder tissue uptake The influence of four different nanoparticulate/micellar formulations of PTX on the overall permeation of PTX into the bladder wall is shown in Figure 29. No differences in PTX tissue concentration were observed between MePEG‐PCL19 micelles and MePEG‐ PCL104 nanospheres as both had very similar profiles. The average tissue levels of drug in bladder tissues incubated with PTX at a concentration of 0.5 mg/mL was significantly higher for MePEG‐PDLLA micelle formulations compared to MePEG‐PCL19 micelles and MePEG‐PCL104 nanosphere formulations (Figure 30). No differences in average tissue levels were observed between tissues incubated with MePEG‐PDLLA micelles and Cremophor EL and EtOH, Cremophor EL and EtOH and MePEG‐PCL19 formulations or between MePEG‐ PCL19 micelles and MePEG‐PCL104 nanosphere formulations. The effect of nanoparticle composition on tissue levels of DTX in the bladder wall is shown in Figure 31. Tissue levels of DTX from MePEG‐PDLLA micelles were higher in bladder tissue compared to Tween 80 micelles and HPG‐C10‐MePEG formulations. Concentration‐depth profiles of Tween 80 and HPG‐C10‐MePEG were very similar. The average tissue levels of drug in bladder tissues incubated with DTX at a concentration of 1 mg/mL is shown in Figure 32. The average tissue levels of DTX from MePEG‐PDLLA micelles did not differ from tissue levels of DTX from Tween 80 and HPG‐C10‐MePEG formulations. No difference in the average bladder DTX tissue levels was observed between tissues incubated with Tween 80 and HPG‐C10‐MePEG formulations.  80  Uro  Tissue levels (μg of PTX/g of tissue)  45  Lam  Mus  40 0.5mg/mL MePEG‐PDLLA PTX  35  0.5mg/mL PTX + Cremophor EL + EtOH 0.5mg/mL MePEG‐PCL19 PTX  30  0.5mg/mL MePEG‐PCL104 PTX  25 20 15 10 5 0 0  250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 Tissue depth (μm)  Figure 29  Tissue level‐depth profiles of PTX in bladder tissue following exposure to 0.5 mg/mL PTX in MePEG‐PDLLA micelles (▲), Cremophor EL + EtOH (●), MePEG‐PCL19 micelles (■) and MePEG‐PCL104 nanospheres (♦).Tissues were incubated for 2 h and sectioned at 60 μm thickness. Uro, Lam and Mus refer to the urothelium, lamina propria and superficial muscle layers. Values are means ± SD (n = 6).  81  Average tissue levels (μg of PTX/g of tissue)  14 12 10  **, ***  0.5mg/mL MePEG‐PDLLA PTX 0.5mg/mL PTX+Cremophor EL+EtOH 0.5mg/mL MePEG‐PCL19 PTX 0.5mg/mL MePEG‐PCL104 PTX  8 6 4 2 0  Figure 30  Average tissue levels of 0.5 mg/mL PTX in whole bladder tissue (60‐3060 μm) following a 2 h incubation. The average tissue levels were determined as the total amount of PTX found in the tissue divided by the total tissue weight. Data are means ± SD (n = 6). ** p < 0.01, MePEG‐PDLLA vs. MePEG‐PCL19 and *** p < 0.001, MePEG‐PDLLA vs. MePEG‐PCL 104  82  Tissue levels (μg of DTX/g of tissue)  Uro  170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0  Lam  Mus  1mg/mL MePEG‐PDLLA DTX 1mg/mL HPG‐C10‐PEG DTX 1mg/mL DTX + Tween 80  0  250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 Tissue Depth (μm)  Figure 31  Tissue level‐depth profiles of DTX in bladder tissue following exposure to 1 mg/mL DTX in MePEG‐PDLLA micelles (▲), Tween 80 (●) and HPG‐C10‐ MePEG (■). Tissues were incubated for 2 h and sectioned at 60 μm thickness. Uro, Lam and Mus refer to the urothelium, lamina propria and superficial muscle layers. Values are means ± SD (n = 4).  83  Average tissue levels (μg of DTX/g of tissue)  60 55  1mg/mL MePEG‐PDLLA DTX  50  1mg/mL DTX + Tween 80  45  1mg/mL HPG‐C10‐MePEG  40 35 30 25 20 15 10 5 0  Figure 32  Average tissue levels of 1 mg/mL DTX in whole bladder tissue (60‐3060 μm) following a 2 h incubation. The average tissue levels were determined as the total amount of PTX found in the tissue divided by the total tissue weight. Data are means ± SD (n = 4).  