EXPLORATION OF AN ELECTRO-MAGNETO-RESPONSIVE POLYMERIC DRUG DELIVERY SYSTEM FOR ENHANCED NOSE-TO-BRAIN DELIVERY OLUFEMI DAVID AKILO A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand in fulfilment of the requirements for the degree of Doctor of Philosophy Supervisor: Professor Viness Pillay University of the Witwatersrand, Department of Pharmacy and Pharmacology, Johannesburg, South Africa Co-Supervisors: Professor Yahya Essop Choonara University of the Witwatersrand, Department of Pharmacy and Pharmacology, Johannesburg, South Africa and Professor Girish Modi University of the Witwatersrand, Division of Neurosciences, Department of Neurology, Johannesburg, South Africa Johannesburg 2016 i DECLARATION I, Olufemi David Akilo, declare that this thesis is my own work. It has been submitted for the degree of Doctor of Philosophy in the Faculty of Health Sciences in the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at this or any other University. ............................................................................. This 27th day of May, 2016 ii PUBLICATIONS 1. Nose-to-Brain Neurotherapeutic Interventions: Olufemi D Akilo, Yahya E Choonara, Pradeep Kumar, Lisa C du Toit and Viness Pillay, Chapter 6 of Advances in Neurotherapeutic Delivery Technologies, OMICS Group eBooks, 80-107, 2015. 2. An in vitro evaluation of a carmustine-loaded Nano-co-Plex for potential magnetic- targeted intranasal delivery to the brain: Olufemi D. Akilo, Yahya E. Choonara, André M. Strydom, Lisa C. du Toit, Pradeep Kumar, Girish Modi and Viness Pillay, International Journal of Pharmaceutics, 500, 196-209, 2016. 3. A Lactoferrin-Galantamine Proteo-Alkaloid Conjugate for the Management of Alzheimer's disease: Olufemi D Akilo, Yahya E Choonara, Pradeep Kumar, Lisa C du Toit and Viness Pillay. Submitted to Medical Hypotheses, January, 2016. 4. Preparation, Physicochemical and Physicomechanical Characterization of an Optimized Nanogel Drug Composite for Potential Intranasal Drug delivery: Olufemi D Akilo, Yahya E Choonara, Pradeep Kumar, Lisa C du Toit and Viness Pillay. Submitted to Biomaterials, February, 2016. 5. In Vitro, Ex Vivo and In Vivo Evaluation of an Electro-Magneto-Responsive Nanogel Composite for Nose-to-brain Delivery. Olufemi D Akilo, Yahya E Choonara, Pradeep Kumar, Lisa C du Toit and Viness Pillay. Submitted to Journal of Pharmaceutical Sciences, March, 2016. iii RESEARCH OUTPUTS AT CONFERENCE PROCEEDINGS 1. Olufemi David Akilo, Yahya Essop Choonara, Girish Modi and Viness Pillay. Preparation and Characterization of a Dual Responsive Polymeric Gel for Intranasal Drug Delivery. (Poster Presentation). School of Therapeutic Sciences Research Day, University of the Witwatersrand, Johannesburg, South Africa, September 10th, 2013. 2. Olufemi David Akilo, Yahya Essop Choonara, Lisa Claire Du Toit, Pradeep Kumar, Girish Modi and Viness Pillay. Synthesis of Carmustine-Loaded Ferrous-Based Nanoconstructs for Targeted Intranasal Delivery to the Brain. (Poster Presentation). 6th Cross-Faculty Graduate Symposium, University of the Witwatersrand, Johannesburg, South Africa, October 27th-31st, 2014. 3. Olufemi David Akilo, Yahya Essop Choonara, Lisa Claire Du Toit, Pradeep Kumar, Girish Modi and Viness Pillay. Preparation and Characterization of a Mucoadhesive-Dual Responsive Polymeric Gel for Intranasal Drug Delivery. (Poster Presentation). International Society of Biomedical Polymers and Polymeric Biomaterials, Orlando, Florida USA, 8th-10th July, 2015. 4. Olufemi David Akilo, Yahya Essop Choonara, Lisa Claire Du Toit, Pradeep Kumar, Girish Modi and Viness Pillay. Synthesis of a carmustine-loaded Polyplex coated iron oxide nanoparticles for targeted intranasal delivery to the brain (Podium Presentation). International Society of Biomedical Polymers and Polymeric Biomaterials, Orlando, Florida USA, 8th-10th July, 2015. 