i DESIGN AND DEVELOPMENT OF AN IMPLANTABLE DRUG DELIVERY POLYMERIC SCAFFOLD FOR THE TREATMENT OF PARKINSON'S DISEASE SAMANTHA PILLAY A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Pharmacy Supervisor: Professor Viness Pillay University of the Witwatersrand Department of Pharmacy and Pharmacology South Africa Co-Supervisors: Professor Michael P. Danckwerts, University of the Witwatersrand, Department of Pharmacy and Pharmacology, South Africa Professor Girish Modi, University of the Witwatersrand, Department of Neurology, Division of Neurosciences, South Africa Professor Sunny E. Iyuke, University of the Witwatersrand, School of Chemical and Metallurgical Engineering, South Africa Johannesburg, 2009 ii DECLARATION I, Samantha Pillay declare that this dissertation is my own work. It has being submitted for the degree of Master of Pharmacy in the Faculty of Health Sciences in the University, Johannesburg. It has not submitted before for any degree or examination at this or any other University. ???????????????. This????.day of May 2009 iii RESEARCH PRESENTATIONS POSTERS Synthesis, Characterization and Preliminary Evaluation of Hydrophilic Nanoparticles and Scaffolds for Controlled Drug Delivery. Samantha Pillay, Viness Pillay, Yahya E.Choonara and Michael P.Danckwerts. Conference of the Academy of Pharmaceutical Sciences, Johannesburg, South Africa, September 2006. Formulation of Polymeric Nanoparticles Intended for Intracranial Implantation. Samantha Pillay, Viness Pillay, Yahya E. Choonara and Michael P.Danckwerts. Conference of the South African Nanotechnology Initiative: NanoAfrica, Cape Town, South Africa, 26th - 29th November 2006. Influence on Drug Molecular Structure on the Formation of Nanotubes. Samantha Pillay, Viness Pillay, Yahya E.Choonara. Conference of the Neurological Association of South Africa: Innovative and Practical Congress, Johannesburg, South Africa, 28th - 31st March 2007. Design and Evaluation of Dopamine (DA) Nanoparticles Dispersed within a Scaffold. Samantha Pillay, Viness Pillay, Yahya E. Choonara and Michael P.Danckwerts. Conference of the Controlled Release Society, Los Angeles, United States of America, 7th - 11th July 2007. Electrospinning of DA-loaded Nanofibers. Samantha Pillay, Viness Pillay, Yahya E.Choonara, Girish Modi and Sunny E.Iyuke. Conference of the Academy of Pharmaceutical Sciences, Cape Town, South Africa, 4th - 7th September 2007. Targeted Delivery of Dopamine to the Brain via an Implantable Nano-enabled Heterogeneously Designed Multi-Polymeric Device, Samantha Pillay, Viness Pillay, Yahya E.Choonara, Girish Modi, Dinesh Naidoo and Sunny E.Iyuke. 9th US-Japan Symposium on Drug Delivery, Maui, Hawaii, 16th ? 20th December 2007. iv PODIUM Determination of Size and Electrokinetic Stability of Dopamine (DA)-loaded Cellulose Acetate Phthalate Nanoparticles. Samantha Pillay, Viness Pillay, Yahya E. Choonara, Girish Modi, Michael P. Danckwerts and Sunny E. Iyuke. Joint Symposium for Chemical and Metallurgical Engineering, University of Pretoria, 3rd - 4th August 2007. An Experimental Design Approach for Optimizing Dopamine-loaded Cellulose Acetate Phthalate Nanoparticles and Barium-Alginate Scaffolds for Intracranial Implantation. Samantha Pillay, Viness Pillay, Yahya E. Choonara, Girish Modi, Michael P. Danckwerts and Sunny E.Iyuke. Therapeutic Sciences Research Day, University of the Witwatersrand, 24th ? 25th August 2007. In Vitro and In Vivo Evaluation of Dopamine-loaded Cellulose Acetate Phthalate Nanoparticles Dispersed within a Scaffold for Intracranial Implantation. Samantha Pillay, Viness Pillay, Yahya E. Choonara, Girish Modi, Dinesh Naidoo and Sunny E. Iyuke. Presentation at the Pharmaceutical Society of South Africa (PSSA) Adcock Ingram Young Scientist Competition 2007, Conference of the Academy of Pharmaceutical Sciences, Cape Town, South Africa, 4th - 7th September 2007. Awarded First Runner-up position. v PATENTS Polymeric Configuration for Site-Specific Drug Delivery. Viness Pillay, Sheri-lee Harilall Samantha Pillay, Yahya E. Choonara, Girish M. Modi, Sunny E. Iyuke and Dinesh Naidoo, South Africa, Patent Application Filed, awaiting priority date. vi ABSTRACT Parkinson's disease, primarily defined as the depletion of dopaminergic neurons in the subtantia nigra of the brain, gives rise to severely debilitating motor symptoms. The pharmacological gold standard treatment for the disease, Levodopa , holds great limitations yet still remains the most effective treatment for the disease for the last 40 years. There has been research into novel drug delivery systems for the treatment of the disease that include the development of implantable devices however none have been introduced onto the market. As the neurodegenerative disorder ravages the younger-aged population so the urgency for the effective chronic treatment of the disease escalates. The field of nanotechnology brings promise for the targeted delivery of drugs which is highly sought after in the treatment of central nervous system disorders. A nano-enabled scaffold device (NESD) incorporating dopamine nanoparticles into a polymeric scaffold for implantation into the brain parenchyma may be able to address and overcome the limitations of the current treatment for Parkinson's disease. Investigations performed cellulose acetate phthalate dopamine-loaded nanoparticles, employing an adopted emulsification- diffusion approach, produced particles with a notably high drug entrapment efficiency (63.05?0.354%) and desirable controlled drug release profiles (16.23% in 24hr). The employment of an experimental design, namely the Box-Behnken design, allowed for the attainment of optimized nanoparticles with high zeta potentials (.34.00mV), minimal particle size (197.20nm) and extended mean dissolution times (40.96). Barium chloride was employed to crosslink calcium-alginate scaffolds formulated in an adopted freeze-drying approach. Highly resilient (63.58?5.13) and porous structures (pore sizes of 100-400?m) were developed. A statistical approach employing the Box-Behnken design resulted in the formulation of a candidate barium-alginate scaffold displaying maximum matrix resilience (82.46%) and minimal matrix erosion (18.23%) over in 30 days. In addition, dopamine-loaded nanoparticles were dispersed within the scaffold that formed the NESD with the desired drug release profiles (5.12% in 168hr). Nanosystems of levodopa, nicotine and dopamine nanofibers were preliminary investigated. Drug release profiles for levodopa (4.21%: in 75hr), nicotine (0.42% in 24hrs) and drug entrapment efficiency for the polymeric nanofibers (75-85%) as well as data from scanning electron microscopy, zetasize analysis and drug release studies proved that these systems hold potential for the treatment of the disease and therefore require further investigation. Ex vivo cytotoxic studies carried out on the NESD and it's separate entities proved that the NESD was biocompatible with the white blood (70-80% cell viability in 24hr) and carcinomic brain cells (25% cell viability in 48hr) despite literature reports of dopamine being highly toxic in vivo. Extensive in vivo studies resulted in the development of a protocol for the surgical implantation of the NESD in the parenchyma of the frontal lobe of the rat brain. Scanning electron microscope images showed the gradual bioerosion (26% in 30 days) of the NESD while histological findings of the brain tissue proved clinically insignificant (absence of ischemia or chronic inflammation). Ultra Liquid Performance Chromatography revealed higher concentrations of dopamine in the CSF of rats which received brain implants of the NESD (28%) than in those administered the oral preparation, Sinemet (0.000012%) in 3 days. vii ACKNOWLEGDEMENTS The author would like to express her gratitude to those without whom this work would not have been possible. To the following people my deepest appreciation: My eternal appreciation goes to the Almighty Lord, for never letting me down in my times of need. I have felt your love even in the most desperate of times and so fueling my belief in your power. To my parents, my world, Rodgers and Elizabeth Pillay, the pages of this dissertation is not merely enough to express my love and gratitude to you. Daddy, thank you for trusting me and allowing me the freedom to pursue a path that I felt was right for me. Mummy, thank you for always pushing me far beyond my limits and keeping me humbled. Thank you to both of you for your sacrifices, for always being the best you can be for me and your unconditional love. I pray God blesses me one day with your wisdom as parents. I will continue to make you proud as I pursue my life?s journey. I love you. My late grandmother, Lutchmama Pillay whose physical presence I was so unfortunate not to experience but whose memories and love I feel everyday of my life. A women whose beauty both inside and out will never be forgotten. To my grandparents, Manikum Pillay and Stella Naidoo, I am so fortunate to spend my life in your guidance and absolute warmthness. God bless you. To my supervisor, Professor Viness Pillay, your guidance and perfection have made this work something I am truly most proud of. My best wishes to you as you continue to inspire all those around you. My advisor and a man to watch out for, Mr. Yahya E. Choonara, the completion of this journey would have been even more difficult without your assistance. My gratitude goes out to my co-supervisors, Professor Michael P. Danckwerts, Professor Sunny E. Iyuke and Professor Girish Modi. Thank you for your guidance. viii A special thank you goes to Dr. Dinesh Naidoo, whose passion for this work was truly inspiring. Thank you for going above and beyond, your expertise added immense value to my research. My deepest gratitude goes to Dr. Daven Compton and the Mintek Group, for allowing me the use of your facilities that added so much to research, for taking me in as one of your own and for always being available for supervision. To Dr Hajierah Davids and Helen Panagiotopoulos for their assistance in the cytotoxic studies and their immense hours in the lab. To my colleagues: Lisa C. du Toit, Neha Singh, Maria Paraskeva, Sheri-Lee Harilall, Sibongile Sibambo, Oluwatoyin A. Kolawole, Bongani Sibeko, Seshni Sewlall, Valence M.K. Ndesendo, Caragh S. Murphy, Deshika Reddy, Priya Bawa, Shivaan Cooppan, Yasien Docrat, Sajida Suleman and Zaheeda Khan- thank you for making the days on campus more bearable, the laughter and advice. My sincere appreciation to Vikesh Rowjee, for being an excellent research partner and always being willing to help. Good luck for your future. My dear friends, Corinne Joseph and Rakhee Patel, you guys have been there for me through so much. My hope is that our relationship grows old with us. A special thank you is also extended to Shanaaz Goolam and Candice Fick. To my extended family: Lekesha Padayachee, Vigie Pillay, Mogie Naidoo and Sheruna Naidoo, Pravin Reddy and Megashni Reddy, Palmer Gengan and Devrani Gengan and family ? thank you for your support and believing in me. My appreciation to the Wits staff, thank you for always being there to lend a helping hand and for fixing the mess we made. To Jonathan Pillay and family, your pride in me is a true inspiration. Thank you for never being able to say no to me. I know I can count on you throughout my life. To the Central Animal Service Staff at Wits, I really gave you guys a hard time yet you went out of your way to accommodate me and always with a smile. ix To my brother, Keegan Pillay, you truly have become someone I can look up to in every sphere of my life. I am humbled by your willingness to always go out of your way for me and your generosity. I am extremely proud of the man you have become and know you are destined for so much more in this lifetime. To Nirisha Pillay, my special thanks for your support. x DEDICATION This work is dedicated to the late Nivash Singh (18/08/1984-16/01/2009): my fianc?, my best friend and the love of my life. I was so blessed to spend the greatest part of my life with you. You showed me how to be a better person even though I may not always have done a good job. Belief is the strongest motivator/ inspiration and that?s what you were and still are to me. Thank you for giving me the strength to complete this work in the time of my greatest sorrow. Your unselfishness allowed me to be the best I can with you. Thank you for always being honest with me and for teaching me how to be more independent. I am what I am because of you. I will miss you every day of my life. God gave me an angel whose short life will never be forgotten. Rest in peace my Varshen. I love you forever and always. xi TABLE OF CONTENTS Page 1. Introduction 1.1. Background to this Study 1 1.2. Rationale for this Study 3 1.3. Aim and Objectives of this Study 6 1.4. Overview of this Dissertation 7 2. Current Strategies and Limitations for Drug Delivery to the Brain in the Treatment of Parkinson's Disease 2.1. Introduction 9 2.1.1. The Physiology of the Blood Brain Barrier 9 2.1.2. Approaches Employed for Drug Delivery to the Brain 10 2.2. Parkinson?s Disease: An Overview 19 2.2.1. Possible theories explicating the aetiology of Parkinson?s disease 19 2.2.2. Diagnosis of Parkinson?s disease 20 2.2.3. Pharmacological treatment of Parkinson?s disease 21 2.2.4. Challenges of current therapy used in the treatment of Parkinson?s disease 23 2.2.4.1. Limitations of Sinemet? for the treatment of Parkinson?s disease 23 2.2.4.2. The gap in current therapy employed in the treatment of Parkinson?s disease 23 2.2.5. Implantable novel polymeric drug delivery systems for the treatment of Parkinson?s disease 24 xii 2.2.5.1. Levodopa/carbidopa loaded microspheres for implantation into the cerebellum 25 2.2.5.2. Dopamine-loaded drug delivery systems: microspheres and a silicone pellet into the striatum 25 2.2.6. Gene therapy 25 2.2.7. Empirical treatments employed in the treatment of Parkinson?s disease 26 2.2.8. Current surgical approaches for the treatment of Parkinson?s disease 28 2.3. Concluding remarks 31 3. Development and Formulation of Dopamine-loaded Nanoparticles 3.1. Introduction 32 3.2. Materials and Methods 34 3.2.1. Materials 34 3.2.1.1. Technology Applied in the Present Study 34 3.2.1.1.1. Application of the Zetasizer? NanoZS Series Instrument for Nanoparticle Analysis 34 3.1.1.1.2. The determination of nanoparticle size 34 3.1.1.1.3. The determination of the zeta potential of nanoparticles 35 3.2.1.2. Rotavapor? R210 (B?CHI Labortechnik AG, Flawil, Switzerland) with heating bath 37 3.2.1.3. Identification of ideal processing conditions for successful formulation of nanoparticles 37 xiii 3.2.1.3.1. Temperature for polymer solubilization 37 3.2.1.3.2. Stirring time and speed for solvent emulsification 37 3.2.1.4. Preparation of dopamine-loaded cellulose acetate phthalate nanoparticles 38 3.2.1.5. Morphological characterization of dopamine-loaded cellulose acetate phthalate nanoparticles 38 3.2.1.6. Determination of polymeric structural variations due to dopamine-loaded cellulose acetate phthalate nanoparticle formation 38 3.2.1.7. Construction of calibration curves for spectrophotometric determination of dopamine release from cellulose acetate phthalate nanoparticles 39 3.2.1.8. Determination of dopamine entrapment efficiency of cellulose acetate phthalate nanoparticles 39 3.2.1.9. In vitro dopamine release studies on cellulose acetate phthalate nanoparticles 39 3.2.1.10. Determination of size and zeta potential of dopamine-loaded cellulose acetate phthalate nanoparticles 40 3.2.11. Formulation and Statistical Optimization of Nanoparticles 41 3.2.11.1. Determination of limitations for variables employed in the Box- Behnken design 41 3.2.11.2. Optimization of dopamine-loaded cellulose acetate phthalate nanoparticles 42 3.3. Results and Discussion 43 xiv 3.3.1. Surface morphology of dopamine-loaded cellulose acetate phthalate nanoparticles 43 3.3.2 Structural variations present in the formation of dopamine-loaded cellulose acetate phthalate nanoparticles 44 3.3.3. Calibration curve for dopamine in phosphate buffer solution (pH 6.8; 37?C) for the determination of the concentration of dopamine release from cellulose acetate phthalate nanoparticles 45 3.3. 4. Dopamine entrapment efficiency of cellulose acetate phthalate nanoparticles 45 3.2.2.5. In vitro dopamine release of cellulose acetate phthalate nanoparticles 46 3.2.2.6. Dopamine-loaded cellulose acetate phthalate nanoparticle size and stability 46 3.2.2.6.1. Size and zeta potential distribution profiles for cellulose acetate phthalate nanoparticles: dopamine- free and dopamine-loaded 47 3.3.7. Experimental design for the optimization of dopamine-loaded cellulose acetate phthalate nanoparticles 49 3.3.7.1. Measured responses for the experimental optimization of dopamine-loaded cellulose acetate phthalate nanoparticles 50 3.3.7.2. Comparison of the experimental and fitted response values calculated for the experimental optimization of dopamine-loaded cellulose acetate phthalate nanoparticles 51 3.3.7.3. Response analysis of Mean Dissolution Time for dopamine-loaded cellulose acetate phthalate nanoparticles 53 xv 3.3.7.4. Response analysis for size of dopamine-loaded cellulose acetate phthalate nanoparticles 55 3.3.7.5. Response analysis for Zeta Potential of dopamine-loaded cellulose acetate phthalate nanoparticles 56 3.3.7.6. The main and interaction effects on the responses: 57 3.3.7.7. Analysis of a Box-Behnken design employed for the optimization of dopamine-loaded cellulose acetate phthalate nanoparticles 62 3.3.7.8. Response optimization of dopamine-loaded cellulose acetate nanoparticles 65 3.3.2.9. Desirability for the measured responses of the optimized dopamine-loaded cellulose acetate phthalate formulation 67 3.4. Concluding remarks 68 4. Formulation of Crosslinked-Alginate Scaffolds for the Design of a Nano-Enabled Scaffold Device 4.1. Introduction 69 4.2. Materials and Methods 72 4.2.1. Materials 72 4.2.2. Processing conditions for the development of crosslinked alginate scaffold 72 4.2.2.1. Temperature for the dissolution of polymer 72 4.2.2.2. Duration required for the efficient post-curing of crosslinked alginate scaffold 73 4.2.3. Biometric Simulation of the Nano-Enabled Scaffold Device employing computer-aided prototyping 73 xvi 4.2.4. Preparation of crosslinked alginate scaffold 74 4.2.5. Textural profile analysis to determine the physicomechanical behaviour of the crosslinked scaffold 74 4.2.6. Morphological characterization of crosslinked alginate scaffold 75 4.2.7. In vitro Matrix Erosion studies on crosslinked alginate scaffold 75 4.2.8. Formulation and Statistical Optimization of the Barium-Alginate Scaffolds 76 4.2.8.1. Determination of limitations for variables employed in a Box-Behnken design 76 4.2.8.2. Optimization of barium-alginate scaffold 77 4.2.9. Corporeal assembly of the Nano-Enabled Scaffold Device 77 4.2.10.2. Assimilation of the dopamine?loaded cellulose acetate phthalate nanoparticles and crosslinked scaffold into the Nano-Enabled Scaffold Device 78 4.2.11. Determination of in vitro dopamine release from the Nano-Enabled Scaffold Device 80 4.2.12. Determination of the thermal transition behaviour of the Nano-Enabled Scaffold Device and it?s constituents 80 4.3. Results and Discussion 81 4.3.1. Computer-aided prototyping for Nano-Enabled Scaffold Device design 81 4.3.2. Matrix Resilience behaviour of crosslinked scaffolds 84 4.3.3. Surface morphology of crosslinked alginate scaffold 86 4.3.4. In vitro Matrix Erosion of the barium-alginate scaffold 87 xvii 4.3.5. Experimental design for the optimization of barium-alginate scaffold 88 4.3.5.1. Measured responses for the experimental optimization of barium- alginate scaffold 89 4.3.5.2. Comparison of the experimental and fitted responses calculated for the experimental optimization of barium-alginate scaffold 90 4.3.5.3. Response analysis for Matrix Resilience of barium-alginate scaffold 91 4.3.5.4. Response analysis for Matrix Erosion of barium-alginate scaffold 91 4.3.5.5. The main and interaction effects on the responses 92 4.3.5.6. Analysis of a Box-Behnken design employed for the optimization of barium-alginate scaffold 95 4.3.5.7. Response optimization of barium-alginate scaffold 98 4.3.5.8. Desirability for the measured responses of the optimized barium- alginate scaffold formulation 99 4.3.6. In vitro dopamine release from the Nano-Enabled Scaffold Device 99 4.3.7. Componential thermal analysis on the Nano-Enabled Scaffold Device 100 4.4. Concluding Remarks 103 5. Investigation into Drug-Loaded Nanostructures for the Employment in Treatment of Parkinson's Disease 5.1. Introduction 104 xviii 5.2. Investigation of Levodopa-loaded Poly (lactic-co-glycolic acid) Nanoparticles 106 5.2.1. Materials and Methods 106 5.2.1.1. Materials 106 5.2.1.2. Preparation of levodopa-loaded poly (lactic-co-glycolic acid) nanoparticles employing a nano- precipitation technique 106 5.2.4. Assimilation of levodopa-loaded poly (lactic-co-glycolic acid) nanoparticles and barium-alginate scaffold into the Nano-Enabled Scaffold Device 107 5.2.5. Determination of levodopa entrapment efficiency of poly (lactic-co-glycolic acid) nanoparticles 107 5.2.6. In vitro levodopa release studies on poly (lactic-co-glycolic acid) nanoparticles and Nano-Enabled Scaffold Device 108 5.2.7. Morphological characterization of levodopa poly (lactic-co-glycolic acid) nanoparticles 108 5.2.8. Determination of polymeric structural variations due to levodopa poly (lactic-co-glycolic acid) nanoparticle formation 108 5.2.8. Determination of size and zeta potential of levodopa-loaded poly (lactic-co-glycolic acid) nanoparticles 109 5.3. Results and Discussion 109 5.3.1. Formulation of levodopa poly (lactic-co-glycolic acid) nanoparticles 109 5.3.2. Levodopa entrapment efficiency of poly (lactic-co-glycolic acid) nanoparticles 110 xix 5.3.2. In vitro levodopa release from poly (lactic-co-glycolic acid) nanoparticles and Nano-Enabled Scaffold Device 110 5.3.4. Morphology of levodopa poly (lactic-co-glycolic acid) Nanoparticles 111 5.3.5. Structural polymeric variation of poly (lactic-co-glycolic acid), levodopa and levodopa-loaded poly (lactic-co-glycolic acid) nanoparticles 113 5.3.6. Levodopa-loaded poly (lactic-co-glycolic acid) nanoparticle size and stability 114 5.4. Investigation of nicotine-loaded cellulose acetate phthalate nanoparticles 115 5.4.1. Material and Methods 115 5.4.1.1. Materials 115 5.4.2. Preparation of nicotine-loaded poly (lactic-co-glycolic acid) nanoparticles 115 5.4.4. Determination of nicotine entrapment efficiency of cellulose acetate phthalate nanoparticles 115 5.4.5. In vitro nicotine release studies on cellulose acetate phthalate nanoparticles 116 5.4.6. Morphological characterization of nicotine-loaded cellulose acetate phthalate nanoparticles 116 5.4.7. Determination of size and zeta potential of nicotine-loaded cellulose acetate phthalate nanoparticles 116 5.5. Results and discussion 117 xx 5.5.1. Calibration curve for nicotine in phosphate buffered solution (pH 6.8; 37?C) for the determination of the concentration of nicotine in cellulose acetate phthalate nanoparticles 117 5.5.2. Nicotine entrapment efficiency of cellulose acetate phthalate nanoparticles 117 5.5.3. In vitro nicotine release from cellulose acetate phthalate nanoparticles 118 5.5.4. Surface morphology of nicotine-loaded cellulose acetate phthalate nanoparticles 119 5.5.5. Nicotine-loaded cellulose acetate phthalate nanoparticle size and stability 119 5.6. Investigation of dopamine-loaded Polymeric Nanofibers 120 5.6.1. Materials and Methods 120 5.6.1.1. Materials 120 5.6.2. Preparation of dopamine-loaded polymeric nanofibers 120 5.6.3. Determination of dopamine entrapment efficiency of polymeric Nanofibers 120 5.6.4. Morphological characterization of dopamine-loaded polymeric Nanofibers 120 5.7. Results and Discussion 121 5.7.1. Dopamine entrapment efficiency of polymeric nanofibers 121 5.7.2. Size and morphology of dopamine-loaded polymeric nanofibers 121 5.8. Concluding Remarks 123 xxi 6. Cytotoxic Evaluation of Nano-Enabled Scaffold Device on Carcinomic Brain and Freshly Isolated, Healthy White Blood Cells 6.1. Introduction 124 6.2. Materials and Methods 127 6.2.1. Routine cell culture 127 6.2.2. Isolation of white blood cells 127 6.2.3. Trypsinisation and cell counting/seeding of cells 128 6.2.4. Exposure of cells to the different compounds 129 6.2.5. Calibration of 3?(4,5?dimethylthiazol-2-yl)?2,5? diphenyltetrazolium bromide assay 129 6.2.6. The 3?(4,5?dimethylthiazol-2-yl)?2,5?diphenyltetrazolium bromide assay 130 6.2.7. Preparation of 3?(4,5?dimethylthiazol-2-yl)?2,5? diphenyltetrazolium bromide solution 131 6.3 Results and Discussion 131 6.3.1. Calibration curve of post-3?(4,5?dimethylthiazol-2-yl)?2,5? diphenyltetrazolium bromide exposure for the determination of the number of viable cells present in the media 131 6.3.2. Cytotoxicity to white blood cells 132 6.3.3. Cytotoxicity directed against the carcinomic SK-N-MC cells 133 6.3.4. Limitations for the employment of 3?(4,5?dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay in the present study 134 6.4. Concluding Remarks 135 xxii 7. In Vivo Evaluation of the Nano-Enabled Scaffold Device upon Implantation into the Parenchyma of the Frontal Lobe of the Rat Brain 7.1. Introduction 136 7.1.1. Biocompatibility of implantable devices 136 7.2. Materials and Methods 138 7.2.1. Materials 138 7.2.2. Technology Applied in the Present Study 138 7.2.2.1. Waters? Acquity Ultra Performance Liquid ChromatographyTM 138 7.2.2.2. Evaporative Light Scattering Detector 139 7.2.3. Development of Methodology for the Surgical Implantation of Nano-Enabled Scaffold Device into the Parenchyma of the Frontal Lobe of the Sprague-Dawley Rat Brain 139 7.2.3.1. Pre-surgical preparation of rats 139 7.2.3.2. Pilot surgical technique for implantation of the Nano-Enabled Scaffold Device into the parenchyma of the frontal lobe of the rat brain 141 7.2.3.3.. End points for experiments that induce illness in rats 141 7.2.3.4. Biological fluid sample collection from the rat model 142 7.2.3.5. Histological analysis of the rat brain post-implantation of the Nano-Enabled Scaffold Device 142 7.2.3.6. Bioerosion studies on the Nano-Enabled Scaffold Device 142 7.2.4. Determination of in vivo drug release from Sinemet? and the Nano-Enabled Scaffold Device using Ultra Performance Liquid Chromatography analysis on biological fluids 143 xxiii 7.2.4.1. Preparation of priming solvents and mobile phases 143 7.2.4.2. Preparation of standards 143 7.2.4.3. Solid Phase Extraction blood and cerebrospinal spinal fluid 143 7.2.4.4. Instrumentation and operating conditions of the Ultra Performance Liquid Chromatography 145 7.2.4.5. Calibration curves and limit of quantification for dopamine in plasma 145 7.2.4.6. Extraction yield, precision and accuracy of the method 145 7.2.4.5. Cerebrospinal fluid and blood preparation for Ultra Performance Liquid Chromatography analysis 146 7.3. Results and Discussion 147 7.3.1. Brain damage in the rat from the surgical implantation of the Nano-enabled Scaffold Device 147 7.3.2. Modifications to surgical implantation procedure 147 7.3.3. Evaluation of modifications to surgical implantation technique 148 7.3.4. Biocompatibility of the Nano-Enabled Scaffold Device systems employed in the study 148 7.3.4.1. Pathological diagnosis of rat brain specimens implanted with dopamine-free Nano-Enabled Scaffold Device 148 7.3.4.2. Pathological diagnosis of rat brain specimens implanted with Nano-Enabled Scaffold Device 149 7.3.5. Bioerosion of the Nano-Enabled Scaffold Device post-implantation 149 7.3.6. Solid Phase Extraction of dopamine from plasma 151 7.3.7. Chromatograms for standards and plasma 151 xxiv 7.3.8. Calibration curve and lower limit of quantification of dopamine 153 7.3.9. Method validation for the analysis of dopamine in plasma employing Ultra Performance Liquid Chromatography analysis 154 7.3.10. In vivo dopamine release profile for the Nano-Enabled Scaffold Device 155 7.4. Concluding Remarks 157 8. CONCLUSIONS AND RECOMMENDATIONS 8.1. Conclusions 158 8.2. Recommendations 160 9. REFERENCES 161 10. APPENDIX 187 xxv LIST OF FIGURES Figure 2.1: Schematic representation of mechanisms of drug delivery to the brain Page 12 Figure 3.1: Schematic representation of a charged particle and its distribution of ions 36 Figure 3.2: Scanning electron microscope images of (a) dopamine-free Nanoparticles 43 Figure 3.3: Transmission electron microscope images of (a) dopamine- free cellulose acetate phthalate nanoparticle and (b) dopamine-loaded cellulose acetate phthalate nanoparticles formed 43 Figure 3.4: Fourier transmission infrared spectra obtained for the formation of dopamine-loaded cellulose acetate phthalate nanoparticles 44 Figure 3.5: Proposed chemical interaction between dopamine and cellulose acetate phthalate 44 Figure 3.6: Dopamine calibration curve at 280nm in phosphate buffered solution (pH 6.8; 37?C) 45 Figure 3.7: Dopamine release profiles from cellulose acetate phthalate nanoparticles in phosphate buffered solution (pH 6.8; 37?C) 46 Figure 3.8: Size distribution profile for dopamine measuring 156.9nm 47 xxvi Figure 3.9: Zetasize profiles of a) and b) size intensity and zeta potential distribution profiles of dopamine-free cellulose acetate phthalate nanoparticles, c) and d) size intensity and zeta potential distribution profiles of dopamine-loaded cellulose acetate phthalate nanoparticles respectively 48 Figure 3.10: The Regression plots for (a) Mean Dissolution Time, (b) Particle Size and (c) Zeta Potential for the calculation of R2 values that determine the similarity between the predicted and experimentally determined values for the responses 51 Figure 3.11: Response surface plots correlating Mean Dissolution Time and (a) cellulose acetate phthalate concentration and poly(vinyl alcohol) concentration and (b) cellulose acetate phthalate concentration and stirring speed 53 Figure 3.12: Release profiles depicting dopamine release from the cellulose acetate phthalate formulations 54 Figure 3.13: Response surface plots correlating Particle Size and (a) emulsifying time and stirring speed and (b) cellulose acetate phthalate concentration and emulsifying time 55 Figure 3.14: Response surface plots correlating Zeta Potential and (a) poly (vinyl alcohol) concentration and stirring speed (b) emulsifying time and stirring speed 56 Figure 3.15: Typical (a) main effects plot and (b) interactions plot of the response values for Mean Dissolution Time 57 Figure 3.16: Typical (a) main effects plot and (b) interactions plot of the response values for Particle Size 59 xxvii Figure 3.17: Typical (a) main effects plot and (b) interactions plot of the response values for Zeta Potential 60 Figure 3.18: The residual plots for the responses (a) Mean Dissolution Time (b) Particle Size and (c) Zeta Potential 62 Figure 3.19: Optimization plots displaying factor levels and desirability values for the chosen optimized formulation 66 Figure 3.20: (a) Size and (b) Zeta distribution profiles for optimized dopamine-loaded cellulose acetate phthalate nanoparticles measuring 197.2nm and -34mV respectively 67 Figure 4.1: Schematic representation of the assimilation of dopamine-loaded cellulose acetate phthalate nanoparticles and barium-alginate scaffold into the Nano-Enabled Scaffold Device 79 Figure 4.2: Three-dimensional prototype images of a) a pre-cured crosslinked-alginate scaffold, b) a barium-chloride post-cured crosslinked-alginate scaffold, and c) dopamine- loaded cellulose acetate phthalate nanoparticles embedded within the cured crosslinked alginate scaffold voids representing the Nano-Enabled Scaffold Device 82 Figure 4.3: Molecular structural models of a) interactions between water molecules in association with acetate and oxygen functional groups of cellulose acetate phthalate and b) cellulose acetate phthalate interactions and dopamine entrapment 83 xxviii Figure 4.4: Graphical models depicting a-e) the stepwise formation of dopamine-loaded cellulose acetate phthalate nanoparticles, f) a single cellulose acetate phthalate adaptation, g) dopamine interaction and wall initiation and h) a dopamine-loaded cellulose acetate phthalate nanoparticle towards completion 84 Figure 4.5: A typical Force-Time profile of a hydrated barium-alginate scaffold for the calculation of resilience 84 Figure 4.6 : Scanning electron microscope images of (a) barium-alginate scaffold that was not subjected to post-curing (b) a typical pore present within the scaffold, (c) a scaffold that underwent post- curing with barium-chloride and (d) the relation of pores in respective to one another 86 Figure 4.7: Matrix Erosion profile for barium-alginate scaffold in phosphate buffered solution (pH 6.8, 37?C) over a period of 72 days 87 Figure 4.8: The Regression plots for (a) Matrix Resilience (%) and (b) Matrix Erosion for the calculation of R2 values that determine the similarity between the predicted and experimentally determined values for the responses 90 Figure 4.9: Response surface plots correlating the scaffold matrix resilience with a) alginate and crosslinker concentration and b) alginate concentration and processing temperature 91 Figure 4.10: Response surface plots correlating scaffold Matrix Erosion with (a) alginate concentration and post-curing time and (b) processing temperature and post-curing time 91 xxix Figure 4.11: Typical (a) main effects plot and (b) interactions plot of response values for Matrix Resilience 92 Figure 4.12: Typical (a) main effects plot and (b) interactions plot of response values for Matrix Erosion 93 Figure 4.13: The residual plots for the responses (a) Matrix Resilience and (b) Matrix Erosion percentage for scaffolds 96 Figure 4.14: Optimization plots displaying factor levels and desirability values for the chosen optimized formulation 98 Figure 4.15: Dopamine release profiles for (a) dopamine-loaded cellulose acetate phthalate nanoparticles and (b) Nano- Enabled Scaffold Device in phosphate buffered solution (pH 6.8; 37?C) 100 Figure 4.16: Temperature modulated differential scanning calorimetry profiling for Nano-Enabled Scaffold Device showing endothermic and exothermic peaks generated 102 Figure 5.1: A schematic representation of a rudimentary electrospinning device employed for the formulation of nanofibers 105 Figure 5.2: Levodopa calibration curve at 285nm in phosphate buffered solution (pH 6.8; 37?C) 110 Figure 5.3: Levodopa release profiles of (a) levodopa-loaded poly (lactic- co-glycolic acid) nanoparticles and (b) Nano-Enabled Scaffold Device in phosphate buffered solution (pH 6.8; 37?C) 110 xxx Figure 5.4: Transmission electron microscope images of (a) an isolated levodopa-loaded poly (lactic-co-glycolic acid) nanoparticle and (b) a cluster of levodopa-loaded poly (lactic-co-glycolic acid) nanoparticles 111 Figure 5.5: Fourier transmission infrared spectra for levodopa, poly (lactic- co-glycolic acid), levodopa-free and levodopa-loaded poly (lactic-co-glycolic acid) nanoparticles 112 Figure 5.6: Proposed dimethyl sulphoxide and poly (lactic-co-glycolic acid) chemical interaction 113 Figure 5.7: Liquid Fourier transmission infrared spectra analysis for poly (lactic-co-glycolic acid) and dimethyl sulphoxide 113 Figure 5.8: Nicotine calibration curve at 269 in phosphate buffered solution (pH 6.8; 37?C) 117 Figure 5.9: Nicotine release profile for poly (lactic-co-glycolic acid) nanoparticles in phosphate buffered solution (pH 6.8; 37?C) 118 Figure 5.10: Transmission electron microscope image of nicotine-loaded cellulose acetate phthalate nanoparticles 119 Figure 5.11: Scanning electron microscope images of (a) dopamine-loaded poly (vinylalcohol) nanofibers revealed longitudinal solid fibers with a minimal length of 20?nm and (b) dopamine-loaded cellulose acetate phthalate nanofibers showed artifacts in the structure 121 xxxi Figure 5.12: Transmission images of dopamine-loaded poly (vinlyalcohol) nanofibers (a) displaying an uneven distribution of poly (lactic- co-glycolic acid) and dopamine along its length and (b) a closer look showed fibers of minimal diameters of 5nm 121 Figure 5.13: Transmission electron microscope images of dopamine-loaded cellulose acetate phthalate nanofibers revealed a diameter of 1.5?m 122 Figure 6.1: Schematic representation of one of the possible mechanisms involved in the metabolism of dopamine leading to cytotoxic and genotoxic events 125 Figure 6.2: In vivo colorimetric reaction that occurs in living cells when exposed to 3?