84  4.2.5 Presence of PTX metabolites by HPLC and stability of tritium label In order to determine whether PTX formed any metabolites in bladder tissue within the 2 hour exposure to drug, pooled and extracted (3H PTX spiked) tissue samples were analyzed by HPLC. Aliquots were collected every minute for 60 min as they eluted off the HPLC column. Figure 33 shows the chromatogram from a 60 min HPLC run of PTX extracted from bladder tissue. PTX was measured at 232 nm. The injection front of the standard was eluted at 1.1 min with a peak area of 13,629 μV∙sec followed by the PTX peak at 2.5 min over a 2‐3 min duration with a peak area of 2,302,224 μV∙sec. PTX from the sample was eluted at 1.3 min over a 1‐3 min duration with a peak area of 45,483,418 μV∙sec. No further peaks were observed beyond 1.3 min suggesting no evidence of PTX metabolites. Furthermore, the eluted samples were counted by liquid scintillation counting (Figure 34). The PTX peak at 1.3 min contained high levels of radioactivity, but no significant counts were detected in any other samples, again suggesting that there was no evidence of PTX metabolism detected and that the tritium label remained attached to the drug.  85  1.279  PTX  Figure 33  Chromatogram of PTX extracted from bladder tissue in ACN by HPLC. Mobile phase: 58:37:5 ACN:H2O:methanol, flow rate: 1 mL/min, injection volume 20 μL, detection at 232 nm.  86  9000 8000 7000  3H CPM  6000 5000 4000 3000 2000 1000 0 0  5  10  15  20  25  30  35  40  45  50  55  60  Time (min)  Figure 34  Eluted samples of 3H PTX from HLPC measured by liquid scintillation counting.  87  4.3 Bladder Tissue Viability 4.3.1 Lactate dehydrogenase assay LDH is a cytoplasmic enzyme present in all cells. When the plasma membrane is damaged, LDH is released. Bladder tissue viability was assessed at 37°C in tyrode buffer over a 24 hour time course by a colorimetric assay used to measure LDH release in the culture supernatant. The levels of LDH released from bladder tissue remained relatively constant up to 8 hours after sacrifice (Figure 35). At 8 hours, an increase in LDH levels was observed. At 24 hours, there was a sizable increase in LDH as these levels were significantly higher than 0 ‐ 5 h. However, levels remained well below the LDH released from 2% Triton X‐100 positive control.  Cytotoxicity LDH release (ΔA/min)  0.8  ***  0.7 0.6  **  0.5 0.4  *  0.3 0.2 0.1 0 0  0.5  1  3  5  8  24  2% Triton‐X  Time (h)  Figure 35  Bladder tissue viability following a 0.5, 1, 3, 5, 8 and 24 h incubated at 37°C in tyrode buffer. Values were measured by UV‐Vis spectroscopy and are expressed as the change in absorbance with time, which represent the amount of LDH released into supernatant. Data are means ± SD (n = 9). *p<0.05, 8 h vs. 0 h, **p<0.01, 24 h vs 0, 0.5, 1, 3 and 5 h and ***p<0.001 vs. all groups 88  4.3.2 Mannitol paracellular permeability assay The paracellular permeability of bladder tissue was measured using the tight junction marker, 3H mannitol (Figure 36). The passive paracellular transport of this sugar has been linked to in vitro cytotoxicity (Konsoula and Barile, 2005). Bladder tissues were incubated with 0.5 mL tyrode buffer with 3H mannitol for 3 and 24 h. Levels of 3H mannitol in bladder tissue following the 3 h incubation were relatively low compared to 0.2% Triton X‐100 (a non‐lytic concentration (Galabova et al., 1996)). However, the amount of 3H mannitol detected in tissue following a 24 h incubation at 37°C was much higher than the 3 h incubation. The findings provided further evidence to support tissue viability over 3 hours.  1200  0.2% Triton X (3h) t = 3h t = 24h  CPM/mg of tissue  1000  800  600  400  200  0 0  250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 Tissue Depth (μm)  Figure 36  Tissue level‐depth profile of 3H mannitol in bladder tissue following a 3 and 24 h incubation. Amount of 3H is expressed as CPM divided by the weight of tissue slices in mg. Values are n = 1.  89  4.4 Bladder Tissue Toxicity of Nanoparticulates 4.4.1 Lactate dehydrogenase assay Using the LDH assay, the toxicity of nanoparticulates (no drug) on bladder tissue was investigated (Figure 37). All formulations were incubated at 37°C with bladder tissue for 2 h and compared to bladder tissue incubated with tyrode buffer. The 3 h incubation with tyrode buffer was performed with a previous LDH assay and used as a control in this experiment. LDH levels from the previous experiment (Figure 35) had the same 2% Triton X‐100 levels in bladder tissue as in this experiment. There was no significant difference in LDH release between the formulations and tyrode buffer at 3 hours. All nanoparticulate formulations had similar levels of LDH release and were well below the LDH released from  Cytotoxicity LDH release (ΔA/min)  2% Triton X‐100.  