5. Olufemi David Akilo, Yahya Essop Choonara, Lisa Claire Du Toit, Pradeep Kumar, Girish Modi and Viness Pillay. Preparation of a Gadolinium based Nanoparticles Coated with Polyethyleneimine-Galantamine-Lactoferrin Complex for Intranasal Drug Delivery in Treating Alzheimer's Disease (Podium Presentation). Faculty of Health Sciences Research Day, University of the Witwatersrand, Johannesburg, South Africa, September 8-9, 2015. iv 6. Olufemi David Akilo, Yahya Essop Choonara, Lisa Claire Du Toit, Pradeep Kumar, Girish Modi and Viness Pillay. An In Vitro Evaluation of Carmustine- Loaded Nano-co-Plex for Potential Magnetically Targeted Intranasal Delivery to the Brain. (Podium Presentation). Academy of Pharmaceutical Sciences of South Africa, Johannesburg, South Africa, September 17-19, 2015. 7. Olufemi David Akilo, Yahya Essop Choonara, Lisa Claire Du Toit, Pradeep Kumar, Girish Modi and Viness Pillay. Synthesis of gadolinium oxide coated with Galantamine-loaded PEG for the management of Alzheimer‟s disease. (Podium Presentation). Academy of Pharmaceutical Sciences of South Africa, Johannesburg, South Africa, March 2-3, 2016. v PATENT FILED A novel drug-loaded Nano-co-Plex incorporated into a mucoadhesive-electro responsive polymeric gel for controlled and magnetically targeted nose-to-brain drug delivery. O. D. Akilo, V. Pillay, Y.E. Choonara L.C. du Toit, P. Kumar. SA 2015/06543. vi ABSTRACT Delivering drugs to the brain for the treatment of brain diseases has been fraught with low bioavailability of drugs due to the Blood-Brain Barrier (BBB). The intranasal (IN) route of delivery has purportedly been given recognition as an alternative route of delivering drugs to the brain with improved bioavailability if the nose-to-brain option is considered. However, drugs administered through the nasal mucosa suffer some challenges such as mucocilliary clearance, enzymatic degradation, inability of a controllable drug release to give a precise dose, resulting in frequent dosing and absorption into the systemic circulation through the blood rich vessels in the mucosa, thus facing the BBB challenge. The aim of this study was to develop a novel Nano-co-Plex (NCP), a magnetic nano-carrier loaded with a therapeutic agent which is further incorporated into a nasal thermosensitive electro-responsive mucogel (TERM) for in situ gelling, for electro- actuated release of the incorporated drug-loaded NCP in a controllable “on-off” pulsatile manner, which is achieved with the aid of an external electric stimulation (ES). The released drug-loaded NCP was then targeted to the brain via a direct nose-to-brain drug delivery pathway with the aid of an external magnetic field (MF) for rapid transportation. The ES was brought about by applying a 5V potential difference (PD) using electrodes on the nose and the external MF would then be applied by placing a magnetic headband on the head of the patient. In this research, the drug-loaded NCP was prepared by firstly synthesizing iron oxide nanoparticles (Magnetite) which were then coated with Polyplex; a polymeric complex fabricated employing polyvinyl alcohol (PVA), polyethyleneimine (PEI) and fIuorecein isothiocyanate (FITC). The coated magnetite was thereafter loaded with Carmustine (BCNU), an effective drug commonly used in brain tumor treatment, to formulate the BCNU-NCP. The TERM was prepared by blending a thermosensitive polymer, Pluronic F127 (F127) with mucoadhesive polymers, chitosan (CS) and hydroxypropyl methylcellulose (HPMC). Polyaniline (PANI) was included in the blend as the electo-active moiety of the formulation. Finally, the BCNU-NCP was incorporated into the gel to form a Nanogel- Composite. A Box–Behnken design model was employed for the optimization of the Nanogel Composite. TERM, BCNU-NCP and Nanogel Composite were characterized employing Thermogravimetric analysis (TGA), Superconducting Quantum Interference Device (SQUID) magnetometry, Fourier Transform Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR), X-ray Diffractometry (XRD), Scanning Electron Microscopy (SEM), Cyclic Voltammetry (CV), Transmission Electron Microscopy (TEM), Rheological, Porositometry, Textural and Zetasize analyses. In vitro drug release, ex vivo permeation and in vivo studies were performed. The BCNU-NCP was found to be paramagnetic with a magnetization value