(4,5?dimethylthiazol-2-yl)?2,5? diphenyltetrazolium bromide 126 Figure 6.3: Diagrammatic representation of the separation of blood that occurs upon it's centrifugation in Ficoll-Hypaque gradient (3:1) tubes (Lifelearn Inc, 2004) 127 Figure 6.4: A schematic representation of the layout of the compounds in the 96-well plate as used in the 3?(4,5?dimethylthiazol-2-yl)? 2,5?diphenyltetrazolium bromide assays on white blood cells and SK-N-MC cells 130 Figure 6.5: 3?(4,5?dimethylthiazol-2-yl)?2,5? diphenyltetrazolium bromide calibration curve for (a) white blood cells and (b) SK-N-MC 131 xxxii Figure 6.6: Graph displaying percentage cell viability of white blood cells after a 24hr exposure period to the various compounds. Cell viability is reported in relation to the control cell sample which was equated to 100%. 133 Figure 6.7: Graphs displaying percentage cell viability of SK-N-MC cells after exposure periods to the various compounds. Cell viability is reported in relation to the control cells which was equated to100%. 134 Figure 7.1: Schematic diagrammatic representation of immunological response to implant within the central nervous system 137 Figure 7.2. Schematic diagram representing the number of rats required for in vivo studies 140 Figure 7.3. Diagrammatic account of the strategy for the optimization of the generic Solid Phase Extraction method 144 Figure 7.4. Photographic representation of the surgical procedure (a) manual drill used for incision, (b) actual perforation of the skull and (c) suturing of the incision 147 Figure 7.5. Light microscope images of histological slides of the site of implantation into the brain, stained using Haematoxylin & Eosin, in (a) control and (b) test brain tissue following 5 days of implantation of Nano-Enabled Scaffold Device 149 Figure 7.6: In vivo bioerosion profile for Nano-Enabled Scaffold Device over a period of 30 days 150 xxxiii Figure 7.7. Light microscope (top) and Scanning electron microscope images (below) of Nano-Enabled Scaffold Device matrix (a) pre-implantation and (b) blood perfused post-euthanasia and removal from rat brain post-7 day implantation showing 16% bioerosion 150 Figure 7.8: A typical chromatograph depicting the peak (Rt = 0.4925) for dopamine employing Ultra Performance Liquid Chromatography at 280nm in double deionised water 152 Figure 7.9: A typical chromatograph depicting the peak (Rt = 1.579) for theophylline(internal standard) employing Ultra Performance Liquid Chromatography at 280nm in double deionised water 152 Figure 7.10: A typical Ultra Performance Liquid chromatogram depicting the distinct separation of dopamine (Rt=0.49) and theophylline (Rt=1.64) at 280nm from the Sprague Dawley rat plasma samples 153 Figure 7.11.: Dopamine calibration curve at 280nm in plasma employing Ultra Performance Liquid Chromatograph in double deionised water 154 Figure 7.12.: In vivo profile for dopamine release from Sinemet? 156 Figure 7.13: In vivo profile for dopamine release from the Nano-Enabled Scaffold Device 156 xxxiv LIST OF TABLES Table 2.1: Selected nano-therapeutic molecules on the market for the treatment of various diseases Page 15 Table 2.2: The diverse architectures of nano-systems 17 Table 2.3: Parkinson?s disease syndrome brain bank diagnostic criteria (United Kingdom) 21 Table 2.4: The current pharmacological treatment for Parkinson?s disease and their limitations 22 Table 2.5 Selected neurorestorative and neuroprotective agents for the treatment of Parkinson?s disease 27 Table 2.6: Surgical strategies employed for the treatment of Parkinson?s disease 29 Table 3.1: Processing conditions for the rotaveporation in formulating dopamine-loaded cellulose acetate phthalate nanoparticles 37 Table 3.2: Illustrates the variations in formulations that were used to identify the limits for a Box -Behnken design to optimize dopamine-loaded cellulose acetate phthalate nanoparticles and their dopamine entrapment efficiency percentage 41 Table 3.3: The variables for the dopamine-loaded cellulose acetate phthalate nanoparticles used in the 4-factor Box-Behnken design 42 Table 3.4: Illustration of variations in formulations employed for a Box- Behnken design for the optimization of dopamine-loaded cellulose acetate phthalate nanoparticles 49 xxxv Table 3.5: Measured responses for the experimental optimization of dopamine-loaded cellulose acetate phthalate nanoparticles 50 Table 3.6: Full ANOVA analysis for the measured responses (Mean Dissolution Time, Particle Size and Zeta Potential for dopamine-loaded cellulose acetate phthalate nanoparticles) 64 Table 4.1: Illustrates alginate-based drug delivery systems that have are being investigated for the treatment of various diseases 70 Table 4.2: Parameters employed to measure the Matrix Resilience of hydrated/unhydrated samples employing the textural analyser 75 Table 4.3: Illustration of variations in formulations that were used to identify the limits for a Box -Behnken design to optimize barium-alginate scaffolds and their Matrix Resilience 76 Table 4.4: The variables for the barium-alginate scaffold used in the 4- factor, 3-level Box-Behnken design 77 Table 4.5: Temperature modulated differential scanning calorimetry settings employed for thermal analysis of the Nano-Enabled Scaffold Device constituents 81 Table 4.6: Percentage Matrix Resilience of crosslinked alginate scaffolds in both hydrated and unhydrated states 85 Table 4.7: Illustrates variations in formulations employed for a Box-Behnken design for the optimization of barium-alginate scaffold 88 Table 4.8: Measured responses for the experimental optimization of barium-alginate scaffold 89 xxxvi Table 4.9: Full ANOVA analysis for the measured responses (Matrix Resilience and Matrix Erosion) 97 Table 4.10: Salient thermal events for dopamine-loaded cellulose acetate phthalate nanoparticles, barium-alginate scaffold and Nano- Enabled Scaffold Device compared to native cellulose acetate phthalate 101 Table 7.2. Illustrates the data obtained to assess the validation of the method 154 37 1 CHAPTER 1 INTRODUCTION 1.1. Background to this Study Parkinson?s disease (PD) is characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the brain (Moos and Jensen, 2004). This results in the loss of striatal dopaminergic terminals and their ability to store and regulate the release of dopamine (DA). DA is a neurotransmitter found in a region of the brain namely the substantia nigra. The transmitter plays a role in behaviour, learning and cognition as well as aids in the execution of controlled muscular movements. DA does not cross the blood brain barrier (BBB) therefore an exogenous supply needs to be orally administered in its levo-form known as Levodopa (L-dopa) (Lai and Yu, 1997). Accordingly, striatal DA receptor activation becomes increasingly dependent on the peripheral availability of an exogenously administered dopaminergic agent (Mrin et al., 2008). As the disease progresses, the patient begins to experience motor abnormalities such as akinesia, a resting tremor, and rigidity. The advancement of the disease results in worsening of these symptoms (Yulmetyev et al., 2006). The disease affects one in every 100 persons above the age of 65 years and is the second most common neurodegenerative disease after Alzheimer's disease (de Rijk et al., 2000). The current treatments for the disease have extensive side-effect profiles and issues with loss of efficacy after prolonged periods of time therefore validating the urgency for new treatment modalities. Drug delivery to the brain remains a highly challenging and essential field of study. Due to the numerous protective barriers surrounding the Central Nervous System (CNS), there is still an urgent need for effective treatment of patients living with neurodegenerative diseases such as PD (Singh et al., 2007). The Blood Brain Barrier (BBB) is a defensive mechanism (Misra et al., 2003). The passage of substances into the brain is highly selective and prevents the entry of high molecular mass hydrophilic compounds. This is a major impediment for drug delivery to the brain as numerous drugs are aqueous in nature and therefore unable to penetrate the BBB (Siepmann, 2006). The alternative is that drugs may be delivered systemically (as in the case with current drug therapy) however only a small percentage of drug reaches the brain due to hepatic degradation as well as the inability to cross the BBB, and the associated side effects 2 related to peak-to-trough fluctuation of plasma drug levels that leads to a lack in patient dose- regimen compliance (Whintey, 2007). High dose parental drug administration is often necessary to reach sufficient concentrations of the drug in the brain parenchyma (Pardridge, 2006) however since neurodegeneration is a chronic disease, this option seems impractical. L-dopa still remains the gold standard for the treatment of PD. Oral administration of L-dopa is rapidly decarboxylated to DA (only 5% DA bioavailability) in extracerebral tissues so that only a small portion (<1%) of a given dose is transported unchanged to the CNS. Due to its significant peripheral metabolism, large doses of L-dopa are required for an adequate therapeutic effect and often produces severe nausea, memory loss and nervousness (Whintey, 2007). Carbidopa inhibits decarboxylation of peripheral L-dopa, increasing the bioavailability of L-dopa by 99% to the brain. It does not cross the BBB nor affects the metabolism of L-dopa within the CNS. Sinemet? (Merck & Co., Inc and Bristol-Myers Squibb, NJ and NY, USA), an immediate release preparation, is a combination of carbidopa and L- dopa and so significantly increases the bioavailability of L-dopa. Sinemet ?CR is a controlled release preparation that allows for a once-a-day dosage. Unlike Sinemet? which is dosed three times a day (Yeh et al., 1989). However there are numerous limitations to the use of L- dopa as well as other anti-parkinson drugs that will be further highlighted in section Chapter 1 Section 1.2 of this dissertation. Nicotine (NT) has received considerable attention for it's possible employment as a neuroprotectant for PD. Smokers have shown a decline in the occurrence of PD as compared to the non-smoker population (Quik et al., 2007). Studies have shown that nicotine may be responsible for an augmentation in vitro as well as in vivo DA release (Rusted et al., 2000). The neuroprotectant effects of NT are further discussed in chapter 2 Table 2.5 of this dissertation. Nanotechnology has a long fairly unexplored history and may overcome limitations posed by current non-targeted drug delivery systems. Nanoparticles are small (mainly polymeric) particles in the nanometer range and are normally spherical depending on the nature of production and can be loaded with various drugs. Due to the small size of nanoparticles, they are able to penetrate the BBB (Liu et al., 2005) and are therefore easily imbibed within cells, allowing for efficient drug accumulation at targeted sites within the brain (Gelperina et al., 2005). The use of biodegradable polymeric material for nanoparticulate formation may also allow sustained drug release at the targeted site over a period of days or even weeks after 3 implantation and ideally would erode in vivo (Yetkin et al., 2000). Furthermore, nano-enabled delivery devices should be able to maintain its physicomechanical properties for the period of drug release and erode with subsequent elimination from the body without producing any toxic by-products (Middleton and Tipton, 1998). Thus, the aim of this research was to combine polymer and nanoscience to design and develop a Nano-Enabled Scaffold Device (NESD) for the delivery of DA to allow for it?s localised and controlled delivery of DA for the treatment of PD. The NESD was implanted into the parenchyma of the frontal lobe of the Sprague-Dawley rat brain. 1.2. Rationale for this Study Polymeric nanotechnology has been researched for it's application in cancer therapy (Alexis et al., 2008). However it has yet to be extensively explored for the treatment of neurodegenerative diseases. The treatment of cancer and neurodegenerative diseases are similar in that they both require targeted drug delivery to optimize bioavailability and reduce systemic side-effects experienced with CNS drugs (Abbot and Ramero, 1996). Nano-drug delivery devices have the potential to (i) maintain therapeutic levels of drug, (ii) reduce harmful side-effects, (iii) decrease the quantity of drug needed, (iv) reduce the number of dosages, and (v) facilitate the delivery of drugs with short in vivo half-lives (Kohane, 2006; Gelperina et al., 2005; Langer, 1998). Drug-loaded nanoparticles can be injected at the site of action but the inclusion of nanoparticles into a biodegradable polymeric scaffold is advantageous for targeted drug delivery as the nanoparticles allow for higher drug loading, due to the high surface area to volume ratio in comparison to other polymeric systems, and are able to facilitate opening of tight junctions between cells for penetrating the BBB (Kreuter, 2001). Furthermore, by employing biodegradable polymers during formulation avoids the need for surgical procedures in order to remove the device once its drug-load has been depleted (Middleton and Tipton, 1998). L-dopa is essentially the levorotatory isomer of dihydroxy-phenylalanine (dopa) which is the metabolic precursor of DA. L-dopa presumably is converted into DA in the basal ganglia. The reason for the formulation and current widespread use of the L-dopa is to enhance transport of the drug across the BBB. Initial therapy with L-dopa significantly restores normal 4 functioning for the patient with PD and every PD-patient will need L-dopa at some time during the course of the disease (Samii et al., 2007). However the major limitation to the use of L- dopa arises after long-term use. The phenomenon is known as the ?end-of-dose wearing-off?, where the therapeutic benefits of each dose of L-dopa lasts for shorter periods (Hely et al., 2000). The patient begins to experience motor fluctuations prior to the time of the next dose. This occurs when the prescribed dose is no longer able to effectively manage the symptoms of the disease. In many patients, ?off? periods of motor immobility are associated with pain, panic attacks, severe depression, confusion and a sense of death (Papapetropoulos and Mash, 2005), which makes the clinical status even more distressing for patients as well as their care-givers. Clinicians attempt to overcome this phenomenon by either increasing the frequency/quantity of the dose or by replacing immediate release preparations with a sustained release preparation for example Sinemet? CR. Increasing the dose places the patient at risk for dyskinesia (the inability to control muscles) which occurs at peak plasma drug levels (Chen and Obering, 2005). The dose also needs to be increased on a regular basis in order to overcome ?the wearing-off? effect which results in an increase in side-effects. Sinemet? CR provides a benefit in that drug plasma levels are maintained over a 24-hour period (Uitti et al., 1997). However, side-effects such as dizziness, insomnia, abdominal pain, dyskinesia, headache and depression are still experienced with sustained release preparations. The inclusion of carbidopa (75-100mg daily) tends to exacerbate psychiatric, gastrointestinal and motor side-effects. Patients also find that while the dosing schedule proves convenient, there is still evidence of dyskinesia (Miyawaki et al., 1997). There have also been reports that, with both Sinemet? preparations, food retards absorption of the drug (Roos et al., 1993). Therefore in order to overcome these limitations, a NESD implanted into the parenchyma of the frontal lobe of the rat brain was developed in this study. The inclusion of DA avoids the need for metabolism to the active (as it is already in its active form) and peripheral loss of the DA thereby increasing its bioavailability. A Nano-Enabled Scaffold Device (NESD) implanted into the parenchyma of the frontal lobe of the brain will ensure DA is delivered in a controlled manner for a prolonged period of time and DA delivery will be targeted to the affected areas of the brain and a lower effective dose would be required with no systemic side-effects. The major benefit of the NESD will be sustained release of DA with increased efficacy in the treatment. This will ensure maintenance of steady-state cerebrospinal fluid (CSF) DA levels which is imperative to prevent the ?end-of-dose wearing-off? phenomenon. The ultimate goal is to increase the patient?s quality of life and restore their daily functioning. Currently, there is no product 5 available on the market that is able to ensure long-term efficiency and control of the motor manifestations of the disease without the aggravation of side-effects such as the psychiatric manifestations and cardiovascular effects (Katzung et al , 2004). The NESD will prove superior in that it serves to overcome the major barriers of current drug delivery systems available for the treatment of PD. Ex vivo and in vivo studies proved essential in this study as the employment of a NESD in the brain is a fairly unexplored field of study and so there are insufficient CNS animal models for correlation purposes. While this study extensively explored in vitro studies performed on the NESD, none served to sufficiently simulate in vivo conditions of the parenchyma of the frontal lobe of the brain. An ex vivo cytotoxic study on brain and white blood cells was developed that allowed for the determination of the potential safety/toxicity of the NESD prior to the undertaking of in vivo animal studies. Animal studies allowed for the determination of the potential CNS effects that the NESD may have within living organisms, unlike the static conditions provided by the in vitro environment. The inclusion of animal studies provided significant data on the DA release kinetics and biodegradation behaviour of the NESD. In order to asses the versatility of the NESD, NT was loaded into the nanoparticles. NT transdermal patches and gums used for the cessation of smoking have proven to be advantageous in stimulating DA release and therefore for the employment in PD (Singh et al., 2007). These findings justify the need for the formulation and thereby investigation into a controlled drug delivery system containing NT. 6 1.3. Aim and Objectives of this Study ? To review current novel drug delivery systems for the treatment of PD and thereby validate the need for the development of a NESD ? To identify a suitable method to develop DA-loaded Cellulose Acetate Phthalate (CAP) nanoparticles that can be incorporated into a crosslinked alginate scaffold ? To identify a suitable method to develop implantable crosslinked alginate scaffolds ? To investigate the physicomechanical and physicochemical properties of the newly formed DA-loaded CAP nanoparticles and crosslinked alginate scaffold ? To synthesis variants of DA-loaded CAP nanoparticles and crosslinked alginate scaffold employing a Box-Behnken Experimental Design followed by physicochemical and physicomechanical analysis ? To establish the optimum parameters necessary for the formulation of the desired DA- loaded CAP nanoparticles and crosslinked alginate scaffold that will form the NESD ? To disperse the DA-loaded CAP nanoparticles into a crosslinked alginate scaffold and thereafter evaluate in vitro DA release and matrix erosion behaviour of the NESD ? To test the cytotoxicity of the NESD on carcinomic brain and white blood cells ? To undertake in vivo animal studies and determine DA release kinetics and ? biocompatibility of the NESD ? To investigate novel configurations for the design of diverse nano-systems that may be employed for the treatment of PD. 7 1.4. Overview of this Dissertation Chapter One outlines the problem and highlights the rationale for the study. It is an introduction to the study that covers the background and introduction. A summary of the aims and objectives is included in this chapter. Chapter Two focuses on drug delivery to the brain and the strategies employed. Furthermore, there is a detailed outline of PD, it's treatment and their limitations. The concepts and definitions of nanotechnology are discussed as well as its application in drug delivery. Chapter Three describes the formulation and development of DA-loaded nanoparticles. Preliminary formulation methods which investigated the stability and size of the nanoparticles are detailed. In addition, formulation parameters were established to allow for the construction of a Box-Behnken followed by optimization that lead to a candidate nanoparticle formulation. Furthermore, physicomechanical and physicochemical properties were analysed as well as in vitro DA release. Chapter Four describes the formulation and development of barium-alginate scaffolds. Preliminary formulation methods highlight the need for increased matrix resilience and decreased mass loss of the scaffold. In addition, formulation parameters were established to allow for the construction of a Box-Behnken followed by optimization that lead to a candidate scaffold formulation. Furthermore, physicomechanical and physiochemical properties were analysed. This chapter also looked at the incorporation of the DA-loaded nanoparticles into the Ba-alginate scaffold and the in vitro release from NESD. Chapter Five is a further development of nanosystems incorporating bioactive compounds such as L-dopa and NT for the treatment of PD. The chapter includes the preliminary in vitro investigation into these systems. Chapter Six is a comprehensive description of the ex vivo cytotoxic testing on brain and white blood cells. Descriptions of the assays employed and the handling of results are contained within the chapter. 8 Chapter Seven contains an explanation of the in vivo animal studies undertaken in the Sprague-Dawley rat model. Development of a protocol for the surgical implantation into the brain, biocompatibility and DA release are the highlights of the chapter. In addition, sample collection of blood and cerebrospinal fluid is described. Chapter Eight presents the conclusions and recommendations for future work. 9 CHAPTER 2 CURRENT STRATEGIES AND LIMITATIONS OF DRUG DELIVERY TO THE BRAIN IN THE TREATMENT OF PARKINSON?S DISEASE 2.1. Introduction 2.1.1. The Physiology of the BBB The BBB serves to protect the brain from the external environment (the rest of the body) by regulating the passage of molecules to the brain. The absence of fenestrations and the presence of tight junctions between the endothelial cells that form the capillaries of the BBB, provide this regulation and protection (Kemper et al., 2004). Approximately 100% of large-molecule drugs and more than 98% of all small, hydrophilic molecules (larger than 400?500 Daltons) do not cross the BBB (Pardridge, 2006). Lipophilic molecules may passively diffuse through the endothelial cells but are generally limited to smaller sized molecules. Other parameters that influence the uptake of a drug in brain tissue are (Summerfield et al, 2007) the degree of ionization of the drug; plasma protein and tissue binding, its affinity for specific carriers as well as local cerebral blood flow. 10 2.1.1.1. Approaches Employed for Drug Delivery to the Brain The diverse mechanisms of drug delivery to the brain are illustrated in Figure 2.1. and briefly discussed hereunder. Passive diffusion of small molecules to the brain The movement of drug from the luminal to the ablumina interface of the BBB, occurs only within lipid-soluble small molecules that have a molecular mass of less than 400 daltons. This is a highly selective and restrictive approach of delivering drug to the brain (Pardridge, 2006). Osmotic disruption of the BBB employing hyperosmolar solutions It is a temporary procedure involving the use of hyperosmolar agents such as mannitol and arabinose (Rapoport, 2000). The administration of these solutions causes the contraction of endothelial cells that constituent the tight junctions. The dysfunction of the cells increases the intracellular spaces thereby allowing access of molecules into the brain (Kemper et al., 2004). This method holds grave consequences in that the disruption is not as temporary as previously stated. The recovery to normal state takes several hours in many instances and may lead to the non-selective entry of toxic substances as well as the rapid influx of fluid into the brain. The employment of osmotic disruption has shown minimal decrease in patient morbidity and it's inevitable risks still hold cause for concern (Kemper et al., 2004). Chemical modification of the drug and it?s penetration through the BBB Lipidization is most commonly used to increase the likelihood of drug passing or bypassing the BBB. However, Pardridge (2006) reported that "to date there is not a single CNS drug prescribed to patients that is an example of a water-soluble drug being converted into a CNS active drug by medicinal chemistry". Furthermore with chemical modification of the drug comes changes in the pharmacokinetic profile as well which could have negative consequences on the biological activity of the drug (Pardridge, 2006; Gaillard and de Boer 2006). Transcranial drug delivery mechanisms There are three major categories for this type of delivery: intracerebroventricular (ICV) infusion, the intracerebral (IC) implantation and the convection-enhanced diffusion (CED) of drug (Pardridge, 2006). These techniques allow for the delivery of drug at the brain parenchyma. In order for the approach to prove beneficial, the drug needs to be placed into the target site to avoid loss of drug through diffusion (Pardridge, 2006). In addition, the CSF 11 turnover rate far supersedes the diffusion rate of drug throughout the brain. CED is the continuous infusion of drug-infused fluid into the brain tissue. This procedure involves serious consequences in that the brain lacks an efficient mechanism to remove this access fluid (Pardridge, 2006; Pathirana et al., 2006). Active efflux of molecules for transportation to the brain An active efflux pump works by ejecting certain molecules from the brain to the systemic circulation. Molecules should have a moiety attached to their structure that would enable inhibition of the efflux transporter, thereby allowing increased brain penetration of the therapeutic drug (Pardridge, 2006; Kusuhara and Sugiyama, 2001). Receptor mediated transport of large molecules through the BBB The attachment of drug onto specific receptors allow for their transportation/endocytosis into the brain. Examples of these include transferrin receptor (holo-transferrin), neonatal Fc (IgG) and receptor type I scavenger receptor (lipoproteins) (Pardridge, 2006; Roberts et al., 1993). Trojan horse receptor mediated transport: an alternative system for the transportation of larger molecules across the BBB This system embodies another approach to deliver larger molecules to the brain. Drug binds to the receptor-specific monoclonal antibody portion of the Trojan horse (a "piggy back" receptor molecule) which binds to an exofacial epitope on the endogenous BBB peptide receptor. Trojan horses allow for the "carrying" of the molecule across the BBB on the endogenous peptide receptor mediated transport system (Pardridge, 2006; Dietz and Bohr, 2004). Carrier mediated transport of water soluble molecules through the BBB These receptors allow for the transportation of water soluble molecules through the BBB. Examples of these carriers include hexose, lactate, glucose and phenylalanine among others. In actuality, the mainstay therapy for PD, L-dopa utilizes the neutral amino acid carrier (used for the transportation of phenylalanine) to cross the BBB. Utilization of the body's intrinsic system to overcome the BBB may be employed to other water soluble drugs. Furthermore, drugs may be converted to the analogues of natural molecules that have transporters within the BBB, thereby allowing entry of drug into the BBB (Tsuji, 2001; Pardridge, 1991). 12 Figure 2.1: Schematic representation of mechanisms of drug delivery to the brain (Rapoport 1976, Pardridge 2002, Pardridge 2005, Abbott et al., 2006) Nasal drug delivery Osmotic disruption Transcranial delivery Passive diffusion Receptor mediated transport Carrier mediated transport Active efflux Transport of lipo-soluble molecules BBB receptor drug nasal epithelium CSF shrinkage of cells hyperosmolar agent BBB CED CEV IC exchanger ATP drug MAb receptor Trojan horse receptor mediated transport Carrier receptor transporting drug into cell 13 Nasal Drug Delivery: an approach for the treatment of central nervous system conditions The administration of drug via the nasal route allows for bypassing the BBB (Mishr, 2004). Drug is transported from the nasal cavity into the olfactory part of the nasal epithelium to enter into the submucus space (adjacent to the olfactory CSF) and then into the CSF compartment of the brain. While drug does not have to pass through the BBB, a prerequisite for nasal drug delivery is a lipophilic drug with a Mw = 400 daltons. Once there is a significant restriction in the drugs that may be delivered using this approach. Another process is to considerably obliterate nasal mucosa by instilling larger molecules (>400Mw) into the nostrils, this causes trauma to the epithelium resulting in the molecules entering through the nasal membrane (Pardridge, 2006; Liu et al., 2001). Nano-therapeutic molecules Nanotechnology comprises ideally of materials with size ranges from 1-100nm. These materials exhibit large surface areas which in turn results in the increase of the particle surface energy and may give rise to biological reactivity (Oberd?rster et al., 2005). Nano- engineered materials and devices aimed at biology and medicine in general and neuroscience in particular are designed to interface with cells and tissues at a fundamental molecular level (Silva, 2007). The process of nano-sizing allows for the potential elimination of obstacles arising from low drug solubility, degradation, fast clearance rates, non-specific toxicity, and inability to cross biological barriers (Kingsley et al., 2006). The site-specific delivery of nano-drugs allows for the maximization of therapeutic effect and minimization of side-effects. Nanodiagnostics: The early recognition and detection of disease states is imperative to the prognosis. Nano-enabled molecular imaging have been made in all imaging modalities including optical, nuclear, ultrasound, computed tomography and magnetic resonance imaging (Caruthers et al., 2007). The increased surface area per volume of nanoparticles ensures that there is no need to load each targeted particle with a high concentration of imaging agent. Examples of these include nanoparticulate iron oxides may be used as contrast agents in magnetic resonance imaging. They monitor gene expression or detection of metastases in prostate cancer, atherosclerotic plaques and brain inflammation (Moghmini, 2005). These imaging agents may further be coupled with a nanosized drug delivery system to allow for multifunctionality allowing for the early detection and treatment of disease (Bawa, 2007). 14 Table 2.1 lists the selected nano-therapeutic molecules on the market for the treatment of various diseases 15 Table 2.1: Selected nano-therapeutic molecules on the market for the treatment of various diseases (adapted from Nijhara and Balakrishnan, 2006, du Toit et al., 2007 and Zhang et al., 2007 Drug name Manufacturer(s) Indications Size (nm) Major benefits FDA Approval Rapamune? (Sirolimus) Wyeth, Elan (Madison, New Jersey, US) Immunosuppressant in kidney transplant patients <200 Enhanced bioavailability, convenient dosage formulation, and extended shelf-life, compared with its microformulated counterpart August 2000 Emend? (Aprepitant) Merck, Elan (New Jersey, US) Delayed nausea and vomiting in chemotherapy patients <1000 First FDA-approved drug for the treatment of delayed nausea and vomiting in chemotherapy March 2003 TriCor? (Fenofibrate) Abbot (Illinois, US) Primary hypercholesterolemia, mixed lipidemia, hypertriglyceridemia <1000 Minimal fasted and fed variability in bioavailability of the drug, when compared with its microformulated counterpart. December 2004 Abraxane? (Abraxane) American Pharmaceutical Partners, Inc. (Illinois, US) Metastatic breast cancer 130 Eliminates the use of toxic solvents that were essential for its microformulated counterpart January 2005 Doxil? (Doxorubicin) ALZA Corporation (California, US) Anti-cancer drug for the treatment of refractory ovarian cancer and Kaposi?s Sarcoma 100 First FDA-approved drug for cancer therapy February 2005 16 Nano-drug delivery systems currently under investigation ? Dalargin poly(butylcyanoacrylate) nanoparticles for central analgesia (Schroeder, 1998); ? Doxorubicin poly(butylcyanoacrylate) nanoparticles coated with polysorbate 80 for the treatment of cancerous tumors (Mitra et al., 2003); ? Diminazenediaceturate (diminazene) polysorbate 80 nanoparticles for the treatment of second stage Human African Trypanosomiasis (Olbrich et al., 2004); ? Amphotericin B chitosan dextran sulfate nanoparticles for the treatment of systemic fungal infection (Tiyaboonchai and Limpeanchob, 2007); ? Delavirdine, stavudine and saquinavir poly (butylcyanoacrylate), methylmethacrylate- sulfopropylmethacrylate, and solid lipid nanoparticles for the treatment AIDs Dementia Complex (Kuo and Su, 2007); ? Indomethacin polylactic acid nanoparticles used for the reduction of fever, pain and inflammation (Z?vi?ov? et al., 2007); ? Insulin fluorescein isothiocyanate nanoparticles for the management of diabetes mellitus (Damg? et al., 2007); and ? Paclitaxel poly (DL-lactide-co-glycolide) nanoparticles for the treatment of cancerous tumors (Feng et al., 2007). Nano-drug delivery systems are by no means limited to nanoparticles and may be manufactured in various configurations. These range from typical nanogels formed from crosslinked hydrophilic polymers to nanofluidics. These structural diversifications are further outlined in Table 2.2. 