1 0.9  ***  0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0  Figure 37  Tissue toxicity of nanoparticulate formulations (no drug) following a 2 h incubation. Values were measured by UV‐Vis spectroscopy and are expressed as the change in absorbance with time, which represent the amount of LDH released into supernatant. Values are means ± SD (n = 3‐9). ***p<0.001 vs. all groups 90  4.4.2 Mannitol paracellular permeability assay Since amphipathic molecules like diblock copolymers may have surfactant‐like effects on tissues, it was considered possible that this drug carrier might permeabilize the bladder tissue and allow for the enhanced penetration of drugs into the tissue through this process. Therefore, the uptake of 14C mannitol (poorly taken up by cells) was used to investigate paracellular transport in the presence of nanoparticulate formulations following a 2 hour incubation. The toxicity of nanoparticulates, MePEG‐PDLLA and the respective control vehicles of Cremophor EL and Tween 80 on bladder tissue was investigated. In the presence of 0.2% Triton X‐100, (sublytic, tissue permeabilizing concentration (Galabova et al., 1996)) mannitol was taken up into tissue at levels almost 4 times higher than those observed for mannitol in tyrode buffer alone (Figure 38). Mannitol dissolved in solutions of either Cremophor EL or Tween 80 penetrated tissues at levels slightly higher than those observed for mannitol in tyrode buffer. However, in the presence of MePEG‐PDLLA, the uptake of mannitol was slightly lower than that observed from mannitol dissolved in tyrode buffer alone.  91  Concentration of mannitol (radioactivity/mg of tissue)  600  Tyrode Buffer  560  MePEG‐PDLLA Cremophor EL + EtOH  520  Tween 80  480  0.2% Triton X‐100  440 400 360 320 280 240 200 160 120 80 40 0 0  250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 Tissue depth (μm)  Figure 38  Tissue level‐depth profile of 14C mannitol in bladder tissue following a 2 h incubation. Mannitol delivered to bladder in tyrode buffer containing nanoparticulate vehicles. Data are means ± SD (n = 3).  92  5  DISCUSSION By nature, the bladder is physiologically adapted to retain large volumes of urine  without transfer of the waste solutes through the bladder wall back into the blood stream. The surface urothelium together with the bound mucous layer maintain tight junction function and act as a natural barrier to the transfer of molecules through the bladder wall. However, despite this barrier effect, superficial bladder cancer is amenable to localized chemotherapy because the tumors are located within the surface urothelial layer and concentrated drug solutions may be readily instilled into the bladder and held for up to 2 to 2.5 hours. Unfortunately, many hydrophilic anticancer drugs such as doxorubicin and mitomycin C do not penetrate the urothelial tissue well (Song et al., 1997; Highley et al., 1999) following intravesical administration and hydrophobic anticancer drugs (such as the taxanes) possess the disadvantage that they cannot be dissolved in water without the use of solubilizing agents. Furthermore, continuous urine dilution reduces drug concentrations significantly prior to first void (Wientjes et al., 1996; Au et al., 2001) and voiding results in complete washout of drugs from the bladder (Wirth et al., 2009). In this work we have solubilized PTX and DTX in biodegradable diblock copolymer (MePEG‐PDLLA) micelles for drug delivery to the bladder wall. Taxanes may be readily solubilized in these systems (Zhang et al., 1996; Burt et al., 1999) and the copolymers offer highly biocompatible vehicles due to the presence of MePEG on the micelle outer surface. The hydrodynamic diameter of these micellar systems was determined to be 20 nm, which did not change upon loading of drug. In vitro, both drugs exhibited controlled and complete release from these micelles over 7 days (Figure 14). Free PTX and DTX was released from the dialysis bags at a rapid rate with complete removal by 24 hours, indicating that the dialysis membrane did not retard drug release from the micelles. An increase in drug loading of the micelles produced greater drug release as expected for a primarily diffusion controlled release process. DTX released more rapidly from the micelles than PTX. Although both DTX and PTX are hydrophobic drugs, the slightly higher aqueous solubility of DTX compared to PTX likely resulted in the faster rate of release for DTX. There is extensive literature demonstrating that hydrophilic drugs release very rapidly from biodegradable polyester matrices as water penetrates the polymer matrix (Lee et al., 2006; Rosenberg et al., 2007; Sheikh et al., 2009). The presence of water molecules at the interface of the corona (shell) – core of the MePEG‐PDLLA micelles may be responsible for 93  the more rapid diffusion and partitioning of DTX into the interface region and subsequently release out of the micelles. In addition, the degree of compatibility between the loaded drug and the nature of the micellar core is known to influence the release rate of encapsulated drug (Letchford et al., 2009). The more hydrophobic PTX may have greater compatibility and interaction with the PDLLA chains of the MePEG‐PDLLA micelle core leading to a slower rate of drug release. Using ex vivo pig bladder tissue, PTX and DTX loaded MePEG‐PDLLA micellar formulations showed penetration of the drugs into the bladder wall (Figures 20 and 23). There was no effect of the two different drug concentrations on penetration levels as both 0.5 and 1 mg/ml concentrations gave similar tissue level‐depth profiles (Figure 20 and 23) for both PTX and DTX. These tissue level‐depth profiles demonstrated taxane levels at about 60 and 100 μg/g for PTX and DTX, respectively, at the urothelium surface decreasing to approximately 30 and 50 μg/g at 1 mm depths. To our knowledge, there are no previous reports of tissue level‐depth profiles for DTX. We found much higher PTX tissue levels for either MePEG‐PDLLA or Cremophor EL/ethanol formulations compared to PTX tissue levels from water, Cremophor EL or DMSO vehicles used by other groups (Song et al., 1997; Knemeyer et al., 1999; Chen et al., 2003). However, there were major differences between these studies. The PTX concentrations placed in the donor chambers in contact with pig bladder tissue were about 20 fold higher than PTX concentrations instilled into dog bladders and there was no urine dilution effect in our studies. The tissue levels achieved in this work might provide an effective antitumor effect in vivo since previously reported data by our group and others have showed significant inhibition of bladder cancer cell growth with just nanomolar concentrations of PTX and DTX (Rangel et al., 1994; Nativ et al., 1997; Hadaschik et al., 2008). The bladder tissue level‐depth profiles for PTX and DTX showed an approximately linear decrease in drug concentration over the initial 200 – 240 μm depth of tissue, corresponding roughly to the urothelial thickness. This was followed by an exponential decline in drug concentrations over the remaining tissue thickness, corresponding to the lamina propria and muscle layers of the bladder that contain blood vessels and lymphatics (Shen et al., 2008). These profiles possessed similar characteristics to those obtained for moxifloxacin in isolated porcine bladder (Kerec et al., 2005) and PTX given intravesically 94  into the bladders of dogs (Song et al., 1997; Knemeyer et al., 1999; Chen et al., 2003). Au, Wientjes and coworkers have examined the bladder tissue pharmacokinetics of PTX in dogs and have described drug transport across the urothelium layer as being diffusion controlled with a linear decline in concentration as a function of urothelial tissue depth, with a first order decrease of concentration through the lower layers of submucosa and superficial muscle tissue (Shen et al., 2008). Tissue level‐depth profiles shown in Figures 20 and 23 were also analyzed by calculating both the average tissue levels and the AUCs under the tissue concentration‐ depth