17 Table 2.2: The diverse architectures of nano-systems Nanosystem Description Image of the nanosystem Nanogels These are crosslinked nanoparticles consisting of hydrophilic polymers that give rise to an increase in viscosity of the material (du Toit et al., 2007) Cabot Corp Nanogel TM, New Hampshire, US, 2003. (www.cabot-corp.com) Nanofibers These are fibers with dimensions <100nm prepared via electrospinning or interfacial polymerization techniques (Yang et al., 2007) Wang et al., 2008 Nanocrystals These are crystalline material of nanosize. Applications include fluorescent semi-conductor nanocrystals employed as probes to allow for imaging and diagnostics (Parak et al., 2003) Shin et al., 2007 Dendrimers These are tree-like macromolecules with branches reaching out from the core. These molecules are of particular interest as their production allows for reproducibility and the formation of highly defined structures. They epitomize the concept of multi-functionality medicinal systems as they permit the attachment of various therapeutic and imaging molecules to their surface (du Toit et al., 2007) Kong and Pan, 2008 Nanoliposomes A lipid layer/s surrounds An aqueous core. These systems may be able to carry both hydrophobic drugs (lipid layer) as well as hydrophilic drugs (aqueous layer). In addition, liposomes may carry charged drugs within its core that will neutralize upon contact to bodily fluids thereby transversing through the surrounding membranous layer (Zaru et al., 2007) University of Washington, 2000-2008 (www.washington.edu) 18 Nanowires These are two dimensional structures with surfaces that can be functionalised to promote targeting, biocompatibility, solubility and controlled drug release (Kwon et al., 2007) Huang et al., 2008 Nanotubes Employment of porous membranes to create nanosized hollow tubes of various dimensions. Functionalization of these tubes allows for the attachment of various drugs to the structure Xiong et al., 2004 Nanofluidics The infamous "lab on a chip". These systems look at the movement of nanoscale drops of fluid into and out of bodily fluids to deliver drug to their target (Hu and Li, 2007) Guo , 2005 19 2.2. Parkinson?s disease: An Overview In 1817, James Parkinson wrote ?An Essay on the Shaking Palsy? which aptly described the clinical manifestations of the disease which later bore his name (Lim, 2005). PD is characterized by the depletion of dopaminergic nigrostriatal neurons. It is the most common movement disorder and second, only to Alzheimer?s disease, as the cause of age-related neurodegeneration (Mosley et al., 2006). As the numbers of the aging population escalates so does the prevalence of neurodegenerative disorders such as PD. Dopamine (DA) replacement therapy is essential to manage the motor manifestations of PD. 2.2.1. Possible theories explicating the aetiology of PD Oxidative stress leading to DA neuronal degeneration It has been reported that the brain has an increased risk (in comparison to the rest of the body) of oxidative stress due to it's inability to counteract radicals (Owen et al., 1996). Furthermore the metabolism of DA leads to the formation of hydrogen peroxide (H2O2) (Czerniczyniec et al., 2007). This hypothesis is further believed as studies have confirmed that (Owen et al., 1996): ? DA turnover is likely to be increased in surviving neurons with a resultant increase in oxidation and thereby increased formation of H2O2; ? Levels of reduced and total glutathione are decreased in the pars compacta of the substantia nigra (SNc) suggesting decreased protection against OH formation; ? Iron concentration is increased in the SNc thereby increasing the likelihood of an interaction with H2O2 and consequent OH formation; and ? Lipid peroxidation is increased in the SNc. Exposure to environmental toxins This theory is based on the symptoms and clinical finding, in animals and humans, when exposed to toxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6- hydroxydopamine (6-OHDA) being the same as in PD. Research has shown that there is a possible link between the exposure to environmental toxins and the development of PD (Rajput, 2001). 20 An inherited disease It has been reported that the lifetime risk in first degree relatives of sporadic cases is estimated to be as high as 17% with the mutated genes, ?-synuclein and parkin, being identified as the genes responsible for the inheritance of PD (Popovic and Brundin, 2005). Increased risk of PD with age The increase in age has an additive effect on cell atrophy that in turn causes the degeneration of specific areas of the brain, detrimental neuropathology and therefore an increase in the prevalence of PD (Elbaz and Tranchant, 2007). However; recent findings have shown that an increase in age may not be significant in the cause of PD (Calne and Kumar, 2007). Pathology of PD: Lewy bodies are considered to be the hallmark feature for PD diagnosis (Lu et al., 2005). These are described as intra-cytoplasmic, single or multiple, spherical or elongated, eosinophilic masses possessing a dense core and a peripheral halo (Wakabayashi et al., 2006). The core consists of microscopic, dense deposits of abnormal protein, formed as products of the mutated ?-synuclein gene. Other factors that may trigger the onset of PD include viral infection; excess iron and manganese in the diet (responsible for additional oxidation and thereby degeneration of the brain cells); CNS disorders (brain tumors, hydrocephalus, encephalitis, meningitis and stroke); traumatic injury to the brain; abuse of antipsychotic drugs and intravenous 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (resulting in mitochondrial dysfunction); and carbon monoxide poisoning (Carson-De Witt, 2003). 2.2.2. The diagnosis of PD Diagnosis and subsequent treatment of PD is sufficiently delayed due to the fact that approximately 80% of striatal nerve terminals and up to 60% of dopaminergic neurons in the substantia nigra have been lost before clinical presentation of symptoms of PD becomes apparent (Agid, 1991). Compensatory mechanisms such as; increased dopaminergic activity in the substantia nigra, down-regulation of DA transporters and up-regulation of postsynaptic DA receptors in the striatum account for the lack of symptoms at an earlier stage of the disease (Nyholm, 2003). There is no definitive test for the diagnosis of PD. Diagnosis is made on the 21 development of symptoms of the disease including gait changes, trembling and difficulty speaking or writing. The United Kingdom PD Society (UK PDS) Brain Bank diagnostic criterion is a guideline tool used to aid in diagnosis (Table 2.3). Table 2.3: Parkinson's disease syndrome brain bank diagnostic criteria (United Kingdom) (extracted from Hughes et al., 1992) Step 1 - Diagnosis of Parkinsonian syndrome Bradykinesia plus at least one of the following: Muscular rigidity Rest tremor Postural instability Step 2 - Exclusion criteria including: History of repeated strokes History of repeated head injury History of definite encephalitis Step 3 - Supportive prospective criteria (at least three required): Unilateral onset Rest tremor present Evidence of progression Persistent asymmetry Excellent response to L-dopa Severe L-dopa-induced chorea L-dopa response for 5+ years Clinical course of 10+ years 2.2.3. Pharmacological treatment of PD Treatment of PD is based on strategies that either prevent dopaminergic cell degeneration and death; stimulate dopaminergic cell proliferation;or compensate depletion of DA (Popovic and Brundin, 2005). Their pharmacological treatment, recommended dosages, side-effects and the limitations are outlined in Chapter 2 Table 2.4 of this dissertation. 22 Table 2.4: The current pharmacological treatment for PD and their limitations (adapted from Cutson et al., 1995, Comella and Tanner, 1995 and Katzung , 2004) Class Drug Dose Side-effects Limitations Dopaminergic L-dopa L-dopa (ratio represent that of carbidopa to L-dopa in mg) 1:10 ratio : 50:200 tds/qid 1:4 ratio: 25:100 L-dopa tds/qid ? Hair loss ? Psychiatric manifestations ? On-off Syndrome ? Multiple dosing (t1/2 = 1.3hrs) ? Sinemet? CR-pulsed rather than constant drug levels Patient variation in absorption results in patient's mood state during the course of the day (United States Patent 4883666, ,1989) MAO inhibitors Selegine Rasagaline Selegine: 5mg bd Rasagaline: 1mg bd or 0.5mg bd(with L-dopa) ? Orthostatic hypertension ? Insomnia ? Impotence ? Hypertensive crisis ? Combination of Rasagaline with L-dopa may possibly increase blood levels of Rasagiline or exacerbate pre-existing dyskinesia (Product information, 2006) Antivirals Amantadine Amantadine: 100mg bd is thought to either, promote the release, prevent the reuptake, or have an influence on the synthesis of DA. ? Slurred speech ? Shortness of breath ? Visual disturbances ? Effect is short lived therefore used as a diagnostic tool COMT-inhibitors Tolcapone Entacapone Tolcapone: 100mg tds (with Sinemet?) Entacapone: 200mg tds (with Sinemet?) ? Dyskinesias ? Hepatotoxicity (talcopone) ? May only be used in combination with L-dopa ? Talcopone - use is limited due to risk of causing potentially fatal, acute fulminant liver failure (should only be used in patients where there is no alternative) Anticholinergics Trihexyphenidyl Benztropine Trihexyphenidyl: 2mg tid Benztropine: 1mg tds Specifically effective against tremor ? Blurred vision ? Difficult or painful urination (especially in older men) ? Dryness of membranes ? There is little to no corrective effect on rigidity or bradykinesia or akinesia ? Increased side-effect profile DA Agonists Ergot derivatives: Bromocriptine Pergolide Non-ergot derivatives: ropinirole pramipexole Pergolide: 1mg tds Bromocriptine: 5mg bd ? Psychiatric disturbances ? Possibility of myocardial infarctions and subsequent death ? Limited to use as adjunctive therapy ? Exacerbation of adverse effects in elderly ? Increased half life will decrease the risk of motor complications but brings increase in price, poorer control of symptoms and DA effects Apormophine Apormorphine: 0.06mg/kg (0.6mL/kg) Alleviates the "off" periods experienced when taking L-dopa ? formation of skin nodules ? nausea ? Short in vivo half-life = 100min ? Impractical administration route (s.c) for chronic use 23 2.2.4. Challenges of current therapy used in the treatment of PD 2.2.4.1. Limitations of Sinemet? for the treatment of PD Sinemet? is currently the leading treatment used for PD but numerous limitations: ? Loss of efficacy: Sinemet? loses it's efficacy in 50% of patients as soon as 5-15 years following continuous administration. Patients begin to experience debilitating dyskinesia ("on-period") and profound tremor, rigidity, and akinesia (?off-periods") (Arica et al., 2005); ? Response fluctuations: Sinemet? has wide distribution throughout the body and patient variation is often documented; ? Accelerated premature metabolism: L-dopa causes delayed gastric emptying, increasing absorption rate in the stomach where L-dopa is absorbed. Dopa decarboxylase is present within the gastric mucosa and will prematurely convert L- dopa into DA which further serves to stimulate DA receptors in the stomach leading to further delays in the gastric emptying rate (Pfeiffer, 2005); ? Minimal BBB transport: approximately 1% of the administered dose is available to the brain; ? Large doses related to side-effects: severe nausea, vomiting and orthostatic hypotension; ? Controlled released preparations: controlled release L-dopa therapy (Sinemet? CR) ? a "once-a-day" treatment results in pulsed rather than constant drug levels (Hely et al., 2002) which alleviates the "wearing-off" phenomenon. In addition, the degree to which Sinemet? CR increases the t1/2 of L-dopa is minimal (40min). Moreover, it has a distinct disadvantage, especially in advanced PD, because its effects are more unpredictable than those of immediate-release L-dopa (Popovic and Brundin, 2005); ? Drug holidays: involves the temporary withdrawal of L-dopa for a predetermined period. This approach is employed as an attempt to resensitize DA receptors in the striatum so that L-dopa therapy can be reintroduced at lower doses with fewer side- effects. This strategy however is highly controversial due to possible serious effects such as neuroleptical malignant-like syndrome and therefore only used as a last resort (Koziorowski and Friedman, 2001); and ? Continuous infusion: the infusion of L-dopa/carbidopa (Duodopa TM, NeoPharma AB, 24 Uppsala, Sweden), into the duodenum or subcutaneous apormorphine infusion over at least 6 months - this seems highly impractical and requires hospitalization for the first series of infusions (Koziorowski and Friedman, 2001). ? Transdermal patches: Neupro? (Schwartz Pharma Neupro ?, Monheim, Germany). The patch allows for sustained release of DA over a 24 hour period resulting in steady state plasma drug levels and essentially the reduction/elimination of the ?on-off? phenomenon. However, the patch needs to be applied and removed once daily and may lead to issues of patient compliance. In addition, there is a limitation to the quantity of drug that may be delivered per square meter of skin (Rascol, 2005) and the resulting continuous stimulation of the receptors may lead to putative desensitivity (Pfeiffer, 2007). 2.2.4.2. The gap in current therapy employed in the treatment of PD Development of novel drug delivery system for the treatment of PD needs to address (Stocchi, 2006) neuroprotection where drug therapy not only provides relief from the immediate symptoms but also serve to restore and repair further neuronal damage in order to inhibit the progression of PD. Furthermore elimination of "end of dose" dyskinesias as with L- dopa and easy administration and favorable dosing schedules need to be achieved with new treatment options. Research into drugs that treat both motor and non-motor symptoms (depression and memory loss) is also encouraged so as to provide holistic treatment for PD. 2.2.5. Implantable novel polymeric drug delivery systems for the treatment of PD Optimization of drug release, stability and sterility as well as size and shape of the implant needs to be scrutinized to ensure the long-term controlled release and biocompatibility of an implanted system (Fournier et al., 2003). Furthermore, an implantable system for any chronic cardiovascular or neurodegenerative disorder should retain the bioactivity of the drug. (Vats et al., 2005). Implantation of a DA-loaded biodegradable polymeric device is necessary in incidences where patients (Taylor and Minger, 2005) show refraction to conventional drug therapy; have a complete loss of DA-producing neurons; require targeted delivery of DA; and/ or wish to avoid ethical and moral consequences that come with the implantation of fetal or tissue cells. 25 2.2.5.1. L-dopa/Carbidopa loaded microspheres for implantation into the cerebellum Arica et al, 2004 conducted studies on these microspheres implanted into the cerebellum of the rat brain. Results showed that test rats had significantly reduced apormorphine rotations as compared to the control group, beginning at week one up to eight post-implantation holding promise as a new treatment for PD. The drawback with the study may have been the employment of two separate costly drugs into the formulation whereas DA-loaded drug delivery systems prove more feasible in that the drug-loaded microspheres system was implanted directly into the brain (Ar?ca et al., 2004). 2.2.5.2. DA-loaded drug delivery systems: microspheres and a silicone pellet into the striatum In two separate studies, DA was loaded into drug delivery systems for the treatment of PD. In the first study, DA-loaded microspheres were implanted into the striatum of the rat brain and released drug in 120min and while it did decrease the rate of release of drug, the quantity was the same as for non-encapsulated, free drug (McRae-Degueurce et al., 1988). In a later study, rats were implanted with DA-releasing pellets which showed a 50% reduction in apomorphine-induced rotational behaviour, and this effect persisted for the 2-month duration of the experiment (Becker et al., 1989). 2.2.6. Gene Therapy The early onset of the disorder may be due to genetic predispositions of PD. Gene therapy is being investigated to slow the progression of the disease. The vector adenoassociated viral (AAV2) not only provides for the attachment of genes but is responsible for the restoration of the Aromatic L-Amino Acid Decarboxylase (AADC) enzymatic function and allows for the conversion of L-dopa to DA, which is significantly reduced in PD. These studies are still underway and have now moved onto clinical trials (Fiandaca et al., 2007). Genes being investigated are: ? AAV2 Glutamic Acid Decarboxylase (GAD): rate-limiting enzyme for synthesis of the major inhibitory neurotransmitter in the brain, ?-amino butyric acid (Fiandaca et al., 2007); ? AAV2 Human Aromatic L-Amino Acid Decarboxylase (hAADC): has an additive effect on the function of AADC and so may decrease the dose of L-dopa required as well as increase its efficacy (Fiandaca et al., 2007); and ? AAV2 neurotrophic factor neurturin (NTN): may prevent the degeneration and provide neuroprotection for the DA-neurons in the striatum (Gasmi et al., 2007). 26 2.2.7 Empirical treatments employed in the treatment of PD Novel symptomatic treatments that target the non-dopaminergic areas will eliminate the motor side effects that arise from the use of dopaminergic agents (Colosimo et al., 2006). Examples of these include -2 adrenergic antagonists, adenosine A2A receptor antagonists, -amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonists, neuronal synchronization modulators and agents that interact with serotonergic systems. In addition, there has been investigation into various agents that may be used to provide neurorestoration and protection in PD, these are outlined in Table 2.5. 27 Table 2.5: Selected neuroprotective and neurorestorative agents under investigation for the treatment of PD Drug Name Mechanism Of Action Short comings/Side-effects Animal model evidence References Co-enzyme Q10 Anti-oxidant Not regulated by the FDA Expensive* Administration of 200mg/kg/day of Co- enzyme Q10 for a period of five weeks resulted in a decrease in striatal DA concentrations in MPTP-lesioned mice Co-enzyme Q10 and nicotinamide are neuroprotective against mitochondrial toxins in vivo (Beal et al., 1994) Creatinine Enhances mitochondrial function and anti-oxidant properties Weight gain, oedema, nausea, vomiting and diarrhea* Administration of 1% creatinine for a period of two weeks resulted in a 10% loss of dopamine neurons in MPTP-lesioned mice in comparison to a 70% loss in untreated mice Creatinine and cyclocreatine attenuate MPTP neurotoxicity (Matthews, 1999). GM1 ganglioside Possible inhibition of cell apoptosis, protection against excitotoxicity and has been hypothesised to reduce the sensitivity of DA-neurons to toxins* Chronic administration may lead to increased serum cholesterol, triglycerides and apolipoprotein B levels* Administration of GM1 to MPTP-lesioned cats resulted in an enhanced sensorimotor behavioral recovery compared to the untreated group. Results show enhanced release and reuptake of DA in GM1-treated animals and an increase in functional DA terminals Differences in release and clearance of extracellular dopamine in the striatum after spontaneous or GM1-ganglioside-stimulated recovery from experimental Parkinsonism (Schneider et al., 2000) Minocycline Might inhibit microglia-related inflammatory events, and also nitric oxide synthase production thereby inhibiting apoptotic (Frankish, 2003 Peschanski, 1994) Anorexia, nausea, vomiting, dizziness,rash, hypersensitivity reactions and headache (Peng et al., 2006) Administration of minocycline to weaver mice decreased the degeneration of DA neurons. Mice (aged 3 weeks) showed a 30% loss of nigral neuronsin comparison to untreated mice who displayed a 50%. Nigrostriatal dopaminergic neurodegeneration in the weaver mouse is mediated via neuroinflammation and alleviated by minocycline administration (Peng et al.,2006 ) NSAIDs Potent inhibitors of cyclooxgenase enzymes resulting in reduction of inflammation involved in PD pathogenesis (Chen et al., 2003) All NSAIDs have shown to be toxic to DA-neurons, except for ibuprofen (Chen et al., 2003) The content of DA in striatum showed significantly decreased after MPTP intoxication in ibuprofen-treated animals compared with control and non-ibuprofen- treated animals Ibuprofen and the mouse model of Parkinson's disease (Kurkowska-Jastrzebska et al., 2006) Nicotine Stimulation of nicotinic receptor evokes the release of DA and is a possible free radical scavenger (Singh et al, 2007) The benefits of the treatment are far overshadowed by the harmful respiratory and addictive effects Nicotine treatment prevented the striatal DA loss after a 6?g 6-OHDA injection when administered 4hr before and 20hr, 44hr and 68hr post- toxin Nicotine prevents striatal dopamine loss produced by 6-hydroxydopamine lesion in the substantia nigra (Costa et al., 2001) Melatonin Potent free radical scavenger which can enhance antioxidant activity (Kadanthode, 2003) Increased drowsiness Chronic administration of melatonin prevented nigral dopaminergic cell death induced by 6- ODHA in the rat model Protective effect of melatonin in a chronic experimental model of Parkinson?s disease (Antol?n et al., 2002) * (National Institute of Neurological Strokes and Disorders, 2005) 28 2.2.8. Current surgical approaches for the treatment of PD The surgical approach for the treatment of PD has lost favour due to complications and the introduction of effective pharmacological treatments such as L-dopa (Walter and Vitek, 2004). However the current failure in the treatment has lead to resurgent interest in this approach (Follett, 2000). Patient selection for surgical intervention (Walter and Vitek, 2004): ? Non-Parkinson's Plus patients: patients who are responsive to pharmacological treatment; ? Age: younger patients seem to tolerate the surgery well; ? Patients who are on medication that still display significantly debilitating symptoms; ? Patients should not display significant neuropsychological dysfunction ; ? Patients should not show any intracranial pathology that could result in surgical complications; and ? Depression or mood disorders adequately controlled with medication. Table 2.6 highlights the three major surgical treatments for PD patients that includes restorative (cell transplantation); ablative (pallidotomy and thalamotomy); and electrophysiological (deep brain stimulation) (Kolchinsky, 2001). 29 Table 2.6: Surgical strategies employed for the treatment for PD Class Description Symptom Improvement Complication/ Limitation Success Rate in Particular Studies Ablation Pallidotomy Performed on the side contralateral to the most debilitating symptoms. Surgical lesioning of the globus pallidus, a deep sub- region of the basal ganglia, situated near the thalamus (Dewey, 2004). Irreversible procedure Tremor L-dopa induced bradykinesia Rigidity (Thompson, 2001) Visual impairment Facial Pareis Hemiparesis Speech and voice Memory impairment (Bilateral pallidotomy gives rise to more serious complications) May exacerbate speech and gait disorders in some patients (Dewey, 2004) Increased success rate <70yrs Bilateral thalamotomies result in a prohibitively high rate of cognitive and speech problems (Dewey, 2004) Thalotomy As per pallidotomy but performed on the thalamus Tremor (Diamond et al., 2007) Speech disturbance Apraxia (Clarke and Moore, 2007) Tremor has reappeared in a few weeks or months in a small percentage of patients (Hallett, 1999) Electrophysiological Deep Brain Stimulation (DBS) A microelectrode is implanted in subthalamic nucleus of globus pallidus (GPi) or in the thalamus. The electrode delivers electrical current at a high frequency that stimulates the tremor control centre resulting in tremor reduction. Non-destructive, non-invasive and reversible (National Institute of Neurological Disorders and Strokes, 2007) Reduction in symptoms of advanced PD (implanted in subthalamic nucleus or GPi) Reduction in tremor (implanted in thalamus) Fatal intracerebral infection Cognition-mania Depression Problems with balance Expensive procedure Generator needs to be replaced every 5-8 years (Panikar and Kishore, 2003) Reduction of PD questionnaire score by 9.5 points and United PD Rating Scale scores by 19.6 in comparison to the pharmacologically treated group, six months post-DBS (Hamani et al., 2005). Thirty months post- surgery there was a lasting decrease in fluctuations by more than 50%, and dyskinesias were reduced by about 70% (Toda et al., 2004) Restorative Embryonic stem(ES) cell transplantation ES cells can be cultured then induced to differentiate into DA-neurons. Initially cells are obtained from developing human embryos, this is a one-time event and so therefore morally acceptable (Walter and Vitek, 2004) All motor symptoms Dystonia and dyskinesia development (1 year or more beyond surgery and continued to have persistent dyskinesia despite reduction or elimination of dopaminergic drugs) Poor survival of the grafts, poor cell purity (University of Pittsburgh Study in Neurosurgery, 2007) Although stem-cell transplants have not yet been used in clinical trials for PD, early animal studies are underway and have shown some ability to produce cells with dopaminergic differentiation 30 Fetal Mesencephalic Dopamine-secreting Cells Ventral mesencephalic tissue from aborted foetuses, after being matched to the patient for ABO blood antigens, can be implanted stereotaxtically through the caudate and putamen via needle tracks in an attempt to replace the lost nigrostriatal neurons (Drucker-Col?n and Verdugo- D?az, 2004) All motor symptoms L-dopa induced dyskinesia Ethical concerns (3-8 embryos are required for a single patient transplantation which does not provide assured success) (Hallett, 1999). Development of "runaway" dyskinesias 30% success rate in patients younger than 60 years old Xenotransplantation May be derived from the ventral mesencephalon tissue from porcine embryos and implanted into the stratium of the human brain (Brevig et al., 2000) All motor symptoms Obsessive compulsive disorder Auditory hallucinations Variance of dyskinesias Worsening of PD symptoms (Peschanski 1994) Fatal or handicapping intracerebral hemorrhages and abscess (Palfi, 2004) Risk of microbe transmission The success rate depends on the gestational period of the donor tissue as well as volume of tissue implanted The survival of newly transplanted cells is very low (Sayles et al., 2004) Unsuccessful integration with the existing neural circuitry, cells may ability to release DA and it?s a complicated non-reproducible procedure (Brevig et al., 2000) Miscellaneous Radiosurgery and the Gamma Knife Allows for the precise location and radiation of specific areas in the brain. Investigation for use as a tool for both thalamotomy and pallidotomy in patients who are not candidates for standard surgery (Swedish Medical Center, 2007) Depends on the type of ablation that is being employed as well as the electrophysiologic al surgery Mortality Weakness Partial paralysis Tissue necrosis Morbidity (Okun et al., 2001) Operated areas were significantly off target (Peschanski et al., 1994) Transcranial Magnetic Stimulation (TMS) Non-invasive technique that employs high frequency magnetic pulses that are placed on specific area on the scalp so as to target affected areas of the brain. An alternate for patients who may not undergo DBS (Erhardt et al., 2004) Motor symptoms (bradykinesia, tremor, postural imbalance) Depressive and visual symptoms (del Olmo et al., 2007) Symptoms may actually worsen with this approach (Boylan et al., 2001) Acute left frontal reverse TMS (20 Hz) was able to modulate dopaminergic neurotransmission release (30% increase) in a rat study (Kanno et al., 2004) 31 2.3. Concluding Remarks This chapter addressed the challenges of delivery of drug to the BBB and the approaches employed to overcome this. Nanotechnology and its applications in drug delivery, in particular to the brain, were investigated. Furthermore, a synopsis of PD was given as well as the current pharmacological treatment used to treat this disorder and the limitations thereof. The employment of implantable and novel drug delivery devices was discussed. Finally, surgical approaches for the treatment of PD were revisited. Overall the urgency for newer and effective treatment was highlighted. 32 CHAPTER 3 DEVELOPMENT AND FORMULATION OF DOPAMINE-LOADED CELLULOSE ACETATE PHTHALATE NANOPARTICLES 3.1. Introduction The size range of <100nm has been the defining term for nanotechnology (Igarashi, 2008). However in DA delivery, this size limitation is not critical and various therapeutically active nano-enabled systems have been used for the treatment of various ailments such as Abraxane? 130nm (Abraxis BioScience Corporate Offices, California, USA), Taxol? 160nm (Bristol-Myers, New York, USA), Adriamycin? 200-400nm (Pharmacia & Upjohn, S.P.A, Milan, Italy) (Farokhzad and Langer, 2006). Nanoparticles may be synthesized employing a number of well established procedures (Peltonen et al., 2004). Salting-out, emulsification-diffusion and nanoprecipitation are among the many examples of these procedures (Olivier, 2005). Salting-out of nanoparticles This involves the combinatory use of an organic and aqueous phase under high magnetic stirring rates for a period of 15-20min to facilitate emulsification. Deionised water is subsequently added to induce the diffusion of the organic phase into the external solvent resulting in nanoparticle formation (Galindo-Rodr?guez et al., 2005). Solvent evaporation/ emulsification-diffusion The polymer and drug are dissolved in an organic phase. The resulting solution is then emulsified by immersing it into an aqueous solution producing an oil in water (o/w) emulsion. Surfactant and/or emulsifying agent may then be added to ensure the stability of the formulation. The organic phase is evaporated by increasing the temperature under reduced pressure. The resultant solution is then centrifuged to produce nanoparticles ( Pi??n- Segundo et al., 1996; Galindo-Rodr?guez et al., 2005). Spontaneous emulsification or solvent diffusion A water soluble solvent and a water insoluble organic phase are mixed to produce an oil phase (used for hydrophobic drugs). An emulsion is then prepared as per the solvent evaporation method and nanoparticles are formed. The size of the particles may be significantly reduced by increasing the concentration of the water-soluble solvent (Mohanraj and Chen, 2006). 33 Supercritical fluid (SF) technology This method limits the toxicity that occurs as a result of surfactants and/or solvents used during formulation. The chosen solute is solubilised in SF. The solution is then pushed through a nozzle allowing the solute to precipitate (completely free of solvent). The disadvantage of this method is that it may only be used for low molecular mass polymers (Pathak et al., 2005). Coercervation or ionic gelation Two diverse aqueous phases are blended (one phase being a hydrophilic polymer solution). The polymers comprise charged groups for electrostatic interaction. The result is the production of nano-size coacervates (Agnihotri et al, 2004). DAs may be encapsulated, covalently attached or adsorbed onto the surface of the nano-carriers (Olivier, 2005). Nanoparticles may be further modified to release DA upon external environmental stimuli such as pH, heat, magnetic fields or chemical changes (Mohanraj and Chen, 2006). 34 3.2. Materials and Methods 3.2.1. Materials Cellulose acetate phthalate (CAP) (Mw=2534.12g/moL), poly(vinyl alcohol) (PVA) (Mw=49,000g/moL), acetone, methanol and dopamine hydrochloride (DA) (Mw=189.64g/moL) were all purchased from Sigma Aldrich (St. Louise, MO, USA). Deionized water was obtained from a Milli-Q water purification system (Milli-Q, Millipore, Billerica, MA, USA). All other reagents were of analytical grade and used as purchased. 3.2.2. Technology Applied in the Present Study 3.2.2.1. Application of the Zetasizer? NanoZS Series Instrument The Zetasizer? Nano Series (Malvern Instruments, Worcestershire, UK) is an evolution in the analysis of nanoparticles. The instrument allows for measurements imperative and exclusive to nanotechnology. Applications include the determination of particle size and zeta potential (which were employed in the present study) as well as the molecular mass of drugs. 3.2.2.2. The determination of the size of the nanoparticles Particle size is determined by dynamic light scattering (DLS) that measures the Brownian motion of the particles. Brownian motion relates to particle size in that smaller particle sizes are expected to move more rapidly than larger particles therefore exhibiting greater Brownian motion. Factors that may affect the rate of particle diffusional speed are: ? Ionic media: a conductive media will increase the electric double layer (Figure 3.1) around the particle, decreasing its speed and thereby increasing its size measurement; ? Surface structure: the arrangement or shape of the particle surface that leads to greater protruding surfaces or reduced, flattened surfaces; and ? Non-spherical particles: the assumption with the DLS technique, employed in this study, is that all particles are spherical. Non-spherical particles may give rise to changes in diffusional speed and therefore size of particles. The particle size is computed as the hydrodynamic diameter calculated by means of the Stokes-Einstein equation (Kusaka and Adachi, 2007): 35 D kTHd pi?3 )( = Equation 3.1 Where d (H) is the hydrodynamic diameter, D is the translational diffusion coefficient, k is Boltzmann's constant, T is the absolute temperature and ? is the viscosity of the liquid/ solvent employed. Parameters, such as temperature and viscosity of the liquid/s used, need to be known as it is imperative as these will affect the calculated hydrodynamic diameter. Temperature needs to be kept constant to ensure that the viscosity of the liquid is maintained throughout the measurement process. 3.2.2.3. The determination of the zeta potential of the nanoparticles The zeta potential, also known as the particle surface charge, may arise from the adsorption of charged surfactant, the ionization of charged groups of DA and CAP and/or surfactants (PVA) employed and the loss of ions from the newly formed nanoparticles. The surface charge influences the rheology, interactions with electrolytes present in the body and stability of the formulation. Stability may be calculated by employing the Derjaguin, Verwey, Landau and Overbeek theory (DVLO) theory (Derjaguin 1940; Vervey and Overbeek, 1948): Vtotal = VR + VA + VS Equation 3.2 Where VS is the potential due to the solvent and VA and VR are the forces of electrostatic born repulsion and van derWaals attraction respectively that exist between the particles. A charge of ?30mV is an indication of a stable formulation that will not aggregate. 36 Figure 3.1: Schematic representation of a charged particle and its distribution of ions An imaginary border exists within the diffuse region, where the sum of the charges of the unstable particles gives rise to the zeta potential measured (Lyklema et al., 1999). The present chapter focuses on the formulation of DA-loaded polymeric nanoparticles with the employment of CAP as the primary polymer. Investigations leading to the development of an experimental design for the DA-loaded CAP nanoparticles was the focal point of this Chapter. The ultimate goal being the identification of a candidate optimized DA-loaded CAP nanoparticle formulation for incorporation into the NESD. In this study, the emulsification-diffusion (ED) method was selected for formulating the DA- loaded CAP nanoparticles. Reasons for employing the ED approach are: ? The ED method allowed for the use of a permutated organic solvent system in which both DA and CAP were soluble in both aqueous and organic solvents. ? Organic solvents employed in the formulation were highly volatile and therefore could easily be removed by the process of rota-evaporation; ? Surfactants could be added in the emulsion to impart stability to the formulation; and ? Noticeable incompatibility between CAP and PVA at high concentrations. The ED method allows for smaller quantities to be added that would maintain a satisfactory DA entrapment of >60%. + + - - - + + + - - - - - - - - - - + Particle with positive charge Stern layer - negative ions strongly attracted to inner core Diffuse region- ions are less strongly attracted 37 3.2.3. Rotavapor? R210 (B?CHI Labortechnik AG, Flawil, Switzerland) with heating bath The apparatus allows for the distillation of organic solvents under reduced pressure thereby concentrating the sample. The reduction of pressure facilitating liquid evaporation at a lower temperature. The distillation tube transfers the distilled solvent into a condenser. A sealed outlet at the end of the distillation tube prevents sample from entering the condenser that may result in contamination. 3.2.4. Identification of ideal processing conditions for the successful formulation of DA-loaded CAP nanoparticles The conditions under which the Rotavap was operated is listed Table 3.1. Table 3.1: Processing conditions for rotaveporation in formulating DA-loaded CAP nanoparticles Processing Condition Settings Temperature (?C) 60 Rotavapor time (min) 60 Rotation speed (rpm) 40 Flask size (mL) 100 Heating media deionized water 3.2.4.1. Temperature for CAP solubilization A magnetic stirrer equipped with a heat adjustable plate (Fried Electric, Haifa, Israel) allowed for the control of temperature during formulation. Heat played a significant role in the rate of CAP dissolution in an acetone: methanol solvent system. A temperature of 30?0.5?C was chosen to provide a balance between the efficient solubilization of CAP as well as inhibition of evaporation and degradation of the highly volatile CAP solution. Parafilm was also used to cover beakers however the volatility of the sample coupled with the high stirring speeds resulted in the degradation of the film and thereby evaporation of solvent. 3.2.4.2. Stirring time and speed for solvent emulsification A minimum emulsification/stirring time of 30min was maintained (Quintanar-Guerrero et al., 1996). This duration was increased to 60min to enable greater agitation and to allow for a further reduction in particle size. The maximum stirring speed 700rpm was chosen to maintain favourable hydrodynamicity of the solution. 38 3.2.5. Preparation of DA-loaded CAP nanoparticles DA-loaded CAP nanoparticles were prepared using an adapted emulsification?diffusion technique, previously reported (Pi??n-Segundo et al., 1996). Briefly, 500mg of CAP and 50mg DA were dissolved in acetone and methanol (3:7 mixture), to which, a 1%w/v PVA solution was added. The solution was agitated for 30min using a magnetic stirrer (Fried Electric, Haifa, Israel) set at 700rpm. A submicronized oil in water emulsion was spontaneously formed due to immediate reduction of the interfacial tension with rapid diffusion of organic solvent into the aqueous phase representative of the Marangoni Effect (Poletto et al., 2008). Excess solvent was evaporated from the suspension using a Rotavap (Rotavapor? R210, Switzerland) at 60?C for 1hr and the resulting solution was centrifuged (Optima? LE-80K, Beckman, USA) at 20,000rpm for 20min. The sediment layer containing DA-loaded CAP nanoparticles was removed and lyophilized for 24hr at 25mtorr to obtain a powder. 3.2.6. Morphological characterization of DA-loaded CAP nanoparticles Morphological characterization of the nanoparticles revealed the shape, surface, structure and size homogeneity and possible degree of aggregation. Surface morphology was characterized by Scanning Electron Microscopy (SEM), (JEOL, JEM 840, Tokyo Japan). Photomicrographs were taken at different magnifications and samples were prepared after sputter-coating with carbon or gold (N=10). Nanoparticle size and shape was further explored using cryo-Transmission Electron Microscopy (TEM) (JEOL 1200 EX, Tokyo, Japan, 120keV) for higher definition and resolution. Samples were prepared by placing a dispersion of nanoparticles in ethanol on a copper grid with a perforated carbon film followed by evaporation and viewing at room temperature (N=10). 3.2.7. Determination of polymeric structural variations due to DA-loaded CAP nanoparticle formation The structure of native CAP, PVA and DA-loaded CAP nanoparticles produced were assessed using Fourier Transmission Infrared (FTIR) spectroscopy to assess the potential for any variations in vibrational frequencies and subsequent CAP and PVA structure as a result of DA, CAP and PVA interactions during DA-loaded CAP nanoparticle formation. Changes in the CAP backbone may alter the inherent stability and therefore affect the physicochemical and physicomechanical properties of the selected polymer type for the intended purpose. Samples of DA-free and DA-loaded CAP nanoparticles were blended with potassium bromide (KBr) in a 1%w/w ratio and compressed into 1?13mm disks using a Beckmann Hydraulic 39 Press (Beckman Instruments, Inc., Fullerton; USA) set at 8 tons. The sample disks were analyzed in triplicate at high resolution with wavenumbers ranging from 4000-400 cm-1 on a Nicolet Impact 400D FTIR Spectrophotometer coupled with Omnic FTIR research grade software (Nicolet Instrument Corp, Madison, WI, USA). 3.2.8. Construction of calibration curves for spectrophotometric determination of DA release from CAP nanoparticles A calibration curve for DA was constructed using a known series of concentrations of DA (0- 0.08mg/mL) in phosphate buffered solution (PBS) (pH 6.8; 37?C). A linear curve was plotted with the observed absorbance on the y-axis and concentration (mg/mL) on the x-axis. The R2 (a statistical value represented the degree at which a function fits a set of values (Phaser Scientific Software, Florida, USA) was calculated to a value more than 0.95. 3.2.9. Determination of DA entrapment efficiency of CAP nanoparticles In order to assess the entrapment efficiency of DA within the CAP nanoparticles, post- lyophilized powdered samples were accurately weighed and completely dissolved in PBS, (pH 6.8; 37?C). The DA content was analyzed by UV spectrophotometry at ?280nm (Hewlett Packard 8453 Spectrophotometer, Germany) and computed from a standard linear curve of DA in PBS (pH 6.8; 37?C) (R2=0.99). Equation 3.3 was utilized to compute the DA Entrapment Efficiency (DEE). 100 tD aDDEE% ?= Equation 3.3. Where DEE% is the DA entrapment efficiency, Da is the actual quantity of DA (mg) measured by UV spectroscopy and Dt is the theoretical quantity of DA (mg) added in the formulation. 3.2.10. In vitro DA release studies on CAP nanoparticles In vitro release studies were performed on the DA-loaded CAP nanoparticle formulations utilizing a shaking incubator (Labex, Stuart SBS40?, Gauteng, South Africa) set at 20rpm. The DA-loaded CAP nanoparticles was immersed separately in 100mL phosphate-buffered saline (PBS) (pH 6.8, 37?C) contained in 150mL glass jars. At predetermine time intervals 3mL samples of the release media were removed, filtered through a 0.22?m Cameo Acetate membrane filter (Millipore Co., Bedford, MA, USA) and centrifuged at 20,000rpm (Redhead, 2001). The supernatant was then removed and analyzed by UV spectroscopy at a maximum 40 wavelength of ?280nm for DA content analysis. DA release was quantified using a linear standard curve (R2=0.99). An equal volume of DA-free PBS was replaced into the release media to maintain sink conditions. In addition, the Mean Dissolution Time (MDT) values were calculated at 8hr, for each of the samples generated in the experimental design, using Equation 3.4. Computing the release data in this manner allowed for the effective model-independent comparison of all formulations in terms of their respective DA release behavior. All release studies were performed in triplicate. Futhermore, the MDT was calculated for formulations generated in the experimental design. 100 M tM-MME% ? ? = ? Equation 3.4 Where Mt is the fraction of dose released in time ti=(ti + ti-1) ? 2 and M? corresponds to the loading dose. 3.2.11. Determination of particle size and zeta potential of DA-loaded CAP nanoparticles The zeta potential value provides an indication of the shelf life stability of the nanoparticles. A high absolute value of zeta potential (-/+30mV) indicates a high electric charge on the surface of the DA-loaded CAP nanoparticles, which may cause strong repellent forces among particles to prevent aggregation in buffered solution. The nanoparticle zeta potential measurements were obtained using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK). Each sample (1%w/v) was appropriately diluted with deionised water, filtered (0.22?m filter Millipore Co., Massachusetts, USA) to maintain the number of counts per second in the region of 600 (Layre et al., 2006) and placed into disposal cuvettes (size) or capillary cells (zeta potential) (Malvern Instruments Ltd, Malvern, Worcestershire, UK. The viscosity and refractive index of the continuous phase were set to those specific to deionized water. Measurements were taken in triplicate with multiple iterations for each run in order to elute size intensity and zeta potential distribution profiles. Futhermore, the particle size and zeta potential was calculated for formulations generated in the experimental design. 41 3.2.11. Formulation and Statistical Optimization of DA-loaded CAP Nanoparticles An experimental strategy, namely the Box-Behnken design, was developed and employed for the statistical optimization of the DA-loaded CAP Nanoparticles. 3.2.11.1. Determination of limitations for variables employed in the Box-Behnken design The formulation variables listed in Table 3.2 were subjected to their higher and lower limits and thereafter DEE was calculated for each formulation. The most influential variables were selected for the employment in the Box-Behnken design. Table 3.2: Illustrates the variations in formulations that were used to identify the limits for a Box -Behnken design to optimize DA-loaded CAP nanoparticles and their DA entrapment efficiency percentage Formulation number Variables DEE% 1 Increased quantity of CAP (0.5-1g) 64.30 2 Increased quantity of DA (0.05-0.1g) 68.97 3 Increased emulsification time (0.5-3hr) 18.39 4 Increased rotavap time (0.5-1hr) 42.19 5 Increased [PVA] (0.5-2%w/v) 18.11 6 Reduced [PVA] (0.5%w/v) 63.20 7 Increased stirring speed (300-700rpm) 65.64 ? Concentration of CAP ? [CAP] was chosen on the basis of [DA] within the formulation. At least 0.5g of CAP was needed to successfully entrap DA. However a limit of 1g was decided on to reduce the formation of DA-free CAP nanoparticles ? Stirring speed - such a parameter is imperative to the size of nanoparticles formed. Stirring speed was set at 300rpm as this allows for minimal agitation while 700rpm was chosen as the upper limit. This was the highest speed at which the vessel could be controlled. ? Emulsification time - a duration of 30min was required to allow for minimal interaction between the aqueous and organic phase while 3hr was chosen as the maximum so as to reduce solvent loss. ? PVA concentration - a concentration less than 0.5%w/v and more than 2%w/v caused precipitation of the PVA and thereby did not produce DA-loaded CAP nanoparticles. 42 A 4-factor (stirring speed, emulsifying time, [CAP] and [PVA]) Box-Behnken design on the measured responses (zetasize analysis and in vitro DA release) was established for formulation optimization. The variables listed in Table 3.3 were selected for the employment in the Box-Behnken design. Furthermore, the measured responses and for the design and the objectives thereof was established. Table 3.3: The variables for DA-loaded CAP nanoparticles used in the 4-factor Box-Behnken design Values Independent Variables Low High CAP (g) 0.5 1 PVA (%w/v) 0.5 2 Stirring speed (rpm) 300 700 Emulsifying time (min) 30 180 Dependent Variables Low High Objective In vitro DA release (MDT) 38 42 Maximize Zeta Potential (mV) -20 -39 Minimize Particle Size (nm) 150 350 Minimize 3.2.11.2. Optimization of DA-loaded CAP nanoparticles Polynomial equations relating the dependent and independent variables were generated, and the formulation process was optimized under constrained conditions to obtain formulations displaying the desirable Particle Size (minimal), Zeta Potential (minimal) and MDT (maximum). 43 3.3. Results and Discussion 3.3.1. Surface morphology of DA-loaded CAP nanoparticles Figures 3.2 and 3.3 are microscopic images that display the morphology and sizes of DA- loaded CAP nanoparticles formulated in an adopted emulsification-diffusion approach Figure 3.2: SEM images of DA-free CAP nanoparticles Figure 3.3: TEM images of (a) DA-free CAP nanoparticles and (b) DA-loaded CAP nanoparticles formed The SEM images (Figure 3.2) show CAP nanoparticles in both DA-free and DA-loaded states. The flocculated particles were uniform in size, spherically shaped and were well- individualised. TEM images of non-DA loaded CAP particles (Figure 3.3a) revealed dense structures with variations in size; however the rounded-shape was maintained. DA-loaded CAP nanoparticles (Figure 3.3b) proved to be slightly transparent with a transient aggregation between them (further explained in 3.2.2.6). Overall both DA-free and DA-loaded CAP nanoparticles showed homogeneous solid matrix structures void of any crystalline surface morphologies. a) a) b) 0.5?m 40nm 40nm 44 3.3.2. Structural variations present in the formation of DA-loaded CAP nanoparticles Chemical structural variations are depicted in Figure 3.4 in the formation of DA-loaded CAP nanoparticles. The variations confirmed the interaction of DA, CAP and PVA in the formation of DA-loaded CAP nanoparticles. Figure 3.4 is the possible chemical reaction that may have occurred between DA, CAP and PVA and therefore provides a further explanation for the structural variations that arose in the formation of the DA-loaded CAP nanoparticles. Figure 3.4: FTIR spectra obtained for the formation of DA-loaded CAP nanoparticles OH OH N O HO O R H2 Figure 3.5: Proposed chemical interaction between DA and CAP .. DA CAP Ionic bonding leading to amine group formation amine group electrophilic oxygen attracted to amine group - (a) PVA (b) DA (c) CAP (d) drug free nanoparticles (e) DA-loaded nanoparticles Wavenumber (cm-1) Tr an sm is si o n 45 FTIR spectra for DA-free CAP nanoparticles (Figure 3.4) revealed a broad stretch band (1070-1242cm-1 and 3200-3600cm-1) representing OH- groups and a stretch band (2926cm-1) indicating alkane moieties while a band at 1731cm-1 revealed the presence of -C=O within the CAP nanoparticle structure. The interpretation demonstrates the definitive presence of impervious CAP in DA-free CAP nanoparticles. The spectra for DA-loaded CAP nanoparticles also confirmed the presence of CAP (bands at 1070, 1242 and 2926cm-1) while the possible interaction of CAP OH- functional groups with the -NH2 group of DA (Figure 3.5) may have resulted in the formation of nitro compounds (1390cm1). The interaction between the H+ of the NH2 group on DA and the O- atom of the OH-group on CAP may have culminated in the proposed physical interactions of the two compounds retarding DA release. 3.3.3. Calibration curve for DA in PBS (pH 6.8; 37?C) for the determination of the concentration of DA release from CAP nanoparticles Figure 3.6 displays a calibration curve obtained for DA in PBS (pH 6.8; 37?C) employing a UV spectrophotometry at ?280 (Hewlett Packard 8453 Spectrophotometer, Germany). Concentration (mg/mL) 0.00 0.02 0.04 0.06 0.08 Ab so rb a n ce 0.0 0.2 0.4 0.6 0.8 1.0 Figure 3.6: DA calibration curve at 280nm in PBS (pH 6.8; 37?C) 3.3.4. DA entrapment efficiency of CAP nanoparticles A DA entrapment value of 63?0.354% was calculated for the DA-loaded CAP nanoparticles. This is considerably high for a nanoparticle formulation which exhibits a large surface area and thereby DA can be easily adsorbed on to the surface instead of being encapsulated within the particle. The DEE value can be attributed to DA having a greater affinity for the aqueous phase of the emulsion therefore significantly increasing the entrapment of DA. y = 13.28x R2 = 0.9964 46 3.3.5. In vitro DA release of CAP nanoparticles The DA-loaded CAP nanoparticles showed minimal release (2%) at t= 0.5hr (Figure 3.7). The release of DA could be ascribed to release of DA that was adsorbed onto the surface of the particles while the steady release at t = 2.5hr could be the result of DA entrapped within the polymeric matrix. Total DA release at 24hr was 16% proving that there was a high degree of prolonged DA delivery. Figure 3.7 is a diagrammatic account of the in vitro release of DA from the CAP nanoparticles over a 25hr period. Time (hrs) 0 5 10 15 20 25 Fr ac tio n a l d ru g re le as e 0.0 0.2 0.4 0.6 0.8 1.0 Figure 3.7: DA release profiles for CAP nanoparticles in PBS (pH 6.8; 37?C), (SD?0.005) 3.3.6. DA-loaded CAP nanoparticle size and stability A nanoparticle z-average size of 241nm and 165nm was recorded for DA-free and DA-loaded CAP nanoparticles, respectively (Figures 3.8a and c). The result was atypical as it was expected that the DA-free CAP nanoparticles would have a smaller size in comparison to the DA-loaded particles due to the absence of DA. However, the zeta potential of DA-loaded CAP nanoparticles displayed increased stability in comparison to the DA-free particles. DA- free particles therefore aggregated more easily, contributing to the relative increase in size. A polydispersity index (PdI) value of 0.030 was calculated for the DA-loaded CAP nanoparticles indicating minimal variation in particle size (165-174nm) and highlighting the uniformity of particle size in the formulation. Zeta potential values of -23.1mV and -35.2mV were recorded for DA-free and DA-loaded CAP nanoparticles respectively (Figures 3.8b and d). While this 47 result was indicative of the desirable lack of particle agglomeration in both DA-free and DA- loaded particles, it also revealed that the DA-loaded CAP nanoparticles displayed superior stability in comparison to DA-free particles. 3.3.6.1. Size and zeta potential distribution profiles for CAP nanoparticles: DA-free and DA-loaded The distribution profiles for size and zeta potential of DA (Figure 3.8), DA-free (Figure 3.9a and b) and DA-loaded (Figure 3.9c and d) CAP nanoparticles were obtained using the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK). A single peak defines a sample with limited distribution in size or zeta potential. 0 5 10 15 20 25 0.1 1 10 100 1000 10000 In te n si ty (% ) Size (d.nm) Figure 3.8: Size distribution profile for DA measuring 156.9nm 48 0 5 10 15 20 25 0.1 1 10 100 1000 10000 Particle Size (d.nm) c) In te n si ty % 0 50000 100000 150000 -200 -100 0 100 200 Zeta Potential (mV) d) To ta l C o u n ts 0 5 10 15 0.1 1 10 100 1000 10000 Particle Size (d.nm) a) In te n si ty % 0 50000 100000 150000 200000 -200 -100 0 100 200 Zeta Potential (mV) b) To ta l C o u n ts a) c) Figure 3.9: Zetasize profiles of a) and b) size intensity and zeta potential distribution profiles of DA-free CAP nanoparticles, c) and d) size intensity and zeta potential distribution profiles of DA-loaded CAP nanoparticles respectively 49 3.3.7. Experimental design for the optimization DA-loaded CAP nanoparticles The Box-Behnken study design, requiring a total of 27 experimental runs (formulation combinations), was generated and analyzed using Minitab? V14 (Minitab Inc, PA, USA). The variables for each formulation are listed in Table 3.4. Table 3.4: Illustration of variations in formulations employed for a Box-Behnken design for the optimization of DA-loaded CAP nanoparticles Formulation number CAP (g) PVA (%w/v) Emulsifying time (min) Stirring speed (rpm) 1 1 1.25 30 500 2 1 1.25 180 500 3 0.75 0.5 180 500 4 0.75 1.25 105 500 5 0.75 1.25 180 30 6 1 1.25 105 30 7 0.75 1.25 105 500 8 0.75 0.5 105 700 9 10.5 0.5 105 500 10 0.75 1.25 105 500 11 0.5 1.25 105 700 12 0.75 1.25 30 700 13 0.75 1.25 180 700 14 0.5 2 105 500 15 0.5 1.25 105 300 16 0.75 2 105 700 17 1 2 105 500 18 0.75 2 105 300 19 0.5 1.25 30 500 20 0.75 2 30 500 21 1 0.5 105 500 22 0.75 0.5 30 500 23 1 1.25 105 700 24 0.75 0.5 105 300 25 0.5 1.25 180 500 26 0.75 1.25 30 300 27 0.75 2 30 500 50 3.3.7.1. Measured responses for the experimental optimization of DA-loaded CAP nanoparticles The measured responses (Zeta Potential, Particle Size and MDT) for each of the 27 formulation employed in the Box-Behnken design are listed in Table 3.5. Table 3.5: Measured responses for the experimental optimization of DA-loaded CAP nanoparticles Formulation number Zeta Potential (mV) Size (nm) (PdI) MDT (DEE %) 1 -22.10 164.47 (0.05) 41.15 (32.71) 2 -14.77 221.83 (0.16) 41.68 (46.43) 3 -1.258 224.87 (0.27) 41.52 (35.33) 4 -28.13 158.93 (0.03) 40.44 (30.48) 5 -32.03 237.83 (0.31) 41.61 (39.56) 6 -25.90 232.93 (0.06) 41.38 (41.34) 7 -22.73 209.33 (0.01) 40.82 (21.13) 8 -23.80 212.70 (0.08) 41.92 (50.36) 9 -10.97 265.27 (0.04) 40.82 (30.04) 10 -35.97 365.93 (0.09) 41.00 (37.68) 11 -12.07 300.13 (0.015) 40.62 (31.34) 12 -26.60 199.00 (0.03) 38.87 (16.97) 13 -30.77 367.40 (0.41) 41.81 (62.10) 14 -17.43 187.60 (0.02) 41.84 (56.57) 15 -26.13 200.43 (0.03) 42.18 (20.11) 16 -28.70 360.10 (0.09) 41.65 (45.33) 17 -27.80 817.20 (0.28) 41.45 (49.52) 18 -29.73 330.23 (0.24) 41.45 (39.50) 19 -32.13 297.63 (0.22) 41.37 (37.23) 20 -23.73 265.03 (0.03) 41.48 (41.38) 21 -18.00 172.60 (0.04) 41.64 (48.55) 22 -28.53 221.83 (0.11) 41.79 (44.88) 23 -33.37 240.27 (0.20) 41.49 (36.71) 24 -17.37 416.53 (0.43) 41.36 (34.96) 25 -38.30 218.73 (0.19) 41.18 (28.59) 26 -30.00 258.03 (0.12) 41.87 (56.48) 27 -31.37 200.63 (0.05) 41.38 (48.13) 51 3.3.7.2. Comparison of the experimental and fitted response values calculated for the experimental optimization of DA-loaded CAP nanoparticles The plots for experimental versus fitted responses for MDT (Figure 3.10a), Particle Size (Figure3.10b) and Zeta Potential (Figure 3.10c) showed R2 values of 81.24, 75.98 and 74.39 respectively. These values show a close agreement between the experimental and predicted values for the responses, proving the Box-Behnken design was robust for the determination of MDT, Particle Size and Zeta Potential. Overall, the plots showed the applicability of the regression models and robustness of the design employed for the optimization of DA-loaded CAP nanoparticles. Formulation no 0 2 4 6 8 10 12 14 16 18 20 22 24 26 M DT 38.5 39.0 39.5 40.0 40.5 41.0 41.5 42.0 42.5 Actual Fitted Formulation no 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Si ze (nm ) 0 200 400 600 800 Actual Fitted a) b) R2 = 81.24 R2 = 75.98 52 Formulation no 0 2 4 6 8 10 12 14 16 18 20 22 24 26 Ze ta Po te n tia l (m V) -40 -30 -20 -10 0 Actual Fitted Figure 3.10: The Regression plots for (a) MDT, (b) Particle Size and (c) Zeta Potential for the calculation of R2 values that determine the correlation between the fitted and experimentally determined values for the formulation responses c) R2 = 74.39 53 3.3.7.3. Response analysis of MDT for DA-loaded CAP nanoparticles The differing DA release profiles for the respective nanoparticulate formulations are represented in Figure 3.12, signifying their variable capabilities to DA release from their matrix structures. A physical incompatibility described by discontinuous aggregation and subsequent clustering between the predominant polymers CAP and PVA was noted. The incompatibility is confirmed in Figure 3.11a where an increase in [CAP] (0.75-1g) and decrease [PVA] (0.5%w/v) led to a desirable MDT value (>41.5) and vice versa. An increase in [PVA] resulted in an increased MDT (Figure 3.11b) therefore stating that the increase in [PVA] (1.5-2%w/v) controlled and limited DA release. Lower stirring speeds (300rpm) also displayed higher MDT values (41.75) presumably due to the efficient entrapment of DA at lower agitation during processing (Figure 3.11b). Figure 3.11: Response surface plots correlating MDT and (a) [CAP] and [PVA] and (b) [CAP] and stirring speed a) b) 54 Time (hrs) 0 5 10 15 20 25 % DA rele a se 0 20 40 60 80 100 1 2 3 4 5 Time (hrs) 0 5 10 15 20 25 % DA rele ase 0 20 40 60 80 100 6 7 8 9 Time (hrs) 0 5 10 15 20 25 % DA relea se 0 20 40 60 80 100 10 11 12 13 Time (hrs) 0 5 10 15 20 25 % DA relea se 0 20 40 60 80 100 14 15 16 17 Time (hrs) 0 5 10 15 20 25 % DA rele as e 0 20 40 60 80 100 18 19 20 21 Time (hrs) 0 5 10 15 20 25 % DA rele ase 0 20 40 60 80 100 22 23 24 25 Figure 3.12: Release profiles depicting DA release from the CAP formulations 55 3.3.7.4. Response analysis for size of DA-loaded CAP nanoparticles Figure 3.13a showed that an increase in stirring speed (300-700rpm) had an unfavourable effect on the Particle Size (150-300nm). Higher stirring speeds may have resulted in greater inter-particle collisions thereby leading to coagulation of particles (Sugih et al., 2007). A prolonged emulsification phase of between 150-180min coupled with a desirable lower stirring speed resulted in the formation of dispersed non-aggregated particles with a reduced Particle Size of maximum 200nm. An interesting observation (Figure 3.13b) was a decrease in [CAP] (0.5g) resulting in an increase Particle Size (200-225nm). DEE results obtained from the experimental design template (Table 3.4) demonstrated that a significantly lower DEE was achieved with an increase in [CAP] which may have resulted in decreased Particle Sizes. (DA=156.9nm as per Figure 3.8). Figure 3.13: Response surface plots correlating Particle Size and (a) emulsifying time and stirring speed and (b) [CAP] and emulsifying time a) b) 56 3.3.7.5. Response analysis for Zeta Potential of DA-loaded CAP nanoparticles The increase in [PVA] (1.5-2%w/v) gave rise to increased, desirable Zeta Potential (-33 to - 35mV) (Figure 3.14a). This was expected as the PVA was added to the formulation due to its ability to act as an absorptive surfactant, decreasing interfacial tension and imparting stability to the formulation. Figure 3.14b showed that an increased in stirring speed (500-700rpm) and decrease in emulsifying time (30min) resulted in high Zeta Potentials (-30 to -35mV). The higher agitation velocity prevented the particles from aggregating and eliminated the possibility of sedimentation or caking of the nanoparticles. Figure 3.14: Response surface plots correlating Zeta Potential and (a) PVA and stirring speed (b) emulsifying time and stirring speed a) b) 57 3.3.7.6. The main and interaction effects on the responses: Mean Dissolution Time The main effects on the MDT (Figure 3.15a) noted that [PVA] had a significant effect (p = 0.052) on MDT, where concentrations that were either < or > 1.25%w/v had a positive effect on the MDT. This showed that the increase in [PVA] (1.5-2%w/v) was able to control and limit DA release. While interaction plots (Figure 3.15b) for MDT displayed that a high MDT value was most significantly influenced by a decrease in stirring speed and time (p = 0.091). Therefore overall, a decreased stirring speed may allow for the adequate homogenation of the formulation components prior to micronisation of the particles. This would significantly increase the DA entrapment efficiency within the polymer thereby controlling release of DA from the CAP nanoparticles. M e a n o f M DT 1.000.750.50 41.6 41.4 41.2 41.0 2.001.250.50 18010530 41.6 41.4 41.2 41.0 700500300 [CAP] [PVA] Emulsifying Time Stirring Speed a) 58 [CAP] Emulsifying Time Stirring Speed [PVA] 2.001.250.50 18010530 700500300 42.0 40.5 39.0 42.0 40.5 39.0 42.0 40.5 39.0 [CAP] 1.00 0.50 0.75 [PVA] 2.00 0.50 1.25 Emulsifying Time 180 30 105 Interaction Plot (data means) for MDT Figure 3.15: Typical (a) main effects plot and (b) interactions plot of the response values for MDT Particle Size [PVA] was once again a significant variable in influencing the Particle Size (p =0.034), with Particle Sizes increasing with an increase in [PVA] being a main effect (Figure 3.16a). However, interaction plots (Figure 3.16b) revealed a higher stirring speed and decreased [PVA] produced smaller particles (<300nm). The rate at which PVA was agitated was sufficient to ensure homogeneity and the impartation of surfactant properties to the formulation thereby reducing the risk of particle attraction that could produce unfavorably larger Particle Sizes. b) 59 M e a n o f P a rti cl e Si ze 1.000.750.50 360 330 300 270 240 2.001.250.50 18010530 360 330 300 270 240 700500300 [CAP] [PVA] Emulsifying Time Stirring Speed [CAP] Emulsifying Time Stirring Speed [PVA] 2.001.250.50 18010530 700500300 900 600 300 900 600 300 900 600 300 [CAP] 1.00 0.50 0.75 [PVA] 2.00 0.50 1.25 Emulsifying Time 180 30 105 Figure 3.16: Typical (a) main effects plot and (b) interactions plot of the response values for Particle Size a) b) 60 Zeta Potential The main effects on Zeta Potential proved that [PVA] has a significant effect on the negative Zeta Potential (Figure 3.17a) and thereby confirmed its capacity to impart stability to the formulation (p= 0.051). In the interaction plots (Figure 3.17b), there was a distinct relationship between a decreased [CAP] and increase Zeta Potential and this was noted in consideration of the physical incompatibility between CAP and PVA. M e a n o f Z e ta Po te n tia l 1.000.750.50 -20 -25 -30 2.001.250.50 18010530 -20 -25 -30 700500300 [CAP] [PVA] Emulsifying Time Stirring Speed a) 61 [CAP] Emulsifying Time Stirring Speed [PVA] 2.001.250.50 18010530 700500300 0 -20 -400 -20 -400 -20 -40 [CAP] 1.00 0.50 0.75 [PVA] 2.00 0.50 1.25 Emulsifying Time 180 30 105 Interaction Plot (data means) for Zeta Potential Figure 3.17: Typical (a) main effects plot and (b) interactions plot of the response values for Zeta Potential b) 62 3.3.7.7. Analysis of the Box-Behnken design employed for optimization of DA-loaded CAP nanoparticles The assessment of the regression models can be undertaken employing residual plots (Figure 3.18). Residual plots are essentially a linear comparison between observed values and model predictions for those observations (Stewardson and Whitfield, 2004). Residual Pe rc e n t 1.00.50.0-0.5-1.0 99 90 50 10 1 Fitted Value Re si du al 42.041.541.040.540.0 0.5 0.0 -0.5 -1.0 Residual Fr e qu e n c y 0.40.0-0.4-0.8 8 6 4 2 0 Observation Order Re s id u a l 2624222018161412108642 0.5 0.0 -0.5 -1.0 Normal Probability Plot of the Residuals Residuals Versus the Fitted Values Histogram of the Residuals Residuals Versus the Order of the Data Residual Plots for MDT a) 63 Residual Pe rc e n t 2001000-100-200 99 90 50 10 1 Fitted Value Re si du al 600400200 300 200 100 0 -100 Residual Fr e qu e n c y 2001000-100 8 6 4 2 0 Observ ation Order Re s id u a l 2624222018161412108642 300 200 100 0 -100 Normal Probability Plot of the Residuals Residuals Versus the Fitted Values Histogram of the Residuals Residuals Versus the Order of the Data Residual Plots for Particle Size Residual Pe rc e n t 100-10 99 90 50 10 1 Fitted Value Re s id u a l -10-20-30 10 5 0 -5 -10 Residual Fr e qu e n c y 1050-5-10 4.8 3.6 2.4 1.2 0.0 Observation Order Re s id u a l 2624222018161412108642 10 5 0 -5 -10 Normal Probability Plot of the Residuals Residuals Versus the Fitted Values Histogram of the Residuals Residuals Versus the Order of the Data Residual Plots for Zeta Potential Figure 3.18: The residual plots for the responses (a) MDT, (b) Particle Size and (c) Zeta Potential b) c) 64 Residual analysis for MDT (Figure 3.18a), Particle Size (Figure 3.18b) and Zeta Potential (Figure 3.18c) showed the indiscriminate distribution of data. The normal plot of residuals formed a linear curve showing normality. The residuals versus fitted plots showed a random pattern of residuals on either side of 0 with no identifiable patterns in the plot thereby indicative of a random scatter and no trends. The histogram supported that the residuals have a normal distribution with zero mean and constant variance. The residuals versus the order of the data was used to identify and non-random error, the plot displayed a negative correlation is indicated by rapid changes in the signs (-/+) of the consecutive residuals. A significant effect is indicated by a p-value of ?0.05 for any factor. The factors and the respective p-values are displayed in Table 3.6. Table 3.6: Full ANOVA analysis for the measured responses (MDT, Particle Size and Zeta Potential for DA-loaded CAP nanoparticles) Term p-value MDT Particle Size Zeta Potential [CAP] [PVA] [ET] [SS] [CAP]2 [PVA]2 [ET]2 [SS]2 [CAP]*[PVA] [CAP]*[ET] [CAP]*[SS] [PVA]*[ET] [PVA]*[SS] [ET]*[SS] 0.687 0.918 0.187 0.091 0.195 0.0520 0.443 0.309 0.292 0.526 0.156 0.882 0.748 0.013 0.412 0.034 0.886 0.352 0.544 0.226 0.823 0.437 0.837 0.916 0.456 0.851 0.031 0.783 0.877 0.051 0.605 0.617 0.293 0.115 0.941 0.698 0.889 0.610 0.222 0.132 0.765 0.777 Where SS = stirring speed, ET = emulsifying time, MDT = mean dissolution time, PVA = poly(vinylalchol) and CAP = cellulose acetate phthalate 65 The complete regression equations generated for MDT, Particle Size and Zeta Potential are indicated below: MDT=-1.049E-13+8.196E-14[CAP]-3.972E-15[PVA]+0.500[ET]+2.031E-16[SS]-1.204E- 14[CAP]2-2.69E-15[PVA]2-2.561E-19[ET]2-1.276E-19[SS]2+1.585E14[CAP*PVA]-8.771E- 16[CAP*ET]+3.723E-18[CAP*SS]+3.370E-17[PVA*ET]-1.596E-17[PVA*SS]- 3.817E-19[ET*SS] Equation 3.5 Particle Size=-131.441-406.807[CAP]+395.560[PVA]+0.985[ET]+0.861[SS] 547.133[CAP]2+124.081[PVA]2-0.002[ET]2+0.0012[SS]2-70.933[CAP*PVA]+0.361[CAP*ET]- 0.974[CAP*SS]-0.216[PVA*ET]-1.027[PVA*SS] - 0.001[SS*ET] Equation 3.6 Zeta Potential=-25.415-53.170[CAP] 25.842[PVA]+0.120[ET]+0.119[SS]+69.820[CAP]2+11.973[PVA]2+5.289E-05[ET]2-3.935E- 05[SS]2-3.467[CAP*PVA]+0.128[CAP*ET]-0.118[CAP*SS]-0.131[PVA*ET]+0.009[PVA*SS]- 8.833E-05 [SS*ET] Equation 3.7 3.3.7.8. Response optimization of DA-loaded CAP nanoparticles This was carried out employing statistical software (Minitab ?, V14, Minitab Inc?, PA, USA) to determine the optimum level for each variable. The optimization process resulted in the attainment of various formulations with a low desirability for all three outcomes therefore a selection of the most influential desired outcome was necessary to the detriment of the other two outcomes. MDT of DA-loaded nanoparticles could be controlled further by the incorporation of these nanoparticles into the scaffold while Zeta Potential could be altered by uniform distribution throughout the scaffold during formulation. Therefore, the DA-loaded CAP nanoparticle formulation displaying the smallest Particle Size with high desirability (>99%) was selected as the optimal formulation. The formulation was optimized for the measured responses of MDT, Particle Size and Zeta Potential (Figure 3.19). Optimization was carried out so as to obtain the levels of [CAP], 66 [PVA], emulsifying time and stirring speed that would maximize the measured responses (Palamakula et al., 2004). Figure 3.19: Optimization plots displaying factor levels and desirability values for the chosen optimized DA-loaded CAP formulation Hi Lo0.72178 D New Cur d = 0.86890 Targ: -30.0 ZETA POT d = 0.99965 Targ: 150.0 PARTICLE d = 0.43291 Minimum MDT y = -26.0671 y = 149.9826 y = 43.5063 300.0 700.0 30.0 180.0 0.50 2.0 0.50 1.0 [PVA] Emulsify Stirring[CAP] [0.5460] [1.9920] [31.1130] [300.0460] Measured response Predicted values Experimental values Desirability (%) Zeta Potential (mV) -26.072 -34.000 76.682 Size (nm) 150.175 197.200 76.154 MDT 43.505 40.956 94.414 67 3.3.7.9. Desirability for the measured responses of the optimized DA-loaded CAP formulation The value for MDT desirability (94.41%) was the most promising outcome and therefore DA release from the CAP nanoparticle system would be controlled and sustained for the period of time desired. With reference to the particle size (possessing a statistical desirability of 76.15%); while the value of 197nm (Figure 3.20a) was not ideal it was within size range of previously marketed medicinal nano-therapeutic systems (Table 2.1). Furthermore, the particles do not need to cross through the BBB and so the size may exceed 100nm. The desirability value of 76.68% obtained for the Zeta Potential optimization (Figure 3.20b) signified that it differed substantially from the fitted value with a superior value of -34.00mV for the optimized system in terms of stability. Overall, the optimized system displayed the desirable DA release, size and stability required for been utilized as an intracranial device for the prolonged and controlled delivery of DA to the brain tissue. 