curves (Figures 21 and 22, 24 and 25). Determination of the AUC provides a measure of exposure of the tissue to the drug. There were no differences in AUC observed between the two PTX (Figure 22) or DTX (Figure 25) initial donor chamber concentrations (0.5 mg/mL and 1 mg/mL), but MePEG‐PDLLA micellar formulations of both drugs produced significantly greater AUCs in urothelial tissue layers than commercial formulations in Cremophor EL (PTX) or Tween 80 (DTX). Assuming diffusion controlled drug transport across the urothelium, the lack of initial drug concentration dependence for any of the formulations suggests that the concentration gradients across the tissue in the diffusion cells were similar for both 0.5 mg/mL and 1 mg/mL donor chamber experiments. A possible explanation is that the drug concentrations released from the 0.5 or 1 mg/mL formulations and available for uptake at the urothelial surface were similar thus leading to similar concentration gradients across the urothelium. Alternatively, other drug transport processes other than diffusion, may have become rate limiting: for example, partitioning into the urothelium or perhaps drug dissolution, if the highly hydrophobic PTX and DTX formed a colloidal precipitate at the mucosal surface. Using equilibrium dialysis methods, Chen et al. (2003) and Knemeyer et al. (1999) reported that Cremophor EL micelles bind PTX very strongly, such that free fractions of PTX decreased to 23% and 11% respectively, in 0.25% and 1% Cremophor. Given that we employed Cremophor concentration as high as 8% this may explain the significantly lower tissue levels for PTX in our work. At both 0.5 and 1 mg/mL incubation concentrations, DTX was taken up into the bladder wall in significantly greater amounts than PTX (p < 0.01). For example at 0.5 mg/mL, the average whole tissue DTX level was 43.9 μg/g and 24.1 μg/g for PTX. A number of studies and reviews have noted that transport of drugs across the urothelium of the human bladder is by passive diffusion and that tissue levels are affected by the 95  physicochemical properties of the drug, drug concentration, duration of exposure, urine volume and pH, patient hydration status (rate of urine production) and urothelial integrity (Highley et al., 1999; Shen et al., 2008; Wirth et al., 2009; Smaldone et al., 2010). Of the physicochemical properties of the drug, the most important parameters are suggested to be molecular weight, hydrophobicity and lipid/water partition coefficient. Pharmacokinetic studies of PTX, doxorubicin and mitomycin C following intravesical administration in dogs have reported that PTX uptake into urothelium was much greater than for doxorubicin or mitomycin C, likely due to the more than 200‐fold higher partition coefficient for PTX (Song et al., 1997). Various values for the octanol/water partition coefficients of PTX and DTX have been reported, depending upon the method of determination. For example, the octanol/water partition coefficient for DTX (using a computer‐based calculation method) has been reported as 281, whereas for PTX (using experimental methods) the values have been determined to be 157 and 99 (Song et al., 1997; Panchagnula et al., 2005; Huynh et al., 2009). Although there is wide variation in these reported values, it is possible that partition coefficient differences between PTX and DTX may account for differences in tissue levels. It is also possible that the slightly smaller molecular size and possibly greater diffusivity of DTX compared to PTX may have been responsible for the greater tissue uptake. The recovered range of concentrations in bladder tissue from formulations of 0.5 and 1 mg/mL PTX and DTX represented 0.1‐1% of the total delivered dose (Figure 26). In the receptor chamber, the less than 0.04% of drug recovered suggested negligible drug penetration through the full thickness of the bladder tissue. Other groups have shown drug uptake into bladder tissue following intravesical administration within the same range (less than 1% of the dose). Song et al. (1997) achieved a 1.2% uptake of PTX in the bladder tissue of dogs using a non‐micellar solution at 25 μg/mL and in a later study by the same group a 0.4% uptake with a Cremophor EL formulation at the same concentration (Knemeyer et al., 1999). Using PTX conjugated to hyaluronic acid to increase its water solubility, Tringali et al. (2008) showed that using a PTX concentration of either 1 mg/mL or 3 mg/mL in rabbit bladders, only 0.68% of the dose was recovered in bladder tissue. There was a rapid initial rate of drug penetration observed for both drugs whereby near maximal penetration was achieved in the urothelium layer after just 1 h for PTX, with maximal tissue uptake for DTX in 2 hours (Figures 27 and 28). However, even after 30 min 96  of exposure to the drug, tissue levels of PTX in the bladder wall were greater than 18 μg/g, a concentration well above the nanomolar concentrations previously determined by others to show significant inhibition on bladder cancer cell growth in vitro (Rangel et al., 1994; Nativ et al., 1997; Hadaschik et al., 2008). The average tissue levels of PTX and DTX were found to increase with incubation time. PTX levels following a 180 min incubation were significantly higher than PTX levels following a 30 min incubation and DTX levels were significantly higher following a 120 min incubation than a 30 and 60 min incubation, suggesting a benefit to prolonging bladder exposure to the drugs. The influence of the nanoparticulate composition on bladder tissue uptake was evaluated. PTX at 0.5 mg/mL was loaded in MePEG‐PDLLA micelles, MePEG‐PCL19 micelles and MePEG‐PCL104 nanospheres, together with PTX in Cremophor EL and ethanol as a control. The average tissue levels of PTX (Figure 30) were highest for MePEG‐PDLLA micelles compared to the other formulations. MePEG‐PCL19 and MePEG‐PCL104 nanoparticles effectively solubilized PTX up to 1.0 mg/mL and 0.5 mg/mL, respectively although these nanoparticles based on PCL core‐forming block were not able to solubilize as much PTX as nanoparticles based on PDLLA core‐forming block (Zhang et al., 1996; Burt et al., 1999; Letchford et al., 2009). PTX release from these nanoparticles was not evaluated as part of this work but were previously carried out and reported by Dr. Kevin Letchford (Letchford et al., 2009) using identical composition nanoparticles. Letchford et al. (2009) did not use the same PTX loading as in this work, but the MePEG‐PCL19 micelles released about 90% of loaded PTX in 7 days, whereas MePEG‐PCL104 released about 60% of loaded PTX in 7 days. It is apparent, therefore, that PTX release profiles from MePEG‐PCL19 micelles was about equivalent to PTX release profiles from MePEG‐PDLLA, but that release from MePEG‐PCL104 was markedly slower. The MePEG‐PCL nanoparticles were about 40 nm and 80 nm for MePEG‐PCL19 and MePEG‐PCL104, respectively and were therefore significantly larger than MePEG‐PDLLA micelles (20 nm). Furthermore, the length of the hydrophilic MePEG block was significantly greater for the MePEG‐PCL nanoparticles (MW of MePEG was 5000 g/mol) than for the MePEG‐PDLLA micelles (MW of MePEG was 2000 g/mol). It is possible that the higher tissue levels for PTX delivered using MePEG‐PDLLA micelles may be due to factors including, greater rate of drug release from these micelles, smaller size and decreased size of hydrophilic MePEG shell allowing for greater penetration of the smaller, more mobile MePEG‐PDLLA micelles into the mucin layer of the 97  bladder tissue. More effective entrapment of the PTX loaded MePEG‐PDLLA micelles within the mucin chains could have resulted in enhanced diffusion and partitioning of PTX into the urothelial cells. We originally hypothesized that delivery of taxanes to bladder tissue using a mucoadhesive drug delivery system should result in increased contact time with the bladder mucosa and enhanced drug uptake into bladder tissue. Other groups have provided some limited evidence that mucoadhesive drug carriers can increase drug uptake into the bladder of animals (Lu et al., 2004; Lee et al., 2005). Using a mucin particle aggregation method as a measure of mucoadhesion showed that none of our formulations were mucoadhesive (Figure 18). Chitosan, a mucoadhesive polymer, used as a positive control showed dramatic increases in mucin particle size