0 5 10 15 20 25 0.1 1 10 100 1000 10000 In te ns ity (% ) Size (d.nm) 0 100000 200000 300000 400000 -200 -100 0 100 200 To ta l C o un ts Zeta Potential (mV) Figure 3.20: (a) Size and (b) Zeta distribution profiles for optimized DA-loaded CAP nanoparticles measuring 197.2nm and -34mV respectively a) b) 68 3.4. Concluding Remarks This chapter served in the development of DA-loaded CAP nanoparticles using an adopted emulsification-diffusion approach. Parameters such as Particle Size, Zeta Potential and MDT were chosen as the significant responses for the formulation due to the ability of these to control DA release. DA entrapment proved significantly high for CAP nanoparticles which compensated for the rapid in vitro release of the DA. Micrographs (TEM and SEM) further revealed the uniform spherical shape and morphology of the DA-loaded CAP nanoparticles. FTIR analysis confirmed the presence of both DA, CAP AND PVA within the DA-loaded CAP nanoparticles as well the ionic interaction. Data from size and zeta potential intensity profiles proved formulations were stable and within the desirable nano-range of 165.4-174.1nm. A Box-Behnken statistical design was employed for the optimization of the formulation. [CAP] and [PVA] as well as the stirring speed and emulsifying time where identified as the significant formulation variables. The design generated diverse formulations giving rise to various differences in responses (MDT, Particle size and Zeta Potential). Evaluation of the statistical significance of the design confirmed the applicability to the present study. Optimization of the formulation resulted in DA-loaded CAP nanoparticles with a Zeta Potential of -34mV, Particle Size of 198nm and MDT value of 40.96. The DA-loaded CAP nanoparticles were foremostly optimized for a maximum MDT value that resulted in controlled DA release. The DA-loaded CAP nanoparticles were incorporated into a Ba-alginate scaffold and further evaluated for in vitro DA release and will be detailed in Chapter 4 of this dissertation. 69 CHAPTER 4 FORMULATION OF CROSSLINKED ALGINATE SCAFFOLDS FOR THE DESIGN OF A NANO-ENABLED SCAFFOLD DEVICE 4.1. Introduction Prototyping technology has created a significant impact in biomedical materials design. Molecular modeling facilitates the design of accurately customized structural models of polymeric devices for various applications (Levy et al., 1997; Ono et al., 1999; Chua et al., 2000; Curodeau et al., 2000; Porter et al., 2001 and Cheah et al., 2002), therefore prompting the adoption of a similar approach to fabricate the NESD with controlled micro-architecture and higher consistency than conventional unsighted techniques. Free-form prototyping technology was used to design the NESD via a three-dimensional (3D) crosslinked alginate scaffold model incorporating DA-loaded CAP nanoparticles. Prototyping provides an alternative that aims to improve the NESD design by employing archetype data manipulation to pre-assemble the complex internal scaffold architectures and nanostructures of the NESD in conjunction with a Box-Behnken statistical design for optimization and an integrated corporeal manufacturing approach that is consistent, reproducible and formulation-specific. While the formulation and development of DA-loaded CAP nanoparticles, for incorporation into a crosslinked-alginate scaffold, was previously discussed in Chapter 3, the present chapter addresses the development of crosslinked-alginate scaffolds which formed part of the NESD. Alginate was selected as the ideal polymer for the formulation of the scaffolds. The polymer is a hydrophilic polymer, has been used extensively in both the food and pharmaceutical industry for it's desirable gelling, degradation and biocompatible properties (Chan et al., 2002). Alginate is a co-polymer extracted from various types of brown algae and is made up of D-mannuronic acid (M) and L-guluronic acid (G). The monomers may be varied in concentration so as to impart various characteristics to the system in which it is employed (Martinsen et al., 1989). Furthermore, alginates may be crosslinked with a selection salts from Hofmeister series such as calcium, barium and zinc, resulting in the ability to form a gel (Ciofani et al., 2007) with decreased degradation kinetics. In terms of bioadhesivity, the alginates are anionic polymers with carboxyl end groups and therefore have excellent 70 mucoadhesivity which has desirable properties for drug absorption and bioavailability (Rastogi et al., 2007). The biocompatibility of these polymers has been extensively discussed in literature and the oral administration thereof proves non-toxic however there is still a significant lack in in vivo data for intravenous as well as implants (Becker et al., 2000; Orive et al., 2005; Ciofani et al., 2007). Alginate-based drug delivery systems have been researched for their possible employment as drug delivery systems for the treatment of various ailments. Table 4.1: Illustrates alginate-based drug delivery systems that have are being investigated for the treatment of various diseases Drug delivery system Treatment Reference Intratumoural injection of hollow chitosan-alginate microspheres Cancer Liu et al., 1997 Ophthalmic drops containing gatifloxacin- loaded alginate/hydroxyl propylmethyl cellulose Bacterial infections Liu et al., 2006 Stomach-specific metronidazole-loaded alginate beads Helicobacter pylori Ishak et al., 2007 Colon-specific delivery of 5- aminosalicylic acid from chitosan calcium alginate microparticles Ulcerative colitis Mladenovska et al., 2007 The appropriate selection of the grade as well as formulation methodology employed may result in variations in pore size, degradation and drug release rates (Ciofani et al., 2007). Biodegradable polymeric scaffolds have been employed extensively in tissue regeneration (Whang et al., 2000) for their ability to interact with cellular tissue and in drug delivery for their ability to control drug release rate (Edlund and Albertsson, 2001). Issues such as drug loading capacity and rate of drug release, degradation and toxicity are imperative in the formulation of these drug delivery devices (Sokolsky-Papkov et al., 2007). Conventional fabrication methods may be adapted so as to impart desired properties onto the scaffold. The following conventional methods have been employed for the formulation of polymeric scaffolds: 71 Gas-foaming under increased pressure Polymers are made into a solid form and placed into an incubator of gas at high pressure for up to 3 days at which point the pressure is rapidly decreased to atmospheric pressure. This method holds favour as there is avoidance of toxic solvents. However the use of heat to compress the polymer may damage the excipients and actives (Mikos and Temenoff, 2000). The average pore size present in such scaffolds is 100?m. Solvent/ particulate leaching of crystals from polymeric solutions A polymeric solution is immersed in a petri dish consisting of a water soluble salt (pyrogen). The solvent is then evaporated and the resultant material is placed in water for upto 2 days to leach out the salt. Modifications of the size of the salt crystals result in changes in pore size while salt concentration results in alterations of porosity. The employment of organic solvents limits the use of therapeutic active agents and may result in toxicity while the duration of the procedure can be extensive (Mikos and Temenoff, 2000). Phase separation/ emulsification followed by lyophilization of the emulsion An emulsion in formed by adding an aqueous phase to an organic phase (with dissolved polymer). The emulsion is then placed into a mould and quenched with liquid nitrogen (Calvin, 2003). The resultant material is then lyophilized to ensure the removal of excess solvent and the formation of a scaffold with almost nanoscopic pore sizes (13-35?m) (Mikos and Temenoff, 2000). The limitation in this technique is that the toxicity of solvents employed is highly questionable. In liquid-liquid phase separation, there is the utilization of a non- solvent and solvent. The polymer is dissolved in the solvent and then subjected to temperature variations and subsequent lyophilized. This method is highly specific and thorough preliminary testing needs to be undertaken prior to formulation (Leong et al., 2003) Bonding of nano-fibers to form meshes The technique of fiber bonding involves the formation of polymer fibers by electrospinning. The fibers are bonded by means of immersion and subsequent evaporation of a polymer solution (separate from the polymeric fibers being formed). The fibrous solution is then heated to a temperature above the melting point of both polymers, allowing the polymers to fill spaces between the fiber network (Mikos and Temenoff, 2000). Excess polymer solution is removed using an appropriate reagent to adsorb the solution. The technique results in the formation of a highly porous matrix with an average pore size of 500?m (Calvin, 2003). The disadvantage is the possible toxicity of the solvent and the high temperature combination. 72 In the present study, lyophilization was chosen for the fabrication of crosslinked-alginate scaffolds. Reasons for employment of this approach include: ? Suitable method for the formation of scaffolds employing a hydrophilic polymer- alginate; ? Porosity of the scaffold could be controlled, in terms of size and shape, which influences the rate of Matrix Erosion (ME) and subsequent DA release; and ? Efficiency and reproducibility of the method is desirable. 4.2. Materials and Methods 4.2.1. Materials Alginate (Protanal? LF10/60; 30% mannuronic acid, 70% guluronic acid residues) was purchased from FMC Biopolymer (Drammen, Norway). Calcium gluconate [(HOCH2 (CHOH)4COO)2Ca], barium chloride (BaCl2), cellulose acetate phthalate (CAP) (Mw=2534.12g/moL), poly(vinyl alcohol) (PVA) (Mw=49,000g/moL),, acetone, methanol and dopamine hydrochloride (DA) (Mw=189.64g/moL) were purchased from Sigma Aldrich (St. Louise, MO, USA). Deionized water was obtained from a Milli-Q water purification system (Milli-Q, Millipore, Billerica, MA, USA). All other reagents used were of analytical grade. A lyophilizer (Virtis, Gardiner, NY, USA)) was used to remove excess moisture and solidify the formulation in order to produce crosslinked alginate scaffolds. 4.2.2. Processing conditions for the development of crosslinked alginate scaffolds A lypholization time of 36hr at 1.2mTorr and condenser temperature of -60?C was established as the optimum conditions for the removal of excess moisture from the alginate scaffold. 4.2.2.1. Temperature for alginate solubilization Heat is an essential component in the solubilization of the alginate powder in deionised water. The gradual addition as well as increase in temperature allowed for the efficient dissolution of the alginate. A minimal temperature of 50?C was chosen to allow for sufficient heat to aid the dissolution process and to refrain from destroying the alginate structure (FMC BioAlginate, USA). In addition, parafilm was used to seal the beakers as to prevent solvent evaporation from the formulation. 73 4.2.2.2. Duration required for the efficient post-curing of crosslinked alginate scaffold Post-curing the alginate scaffold, which entailed the further crosslinking of the [(HOCH2(CHOH)4COO)2Ca]-crosslinked alginate scaffolds in salt solutions (2%w/v), resulted in slow erosion rate of the scaffold in PBS (pH 6.8, 37?C). While a prolonged post-curing period holds positive implications for Matrix Resilience (MR), it can be highly detrimental to the DEE of the system and therefore a maximum post-curing time of 3hr was established. Furthermore, scaffolds were cured for up to 12hr with no significant effect (SD = 2.65) onMR. N=3. 4.2.3. Biometric simulation of the NESD employing computer-aided prototyping The implicit design of the NESD required customization of the crosslinked-alginate scaffold for embedding the DA-loaded CAP nanoparticles with the ability to support bioadhesion and the physicomechanical stability for intracranial implantation of the device. CAP and [(HOCH2(CHOH)4COO)2Ca]-crosslinked alginate were selected for producing the DA-loaded CAP nanoparticles and scaffold components of the NESD respectively. The crosslinked scaffold was subsequently cured in a BaCl2 solution as a secondary crosslinking step. The componential NESD properties were modulated through computational prototyping to produce a viable scaffold embedded with stable DA-loaded CAP nanoparticles. The fundamental design parameters were pivoted on the polymer assemblage, curing methods, surface properties, macrostructure, physicomechanical properties, nanoparticle fixation and biodegradation of the NESD. In order to incorporate fine control within the complexities of three-dimensional (3D) design, the physical properties of the crosslinked alginate scaffold such as the pore size, shape, wall thickness, interconnectivity and networks for nanoparticle diffusion was regulated to produce a 3D prototype NESD model. The NESD topography was predicted for intracranial implantation with pre-defined micro-architecture and physicomechanical properties equilibrating frontal lobe brain tissue as the site of implantation to provide mechanical support during sterilizability prior to function. A suppositional 3D graphical model with potential inter-polymeric interactions during formation was generated on ACD/I-Lab, V5.11 Structure Elucidator Application (Add-on) biometric software (Advanced Chemistry Development Inc., Toronto, Canada, 2000) based on the step-wise molecular mechanisms of scaffold and nanoparticle formation, polymer interconversion and DA-loaded CAP nanoparticle fixation as envisioned by the chemical behavior and physical stability. A combination of a computationally rapid Neural Network (NN) and a modified Hierarchal Organization of Spherical Environments (HOSE) code approach were employed as the fundamental algorithms in designing the prototype NESD. The associated energy 74 expressions were chemometrically designed based on the assumption of the scaffold behaving initially as a gel-like structure with higher states of combinatory energy for the complete NESD. 4.2.4. Preparation of crosslinked alginate scaffold A 2%w/v alginate solution in deionized water (Milli-DI? Systems, Bedford, MA, USA) was prepared at 50?C and a primary 0.4%w/v [HOCH2(CHOH)4COO]2Ca crosslinking solution was added and agitated until a homogenous mixture was obtained. The resulting mixture was then placed in Teflon moulds and lyophilized for 24hr at 25mtorr (Virtis, Gardiner, NY, USA). Thereafter the lyophilized structures were immersed in a secondary 2%w/v ZnCl2, CaCl2 or BaCl2 crosslinking solution for 3hr as a curing step followed by a further lyophilization phase of 24hr at 25mtorr. The resultant cured scaffolds were removed from the moulds, washed with 3?100mL deionized water to leach out unincorporated salts and air-dried under an extractor. 4.2.5. Textural profile analysis to determine the physicomechanical behaviour of the crosslinked alginate scaffold One of the key approaches to intricate crosslinked polymeric scaffold engineering is the assessment of the physicomechanical properties of the scaffold matrix following 3D prototyping and prior to sterilization and implantation. Textural profile analysis was therefore conducted to characterize the 3D salient core regions of the crosslinked alginate scaffold using a Texture Analyzer (TA.XTplus Stable Microsystems, Surrey, UK) in terms of the scaffold matrix resilience (MR). Hydrated samples of the crosslinked alginate scaffold were analyzed. Serial Force-Time profiles were sufficient to perform the necessary computations of MR (N=5). The MR f the crosslinked scaffolds was calculated employing the TA, the settings of which are outlined in Table 4.2. Futhermore, the MR was calculated for formulations generated in the experimental design. 75 Table 4.2: Parameters employed to measure the MR of hydrated/unhydrated samples employing the texture analyzer Parameter Settings Test Mode Pre-Test Speed Test Speed Post-Speed Speed Target Mode Strain Trigger Type Trigger Force Compression 1.0mm/sec 1.5mm/sec 1.5mm/sec Strain 50% Force 0.05N 4.2.6. Morphological characterization of crosslinked alginate scaffold Morphological characterization of the crosslinked alginate scaffold was instituted. Scaffold parameters such as the micro-structure, pore length, pore distribution and inter-pore wall thickness was also examined. The surface morphology of the cured and un-cured crosslinked alginate scaffolds were also characterized to assess the influence of crosslinking and subsequent curing on potential surface morphological transitions (N=10). SEM, (JEOL, SEM 840, Tokyo Japan) was employed and photomicrographs were captured at various magnifications for analyzing the scaffold samples that were prepared after sputter-coating with carbon or gold. 4.2.7. In vitro matrix erosion studies of crosslinked alginate scaffold Samples of the biodegradable crosslinked alginate scaffolds were immersed in 100mL PBS (pH 6.8, 37?C) and agitated at 20rpm in a shaking incubator (Labex, Stuart SBS40?, Gauteng, South Africa). At pre-determined time intervals samples were removed, blotted on filter paper and dried to a constant mass at 40?C in a laboratory oven. Equation 4.1 was then used to compute the extent matrix erosion (ME) after gravimetrical analysis. Futhermore, the ME was calculated for formulations generated in the experimental design. 100 0M tM-0MME% ?= Equation 4.1 Where ME% is the extent of scaffold matrix erosion, Mt is the mass of the scaffold at time t and M0 is the initial mass of the scaffold. 76 4.2.8. Formulation and Statistical Optimization of the Ba-Alginate Scaffold An experimental strategy, namely the Box-Behnken design, was developed and employed for the statistical optimization of the Ba-alginate scaffold. 4.2.8.1. Determination of limitations for variables employed in a Box-Behnken design The formulation variables listed in Table 4.3 were subjected to their higher and lower limits and thereafter MR was calculated for each formulation. The most influential variables were selected for the employment in the Box-Behnken design. Table 4.3: Illustrates the variations in formulations that were used to identify the limits for a Box -Behnken design to optimize Ba-alginate scaffold and their MR Formulation number Variables MR % 1 Increased [alginate] (1-3% w/v) 78.72 2 Increased lyophilization time (24-48hr) 73.43 3 Increased post-curing time (0.5-1.5hr) 80.34 4 Reduced [alginate] (1%w/v) 73.05 5 Reduced [(HOCH2(CHOH)4COO)2Ca] (0.2%w/v) 80.11 6 Increased temperature to (50-70?C) 36.47 7 Increased [BaCl] to (0.2-0.6%w/v) 67.06 ? [Alginate]- concentrations between 1-3%w/v was selected. Concentration of <1%w/v and <3% would not provide desirably crosslinking and >4%w/v, the solution crosslinked too extensively to place into moulds (also produced a heterogeneous solution). ? [BaCl2]- concentrations between 0.2-0.6% w/v were selected. A concentration of greater 0.6% w/v gave rise to excessive crosslinking. ? Heat-the temperature limits were set 50-70?C. The solution required at least 50?C to allow for the alginate to dissolve in the deionised water. A temperature exceeding 70?C would destroy the polymer's integrity. ? Post-curing time-a minimum of 30min was required in order to facilitate the crosslinking of scaffolds however it was found that scaffolds post-cured for 6hr or longer even 12hr showed no greater MR in comparison to those crosslinked for 1.5hr. This study design, requiring a total of 27 experimental runs (formulation combinations), was generated and analyzed using Minitab? V14 (Minitab Inc?, PA, USA). A 4-factor, 3 level (post- curing time, [Alginate], [(HOCH2(CHOH)4COO)2Ca] and heat) Box-Behnken design on the 77 measured responses (MR and ME %) was established for this optimization. The independent factors and the dependent variables used in the design are listed in 4.7. The variables listed in Table 4.4 were selected for the employment in the Box-Behnken design. Furthermore, the measured responses and for the design and the objectives thereof was established. Table 4.4: The variables for Ba-alginate scaffold used in the 4-factor, 3-level Box-Behnken design Values Independent Variables Low High Alginate (%w/v) 1 3 [(HOCH2(CHOH)4COO)2Ca] (%w/v) 0.2 0.6 Temperature (?C) 50 70 Post-curing time (min) 30 90 Dependent Variables Low High Objective MR (%) 86 94 Maximize ME (%) 3 59 Minimize 4.2.8.2. Optimization of Ba-alginate scaffold Polynomial equations relating the dependent and independent variables were generated, and the formulation process was optimized under constrained conditions for to obtain formulations displaying the desirable percentage ME (minimal) and percentage MR in the hydrated state (maximum). 4.2.9. Corporeal assembly of the NESD Production of the NESD required the initial componential preparation of the DA-loaded CAP nanoparticles and the Ba-alginate scaffold. Once the two components were optimized (Figure 4.11; scaffolds and Chapter 3, Figure 3.20 of this dissertation) the DA-loaded CAP nanoparticles were incorporated via intermittent blending and lyo-fusion (spontaneous freezing followed by lyophilization) into the [(HOCH2(CHOH)4COO)2Ca]-crosslinked and subsequently Ba-alginate scaffold. 78 4.2.9.1. Assimilation of DA?loaded CAP nanoparticles and Ba-alginate scaffold into the NESD The NESD was assembled by a lyo-fusion process (Figure 4.1). Briefly, the optimally defined DA-loaded CAP nanoparticles (200mg) were placed into moulds containing a [HOCH2(CHOH)4COO]2Ca and alginate solution (2mL) obtained in accordance with set optimization constraints. The mixture was agitated and spontaneously frozen at -70?C for 24hs. Thereafter the structures were lyophilized for 48hr at 25mtorr and immersed in a 2%w/v BaCl2 crosslinking solution for 3hr as a curing step followed by a further lyophilization phase of 24hr at 25mtorr to induce fusion of the DA-loaded CAP nanoparticles and the Ba-alginate scaffold. 79 Figure 4.1: Schematic representation of the assimilation of DA?loaded CAP nanoparticles and Ba-alginate scaffold into the NESD PVA (2%w/v) solvent feed solid feed high speed stirrer (300rpm) for 30min rotavapor for the evaporation off solvents centrifuge and collection of sediment lyophilisation for 24hrs to formulate solid dry nanoparticles dry nanoparticles weighed and placed into alginate- gluconate moulds filled moulds placed in freezer @ 60?C lyophilisation for 36hrs post-curing of scaffolds in Ba (3%w/v) scaffolds removed from moulds lyophilisation for 12hrs Scaffolds rinsing of scaffolds in deionised water followed by drying 80 4.2.11. Determination of in vitro DA release from the NESD In vitro release studies were performed on the the final NESD utilizing a shaking incubator (Labex, Stuart SBS40?, Gauteng, South Africa) set at 20rpm. The NESD was immersed separately in 100mL phosphate-buffered saline (PBS) (pH 6.8, 37?C) contained in 150mL glass jars. At predetermine time intervals 3mL samples of each release media were removed, filtered through a 0.22?m Cameo Acetate membrane filter (Millipore Co., Bedford, MA, USA) and centrifuged at 20,000rpm (Reading, 2001). The supernatant was then removed and analyzed by UV spectroscopy at a maximum wavelength of ?280nm for DA content analysis. DA release was quantified using a linear standard curve (R2=0.99). An equal volume of DA-free PBS was replaced into the release media to maintain sink conditions. 4.2.12. Determination of the thermal transition behaviour of the NESD and it?s constituents The inherent and sequential transient thermal behavior of polymers may influence the physicochemical and physicomechanical properties as well as the final performance of the system (Liu et al., 2003). Temperature Modulated Differential Scanning Calorimetry (TMDSC) was therefore performed to provide a distinct interpretation of the polymeric thermal transitions with improved sensitivity and the ability to separate reversible glass transition temperatures (Tg) that have minimal changes in heat capacity (?H) from overlapping non- reversible relaxation endotherms (Reading, 1993) (Ferrero et al., 1999) (Sandor et al., 2002). Thermal analysis was therefore undertaken on the DA-loaded CAP nanoparticles, the Ba- alginate scaffold and the assimilated NESD in order to assess thermal behavior using TMDSC (Mettler Toledo DSC1, STARe System, Switzerland). Thermal transitions were assessed in terms of the Tg, measured as the reversible heat flow due to variation in the magnitude of the Cp-complex values (?Cp); melting temperature (Tm) and crystallization temperature (Tc) peaks that were consequences of irreversible heat flow corresponding to the total heat flow. The temperature calibration was accomplished with a melting transition of 6.7mg indium. The thermal transitions of native CAP were compared to the CAP nanoparticles. Samples of 5mg were weighed on perforated 40?L aluminum pans and ramped within a temperature gradient of 150-500?C under a constant purge of N2 atmosphere in order to diminish oxidation. The instrument parameter settings employed comprised a sine segment starting at 150?C with a heating rate of 1?C/min at an amplitude of 0.8?C and a loop segment incremented at 0.8?C and ending at 500?C. The instrument parameter settings employed are shown in Table 4.5. 81 Table 4.5: TMDSC settings employed for thermal analysis of the NESD constituents Segment Type Parameter Setting SINE Start 150?C Heating rate 1?C /min Amplitude 0.8?C Period LOOP To segment Increment End Count 0.8?C 1 0.8?C 500 436 4.3. Results and Discussion 4.3.1. Computer-aided prototyping for NESD design An output format of serial bitmap images generated via the prototyping technology employed enabled the step-wise 3D volumetric construction of the NESD model. 3D construction was initiated by ascribing an assumed height to each image in order to represent a volume unit or a stacked voxel depicting a prototype model of the NESD described by the grayscale intensity threshold images shown in Figure 4.2. Prototyping of the NESD device revealed that the functional properties of the NESD depended on the characteristics of the polymeric materials employed, the processing technique, and the subsequent interaction of fixated DA- loaded CAP nanoparticles within the crosslinked alginate scaffold. The 3D prototype design of the device permitted the porosity, surface area, and surface characteristics to be semi- optimized in the pre-cured and post-cured phases with BaCl2 for each component of the NESD (Figure 4.2a). Fine control of the micro-architectural characteristics influenced the mechanical properties of the scaffold that was significant for nanoparticle fixation and mechano-transduction in order to control the release of DA. A significant advantage of employing prototyping technology to develop the NESD was the elimination of reliance on individual skills that are required for conventional techniques of device fabrication. Commencing with a limited range of fundamental structural units a NESD with precise micro- architectures was designed using prototyping technology with internal channels or cavities resembling the negative image of the final required NESD as depicted in Figures 4.2a, b and c. Visibly, the scaffold models depicted channels that extended through the entirety of the tetragon matrices in both horizontal and vertical axes with consistency in the strand layout after DA-loaded CAP nanoparticle fixation. At the periphery of the matrix, a region of thick 82 and blurred pore deposition was visible after curing the alginate scaffold in BaCl2 (Figure 4.2b). This entire matrix region was approximately 5?3mm at the edge of the tetragon (Figure 4.2. enlarged for clarity). SEM images confirmed the strut and pore widths to be in the range of 100-200?m. Furthermore, the unconnected pore space, when inspected qualitatively, comprised diminutive cavities within the matrix for controlling the outward diffusion of the DA- loaded CAP nanoparticles from the crosslinked alginate scaffold. Figure 4.2: Three-dimensional prototype images of a) a pre-cured crosslinked-alginate scaffold, b) a BaCl post-cured crosslinked-alginate scaffold, and c) DA-loaded CAP nanoparticles embedded within the cured crosslinked alginate scaffold voids representing the NESD The computational design process revealed that curing of the crosslinked alginate scaffold in BaCl2 involved the residual crosslinking of open, approachable and chemically reactive molecular functional groups that possessed chemical affinity towards BaCl2 as the secondary crosslinker and produced an equivalent of edging and interlocking of the matrix surface functional groups with a superiorly compact matrix structure (Figure 4.2b). Furthermore DA was not covalently bonded to the CAP with no amide bond formation but interacted ionically via physical associations involving H-bonding and smaller force interactions through the influence of the external crosslinking medium. Figure 4.3a represents a structural model of the interactions between H2O molecules in association with CH3COO- and O2 functional groups of strongly hydrophilic CAP sites. DA, other ionic species and molecules revealing an interactive model of CAP and DA entrapment constituents are also depicted in Figure 4.3b. a) b) c) . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . .... . .. . . . . . . ... . .. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .... . . . . . . .. . . . . . . . . . . . . . . BaCl2 CAP Nanoparticles 5mm 3mm (HOCH2 (CHOH)4COO)2Ca-ALG 83 Figure 4.3: Molecular structural models of a) interactions between H2O molecules in association with CH3COO- and O2 functional groups of CAP and b) CAP interactions and DA entrapment Figure 4.4a-e depicts a step-wise single CAP chain structural model under the influence of surrounding interactive forces within the emulsified medium such as solvent molecules at the periphery, PVA as the surfactant and DA. The affinity interactions with explicit lipophilic and hydrophilic orientations towards the formation of a nanoparticle wall are also shown (Figure 4.4f-h). CAP was initially suspended in the binary acetone: methanol solvent system as unorganized random orientations with irregular lipophilic rings (Figure 4.4a). The addition of DA and ionic or physical interactions with the hydrophilic functional groups of CAP and free DA molecules resulted in CAP conforming to orientations of the affinity-wise molecular sites in terms of lipophilicity and hydrophilicity of the medium (Figure 4.4b). DA also influenced the overall polarity spectrum of the medium. The addition of PVA as a surfactant produced strong molecular associations and crosslinker ions with the subsequent energy supplied via agitation and processing temperatures contributing to surface interactions that produced CAP molecules pivoted toward surface minimization, compactness and orientations of the lipophilic regions (Figure 4.4c). The stronger energetic orientations and the presence of PVA as the surfactant tended to sphericalize the CAP strands (Figure 4.4d). The CAP strands sphericalized completely to produce nanoparticles under the primary influence of solvent diffusion phenomena and the presence of PVA with the inner core containing DA molecules and lipophilic regions of CAP conforming toward the periphery as the boundary between the outer hydrophilic medium (Figure 4.4e). Thus, DA molecules orientated within the hydrophilic voids of the nanoparticles shielded by the lipophilic boundary to form stable CAP nanoparticles. H2O H2O H2O H2O H2O O2 O2 O2 O2 O2 O2 DA DA DA DA CAP Strand CAP Strand 84 . . . . . . . . . . . . . . . . ... . . . . . . . . .... . . . . . . . . .. . . . . . . . . . . . . . . a) b) c) d) e) f) g) h) Figure 4.4: Graphical models depicting a-e) the stepwise formation of DA-loaded CAP nanoparticles, f) a single CAP adaptation, g) DA interaction and wall initiation and h) a DA- loaded CAP nanoparticle towards completion 4.3.2. MR behaviour of crosslinked scaffolds Figure 4.5 depicts a typical Force-Time profile used to calculate the MR which is represented as a percentage of the ratio between the Area Under the Curve (AUC) of anchors 1 and 2 (AUC 1 and 2), and2 and 3 (AUC 2 and 3) Figure 4.5: A typical Force-Time profile of a hydrated Ba-alginate scaffold for the calculation of MR DA DA CH3OH CH3COO- CAP CAP CAP CH3COO- CH3OH CH3COO- CH3OH AUC1, 2 AUC2, 3 85 The Force-Time profiles for each sample, in both the hydrated and unhydrated states, was used to calculate the MR and thereby establish the most integral crosslinker reagent for the formulation of [(HOCH2(CHOH)4COO)2Ca]-crosslinked alginate scaffolds. Table 4.6: Percentage MR of crosslinked alginate scaffolds in both hydrated and unhydrated states. MR (%) Formulation type Hydrated Unhydrated Zn-Alginate 9.34 32.68 7.29 26.18 10.44 29.59 Ba-Alginate 62.34 66.44 59.18 72.21 69.21 66.23 Ca-Alginate 27.51 41.88 33.15 39.35 29.98 38.53 TA results proved that the Ba-alginate scaffolds were relatively the most resilient in both the hydrated and unhydrated states. The unhydrated samples for both Ca-alginate and Zn- alginate scaffolds showed greater integrity than the hydrated. The Ba-alginate scaffolds showed only a increase (?5%) in MR in the hydrated state in comparison to the unhydrated state, indicating that the scaffold may maintain its integrity in both formulation and in vivo conditions. MR has important implications for not only DA delivery but product storage as well. In drug delivery, the MR profile of a drug delivery device provides data for the possible in vitro degradation and in turn the drug release behaviour of the system. In this formulation, it is particularly imperative to have a rigid scaffold so as to be able to withstand the demands of further formulation processes involved during the incorporation of DA-loaded CAP nanoparticles. Ba-alginate scaffolds were chosen as the candidate formulation, due to the superior MR in both the hydrated and unhydrated states, for the dispersal of DA-loaded CAP nanoparticles. 86 4.3.3. Surface morphology of crosslinked alginate scaffold Figures 4.6 are microscopic images that display the morphology and pore sizes of Ba- alginate scaffolds formulated in an adopted freeze-drying approach. Figure 4.6:Scanning electron microscope images of (a) Ba-alginate scaffold that was not subjected to post-curing (b) a typical pore present within the crosslinked alginate scaffold, (c) scaffold that underwent post-curing with BaCl and (d) the relation of pores in respective to one another The crosslinked alginate scaffold displayed an average pore size of 100-400?m with a wall thickness calculated at an average of 10?1.04?m. (Figure 4.6d). Scaffolds that were not subjected to post-curing in a secondary crosslinking BaCl2 solution revealed a "tissue-like" appearance (Figure 4.6a) in comparison to the evenly distributed porous granular yet compact appearance of post-cured scaffolds (Figure 4.6b) Ba-alginate scaffolds revealed a granular yet intact appearance with an even distribution of pores along its surface (Figure 4.6c). a) b) c) d) 100?m 10?m 100?m 100?m 87 4.3.4. In vitro ME of the Ba-alginate scaffold Minimal ME (12.1%) of the Ba-alginate scaffolds occurred within the first 60 days (Figure 4.7) thereafter there was a mere ME loss of 2.96% in 12 days. Results confirmed that further crosslinking (post-curing) provided robust scaffolds. Time (days) 0 20 40 60 M a tri x Er os io n (% ) 0 2 4 6 8 10 12 14 16 Figure 4.7: ME profile for Ba-alginate scaffold in PBS (pH 6.8, 37?C) over a period of 72 days (SD?0.23) 88 4.3.5. Experimental design for the optimization of Ba-alginate scaffold The Box-Behnken study design, requiring a total of 27 experimental runs (formulation combinations), was generated and analyzed using Minitab? V14 (Minitab? Inc, PA, USA). The variations of the variables for each formulation are listed in Table 4.7. Table 4.7: Illustration of variations in formulations employed for a Box-Behnken design for the optimization of Ba-alginate scaffold Formulation number Alginate (%w/v) Stirring Temperature (?C) [(HOCH2(CHOH)4COO)2Ca] (%w/v) Post Curing Time (min) 1 3 60 0.2 60 2 3 60 0.6 60 3 2 50 0.6 60 4 2 60 0.4 60 5 2 60 0.6 30 6 3 60 0.4 30 7 2 60 0.4 60 8 2 50 0.4 90 9 1 50 0.4 60 10 2 60 0.4 60 11 1 60 0.4 90 12 2 60 0.2 90 13 2 60 0.6 90 14 1 70 0.4 60 15 1 60 0.4 30 16 2 70 0.4 90 17 3 70 0.4 60 18 2 70 0.4 30 19 1 60 0.2 60 20 2 70 0.2 60 21 3 50 0.4 60 22 2 50 0.2 60 23 3 60 0.4 90 24 2 50 0.4 30 25 1 60 0.6 60 26 2 60 0.2 30 27 2 70 0.6 60 89 4.3.5.1. Measured responses for the experimental optimization of Ba-alginate scaffold The measured responses (MR and ME) for each of the 27 formulation employed in the Box- Behnken design are listed in Table 4.8. Table 4.8: Measured responses for the experimental optimization of Ba-alginate scaffold Formulation number MR (%) ME (%) in 30 days 1 82.95 24.12 2 85.06 25.03 3 84.85 58.92 4 83.73 9.21 5 87.89 12.183 6 94.37 6.93 7 83.73 9.21 8 94.53 15.72 9 84.813 20.68 10 83.73 9.21 11 82.50 3.57 12 81.17 23.14 13 84.78 27.63 14 86.53 6.83 15 81.4 37.91 16 82.38 49.42 17 93.45 3.21 18 86.23 34.89 19 83.33 8.99 20 81.89 14.09 21 81.10 62.19 22 76.81 5.83 23 80.75 6.83 24 94.75 18.143 25 85.07 51.26 26 78.50 11.30 27 84.83 38.81 90 4.3.5.2. Comparison of the experimental and fitted responses calculated for the experimental optimization of Ba-alginate scaffold The plots of the experimental versus fitted responses for MR (Figure 4.8a) and ME (Figure 4.8b) of Ba-alginate scaffold showed R2 values of 72.23% and 68.86% respectively. These values show a satisfactory correlation between the experimental and fitted values for the responses, proving the experimental design was robust in the undertaking of this research. Formulation no 0 2 4 6 8 10 12 14 16 18 20 22 24 26 M at rix Re sil ie n ce (% ) 70 75 80 85 90 95 Actual Fitted Formulation no 0 2 4 6 8 10 12 14 16 18 20 22 24 26 M at rix Er os io n (% ) 0 10 20 30 40 50 60 Actual Fitted Figure 4.8: The Regression plots for (a) MR (%) and (b) ME for the calculation of R2 values that determine the similarity between the predicted and experimentally determined values for the responses a) b) R2=0.722 R2=0.689 91 4.3.5.3. Response analysis for MR of Ba-alginate scaffold An increased MR was seen at higher alginate (2-3%w/v) and [HOCH2(CHOH)4COO]2Ca concentrations (0.3-0.4%w/v) (Figure 4.9a). This was expected as at higher [alginate] an advanced degree of crosslinking occurs producing a superiorly robust and interconnected polymeric networked structure with the increased availability of [HOCH2(CHOH)4COO]2Ca. Higher processing temperatures (60-70?C) and lower [alginate] (1%w/v) also provided a desirable MR value (Figure 4.9b). This was attributed to the enhanced molecular mobility of alginate polymeric chains at higher temperatures that induced participation in the crosslinking reaction resulting in the preferred micromechanical behavior. Figure 4.9: Response surface plots correlating the MR with a) alginate and [crosslinker] and b) [alginate] and processing temperature 4.3.5.4. Response analysis for ME of Ba-alginate scaffold An increase in [alginate] (2-3%w/v) resulted in reduced ME (Figure 4.10a) as a result of a superiorly compact scaffold produced from a precursor solution of increased viscosity Furthermore an increase in [HOCH2(CHOH)4COO]2Ca resulted in a greater degree of crosslinking thereby increasing the scaffold rigidity and retarding ME. An increase in temperature (60-70?C) and post-curing time (60-90min) retarded ME of the scaffold (Figure 4.10b). Higher temperatures enhanced the aqueous solubility of [HOCH2(CHOH)4COO]2Ca (Material Data Safety Sheet, www.sciencelab.com) in water. A prolonged period of post- curing time will allow for the optimal level of crosslinking within the formulation thereby reducing ME. a) b) 92 Figure 4.10: Response surface plots correlating scaffold ME with a) [alginate} and post- curing time and b) processing temperature and post-curing time 4.3.5.5. The main and interaction effects on the responses: Matrix Resilience The concentration of [HOCH2(CHOH)4COO]2Ca had the most significant effect (Figure 4.11a and 4.11b) in terms of achieving superior MR (p?0.05) with increased concentrations providing higher MR values, while the processing temperature displayed the most significant role in matrix design (p?0.05). A processing temperature of 50?C also provided desirable MR values. However this was not relevant for post-curing times of 60 minutes. M e a n o f M a tri x Re si lie n ce 321 88 86 84 82 80 706050 0.40.30.2 88 86 84 82 80 906030 [ALGINATE] Process Temp [CROSSLINKER] Post Curing Time Main Effects Plot (data means) for Resilience a) a) b) 93 [ALGINATE] [CROSSLINKER] Post Curing Time Process Temp 706050 0.40.30.2 906030 96 88 80 96 88 80 96 88 80 [ALGINATE] 3 1 2 Process 70 Temp 50 60 [CROSSLINKER] 0.4 0.2 0.3 Figure 4.11: Typical (a) main effects plot and (b) interactions plot of response values for MR Matrix Erosion The main effects plots showed that an increase in [crosslinker] promoted ME (p=0.098) (Figure 4.12a). This was unexpected however this could have resulted from sequestration of ions where the phosphate ions in the buffer caused the sequestration of the calcium ions ion the crosslinker, thereby resulted in the rapid dissolution of the highly water soluble crosslinker from the scaffold, decreasing scaffold mass. A higher temperature coupled with decreased [alginate] gave rise to reduced ME (Figure 4.12b) shown in the interaction plots. Higher temperatures resulted in more efficient annealing of the alginate which ultimately improved mechanical integrity of matrix with resultant decreased ME. b) 94 M e a n o f M a tri x Er o si o n 321 35 30 25 20 15 706050 0.40.30.2 35 30 25 20 15 906030 [ALGINATE] Process Temp [CROSSLINKER] Post Curing Time Main Effects Plot (data means) for Erosion [ALGINATE] [CROSSLINKER] Post Curing Time Process Temp 706050 0.40.30.2 906030 50 25 0 50 25 0 50 25 0 [ALGINATE] 3 1 2 Process 70 Temp 50 60 [CROSSLINKER] 0.4 0.2 0.3 Figure 4.12: Typical (a) main effects plot and (b) interactions plot of response values for ME b) a) 95 4.3.5.6. Analysis of a Box-Behnken design employed for the optimization of Ba- alginate scaffold The assessment of the regression models can be undertaken employing residual plots (Figure 4.13). Residual plots are essentially a linear comparison between observed values and model predictions for those observations (Stewardson and Whitfield, 2004). Residual Pe rc e n t 1050-5-10 99 90 50 10 1 Fitted Value Re s id u a l 92888480 6 3 0 -3 -6 Residual Fr e qu e n c y 86420-2-4-6 8 6 4 2 0 Observ ation Order Re s id u a l 2624222018161412108642 6 3 0 -3 -6 Normal Probability Plot of the Residuals Residuals Versus the Fitted Values Histogram of the Residuals Residuals Versus the Order of the Data Residual Plots for Matrix Resilience a) 96 Residual Pe rc e n t 30150-15-30 99 90 50 10 1 Fitted Value Re si du al 483624120 20 10 0 -10 -20 Residual Fr e qu e n c y 20100-10-20 8 6 4 2 0 Observ ation Order Re s id u a l 2624222018161412108642 20 10 0 -10 -20 Normal Probability Plot of the Residuals Residuals Versus the Fitted Values Histogram of the Residuals Residuals Versus the Order of the Data Residual Plots for Matrix Erosion Figure 4.13: The residual plots for the responses (a) MR and (b) ME % for Ba-alginate scaffold Residual analysis for MR (Figure 4.13a) and ME (Figure 4.13b) showed the casual distribution of data. The normal plot of residuals displayed slight curvatures of the lines which occurred due to the decreased observation points (less than 50) however the plot still showed normal distribution of the data. The residuals versus fitted plot showed randomly scattered data points around the horizontal line (residual = 0), with some fanning indicative of a degree of non-constant variance, and were within 3 standard deviations of the mean, i.e., zero. The histogram supported that the residuals have a normal distribution with zero mean and constant variance. The residuals versus the order of the data was used to identify non- random error, the plot showed a both a positive (clustering of formulations 4-12) and a negative correlation indicated by rapid changes in the signs (-/+) of the consecutive residuals thereafter. b) 97 A significant effect is indicated by a P value of ? 0.05 for any factor. The factors and the respective P-values are displayed in Table 4.9. Table 4.9: Full ANOVA analysis for the measured responses (MR and ME) Term p-value MR ME [ALG] [PT] [CRL] [PCT] [ALG]2 [PT]2 [CRL]2 [PCT]2 [ALG]*[PT] [ALG]*[CRL] [ALG]*[PCT] [PT]*[CRL] [PT]*[PCT] [CRL]*[PCT] 0.366 0.940 0.048 0.443 0.760 0.387 0.420 0.314 0.449 0.877 0.376 0.354 0.530 0.563 0.740 0.657 0.098 0.710 0.656 0.380 0.391 0.649 0.561 0.338 0.304 0.648 0.979 0.744 The complete regression equations generated for MR and ME are indicated below: MR=83.391-9.883[ALG]-1.703[PT]+313.375[CRL]+0.290[PCT]+0.692[ALG] 2+0.019[PT] 2- 184.833[CRL]2+0.003[PCT]2+0.199[ALG*PT]+4.050[ALG*CRL]-0.078[ALG*PCT]-2.463 [PT*CRL]-0.005[PT*PCT]-0.507[CRL*PCT] Equation 4.2 ME=344.335+21.368[ALG]-10.984[PT]+16.183[CRL]-1.180[PCT]+ 5.605[ALG]2+0.112[PT]2+1095.29[CRL] 2+0.006[PCT] 2-0.617[ALG*PT]- 103.325[ALG*CRL]+0.37[ALG*PCT]-4.833[PT*CRL]-9.083E-04[PT*PCT] -1.146[CRL*PCT] Equation 4.3 98 4.3.5.7. Response optimization of Ba-alginate scaffold This was carried out employing statistical software (Minitab?, V14, Minitab Inc?, PA, USA) to determine the optimum level for each variable. The optimization process resulted in the attainment of various formulations with a significantly low desirability for all three outcomes therefore a selection of the most influential desired outcome was necessary to the detriment of the other two outcomes. MR and ME were the most important and essential characteristics for the scaffold and so a scaffold formulation displaying both characteristics at optimal level was selected. The formulation was optimized for the measured responses of MR and ME. Optimization was carried out so as to obtain the levels of [CAP], [PVA], emulsifying time and stirring speed that would maximize the measured responses (Palamakula et al., 2004). Figure 4.14: Optimization plots displaying factor levels and desirability values for the chosen optimized formulation Hi Lo1.0000 D Optimal Cur d = 1.0000 Maximum Textural d = 1.0000 Minimum Erosion y = 92.6650 y = 21.9500 30.0 90.0 0.20 0.40 50.0 70.0 1.0 3.0 Process [CROSSLI Post Cur[ALGINAT [3.0000] [70.0000] [0.20] [30.0] Measured response Predicted values Experimental values Desirability (%) MR (%) 92.6650 82.455 88.982 ME (%) 21.9500 18.23 83.052 99 4.3.5.8. Desirability for the measured responses of the optimized Ba-alginate scaffold formulation With reference to the optimized Ba-alginate scaffold, the MR of the experimental formulation (82.46%) displayed favorability to the fitted formulation (88.98%). While the experimental formulation had slightly lower MR than fitted, this was counteracted as the ME was lower than predicted (only 18.23% after 7 days) (Figure 4.14). The optimized formulation proved have the desired characteristics of increased MR and decreased ME. 4.3.6. In vitro DA release from the NESD DA release from the NESD could be separated in two phases: (1) diffusion of nanoparticles. Upon contact with this media, the scaffold will become infused with PBS thereby releasing nanoparticles through the pores. The gradual ME of the scaffold will result in an increased diffusion rate of nanoparticles out of the matrix and (2) dissolution of the nanoparticles in PBS thereby releasing DA. The nanoparticles (Figure 4.15a) showed a release of 0.25% in the first 2.5hr, however the release profile for DA-loaded CAP nanoparticles incorporated into the scaffold (Figure 4.15b) showed a lag phase for the initial hr (prior to 6hr) of the study followed by the steady increase in the release of DA thereafter. A release of 5.12% was noted after 168hr. This type of release was attributed to the degradation and increase in pore formation in the scaffold. The DA release pattern was attributed to the ME behaviour of the scaffold (Figure 4.7). The release of DA was highest at the point where ME of the scaffold was at maximum (12-30hr) thereafter both DA release and ME maintain a constant profile. SEM images showed an increase in the number of pores and the size post-ME studies (30 days) allowing for the increased flow of buffer into the pores thereby facilitating a further diffusion of DA out of the pores. Zeta potential and size of the nanoparticles also aided in DA release. DA-loaded CAP nanoparticles had high zeta potentials (-35.2mV) and reduced particle sizes (165.4nm) illustrated in Chapter 3 Figure 3.20 of this dissertation. The charges on the particles would electrostatically repel them from one another assisting in their molecular movement through the scaffold. 100 Time (hrs) 0 5 10 15 20 25 Fr ac tio n al dr u g re le as e 0.0 0.2 0.4 0.6 0.8 1.0 Time (hrs) 0 20 40 60 80 100 120 140 160 Fr ac tio n a l d ru g re le a se 0 1 2 3 4 5 6 Figure 4.15: DA release profiles for (a) DA-loaded nanoparticles (SD?0.02) and NESD in PBS, pH 6.8; 37?C), (SD?0.16) 4.3.7. Componential thermal analysis on the NESD TMDSC profiles portrayed the paradigms of the thermal behavior in the three componential elements of the NESD that included the CAP nanoparticles, the crosslinked alginate scaffold and the NESD as shown in Figures 4.16a, b and c. The changes in Tg, Tm and Tc that occurred upon the formation of DA-loaded CAP nanoparticles, the crosslinked alginate scaffold and the assimilated NESD when compared to native CAP employed for nanoparticle fabrication is depicted in Figures 4.16a-c. a) b) 101 Table 4.10: Salient thermal events for DA-loaded CAP nanoparticles, Ba-alginate scaffold and NESD compared to native CAP Temperature transition points (?C) Formulation Component Tg Tc Tm DA-loaded CAP nanoparticles 260 160 268 262 378 Crosslinked alginate scaffold 210 158 390 238 330 NESD 260 158 390 315 358 Native CAP 160-170 180 192 Tg = glass transition temperature; Tc = crystallization temperature and Tm = melting temperature All components presented with triple exothermic peaks depicting a coincidental similarity in crystallization behaviors (Tc) (Figures 4.16a, b and c). The similarity in thermal behavior between the crosslinked alginate scaffolds and NESD portrayed a direct indication of the high degree of crystallinity imparted by the secondary crosslinker BaCl2 that was employed as a curing step for scaffold formation. Noteworthy was the significantly large variation in Tg and Tm between the native CAP (Tg=160-170?C; Tm=192?C) and the DA-loaded CAP nanoparticles (Tg=260?C; Tm=268?C). The apparent shifts in Tg and Tm elucidated a possible interfacing between CAP and DA molecules that contributed to the formation of physical interactions culminating into the thermal behaviour observed. The large positive shifts in thermal events may have also influenced the release of DA from the CAP nanoparticles as supported by the initial prototyping technology employed and DA release profiles discussed later on. The presence of transient melting endothermic peaks and further shifts in Tg observed on the TMDSC signals of the NESD samples clearly reflected the effect of altered thermal properties produced by initial crosslinking between [HOCH2(CHOH)4COO]2Ca and alginate and further the dispersion of DA-loaded CAP nanoparticles within the BaCl2 solution as a post-curing process. The altered thermal behaviour influenced the physicomechanical behaviour as supported by the earlier morphological, textural profile and FTIR analysis. 102 Overall, the thermal behavior observed may be due to variation in the ?H involved, ability to attain near-equilibrium conditions during measurement, and the rapid rate of change in molecular rearrangement compared to the ?T. These pertinent intermolecular interactions, which resulted in the observed thermal transitions (Figures 4.16a, b and c), may have also contributed substantially to the superior control of DA released from the NESD. Figure 4.16: Temperature modulated differential scanning calorimetry profiling for NESD showing endothermic and exothermic peaks generated 103 4.4. Concluding Remarks In this chapter, alginate was chosen as the polymer for the employment in scaffolds. The crosslinking of this with various agents resulted in Ba-alginate scaffolds being selected as the ideal candidate for it's MR. A porous structure yet high integrity was revealed in the micrographs of the Ba-alginate scaffolds. A ME of 15% was calculated only at 60hr, showing minimal rapid degradation throughout the period of investigation. The Box-Behnken design was employed to optimize the formulation and gave rise to 27 formulations where [alginate] and [Ba] were altered as well as processing factors such as post-curing time and heat. MR and ME % were chosen as the imperative properties to be optimized. A single candidate optimal formulation displaying MR of 82.455% and a ME of 18.23% in 30 das was selected. In this chapter, the DA-loaded CAP nanoparticles were incorporated into the Ba-alginate scaffold (optimal formulations) and analysed, generating for in vitro release profiles. The addition of the nanoparticles into a Ba-alginate scaffold significantly reduced DA release (5.12% release in 168hr) and was established as the main contributor to the controlled DA release. Significant shifts in thermal events noted with TMDSC analysis of the DA-loaded CAP nanoparticles and NESD supported the mechanism by which modulated release of DA occurred from the device. Biometric simulation and prototyping technology in conjunction with Box-Behnken statistical experimental designs as preparation and optimization strategies for the scaffold and nanoparticles proved robust in selecting optimal components for assembling the NESD. 104 CHAPTER 5 INVESTIGATION INTO DRUG-LOADED NANOSTRUCTURES FOR THE EMPLOYMENT IN TREATMENT OF PARKINSON'S DISEASE 5.1. Introduction Nanotechnology has widened the horizon for novel therapeutic opportunities for agents that cannot be formulated using conventional drug techniques; due to poor drug instability and/or bioavailability allowing for site specific delivery of medicine to diseased tissues and cells thereby dramatically increase the chances of healing in affected individuals. Polymeric nanofibers, an example of a nanosystem, are uniquely shaped two dimensional structures that may be formed employing a simple yet reproducible electrospinning technique. Examples of nanofiber drug delivery systems include dexamethasone-loaded PLGA nanofibers in neural prosthetics (Abidian et al., 2006) and ketaprofen-loaded PVA nanofibers for inflammation (Kenawy et al., 2007).Furthermore, the polymer fibers may be coated with an electro-conductive polymer which can be stimulated by an external electrical current. These fibers may contract/dilate according to the stimuli received so as to reduce/increase drug delivery at their target site. An example of this system is poly (3, 4- ethylenedioxythiophene) for implantation into the brain (Richardson-Burns et al., 2007). Electrospinning involves the application of electricity to form nanofibers (Reneker and Yarin, 2008). A polymeric solution of the appropriate viscosity is placed in a capillary tube/pipette. A high voltage is sent through an electrode that is placed within the capillary tube. This gives rise to an electric field which opposes the surface tension (present at the end of the capillary tube). As a result, a conical shaped protrusion known as the Taylor cone (Figure 3.1). The polymeric solution forms at the end of the tube (Welle et al., 2007). A jet of polymeric solution is ejected from the tip, which is attracted to a grounded metal screen lined with foil, resulting in nanofibers. The following variables influence the length and uniformity of the fibers and can be controlled (Welle et al., 2007): Viscosity of the polymeric solution; flow rate of the polymeric solution; distance of the capillary end from screen; and voltage from the external supply. 105 Figure 5.1: A schematic representation of a rudimentary electrospinning apparatus employed for the formulation of nanofibers V stirrer dissolution of polymer in appropriate solvent pipette filled with polymeric solution spinning of fibers Taylor cone voltmeter aluminium screen polymer solution electrode 106 The impetus of this chapter greatly revolves around the development of a controlled drug delivery systems for pharmacological agents used in the treatment of PD that reduces the dosages thereby reducing the ?off? period experienced and reducing the side effect profile. This will increase compliance and most of all the development of a common drug delivery platform that can be used to deliver both previously and newly formulated drugs in a targeted approach to treat CNS disease states. This chapter sought to address the need for alternate nanosystems, L-dopa and NT nanoparticles and DA nanofibers, which may be employed for the treatment of PD. 5.2. Investigation of L-dopa-loaded Poly (lactic-co-glycolic acid) Nanoparticles 5.2.1. Materials and Methods 5.2.1.1. Materials 3-(3,4-dihydroxy phenyl)-l-alanine, (L-dopa), (Mw=197.19g/mol), cellulose acetate phthalate (CAP) (Mw=2534.12g/moL), poly(vinyl alcohol) (PVA) (Mw=49,000g/moL) was purchased from Sigma Aldrich (St. Louise, MO, USA), hydrochloric acid (HCl), 32%, was purchased from SAARCHEM (Johannesburg, South Africa). Poly (DL-lactide-co-glycolide) (PLGA), resomer RG504 (Mw=48000g/mol) purchased from Boehringer Ingelheim (Ingelheim, Germany) and dimethyl sulphoxide (DMSO) (Mw=78.13g/mol) from obtained from Merck (Johannesburg, South Africa). Deionized water was obtained from a Milli-Q water purification system (Milli-Q, Millipore, Billerica, MA, USA). All other reagents were of analytical grade and used as purchased. 5.2.2. Preparation of L-dopa-loaded PLGA nanoparticles employing a nano- precipitation technique The precipitation method adopted in this study is based on work previously reported by Fessi et al in 1989 which involved a lipophilic drug/polymer/acetone or ethanol (solvent)/ water (non-solvent) system which was then altered to a more versatile solvent-non-solvent scheme (Bilati et al., 2005). The limited solubility of the polymer (PLGA) and drug (L-dopa) led to the employment of the nano-precipitation rather than the previously adopted emulsification- diffusion method stated in Chapter 3, Section 3.2.5. of this dissertation. The selection of this method also allowed for the investigation of versatility of the NESD by means of incorporating drug-loaded polymeric nanoparticles, produced employing different techniques, into the Ba- alginate scaffold. 107 Preparation of the non-solvent system: Two separate solvent systems were used; (1) consisted of buffer (pH 6.8, 20mL) and (2) consisted of buffer (pH 6.8, 19mL) and PVA (1%w/v, 1mL). Preparation of the solvent system: A PLGA (0.5 %w/v) solution was prepared by dissolving PLGA (1g) in DMSO (20mL). Three separate solutions of this PLGA (0.5% w/v) were prepared, to which 0.1% w/w, 0.3% w/w and 0.5% w/w of L-dopa to polymer was added. Drug-free nanoparticles were prepared in a similar manner. Incorporation of non-solvent to solvent system to formulate L-dopa-loaded PLGA nanoparticles The solvent system was infused into the non-solvent system with a syringe and a needle of varying gauge (25G and 21G). Needles of different gauge sizes were employed to investigate the effect that friction has on the characterization of nanoparticles (will be hereon referred to as nanoparticles). The ratio of non-solvent to solvent was maintained at 20:1. In each case the infusion process took place either under moderate magnetic stirring at 300rpm (Hibi? magnetic stirrer, Gauteng, S.A) or utilising a three blade propeller overhead stirrer at 1000rpm (Heidolph?, Lebotec, Gauteng, S.A). The suspended precipitate was collected, centrifuged at 8000rpm (Optima? LE-80K, Beckman, USA). The sediment was collected and subsequently lyophilised (Virtis lyophiliser, Virtis?, Gardiner, NY, USA), refer to Chapter 4, Table 4.3 of this disseration for parameter and settings, for a 24hr period. 5.2.3. Assimilation of L-dopa-loaded PLGA nanoparticles and Ba-scaffold into the NESD L-dopa loaded nanoparticles (200mg) were placed into moulds containing a Ba-alginate solution (2mL) vigorously stirred and immediately placed in a freezer set at -70?C for 24hr. Thereafter the scaffolds were freeze dried for 48hr and crosslinked with calcium gluconate as per the method in Chapter 4 Section 4.2.9.1 of this dissertation. 5.2.4. Determination of L-dopa entrapment efficiency of PLGA nanoparticles A calibration curve for L-dopa was determined using a known series of concentrations of L- dopa in PBS pH 6.4. Samples were accurately weighed and completely dissolved in 0.1M HCl (100mL) and left under vigorous magnetic stirring (700rpm) for a period of one week. The L-dopa content was analyzed by UV spectrophotometry at ?280nm (Hewlett Packard 8453 Spectrophotometer, Germany) and computed from a standard linear curve of DA in PBS (pH 108 6.8; 37?C) (R2=0.99). Chapter 3, Equation 3.1 of this dissertation was utilized to compute the Drug Entrapment Efficiency (DEE). 5.2.6. In vitro L-dopa release studies on PLGA nanoparticles and NESD In vitro release studies were performed on the L-dopa-loaded PLGA nanoparticles and NESD utilizing a shaking incubator (Labex, Stuart SBS40?, Gauteng, South Africa) set at 20rpm. The L-dopa loaded nanoparticles and NESD was immersed separately in 100mL PBS (pH 6.8, 37?C) contained in 150mL glass jars. At predetermine time intervals 3mL samples of each release media were removed, filtered through a 0.22?m Cameo Acetate membrane filter (Millipore Co., Bedford, MA, USA) and centrifuged at 20,000rpm (Redhead et al., 2001). The supernatant was then removed and analyzed by UV spectroscopy at a maximum wavelength of ?280nm for L-dopa content analysis. L-dopa release was quantified using a linear standard curve (R2=0.99). An equal volume of DA-free PBS was replaced into the release media to maintain sink conditions. 5.2.7. Morphological characterization of L-dopa-Loaded PLGA nanoparticles Morphological characterization of the nanoparticles revealed the shape, surface, structure and size homogeneity and possible degree of aggregation. Surface morphology was characterized by Scanning Electron Microscopy (SEM), (JEOL, JEM 840, Tokyo Japan). Photomicrographs were taken at different magnifications and samples were prepared after sputter-coating with carbon or gold (N=10). Nanoparticle size and shape was further explored using cryo-Transmission Electron Microscopy (TEM) (JEOL 1200 EX, Tokyo, Japan, 120keV) for higher definition and resolution. Samples were prepared by placing a dispersion of nanoparticles in ethanol on a copper grid with a perforated carbon film followed by evaporation and viewing at room temperature (N=10). 5.2.8. Determination of polymeric structural variations due to L-dopa-loaded PLGA nanoparticle formation The structure of native PLGA, L-dopa and the PLGA nanoparticles produced were assessed using Fourier Transmission Infrared (FTIR) spectroscopy to assess the potential for any variations in vibrational frequencies and subsequent polymer structure as a result of L-dopa- polymer interactions during nanoparticle formation. Changes in the PLGA backbone may alter the inherent stability and therefore affect the physicochemical and physicomechanical properties of the selected polymer type for the intended purpose. Samples of L-dopa-free and L-dopa loaded PLGA nanoparticles were blended with potassium bromide (KBr) in a 1%w/w 109 ratio and compressed into 1?13mm disks using a Beckmann Hydraulic Press (Beckman Instruments, Inc., Fullerton; USA) set at 8 tons. The sample disks were analyzed in triplicate at high resolution with wavenumbers ranging from 4000-400 cm-1 on a Nicolet Impact 400D FTIR Spectrophotometer coupled with Omnic FTIR research grade software (Nicolet Instrument Corp, Madison, WI, USA). Liquid FTIR analysis: The FTIR analysis of the liquid (DMSO) used in the formulation of L-dopa-loaded nanoparticles identified their chemical interaction on a molecular level that took place prior to precipitation. These were performed on PLGA and DMSO solutions employing a Bruker Tensor 27 (OPUS software) FTIR instrument (Bruker Daltonik GmbH, Germany) with frequency range 4000 to 400 cm-1. 5.2.9. Determination of size and zeta potential of L-dopa-loaded PLGA nanoparticles The nanoparticle zeta potential measurements were obtained using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK). Each sample (1%w/v) was appropriately diluted with deionised water, filtered (0.22?m filter Millipore Co., Massachusetts, USA) to maintain the number of counts per second in the region of 600 (Layre et al., 2006) and placed into disposal cuvettes (size) or capillary cells (zeta potential) (Malvern Instruments Ltd, Malvern, Worcestershire, UK. The viscosity and refractive index of the continuous phase were set to those specific to deionized water. Measurements were taken in triplicate with multiple iterations for each run in order to elute size intensity and zeta potential distribution profiles. 