caused by aggregation. The addition of excessive amounts of chitosan resulted in deaggregation of mucin particles caused by the chitosan polymer chains forming a multilayer around the mucin particles leading to repulsion and a decrease in solution turbidity (Sogias et al., 2008). It seems possible that the size of the nanoparticulate drug carrier rather than mucoadhesion may be a factor in allowing for penetration of the carrier between the protruding mucin chains and into closer contact with the urothelial tissue layer, although further studies would be required to examine the effect of particle size. Furthermore, the use of a diffusion cell technique to evaluate the role played by mucoadhesion of the drug carrier is not ideal. In the diffusion cell, the bladder tissue is static and the drug loaded nanoparticles placed in the donor chamber are in continuous contact with the bladder tissue for the 2 hour incubation. This is clearly quite unlike the dynamic in vivo situation where there is continuous bladder filling with urine and where adhesion between the drug carrier and the mucin layer may play an important role. In vivo, PTX has been shown to undergo extensive metabolism when administered intravenously (Huizing et al., 1995b; Malingre et al., 2000), however, no evidence of PTX metabolism in bladder tissue was detected. The stability of radiolabelled PTX was confirmed since all the radioactivity was located in the PTX HPLC peak with no evidence of radioactivity in any eluted samples following the PTX peak. In viability studies, the pig bladder tissue was determined to be viable for up to 8 hours in the Franz diffusion cell as measured by the release of lactate dehydogenase (LDH) (Figure 35). Furthermore, the paracellular permeability of bladder tissue was measured 98  using mannitol (Figure 36). Mannitol is a well known tight junction marker. The passive paracellular transport of this sugar has been linked to in vitro cytotoxicity (Konsoula and Barile, 2005). Konsoula and Barile (2005) were able to correlate in vitro cytotoxicity with paracellular permeability of Caco‐2 cells. Cytotoxicity was determined using the methylthiazol tetrazolium (MTT) cell viability assay and paracellular permeability was measured using transepithelial electrical resistance (TEER) measurements, as well as passage of 3H mannitol in culture inserts. The group found that the MTT assay indicated that cytotoxicity occurred before TEER was compromised and that the passive paracellular transport of 3H mannitol correlated with the IC50s determined with the viability assays and TEER measurements (Konsoula and Barile, 2005). The results from this assay mirrored the data shown for LDH release. Although many groups have reported that porcine bladder tissue is viable for 5 h, we found little evidence to support this (Grabnar et al., 2003; Kerec et al., 2005; Kos et al., 2006). Therefore, our findings demonstrate viability of the bladder tissue during the time frame of our studies. Since amphipathic molecules like diblock copolymers may have surfactant‐like effects on tissues, it was considered possible that this drug carrier might permeabilize the bladder tissue and allow for the enhanced penetration of drugs into the tissue through a paracellular process. The sugar mannitol is not readily taken up into cells and may be used to study paracellular transport effects (Soler et al., 1999; Konsoula and Barile, 2005) (i.e. tissue permeabilization). Using radioactive mannitol solutions, we demonstrated that the surfactant Triton X‐100 (used at a non lytic concentration of 0.2% (Galabova et al., 1996)) allowed for 4 fold increases of mannitol penetration into bladder tissues as compared to control (Figure 38). However, mannitol penetration in the presence of the diblock copolymers was slightly lower than those observed in control samples establishing that taxane penetration from micellar formulations probably did not arise from any permeabilization effect of the polymeric excipient. Interestingly, both Cremophor EL and Tween 80 carriers caused increases in mannitol penetration into bladder tissue suggesting a small tissue permeabilization effect of these agents. Therefore, it is likely that the low levels of taxane penetration observed with these carriers may arise from sequestration of the drug in the micelles of these agents and reduced free drug to penetrate tissues. The toxicity of nanoparticulate formulations on bladder tissue was also investigated using LDH (Figure 37). No significant toxicity was observed as all formulations had similar levels of 99  LDH release below levels measured from 2% Triton X‐100. The assay provided further evidence to support minimal cell death caused by formulations prepared for 0.5 and 1 mg/mL PTX and DTX delivery. In this project the use of micellar formulations of taxanes allowed for PTX and DTX solubilization at high concentrations in stable, biocompatible formulations of the drugs. The excellent biocompatibility of this formulation may arise, in part, from the outer layer of MePEG which is known to shield unwanted surface effects with cells (Gref et al., 2000). The MePEG‐PDLLA micellar formulation of PTX had no adverse toxicity following intravenous (Burt et al., 1999) or intravescial administration in mice at higher concentrations (5mg/ml) (Hadaschik et al., 2008) than concentrations used in this study. Whilst the outer MePEG surface of micelles may allow for improved biocompatibility (Lee et al., 2001), it is also possible that the MePEG chains associated or interpenetrated the mucin chains of the urothelium to effectively “entrap” the formulation at the urothelial surface. Overall, it is likely that the rapid and high levels of PTX and DTX penetration observed in bladder tissue using the MePEG‐PDLLA micellar formulation of these drugs arose from three factors: the ability to use high concentrations of drugs with these polymeric micelles, greater free drug released from the MePEG‐PDLLA micelles and the ability to increase the contact of the formulation at the bladder wall surface allowing for improved partitioning of the drug into the tissues. We have previously shown efficacy following intravesical administration of MePEG‐PDLLA PTX at 5 mg/ml in mice bearing xenograft human bladder tumors without carrier‐induced local toxicity (Hadaschik et al., 2008). Taken together, our studies support the use of the micellar formulation for taxane delivery to superficial bladder tumors.  100  6  SUGGESTIONS FOR FUTURE WORK To further support our findings on the distribution of taxanes into bladder tissue, it  would be interesting to characterize the permeability of the urothelium to nanoparticles after expansion and contraction of the bladder mucosa. The natural physical and biological changes that occur as the bladder wall unfolds and folds during filling and voiding may impact uptake and distribution of nanoparticulate delivery systems. With the processes of endocytosis and exocytosis occurring during bladder filling and voiding, a closer look at the possible pathways to drug absorption into the bladder wall should be investigated further. Marking the drug and delivery system with a fluorescent or radio‐tag and tracking their movement into the tissue would ultimately provide a better understanding of the possible mechanisms of drug uptake. Furthermore, the distribution of drug in tumor tissue is likely different from that of normal tissue due to the abnormal vasculature of the epithelial wall. Thus, a study comparing drug uptake levels in tumour and normal tissue should be determined. Finally, it is known that the pH of urine may range from 4.5 to 8 and thus have an effect on the delivery of drugs into the bladder wall. Therefore, by changing the pH of the aqueous environment within the bladder, the effect of pH on drug uptake can be performed. Tissue distribution studies should be repeated in more acidic and basic environments.  101  REFERENCES Aapro, M., and Bruno, R. (1995). Early clinical studies with docetaxel. Docetaxel Investigators Group. Eur J Cancer 31A Suppl 4, S7‐10. Adams, J.D., Flora, K.P., Goldspiel, B.R., Wilson, J.W., Arbuck, S.G., and Finley, R. (1993). Taxol: a history of pharmaceutical development and current pharmaceutical concerns. J Natl Cancer Inst Monogr, 141‐147. 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