5.3. Results and Discussion 5.3.1. Formulation of L-dopa-loaded PLGA nanoparticles L-dopa is slightly soluble in the non-solvent system. Hence the pH was altered to 6.7 by adding HCl to the solution and thereby decreasing the solubility of L-dopa even further. At pH 6.7, L-dopa is almost unionized in the non-solvent system and therefore remains in the solvent system, thereby increasing the DEE within the polymer during the process of precipitation at the step of infusion. Equation 5.1 further motivates the employment of PBS pH 6.7: ( ) x xpKapH ??= 100log Equation 5.1 110 Where pH is that of the PBS in which L-dopa is unionized, pKa = 9.7 and is that of the grade of L-dopa being employed and x = 0.1 and is the % ionization of L-dopa that occurs at pKa 9.7. 5.3.2. L-dopa entrapment efficiency of PLGA nanoparticles A calibration curve was set up as shown in Figure 5.2 and the DEE for L-dopa-loaded nanoparticles was calculated to be 61.22%. L-dopa is slightly soluble in water, 66mg in 40mL, (Budavari, 1996) water during the infusion process the L-dopa dissolved into the non-solvent (water) from the solvent system, this is referred to as the leaking of L-dopa. Hence the solvent system was adjusted to pH 6.7 which would prevent the L-dopa from going into dissolution in the non-solvent system, thus enhancing the DEE (14.8?0.7). Concentration (mg/mL) 0.00 0.02 0.04 0.06 0.08 Ab so rb a n ce 0.0 0.2 0.4 0.6 0.8 Figure 5.2: L-dopa calibration curve at 285nm in PBS (pH 6.8; 37?C) 5.3.3. In vitro L-dopa release from PLGA nanoparticles and the NESD L-dopa release from the nanoparticles over a 75hr period was 4.21% (Figure 5.3a). The second hour saw the majority of L-dopa release (0.019%), in comparison to the total amount of L-dopa release. L-dopa release over the sampling period occurred gradually with minimal increment of L-dopa release. The scaffold retarded the release of L-dopa significantly which can be attributed to the Mw of PLGA (Mw=48000) and the hydrophobicity of it's constituents (PLA and PGA) which imparts high erosion resistance (von Burkersroda et al., 2002). The capacity of the PLGA polymer to undergo hydrolysis and consequently biodegradation is influenced by the crystalline properties of the PLGA polymer (Jain, 2002). L-dopa release from the PLGA nanoparticles dispersed within the scaffold (Figure 5.3b) displayed a lag y = 11.383x R2 = 0.9997 111 phase of 2hrs compared to the native nanoparticles. It can therefore be deduced that the scaffold aided in controlling DA release by creating diffusion resistance pathways. Time (hrs) 0 10 20 30 40 50 60 Fr ac tio n al dr u g re le as e 0.0 0.2 0.4 0.6 0.8 1.0 Time (hrs) 0 20 40 60 80 Fr ac tio n al dr u g re le a se 0.0 0.2 0.4 0.6 0.8 1.0 Figure 5.3: L-dopa release profiles from (a) L-dopa-loaded PLGA nanoparticles (SD?0.0012) and (b) NESD in PBS (pH 6.8; 37?C); (SD?0.006) 5.3.4. Morphology of L-dopa-loaded PLGA nanoparticles Figure 5.4a shows uniformly distributed L-dopa-loaded PLGA nanoparticles and presence of aggregation. Figure 5.4b depicted an isolated L-dopa-loaded PLGA nanoparticle indicating PLGA and encapsulated L-dopa Figure 5.4: TEM images of (a) an isolated L-dopa-loaded PLGA nanoparticle and (b) a cluster of L-dopa-loaded PLGA nanoparticles b) a) b) a) 112 5.3.5. Structural polymeric variation of polymer, L-dopa and L-dopa-loaded PLGA nanoparticles L-dopa is decarboxylated to DA by the enzyme dopa decarboxylase. The only structural difference between L-dopa and DA is the presence of a -COOH (2500cm-1-3000cm-1), positioned on the terminal group of alanine, on L-dopa displayed in the FTIR (Figure 5.5). The -C=O that forms part of -COOH group is displayed at 1748.93cm-1. These groups indicated the presence of L-dopa in the L-dopa-loaded PLGA nanoparticles. The primary amine and the aromatic -OH groups present on both molecules L-dopa and DA are essential for activity of the molecules. FTIR of both the native L-dopa and L-dopa-loaded PLGA nanoparticles have peaks of 1087.36cm-1 which falls in the range in which an aliphatic -C-N stretch occurs. However, the peak of the aromatic -OH in the L-dopa-loaded PLGA nanoparticles group which occurs very strongly in the 3200-3600cm-1 range, could be masked by the peaks occurring in the polymer over the same wavelength range. The FTIR spectra indicated that PLGA and L-dopa (native) maintained its backbone structure in the formation of L-dopa-loaded PLGA nanoparticles. There was an absence of any newly formed compounds that may have aided in retarding L-dopa release. However the FTIR spectra confirmed that nanoparticles were made up of both L-dopa and PLGA. Figure 5.5: FTIR spectra for L-dopa, PLGA, L-dopa free and L-dopa-loaded PLGA nanoparticles 113 Proposed PLGA and DMSO employing liquid FTIR analysis c o o.. .. PLGA + S 0 H3 CH3C DMSO C O. . H CH 3 S 0 CH3 O. . S C OH.. O. S O CH CH 3 3 2 3 CH CH O S.O . .OH C H.. ..H C O. S O CH CH 3 2 O +OH.. Figure 5.6: Proposed PLGA and DMSO chemical interaction Figure 5.7: Liquid FTIR spectra for PLGA and DMSO 114 In Figure 5.7, the broad band in the 3200-2600 cm-1 region is due to the oxygen-hydrogen (O -H) stretch of the hydroxyl group. The IR spectra of carboxylic acid derivatives resulted in the formation a strong C=O stretch in the 1690-1760 cm-1 region (as indicated in the 1765.14 cm- 1 band). The C-O bond occurred over the 1080-1300 cm-1 region (as indicated in the 1170.64 cm-1 band). The thiocarbonyl (C=S) and S-OR ester possessed a stretched frequency at 1050-1200 cm-1 and 700 and 900 cm-1 region respectively. These spectra are supported by the chemical reaction in Figure 5.6. 5.3.6. L-dopa-loaded PLGA nanoparticle size and stability The L-dopa-loaded nanoparticles showed a smaller size (113nm), and zeta potential (- 32.8mV) in comparison to L-dopa-free nanoparticles (175nm, 0.019 and -23mV). The reduced size of the L-dopa-loaded nanoparticles could be attributed to the disturbance of interfacial turbulence due to the presence of the L-dopa. The principles of interfacial flow and turbulence is explained by the Maragoni effect (Fessi et al., 1989) in that liquids with high surface tensions will pull on their surrounding media more strongly in comparison to liquids with low surface tension. Turbulence arises from the spontaneous agitation of these liquids with different surface tensions resulting in changes in flow. The negative zeta potential may be attributed to the presence of carboxyl groups of PLGA. The use of the overhead stirrer was seen to have the potential to decrease particle size (143.2nm) in relation to the magnetic stirring, nanoparticle size (194.5nm), but also resulted in an irregular zeta potential value at 0.072mV. The addition of PVA under magnetic stirring did not produce favourable results on particle size (213.2nm) and zeta potential (-14.5mV). It however stabilized the irregular zeta potential values that resulted from the use of the overhead stirrer. The irregularities that occurred in particle size range was due to the surfactant that suppressed interfacial flow, which is a crucial stage in the nano precipitation method. The effect of varying needle gauge size had no significant effect on nanoparticle size, distribution and zeta potential. 115 5.4. Investigation of NT-loaded CAP Nanoparticles 5.4.1. Material and Methods 5.4.1.1. Materials Cellulose acetate phthalate (CAP) (Mw=49,000g/moL), poly(vinyl alcohol) (PVA), acetone, methanol and NT ((?)-1-Methyl-2-(3-pyridyl)pyrrolidine) (Mw=162.23g/mol) (NT) were all purchased from Sigma Aldrich (St. Louise, MO, USA). All other reagents were of analytical grade and used as purchased. 5.4.2. Preparation of NT-loaded CAP nanoparticles NT-loaded nanoparticles were prepared using an adapted emulsification?diffusion technique, previously reported (Pi??n-Segundo et al., 1996). Briefly, 500mg of CAP and 50mg NT were dissolved in acetone and methanol (3:7 mixture), to which, a 1%w/v PVA solution was added. The solution was agitated for 30min using a magnetic stirrer (Fried Electric, Haifa, Israel) set at 700rpm. A submicronized oil in water emulsion was spontaneously formed due to immediate reduction of the interfacial tension with rapid diffusion of organic solvent into the aqueous phase representative of the Marangoni Effect (Poletto et al., 2008). Excess solvent was evaporated from the suspension using a Rotavap (Rotavapor? R210, Switzerland) at 60?C for 1hr and the resulting solution was centrifuged (Optima? LE-80K, Beckman, USA) at 20,000rpm for 20min. The sediment layer containing nanoparticles was removed and lyophilized for 24hr hours at 25mtorr to obtain a free-flowing powder. 5.4.3. Determination of NT entrapment efficiency of CAP nanoparticles A calibration curve for NT was determined using a known series of concentrations of NT in PBS pH 6.8. In order to assess the entrapment efficiency of NT within the CAP nanoparticles, post-lyophilized powdered samples were accurately weighed and completely dissolved in PBS (pH 6.8; 37?C). The L-dopa content was analyzed by UV spectrophotometry at ?269nm (Hewlett Packard 8453 Spectrophotometer, Germany) and computed from a standard linear curve of DA in PBS (pH 6.8; 37?C) (R2=0.99). Chapter 3, Equation 3.3 of this dissertation was utilized to compute the Drug Entrapment Efficiency (DEE). 116 5.4.4. In vitro NT release studies from CAP nanoparticles In vitro release studies were performed on the NT-loaded nanoparticles utilizing a shaking incubator (Labex, Stuart SBS40?, Gauteng, South Africa) set at 20rpm. The NT-loaded nanoparticles were immersed separately in 100mL PBS (pH 6.8, 37?C) contained in 150mL glass jars. At predetermine time intervals 3mL samples of each release media were removed, filtered through a 0.22?m Cameo Acetate membrane filter (Millipore Co., Bedford, MA, USA) and centrifuged at 20,000rpm (Redhead et al., 2001). The supernatant was then removed and analyzed by UV spectroscopy at a maximum wavelength of ?269nm for NT content analysis. NT release was quantified using a linear standard curve (R2=0.99). An equal volume of NT-free PBS was replaced into the release media to maintain sink conditions. 5.4.5. Morphological characterization of the NT-loaded CAP nanoparticles Morphological characterization of the nanoparticles revealed the shape, surface, structure and size homogeneity and possible degree of aggregation. Surface morphology was characterized by Scanning Electron Microscopy (SEM), (JEOL, JEM 840, Tokyo Japan). Photomicrographs were taken at different magnifications and samples were prepared after sputter-coating with carbon or gold (N=10). Nanoparticle size and shape was further explored using cryo-Transmission Electron Microscopy (TEM) (JEOL 1200 EX, Tokyo, Japan, 120keV) for higher definition and resolution. Samples were prepared by placing a dispersion of nanoparticles in ethanol on a copper grid with a perforated carbon film followed by evaporation and viewing at room temperature (N=10). 5.4.6. Determination of size and zeta potential of NT-loaded CAP nanoparticles The nanoparticle zeta potential measurements were obtained using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK). Each sample (1%w/v) was appropriately diluted with deionised water, filtered (0.22?m filter Millipore Co., Massachusetts, USA) to maintain the number of counts per second in the region of 600 (Layre et al., 2006) and placed into disposal cuvettes (size) or capillary cells (zeta potential) (Malvern Instruments Ltd, Malvern, Worcestershire, UK. The viscosity and refractive index of the continuous phase were set to those specific to deionized water. Measurements were taken in triplicate with multiple iterations for each run in order to elute size intensity and zeta potential distribution profiles. 117 5.5. Results and Discussion 5.5.1. Calibration curve for NT in PBS (pH 6.8; 37?C) for the determination of the concentration of NT in CAP nanoparticles Figure 5.8 displays a calibration curve obtained for NT in PBS (pH 6.8; 37?C) employing a UV spectrophotometry at ?269 (Hewlett Packard 8453 Spectrophotometer, Germany). Concentration (mg/ml) 0.00 0.01 0.02 0.03 0.04 Ab so rb an ce 0.00 0.05 0.10 0.15 0.20 Figure 5.8: NT calibration curve at 269 in PBS (pH 6.8; 37?C) Series concentrations of NT were made up ranging from 0.01mL to 0.04mL. A calibration curve for NT was constructed (Figure 5.7) and employed for the determination of DEE%. 5.5.2. NT entrapment efficiency of CAP nanoparticles The DEE was calculated at 58%?2.47, this was much lower in comparison to the other nanosystems formulated in this study. This may be attributed to the water solubility of NT (50mg/100mL) being lower than that of DA (100mg/mL), causing the NT to diffuse into the organic phase thereby decreasing the DEE. y = 5.12 x R2 = 0.9991 118 5.5.3. In vitro NT release from CAP nanoparticles Figure 5.9 is illustrates the in vitro release profile of NT from the CAP nanoparticles over a 25hr period. Time (hrs) 0 5 10 15 20 25 Fr ac tio n al dr ug re le as e 0.0 0.2 0.4 0.6 0.8 1.0 Figure 5.9: Profile of NT release from CAP nanoparticles (SD?0.01) in PBS (pH 6.8; 37?C) The release profiles for NT further confirmed the decreased solubility of NT in PBS (pH 6.8; 37?C). The drug delivery system showed enhanced control release of NT (0.415% release in 24hr) as the phenomenon of NT release was regulated by dissolution of the polymer and thereafter NT. This process occurred at a slower rate due to poor solubility of NT in an aqueous environment. 119 5.5.4. Surface morphology of NT-loaded CAP nanoparticles Figure 5.10 is a microscopic image that displays the morphology and sizes of NT-loaded CAP nanoparticles formulated in an adopted emulsification-diffusion approach Figure 5.10: TEM image of NT-loaded CAP nanoparticles TEM images (Figure 5.10) of NT-loaded CAP formulations revealed uniform spherical shaped NT-loaded CAP nanoparticles with a size range of 25-100nm and embedded NT particles with a size range of 2-20nm within their structure. A helical arrangement/ association was present in the images and this instability was further confirmed by the undesirable zeta potential. 5.5.5. NT-loaded CAP nanoparticle size and zeta potential Results revealed a particle size of 172nm, this shows that while the particle size was in the desirable range (<200nm), the variation in size (PdI value) was significantly high. The zeta potential profile for NT was erratic displaying two peaks in the distribution curves with a value of -15.4mV revealing that the formulation did not display suitable stability. The method employed for the formulation of NT nanoparticles was not reproducible in that there was an absence in uniformity of particle size which could be attributed to the low zeta potential that may have caused the particles to agglomerate resulting in a variation in particle size. 100nm 120 5.6. Investigation of DA-loaded Polymeric Nanofibers 5.6.1. Materials and Methods 5.6.1.1. Materials Cellulose acetate phthalate (CAP) (Mw=2534.12g/moL), poly(vinyl alcohol) (PVA) (Mw=49,000g/moL),, acetone, methanol and dopamine hydrochloride (DA) (Mw=189.64g/moL) were all purchased from Sigma Aldrich (St. Louise, MO, USA). Deionized water was obtained from a Milli-Q water purification system (Milli-Q, Millipore, Billerica, MA, USA). All other reagents were of analytical grade and used as purchased. 5.6.2. Preparation of DA-loaded polymeric nanofibers DA (0.9g) was added to a PVA solution (9%w/v) and a CAP in acetone solution (0.16%w/v) and agitated for 15min @50?C using a magnetic stirrer (Fried Electric, Haifa, Israel) to produce DA-loaded PVA and DA-loaded CAP nanofibers respectively. Polymeric solutions (1mL), DA- free and DA-loaded, were placed into 5mL pipettes and secured to a custom-built electrospinning device equipped with a voltmeter. The voltage was increased at 0.5Kv/sec until the Taylor Cone was visible and allowed for the spinning of fibers which were collected on an aluminum screen for analysis. The voltage was set at 12kV at a distance of 3cm and 14Kv at a distance of 1cm, from the pipette stand to the aluminium screen, for the DA-loaded PVA and DA-loaded CAP nanofibers respectively. DA-free polymeric nanofibers were formed in a similar manner. 5.6.3. Determination of DA entrapment efficiency of polymeric nanofibers In order to assess the entrapment efficiency of DA within the CAP and PVA nanofibers, electrospun samples were accurately weighed and completely dissolved in PBS (pH 6.8; 37?C). The DA content was analyzed by UV spectrophotometry at ?280nm (Hewlett Packard 8453 Spectrophotometer, Germany) and computed from a standard linear curve of DA in PBS (pH 6.8; 37?C) (R2=0.99). Chapter 3, Equation 3.3 of this dissertation was utilized to compute the Drug Entrapment Efficiency (DEE). 5.6.4. Morphological characterization of DA-loaded polymeric nanofibers Morphological characterization of the nanofibers revealed the shape, surface, structure and size homogeneity and possible degree of aggregation. Surface morphology was characterized by Scanning Electron Microscopy (SEM), (JEOL, JEM 840, Tokyo Japan). Photomicrographs were taken at different magnifications and samples were prepared after sputter-coating with carbon or gold (N=10). Nanofiber size and shape was further explored 121 using cryo-Transmission Electron Microscopy (TEM) (JEOL 1200 EX, Tokyo, Japan, 120keV) for higher definition and resolution. Samples were prepared by placing a dispersion of nanofibers in ethanol on a copper grid with a perforated carbon film followed by evaporation and viewing at room temperature (N=10). 5.7. Results and Discussion 5.7.1. DA entrapment efficiency of polymeric nanofibers DEE was calculated as 85%?3.63 and 78%?2.60 for the DA-loaded PVA and CAP nanofibers respectively. This result proved that DA had a greater likelihood to be entrapped into the PVA fibers due to its hydrophilic nature. 5.7.2. Size and morphology of DA-loaded polymeric nanofibers Figures 5.11-5.13 are microscopic images that display the morphology and sizes of DA- loaded PVA and CAP nanofibers formulated in an employing an electrospinning technique. Figure 5.11: SEM images of (a) DA-loaded PVA nanofibers revealed longitudinal solid fibers with a minimal length of 20?m and (b) DA-loaded CAP nanofibers showed artifacts in the structure Figure 5.12: TEM images of DA-loaded PVA nanofibers (a) displaying an uneven distribution of PVA and DA along its length and (b) a closer look showed fibers of minimal diameters of 5nm a) b) b) a) 1?m 0.5?m 50nm 10nm 122 Figure 5.13: TEM image of DA-loaded CAP nanofibers revealed a diameter of 1.5?m SEM and TEM images indicated a larger diameter in the DA-loaded CAP (Figure 5.11b and Figure 5.13) nanofibers in comparison to the DA-loaded PVA nanofibers (Figure 5.11a and Figure 5.12) this could possibly be due to the presence of an ideal voltage in the DA-loaded PVA formulation that lead to an increased elongation of nanofibers. The employment of a highly volatile organic solvent (acetone) which resulted in the rapid evaporation of the acetone and the clumping of the polymeric material thereby increasing diameter. Despite the evaporation of the acetone, TEM images of DA-loaded CAP nanofibers (Figure 5.13) showed a greater degree of uniformity in structure in comparison to DA-loaded PVA nanofibers (Figure 5.12a and b) which showed an uneven distribution of mass along its length, this could be due to the continuous replacement of the DA-loaded CAP solution that took place in formulation. 100nm 123 5.8. Concluding Remarks L-dopa-loaded PLGA nanoparticles were formulated employing an adopted nanoprecipitation method overcoming difficulties such as limited solubility of the L-dopa. NanoZS analysis disclosed particles, formulated under different processing techniques, of 113-213.2nm. FTIR, both solid and liquid, was conducted on the formulations that showed the various reactions that took place at the time of formulation. DEE was calculated as 61.244% as a result of the L-dopa affinity to the solvents modified in the development of the methodology of the system. TEM images sought to identify the aggregation of the particles with high yields. In vitro L- dopa release from the PLGA nanoparticles and NESD again proved that the scaffold provided the foundation for controlled delivery of L-dopa. NT-loaded CAP nanoparticle formulations showed a decrease in DEE% in comparison to the other nanosystems evaluated. The particles displayed promising particle sizes however zeta size profiles and TEM images revealed aggregation and thereby instability of these formulations. Electrospinning technology was employed in the formation of DA-loaded PVA and CAP nanofibers of minute diameters and smooth morphologies (indicated from SEM/TEM images). DEE was noted as 85% and 78% for DA-loaded PVA and CAP fibers respectively. The novel approach and ideal properties of these fibers showed great promise for their prospective employment in drug delivery systems however issues such as instability and low yield need to be addressed. Overall, the results for all formulations showed that the methodology requires further optimization prior to it's evaluation in the in vivo environment however the drug delivery system showed potential for the employment as an alternative treatment for various diseases. 124 CHAPTER 6 CYTOTOXIC EVALUATION OF NANO-ENABLED SCAFFOLD DEVICE ON CARCINOMIC BRAIN AND FRESHLY ISOLATED, HEALTHY WHITE BLOOD CELLS 6.1. Introduction Cytotoxicity is a measure of the degree to which a compound is toxic to cells, possibly resulting in cell death. In vitro cytotoxic testing methods include the 3?(4,5?dimethylthiazol-2- yl)?2,5?diphenyltetrazolium bromide (MTT), trypan blue (TB), sulforhodamine B (SRB) and (4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) (WST) assays and clonogenic assays (Matsuoka et al., 2000). These tests are vital in the research and development process and, within the context of the current study, allows for cytotoxicity levels to be evaluated prior to in vivo studies. The nigrostriatal depletion of dopaminergic neurons that defines PD results in the augmentation of DA to rectify the neurotransmitter imbalance (Lai and Yu, 1997). An increase in DA concentration leads to an escalation in the production of hydrogen peroxide and thereby apoptotic events. Exogenous treatment for PD (L-dopa and DA) has been postulated to exacerbate the degeneration present in this condition (Lai and Yu, 1997) as explained in Chapter 2 in this dissertation. DA, both endogenous and exogenous, is highly neurotoxic in a dose-dependent manner (Stokes et al., 1999). The metabolism of DA results in the production of reactive oxygen species which causes cytotoxicity in cells (Stokes et al., 1999). DA-induced apoptosis has been identified in chick embryonic sympathetic nerve cells (Ziv et al., 1994) at high concentrations (100-300?M) after a 24hr exposure period, and rat neuronal cells (Walkinshaw and Waters, 1995). In vivo studies in rats have also shown that intrastriatal DA injections (0.05-1.0?M) produced dose-dependent neurotoxic events such as neuronal loss and gliosis in various areas of the brain after a 24hr exposure period (Hastings et al., 1996). The auto-oxidation of DA (Figure 6.1) leads to the formation of a highly reactive quinone, a molecule that has the potential to alter cellular components such as DNA, lipids and proteins (Stokes et al., 2000). The oxidation is further accelerated by the presence of metals such as copper and iron, which, in the clinical setting, is elevated in PD. Enzyme-catalysed oxidation 125 of DA is also responsible for quinone formation. The excess chronic production of the reactive molecule may thus have significant consequences in terms of cell viability. NH HO HO 2 Figure 6.1: Schematic representation of one of the possible mechanisms involved in the metabolism of DA leading to cytotoxic and genotoxic events (Stokes et al., 1999) There are numerous methods used to assess of the viability of cells; the 3?(4,5? dimethylthiazol-2-yl)?2,5?diphenyltetrazolium bromide (MTT) assay being one of them. The MTT assay is colorimetric in that it determines the survival and growth of cells (upon exposure to the compound, in this case) by measuring the cells ability to reduce the MTT salt (yellow) to it?s formazan crystal form (purple) (Freimoser et al., 1999). The reduction process is indicative of the functioning of the cell?s mitochondrial succinate dehydrogenase and thus indicates cell viability (Yedjou et al., 2006). The reaction is outlined in Figure 6.2 as follows: autooxidation enzymatic oxidation tyrosine prostaglandin H synthase xanthine oxidase DA QUINONE protein modification and inactivation genotoxic effects cell apoptosis NH 2 O O Increased in presence of iron and copper (as in PD) 126 N NN N + S N CH CH3 3 Br-3 MTT (yelow ) succinate dehydrogenase NH N N N S N MTT FORMAZAN (purple ) Figure 6.2: In vivo colorimetric reaction that occurs in living cells when exposed to MTT (Dojindo Laboratories, 2000) At the outset, this chapter sought to address the possible cytotoxicity of the NESD and it's components in carcinomic SK-N-MC as well as healthy white blood cell lines. The determination of the cytotoxic activity of these compounds allowed for the progression of the study into in vivo implantation of the NESD in the rat brain. 127 6.2. Materials and Methods 6.2.1. Routine cell culture The SK-N-MC cells (ATCC, U.S.A.) are derived from a neuroepithelioma of human origin. The cells were routinely maintained in complete growth medium consisting of Minimum Essential medium (Eagle) with 2mM L-glutamine and Earl?s Balanced Salt Solution adjusted to contain 1.5g/L sodium bicarbonate, 0.1mM non-essential amino acids and 1mM sodium pyruvate, further supplemented with 10% heat-inactivated foetal bovine serum. All products were purchased from Invitrogen (BD Bioscience, California, USA). The cells were incubated at 37?C in a humidified incubator circulated with 95% O2 /5% CO2 (Afrox, South Africa). 6.2.2. Isolation of white blood cells Blood (6mL) was collected by venous puncture from healthy individuals (Human Ethics Clearance no. M070519, see Appendix of this dissertation). White blood cells (WBCs) were isolated using a Ficoll- Hypaque gradient (3:1) tube (Latimer et al., 1989, US Patent 4591557). The tubes were centrifuged for 30min at 1500rpm (Sorvall T6000D, GMI Incorporated, Minnesota, USA). Upon centrifugation, blood separates according to the weights of the different components (Figure 6.3) thereby allowing for the separation of the specific cells. The buffy coat, containing WBCs, was used in the WBCs cytotoxicity assay. Figure 6.3: Diagrammatic representation of the separation of blood that occurs upon it's centrifugation in Ficoll- Hypaque gradient (3:1) tubes (Lifelearn Inc, 2004) 128 The buffy coat was removed using a pipette (1mL) and washed three times with sterile red cell lysis buffer (500?L) containing ammonium chloride (0.15M), potassium carbonate (1mM) and ethylenediaminetetraacetic ethylenediaminetetraacetic acid (0.1mM). The buffy coat and red cell lysis buffer were inverted five times and thereafter placed in an incubator (HERAcell? 240 CO2 Incubator, Thermo Scientific, USA) (5% CO2; 37?C) for 5min. The solution was centrifuged for 10min at 5500rpm (Jouan MR1812, Scientific Products, South Africa), the supernatant removed and the process repeated until a clear solution (pure WBC) was obtained. The WBC sediment was resuspended in fresh RPMI-1640 media and counted using a hemocytometer (Neubauer 0.025mm, depth 0.1mm). WBCs were seeded into sterile 96-well plates at a density of 30000cells/well, to a final volume of 180 ?L. 20?L of each treatment was added to the wells (Section 6.2.4) and incubated at 37?C for 24hr. The MTT assay was then conducted to determine cell viability. 6.2.3. Trypsinisation and cell counting / seeding of cells The SK-N-MC cells were trypsinised using 500?L 0.25% trypsin containing 0.1% EDTA (Highveld Biologicals, SA) and incubated at 37?C for 10min. The cells were resuspended in fresh complete medium and centrifuged at 1000 rpm for 5min to pellet the cells. The cell pellet was then resuspended in ~5 mL medium. The number of cells per mL was determined using the trypan blue exclusion assay in which viable cells appear yellow, while non-viable cells stain blue. Only cell suspensions with a viability in excess of 95% were used in the assays. Cells (180?L) were seeded into sterile 96-well plates at a density of 15 000 cells per well. Cells were incubated overnight to facilitate attachment to the wells. Thereafter, 20?L of each treatment was added to the wells as described in Chapter 6 Section 6.2.4 in this dissertation, and incubated at 37?C for 24 and 48hr. The MTT assay was then conducted to determine cell viability. 129 6.2.4. Exposure of cells to the different compounds Cells were exposed to the following compounds for 24hr (WBCs) and 48hr (SK-N-MC cells): a) DA-free NESD (0.5mL alginate) b) NESD (5mg DA in 1mL of alginate) c) DA (3.79mg) d) DA-loaded CAP nanoparticles (1mg) e) 5?FU (260.16mg) f) Control (cells with medium only) All assays were performed in triplicate. 6.2.5. Calibration of MTT assay Cells were centrifuged at 1000rpm (Optima? LE-80K, Beckman, USA) for 3min. The cell pellet was resuspended in 20mL of media. 200?L of each dilution was added to eppendorfs, to which 50?L MTT (5mg/mL) was added. The samples were then incubated for 2hr at 37?C. The samples were then centrifuged at 1000rpm (Optima? LE-80K, Beckman, USA) for 5min and the supernatant removed. The cell pellet was resuspended in 200?L DMSO and the absorbance was measured at 540nm with a microplate reader (Absorbance Labsystems Multiskan MS Version 2.4). Equation 6.1 was used to determine the number of viable cells in each sample. % cell viability= [no of viable treated cells]/ [no of viable control cells] x 100% Equation 6.1 130 6.2.6. The MTT assay The WBCs and SK-N-MC cells were each treated with compounds (20?L) as shown in Figure 6.4. Figure 6.4: A schematic representation of the layout of the compounds in the 96-well plate as used in the MTT assays on WBCs and SK-N-MC cells After the incubation period, 50?L MTT was added to each well. The plates were incubated at 37oC for 2hr. Thereafter, the plates were centrifuged at 3000rpm for 10min and the supernatant removed. Formazan crystals were dissolved in 200?L DMSO, and the absorbance read at 540nm using a microplate reader (Absorbance Labsystems Multiskan MS Version 2.4) DA - fre e N ES D NE SD DA DA - lo ad ed CA P n ao pa rt ic le s 5' FU DA - fre e N ES D NE SD DA DA - lo ad ed CA P n ao pa rt ic le s 5' FU Co n tr o l Co n tr o l SK-N-MC cells WBCs 131 6.2.7. Preparation of MTT solution MTT (0.5%w/v) was prepared as per United Scientific Protocol (USA). MTT (5g), obtained from Sigma (Johannesburg, South Africa), was dissolved in 1000mL PBS (pH 7.4). The solution was filter-sterilised using a 0.22?m Cameo acetate membrane filter (Millipore Co., Bedford, Massachusetts) and stored in the dark at 4?C until use. 6.3 Results and Discussion 6.3.1. Calibration curve of post-MTT exposure for the determination of the number of viable cells present in the media All experiments were conducted in triplicate and the results represented as mean ? standard error (SE). The statistical significance of all treatments were evaluated using the GraphPad Prism3 Instat package, using ANOVA, Student-Newman-Keuls test. A probability limit of p<0.05 was considered to be significant. The calibration curve obtained for both WBC and SK-N-MC cells (Figure 6.5a and b, respectively) showed a linear correlation (R2 = 0.99). This means that the resultant absorbance obtained at 540nm is directly proportional to the number of viable cells present in each well. The absorbance readings were thus used to calculate the number of viable cells present in the wells after the respective exposures to the different compounds. Cell number 0 20000 40000 60000 80000 Ab so rb an ce at 54 0n m 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 y = 4E-06x R2 = 0.9879 a 132 Cell number 0 20000 40000 60000 80000 Ab so rb an ce at 54 0n m 0.00 0.05 0.10 0.15 0.20 Figure 6.5: MTT calibration curve for (a) WBCs and (b) SK-N-MC cells 6.3.2. Cytotoxicity to WBCs DA-free and DA-loaded NESD reduced cell viability to 70-80% (Figure 6.6). The DA-loaded CAP nanoparticles did not adversely affect cell viability as ?100% cell viability was noted, while DA appeared to have increased the number of cells by approximately ?15%. It was noted that the DA and DA-loaded CAP nanoparticles showed similar cell viability profiles proving that the inclusion of CAP into the formulation did not have a detrimental effect of cell growth. Once again, 5?FU showed a cytotoxic effect (50% cell growth) on the WBCs confirming its purpose in the study as a positive control. y = 3E-06x R2 = 0.9929 b 133 % ce ll g ro w th 0 20 40 60 80 100 120 140 Figure 6.6: Graph displaying percentage cell viability of WBCs after a 24hr exposure period to the various compounds. Cell viability is reported in relation to the control cell sample which was equated to 100%. 6.3.3. Cytotoxicity directed against the carcinomic SK-N-MC cells After a 24hr incubation period (Figure 6.6), the DA-loaded and DA-free NESD significantly (p <0.05) reduced the viability of the SK-N-MC cells (? 25% viability). This decrease was also significantly (p<0.05) greater than the exposure to 5'-FU. DA did not adversely affect cell viability, as 100% viability was noted, while the DA-loaded CAP nanoparticles only reduced cell viability to ? 75%. After 48hr (Figure 6.7), cell viability of the DA-free and DA-loaded particles did not differ significantly (p>0.05 for both) from the results obtained after the 24hr exposure period, while a significant reduction in cell viability was noted after 48hr exposure to the DA-loaded CAP nanoparticles (p<0.05). This is also seen for the 5'-FU-treated cells. DA did not significantly reduce cell viability after 48 hr. NE SD DA - fre e NE SD DA 5? FU DA - lo ad ed CA P n an op ar tic le s 134 0 20 40 60 80 100 120 24hrs 48hrs Figure 6.7: Graphs displaying percentage cell viability of SK-N-MC cells after exposure periods to the various compounds. Cell viability is reported in relation to the control cells which was equated to 100%. 6.3.4. Limitations for the employment of MTT assay in the present study The following effects could have interfered with the MTT assay and resulted in erroneous results: ? The colour of the media discoloured upon addition of the DA-loaded CAP nanoparticles and thus was noted to have lowered the pH of the media. Attempts to change the solvent for this sample failed to cause dissolution of the particles. ? Upon its addition to the media, the NESD matrix absorbed some of the media. ? The NESD size may have hindered growth of the cells competing with the cells for space by occupying the majority of the space within the plates and thereby space for the cells to grow. ? Some of the MTT was absorbed onto the NESD, thereby preventing interaction with the mitochondria in the viable cells. NE SD DA - fre e NE SD DA 5? FU DA - lo ad ed CA P n an op ar tic le s % ce ll g ro w th 135 6.4. Concluding Remarks This chapter sought to address the possible cytotoxic effects of the NESD on WBCs and human carcinomic SK-N-MC cells. The use of the colorimetric MTT assay allowed for the determination of cell growth upon exposure to the treatment compounds. Results for the assays on the WBCs and SK-N-MC cells revealed that DA-free and DA-loaded NESD were the most cytotoxic to the cells. It was later established that these results could be attributed to the absorptive nature of the NESD, causing them to soak up the cell media, thereby hindering cell growth. In earlier studies, (Weingarten and Zhou, 2001; Lai and Yu, 1999, Clement et al., 2002) DA had been reported to be cytotoxic to cells at higher doses (mM). In the present study, the concentrations of DA employed were much lower (nM) and so could have attributed to the growth of both carcinomic and healthy cells. DA-loaded CAP nanoparticles were confirmed to be detrimental to carcinomic cell growth with an increase in the exposure duration whereas results from the healthy WBC study showed that DA-loaded CAP nanoparticles promoted cell growth. The DA-loaded CAP nanoparticles may have a selective anti-cancer effect on the cells. Overall, results for the MTT assays on both carcinomic WBCs and SK-N-MC cells revealed that the NESD does not display any noteworthy unfavorable effects on cell survival and growth. 136 CHAPTER 7 IN VIVO EVALUATION OF THE NANO-ENABLED SCAFFOLD DEVICE UPON IMPLANTATION INTO THE PARENCHYMA OF THE FRONTAL LOBE OF THE RAT BRAIN 7.1. Introduction Animal models are an invaluable tool in experimental medical science because they enable one to study the pathogenetic mechanisms and thereby the possible therapeutic strategies of human diseases. Once the causative mechanism is understood, PD does not naturally occur in animals however scientists have found neurotoxins that sufficiently mimic the disease (Gerlach, 1996). Mice have become the most established animal model for research into PD however there are several drawbacks in the lack of anatomical similarity between the mouse model and humans. For this reason, the monkey model has received much favor in that the anatomical structures present within the animal are most similar to that of the human. However, tremendous ethical constraints limit the use of the monkey model in in vivo drug studies (Halbach, 2005). The Sprague-Dawley rat model was selected for use in this study. The rat model held the advantage of greater brain volume for implantation of the NESD than the mouse model. 7.1.1. Biocompatibility of implantable devices "The ability of a material to perform with an appropriate host response in a specific application" (Williams, 1999). The interest in biocompatible polymeric devices for implantation into the body has gained a surge of interest in the fields of orthopedics, cardiovascular and ophthalmics to name a few (Katz, 2001). The concept of biocompatibility is further explained in Figure 7.1. 137 Figure 7.1: Schematic diagrammatic representation of immunological response to implant within the CNS (adapted Fournier et al., 2003; Polikov et al., 2005) Slow degradation rate of implant Acute Response Vessels dilate Stagnation of bodily fluids and blood clots Cell non-specific protein infiltration and adsorption onto implant (fibrinogen, immunoglobulin G, albumin Chronic Response Phagocytes infiltrate Fibroblasts (FBCG) encapsulate implant (Fibrous Capsule) Granuloma- oedema and pain + Toxicity develops Cell Necrosis May decrease drug release rate Biocompatible Device 138 This chapter sought to develop a protocol for the surgical implantation of the NESD into the Sprague-Dawley rat brain model. Furthermore, issues of biocompatibility and bioerosion of the device were addressed while the elucidation of it's in vivo drug release profile formed the crux of this chapter. 7.2. Materials and Methods 7.2.1. Materials Solvents used for UPLC?MS/MS measurements were of UPLC grade, and all other reagents were of analytical grade. Double deionized water was obtained from a Milli-Q system, (Milli- Q, Millipore, Johannesburg). Oases HLB were provided by Waters (Milford, MA, USA). Control blank rat plasma was supplied by healthy donors. Ammonium acetate (Mw=77.08g/mol), acetic acid (CH3COOH) (Mw=60.05g/mol), sodium hydroxide (Mw=40.00g/mol), dopamine hydrochloride (DA) (Mw=189.64g/mol) theophylline (Mw=180.17g/mol) from Sigma Aldrich (St. Louise, MO, USA). Healthy adult Sprague Dawley rats were used for the in vivo release study 7.2.2. Technology Applied in the Present Study 7.2.2.1.. Waters? Acquity Ultra Liquid Performance ChromatographyTM (UPLC) The UPLC allows for samples to be run with the shortest run time, highest sensitivity, but without compromising the selectivity of the assay (Dongre et al., 2008). Typically, UPLC refers to applications using porous sub-2 ?m particles at high linear velocities (Waters supplies catalogue, 2008). UPLC parameters such as pH, flow rate, column type, and buffer concentration can be optimized to achieve the best sensitivity, peak shape and selectivity (Everley and Croley 2008). The UPLC allows for the identification of various compounds and the determination of their concentrations. Its application is with compounds that are non- volatile in comparison to their mobile phase at the time of analysis. The concentration of the compounds is calculated by means of Beer-Lamberts Law: A = ?bc Equation 7.1 Where A is the absorbance (calculated for each diode of the system) of the sample measured, ? is the molar absorbtivity of the sample, b is the pathlenght of the cuvette (1cm) and c is the concentration of the sample in moles per liter. 139 7.2.2.2. Evaporative Light Scattering (ELS) Detector The UPLC is coupled with an ELS detector which is responsible for the qualitative and quantitative analysis of drug loaded samples. The process ELS detection has three stages: ? Nebulization: the effluent is transformed into a fine aerosol using nitrogen as a carrier gas; ? Desolvation: the mobile phase is evaporated and condensed thereafter moving to the detector phase. The mobile phase should have a low viscosity and high volatility so as to increase the rate of evaporation thereby increasing the quality of the sample; and ? Detection: the light source is exposed onto the particles, the particle size determines the intensity measured. The detector may be further coupled with a mass spectrophotometer or absorbance detector to assess the quality of the samples and ensure that all compounds present are identified. 7.2.3.. Development of Methodology for the Surgical Implantation of the NESD into the Parenchyma of the Frontal Lobe of the Sprague-Dawley Rat Brain 7.2.3.1. Pre-surgical preparation of rats Healthy male Sprague?Dawley rats (350?20g) were employed in this pilot study (Figure 7.2.). Rats were housed in cages, maintained under a 12hr light/dark cycle, fed a commercial diet, and given water ad libitum. 140 Figure 7.2: Schematic diagram representing the number of rats required for in vivo studies 45 rats 30 rats 15 rats 15 rats 15 rats Administration of Sinemet ? via gastric lavage Implantation of placebo carrier into the parenchyma of the frontal lobe of the brain Implantation of NESD into the parenchyma of the frontal lobe of the brain post-surgical care blood sampling euthanasia CSF collection and removal of entire brain anaesthesia for sampling anaesthesia 141 Note: Rats had undergone surgical implantation with device at day 0. Sampling had taken place on days 3, 7, 14, 21 and 30 followed by euthanasia (n = 3). 7.2.3.2. Pilot surgical technique for implantation of the NESD into parenchyma of the frontal lobe of the rat brain Animal Ethics no. 2007/76/4 (see Appendix of this dissertation).The technique for the insertion is analogous to the technique used for the insertion of an intracranial pressure monitor in the human subject. The rat was anaesthetized for the duration of the procedure which was performed in an aseptic manner. Rats were anesthetized with a mixture of ketamine (65mg/kg) and xylazine (7.5mg/kg). A small (0.5-1cm) para-midline right sided scalp skin incision was made. A hand-held twist drill was then used to make a controlled perforation of the skull. The resultant skull opening was approximately 0.5mm in diameter. The skull opening was followed by sharp incision of the dural lining; this was likely to be associated with a small volume of self-limiting bleeding. The brain parenchyma was then ready for insertion of the implant. The cylindrically shaped implant had a volume of approximately 0.000354cm3 (length = 0.5cm, diameter = 0.3cm). The volume of the rat brain varies from 0.865 ? 0.026cm3 to 1.165 ? 0.071cm3 depending of the age of the rat i.e. implant was less than 20% of the rat brain volume. The skull defect was sealed with wax and the scalp insertion was closed with a single layer of appropriately sized non-absorbable suture. The rat then received Temgesic: (0.1mL diluted to 1mL with ultra purified water), at a dose of 1mL post-operatively for pain relief with a rehydration treatment of 5% glucose in 0.9% saline. A series of behavioral asymmetry tests were performed on the rats to assess any degree of motor dysfunction present 7.2.3.3. End points for experiments that induce illness in rats Rats that had experienced any degree of distress were removed from the study. Weight, eating and grooming habits were monitored periodically. Any significant fluctuations in these areas led to the removal of the rat affected from the study. The inclusion of a placebo NESD group allowed for the comparison against the experimental group in terms of the assessment of the degree of distress experienced by the rats. 142 7.2.3.4. Biological fluid sample collection from the rat model Rats received anesthesia with ketamine/xylazine mixture (as per 7.2.1.2)) prior to the sample collection. Blood: Sequential blood samples (5mL) were collected via cardiac puncture into heparin- containing test tubes at predetermined time intervals. Plasma was separated by centrifugation for 8min at 1000g and stored at ?70 ?C until analysis. Cerebrospinal Fluid (CSF): The technique being analogous to that employed in Ethics Application 2005/86/5. The rats were euthanized prior to the procedure with sodium pentobarbitone (200mg/kg) and the skin overlapping on the neck (this is the skin where the head and neck meet to allow for insertion of the needle) was shaven. A device was specially made up consisting of a 25G needle attached to polyethylene tubing at one end and a 1mL syringe at the other end. The device was inserted into the cisterna magna to obtain the CSF (100-150?L). CSF samples were transferred to ependorfs (1.5mL) and placed on ice. Thereafter, the samples were centrifuged @10 000rpm (Optima? LE-80K, Beckman, USA) for 20min, the supernatant was removed and stored in refrigerator @ -70?C. Brain Tissue: The entire brain was removed and placed in formalin solution (10%v/v). 7.2.3.5. Histological analysis of the rat brain tissue post-implantation of the NESD Rats were euthanized with sodium pentobarbitone and decapitated. Brain tissue was removed and placed in 10%v/v neutral buffered formalin. The tissue was then dehydrated, infiltrated and embedded in paraffin followed by transversion into 400?m slices and stained with hemotoxylin and eosin. The slides were then viewed microscopically to assess the presence/absence of inflammatory cell infiltration and markers of necrosis. 7.2.3.6. Bioerosion studies on the NESD Devices were removed at various predetermined time intervals (5, 7, 14, 21 and 30 days) and their bioerosion was assessed using SEM (JEOL, JEM 840, Tokyo Japan). Photomicrographs were taken at different magnifications and samples were prepared after sputter-coating with carbon or gold. 143 7.2.4. Determination of in vivo drug release from Sinemet? and the NESD using UPLC analysis on biological fluids 7.2.4.1. Preparation of priming solvents and mobile phases Strong Wash: Acetonitrile (90% v/v) and water (10% v/v). Weak Wash: Acetonitrile (10% v/v) and water (90% v/v). Mobile Phase A (98%): Ammonium acetate (7.09g) was dissolved in 1L of water, to give a 0.1M solution. acetic acid was added to adjust the pH to 5.0 Mobile Phase B (2%): Acetonitrile (100%v/v). Double deionised water (Milli-Q, Millipore, Johannesburg) of 18.2 M? cm?1 resistivity was used. The mobile phases were filtered a 0.22?m pore size Cameo Acetate membrane filter (Millipore Co., Bedford, Massachusetts). 7.2.42. Preparation of standards Primary stock solutions of DA (100mg/mL) were prepared in doubly deionised water. The stock solutions were stored in polypropylene tubes with screw caps and kept in a refrigerator at 5 C. Working standard solutions of DA were prepared by diluting the primary solution with doubly deionised water. Drug-free human plasma (0.4mL) was spiked with appropriate working solutions of DA to produce final DA concentrations ranging from 0.0008-15.00?g/mL. Solutions of theophylline (internal standard, IS)) was constituted in doubly distilled water (1mg/mL). All solutions were filtered with a 0.22?m pore size Cameo Acetate membrane filter (Millipore Co., Bedford, Massachusetts). 7.2.4.3. Solid Phase Extraction of blood and CSF The Generic Oasis? HLB Solid Phase Extraction (SPE) method was employed to determine the concentrations of methanol that would increase the purity of the analyte (DA) thereby allowing the determination of drug concentration in the samples. Oasis? HLB cartridges enabled with the Visiprep Vacuum Manifold and Standard Lid (Waters,) were conditioned with 144 methanol (1mL) and deionized water (1mL). The prepared calibration standards and plasma samples (as in 7.2.1.3.2.) were loaded onto the cartridges post-conditioning followed by a mild wash with 5% methanol (1mL). The strong wash entailed the employment of a gradient of concentrations (10-100%) of methanol (Figure 7.3) which were collected in separate testtubes (5mL) to which theophylline (0.1mL) was added. The samples were then placed in Waters certified vials (1.5mL) for analysis (Li et al., 2005). Figure 7.3: Diagrammatic account of the strategy for the optimization of the generic SPE method acidified with phosphoric acid (2%v/v) conditioned HLB cartridge with methanol (1mL) and water (1mL) loaded sample (1mL) prepared 10mL drug-loaded (10?L) sample Basic Study Acidic Study 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 methanol concentration (%) added NaOH (2%v/v) methanol concentration (%) added acetic acid (2%v/v) 145 7.2.4.4. Instrumentation and operating conditions of the UPLC system UPLC analyses were performed with a Waters Acquity Ultra Performance LC system (Waters, Milford, MA, USA) coupled with a PDA detector. UPLC separation was achieved on an Acquity UPLC BEH C18 column (50 mm ? 2.1mm, i.d., 1.7?m particle size, Waters) maintained at 25 ?C. The compounds were injected in the mobile phase with an injection volume of 3.7?l (partial-loop injection). Run time of 2min and flow rate of 0.500mL/min was calculated for the study. The equipment was primed with the washes and mobile phases for 10 cycles of 5min each. Acetonitrile (100%v/v) was injected into the system so as to remove residue sample material from the column prior to the sample runs. Aliquots (1?L) of samples were injected into the UPLC system and thereafter processed. 7.2.4.5. Calibration curves and limit of quantification for DA in plasma Analyte standard solutions (0.4mL) at 8 different concentrations, were added to 0.4mL of blank plasma. Acetonitrile (0.4mL) was added to each sample and centrifuged (Optima? LE- 80K, Beckman, USA) at 15000rpm for 10min. The supernatant of these samples were removed, subjected to the SPE procedure as detailed in 7.4.2.4. and injected into Waters Acquity Ultra Performance LC system (Waters, Milford, MA, USA). The analyte/IS peak area ratios were plotted against the corresponding analyte concentrations (expressed as ng/mL?1). Linearity equations and correlation coefficients (rc) were obtained by means of the least square method. The limit of quantitation (LOQ) is defined as the analyte concentrations, which gives rise to chromatographic peaks whose height was equal to 10 and 3 times to that of the baseline noise (Mandriol et al., 2006) and was calculated for DA. 7.2.4.6. Extraction yield, precision and accuracy of the method Standard solutions (N=3) at three different concentrations of DA were added to 0.4mL of blank plasma, in order to obtain plasma analyte concentrations of 150000?g, 15000 ?g and 0.8ng for DA; these mixtures were subjected to the SPE procedure and injected into the UPLC system. 146 Percentage extraction yield for the samples was calculated by comparing the peak areas of analytes obtained employing the SPE method to those obtained from standard solutions at the same theoretical concentration. Intra-day (multiple injections of samples during a 24 hr period N=3) and inter-day (multiple injections of samples over 3 consecutive days, N=3 for each day) sampling provided the data for precision and accuracy. 7.2.4.7. CSF and blood preparation for UPLC analysis Samples (plasma and CSF) were removed from the freezer (-70?C). Acetonitrile (0.4mL) was added to each sample and placed in the centrifuge (Optima? LE-80K, Beckman, USA) @15000rpm for 10min. The supernatant was removed, placed in testtubes and centrifuged @5000rpm for a further 5min. The resultant was thereafter subjected to the SPE procedure as detailed in 7.4.3. and spiked with internal standard (0.1mL). The samples were then placed in Waters certified vials (1.5mL) for analysis (Li et al., 2005) and analysed on the Waters Acquity Ultra Performance LC system (Waters, Milford, MA, USA). 147 7.3. Results and Discussion 7.3.1. Brain damage in the rat from the surgical implantation of the NESD The procedure (Figure 7.4) was associated with a small amount of cortical contusion. However when damage to the brain was found to be of no clinically detectable neurological deficit. It was found that a significant number of rats suffered severe and even lethal complications from the surgical procedure leading to the reevaluation and development of the implantation procedure. Figure 7.4: Photographic representation of the surgical procedure (a) manual drill used for incision, (b) actual perforation of the skull and (c) suturing of the incision 7.3.2. Modifications to surgical implantation procedure The rats were placed in a Kopf stereotaxic frame and a straight midline incision was made from nasion to occiput. The skin and perisoteum was reflected exposing the dorsal surface of the skull. This was done in order to facilitate identification of the cranial sutures and to ensure the skull trephination was made in the frontal bone. An electric drill was used to decrease the pressure exerted on the scalp so as to minimize any excess bleeding. The mass of rats was increased (350-400g) as it was found that smaller rats had more complications as a result of surgery. a) c) b) 148 7.3.3. Evaluation of modifications to surgical implantation technique The employment of a stereotaxic frame allowed for the accurate implantation of the implant into the parenchyma beneath the frontal bone thereby avoiding motor and eliminating the outcome of paralysis in the rats. Furthermore, larger rats exhibited a more positive effect to the implant due to their increased brain volume. Overall, rats exposed to the modified surgical technique displayed good health (weight and grooming techniques). 7.3.4. Biocompatibility of the NESD systems employed in the study 7.3.4.1. Pathological diagnosis of rat brain specimens implanted with DA-free NESD Histological analysis of all specimens was representative of cortical grey and white matter as well as the ventricular and cerebellum regions of the rat brain. Day 5 and 7: There was no evidence of inflammatory cells within the brain parenchyma and Virchow's Robin spaces. No evidence of ischemia or morphological features of demyelinating disorder. Day 14: Extremely sparse and scanty inflammatory lymphocyte were scattered in the white matter. Minute inflammation was present in the Virchow?s Robin spaces. There was no indication of ischemic injury and demyelinating disorder. Day 21: There was mild inflammation within the meningeal covering, neural parenchyma and in the periventricular area. Once again, there were no findings of inflammation in the Virchow?s Robin spaces. Mature lymphocytes were found sparsely dispersed in the white and grey matter. In addition congested blood vessels were present in the meninges. Day 30: Mild inflammation was identified in the cortical white and grey matter as well as periventricular areas. There was also mild chronic inflammation in the meninges and congested blood vessels. 149 7.3.4.2. Pathological diagnosis of rat brain specimens implanted with NESD The histological findings (Figure 7.5) in the rat brain for the NESD showed the same pathological features as in 7.3.5.1. Figure 7.5: Light microscope images of histological slides of the site of implantation of the NESD into the brain, stained using H & E, in (a) control and (b) test brain tissue following 5 days of implantation of the NESD 7.3.5. Bioerosion of NESD post-implantation Figure 7.6 represents the mass loss from the NESD over a period of 30 days post- implantation. Bioerosion was 16% at 7 days (Figure 7.7) and thereafter minimal erosion took place (26% bioerosion at 30 days). The erosion profile proved that the device should remain intact throughout the treatment period (at least 30 days) and thereby possibly contribute to controlled drug release. In addition, results confirmed that the device was in fact biodegradable and so there was no need for a second surgical to remove the device once the drug is depleted. b) a) 150 Time (days) 0 5 10 15 20 25 30 Bi oe ro sio n (% ) 0 5 10 15 20 25 Figure 7.6: In vivo bioerosion profile for NESD over a period of 30 days (SD?0.78) Figure 7.7: Light microscope (top) and SEM (bottom) images of NESD matrix (a) pre- implantation and (b) blood perfused post-euthanasia and removal from rat brain post-7 day implantation showing 16% bioerosion 1.49cm 1.24cm a) b) 151 7.3.6. SPE of DA from plasma The optimized SPE technique was selected to remove plasma constituent interferences and isolate the drug in the CSF and blood samples. The employment of HLB cartridges for the extraction offered the advantage of retaining polar compounds, as is DA, and thereby removing impurities. Serial dilutions of methanol solutions (5-100%v/v), represented in Figure 7.6, with either the addition of an acid (CH3COOH) or a base (NaOH) was employed in the SPE technique. It It was noted that during the acidic phase (CH3COOH) higher integral UPLC peaks and extraction yields were obtained as compared to the basic phase (NaOH), in particular, at 70%v/v methanol with 2%v/v acetic acid. An additional wash-step of 45%v/v methanol produced even larger recoveries and level chromatographic baselines. 7.3.7. Chomatograms for standards and plasma A typical chromatogram of a standard solution of DA (Figure 7.8), theophylline (Figure 7.9) and a blood sample obtained from the cardiac vein in the rat model post-implantation of the NESD is displayed in Figure 7.8. The chromatograms proved that the method for extraction was successful in eluting dopamine and internal standard. 152 0. 28 3 0. 39 3 0. 49 2 0. 76 5 0. 91 6 1. 11 8 1. 33 7 1. 58 0 1. 73 4 1. 86 7 0.00 1.00 2.00 0.00 0.50 1.00 1.50 2.00 Figure 7.8: A typical chromatograph depicting the peak (Rt = 0.4925) for DA employing UPLC at 280nm in double deionised water 0. 10 8 0. 27 7 0. 50 5 0. 68 0 0. 88 8 1. 06 5 1. 20 1 1. 34 5 1. 57 9 1. 87 2 2. 05 2 2. 24 5 AU 0.00 1.00 2.00 0.00 0.50 1.00 1.50 2.00 2.50 Figure 7.9: A typical chromatograph depicting the peak (Rt = 1.579) for theophylline (internal standard) employing UPLC at 280nm in double deionised water Retention time (min) AU AU Retention time (min) 153 Retention Time (min) Au 1. 63 8 0. 49 9 1.00 0.00 0.00 0.50 0.50 1.00 1.50 2.00 2.50 DA THP Figure 7.10: A typical UPLC chromatogram depicting the distinct separation of DA (Rt=0.49) and theophylline (Rt=1.64) at 280nm from the Sprague Dawley rat plasma samples 7.3.8. Calibration curve and lower limit of quantification of DA Figure 7.11. displays the calibration curve for DA in plasma samples employing the UPLC (Waters, Milford, MA, USA) and PDA detector set at 280nm. Concentrations of 150000- 0.8ng/mL offered good linearity (R2 = 0.9811). AU 154 Concentration (ng/mL) 0.0 2.0e+4 4.0e+4 6.0e+4 8.0e+4 1.0e+5 1.2e+5 1.4e+5 AU C D A/A UC IS 0.0 0.1 0.2 0.3 0.4 0.5 Figure 7.11: DA calibration curve at 280nm in plasma employing UPLC at 280nm in double deionised water The lower limit of quantification for this study was noted as 0.8ng/mL. 7.3.9. Method validation for the analysis of DA in plasma employing UPLC analysis The method validation data (extraction efficiency, intra- and inter-day variation) for the analyte are summarized in Table 7.1. The extraction recoveries ranged from 95.89 to 101.02%, while the precision values ranged from 1.5 to 7.4% over the three concentrations (i.e., 0.8, 15000 and 150000ng/ml) evaluated over 3 consecutive days. These results indicated that the implemented solid phase extraction method has an acceptable accuracy and precision. Table 7.1: Illustrates the data obtained to assess the validation of the method Intra-day Inter-day Concentration added (ng/mL) Extraction yield (%) SD CV SD CV 0.8 95.89 0.000007 0.036 0.000014 0.074 15000 98.98 0.0014 0.015 0.0021 0.022 150000 101.02 0.0021 0.041 0.0028 0.055 y = 4E-06x R2 = 0.9811 155 7.3.10. In vivo DA release profile for NESD The in vivo drug release profiles for the control preparation (Sinemet ?) (Figure 7.12) and the NESD (Figure 7.13) showed indirect proportionality to each. The drug release for the group receiving Sinemet? was carried out over a period of 10hr while the drug release study for the test group was carried over a month. The drug release from Sinemet ? (Figure 7.12) was quite clearly substantially reduced, approximately 10-fold, in the CSF in comparison to the plasma. And while Cmax = 0.000053% was noted at 2hr in the plasma, the peak plasma concentrations in the CSF only took place at 8hr with 0.000012% of DA being released. The drug release for the NESD (Figure 7.13) produced a peak at 3 days for both the CFS and plasma, the CSF concentration of DA being 28% while the plasma concentration was only 1.2% of the total concentration administered. The DA release profile for plasma maintained low drug release throughout the 30 days of the study whereas the CSF concentration of DA peaked at 3 days and thereafter maintained reduced drug release for the remainder of the study. Overall, the NESD was implanted at the site of action and therefore drastically improved the delivery of DA to the brain tissue comparatively to the Sinemet ?. In addition, DA concentrations found in the plasma were minimalistic and therefore could result in the alleviation of the systemic side-effects from the oral administration of levodopa preparations. 156 Time (hrs) 0 2 4 6 8 10 Lo g co n ce n tra tio n (ng /m L) 1e-7 1e-6 1e-5 1e-4 plasma CSF Figure 7.12: In vivo profile for DA release from Sinemet? (SD?0.000001) Time (Days) 0 3 6 9 12 15 18 21 24 27 30 Lo g D A Co n ce n tra tio n (ng /m L) 0.0001 0.001 0.01 0.1 DA in CSF DA in plasma Figure 7.13: In vivo profile for DA release from the NESD (SD?0.003) 157 7.4. Concluding Remarks The in vivo chapter sought to address the biocompatibility and in vivo DA release of the NESD in the rat brain employing novel surgical approaches, SEM and UPLC techniques. A surgical protocol for implantation of the NESD into the parenchyma of the frontal lobe of the rat brain was developed that resulted in only minor clinical implications for the rat model post- surgery. Biocompatibility was addressed in this chapter and findings proved that the placebo as well as the NESD had no significant effects on the brain tissue with no evidence of ischemia or morphological events in the brain cells. The bioerosion profile of the NESD mimicked that of the in vitro degradation studies with minimal erosion post day 7 of implantation proving the device would remain intact throughout the treatment period but also retained it's biodegradation characteristics. UPLC analysis was undertaken for the determination of DA in the plasma and CSF. Method development and validation studies were undertaken in the establishment of the technique. DA release profiles illustrated concentrations of DA in the plasma and the CSF of both the group receiving Sinemet ?and the NESD. The NESD proved to be superior in terms of drug release to the brain tissue and also reduced systemic circulation of DA thereby possibly decreasing side-effects experienced from the drug. Overall, results obtained in the chapter showed that the NESD was biocompatible, biodegradable and had a positive effect of the concentrations of DA in the brain. 158 CHAPTER 8 CONCLUSIONS AND RECOMMENDATIONS 8.1. Conclusions In January 2008, the cause of PD still remains unknown (Richter et al., 2008) and with chronic treatment being limited and ineffective, the disease holds grave consequences for its sufferers. A cure is nowhere in sight and so pharmacological and non-pharmacological agents are the sole promise of a normal life for these patients. The R & D process is a lengthy one and with gene therapy still in the early stages of human clinical trials (Lewis and Standaert, 2008) and advancements in non-pharmacological treatment being leisurely, researchers need to look at the development of other possible agents to treat this rapidly growing disease. Young-onset/ baby boomers PD now affects 5?10% of patients while 1% of the aging population (60 yrs and older) will suffer from PD (Samii et al., 2004) and it's number of victims continue to climb. Patient's medical needs increase significantly for the years following the onset of PD (Fiandaca et al., 2008) and depression is now a common symptom (Barrero et al., 2005). The suffering continues to the patient's family and caregiver/s and so the disease knows no end. This research undertook to develop a drug delivery system consisting of DA-loaded CAP nanoparticles dispersed within Ba-alginate scaffolds for implantation into the parenchyma of the frontal lobe of the brain. The concept is novel in that it incorporates two unique drug delivery systems that complement each other in the achievement of controlled release. Extensive in vitro testing resulted in the establishment of the NESD. Preliminary investigations on both the Ba-alginate scaffold and DA-loaded CAP nanoparticles allowed for the selection of appropriate crosslinkers and polymers to be employed in their formulation. DA-loaded CAP nanoparticles were subjected to vigorous in vitro release studies to determine MDT and zetasize analysis to determine their stability and quintessentially their particle size and zeta potential. While the structural integrity and matrix erosion kinetics were imperative characteristics for Ba-alginate scaffold design. 159 Formulations of the Ba-alginate scaffold and DA-loaded CAP nanoparticles were both optimised employing the Box-Behnken design which saw the in vitro testing (Matrix Resilience and Matrix Erosion for the Ba-alginate scaffold and MDT, Particle Size and Zeta Potential for DA-loaded CAP nanoparticles) of 27 formulations each. A single candidate formulation was chosen for each system. The combinatory NESD was then subjected to simulated CSF and it's in vitro drug release was confirmed. The promise of the NESD lead to it's evaluation in vivo. The parenchyma of the frontal lobe of the brain of rat models were implanted with the NESD followed by histological, bioerosional and in vitro DA analysis at predetermined time intervals. Futhermore, preliminary in vitro evaluation was undertaken on other possible nanosystems that may be used to treat PD. Characterization and in vitro release of L-dopa-loaded PLGA and NT-loaded CAP nanoparticles as well as DA-loaded polymeric nanofibers showed that there is a possibility for these systems to be employed in the treatment of PD and that further testing should be undertaken. While this drug delivery system has been developed with the incorporation of DA in the treatment of PD, it is not exclusive to this disease as the NESD may be applicable to numerous other chronic CNS conditions. The addition of different combinations of polymers and drug may impart a range of different properties to the device thereby allowing it to be used for the treatment of other ailments. The strategic implantation of the device into the brain will circumvent the allows for targeted drug delivery and thereby allow numerous other CNS drugs to be delivered to the brain tissue, mostly unchanged and eliminating systemic side effects. 160 8.2. Recommendations Nanosystems (L-dopa-loaded PLGA and NT-loaded CAP nanoparticles and DA-loaded polymeric nanofibers) need to be more extensively researched both in vitro and in vivo so as to provide potential alternatives for the treatment of PD. Major variations in drug-loaded polymeric nanoparticle size were noticed for all formulations, methods and mechanisms need to be investigated so as to limit and even provide uniformity to particle size. In vitro drug release in the brain is still in it's early stages and therefore lacks a standard protocol for the conditions that need to be maintained to simulate the in vivo environment. Animal studies in the rat model have shown great promise and so these studies need to move to research in larger mammalian models such as apes. These results will be more significant in that they will more closely represent the human model (human clinical trials being the next stage). Fluorescent markers should be attached to the drug-loaded polymeric nanoparticles in vivo, so as to determine their transportation and distribution throughout the brain and even the body. This will allow us to evaluate the exact numbers and mechanisms of nanoparticulate drug delivery. The minute size of the NESD for in vivo implantation posed limitations for bioerosion studies. 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