of 61emu/g, possessing a mixture of spherical and hexagonal shaped core-shell nanoparticles of size 30-50nm with zeta potential of +32 ±2mV. The NCP displayed a high degree of crystallinity with 32% Polyplex coating. The loading capacity of NCP was 176.86µg BCNU/mg of the carrier and maximum release of 75.8% of the loaded BCNU was achieved after 24 hours. FTIR and NMR confirmed the conjugation of PVA and PEI of the Polyplex at a ratio of 1:4. Cytotoxicity of the BCNU-loaded Nano-co-Plex displayed superiority over the conventional BCNU towards human glioblastoma (HG) A170 cells. Cell studies revealed enhanced uptake and internalization of BCNU-NCP in HG A170 cells in the presence of an external MF. BCNU-NCP was found to be non-toxic to healthy brain cells. A thermally stable gel with desirable rheological and mucoadhesive properties was developed. The results revealed gelation temperature of 27.5±0.5°C with a porous morphology. Nanogel Composite possesses electroactive properties and shows response to ES and releases incorporated BCNU-NCP in an “on-off” pulsatile drug release profile upon application of a 5V PD. The in vitro release studies showed an average release of BCNU-NCP per release cycle to be 10.28%. Ex vivo permeation studies were performed using a freshly excised nasal tissue of the New Zealand white rabbit; the results showed that BCNU-NCP was able to permeate through the nasal tissue at a 6 times greater amount in the presence of a MF than in the absence of MF. BCNU concentration was found to be high in the brain and CSF of rabbit when the Nanogel Composite is intranasally administered compared to the IV injection of the conventional BCNU. Furthermore, application of the MF was found to increase the concentration of BCNU in the brain and CSF of the rabbit. The result of Field Emission Electron Probe Micro Analyzer (FE EPMA) was further used to confirm the presence of NCP in the rabbit brain tissue. Histopathological results indicated mild lesions in the nasal mucosa of the rabbit after IN administration of Nanogel Composite. The results of the in vitro, ex vivo and the in vivo proved that the Nanogel Composite is superior in delivering BCNU into the brain than the conventional drug delivery system for the treatment of brain tumor as it was able to release the therapeutic agent in a controllable manner. The MF applied aided drug to be targeted and rapidly transported to the brain via a direct nose-to-brain pathway thereby circumventing the BBB and increasing bioavailability of drug in the brain. This vehicle may also be used to deliver other similar therapeutic agents into the brain for the treatment of various brain diseases. vii ACKNOWLEDGEMENTS I am grateful to God, The Almighty. I thank my wife and children for their love and cooperation. I sincerely acknowledge and appreciate the invaluable role of my supervisor, Professor Viness Pillay whose mentorship and guidance have been most inspiring and motivating. I am highly impressed by his prompt and swift responses to situations which is second to none. I am so ever grateful for giving me the opportunity to be part of WADDP. I will also like to acknowledge my co-supervisors, Professor Yahya Essop Choonara and Professor Girish Modi who were always willing to provide the necessary assistance and direction at all times. I will like to thank Prof. Paul Danckwerts for his assistance whenever needed. My profound gratitude goes to Professor Lisa du Toit and Pradeep Kumar. The time and effort you invested has only made the completion of this thesis so much easier. Your valuable feedback and suggestions helped me improve both my lab experiments and my thesis in many ways. I wish you all success in your future endeavours. My gratitude also goes to Prof. Andre Strydom, Prof. and Dr. Mrs. Amadi Ihunwo and Dr. Ndidi Ngwuluka, for their assistance in various dispensations. To my friends, colleagues and fellow researchers, Kenny Debayo-Sanusi, Dr. Patrick Adebola Dr., Joseph Eguale, Maluta Mufamadi, Olatunbosun Olaleye, Samson Adeyemi, Martina Manyikana, Kealeboga Mokolobate, Jonathan Pantshwa, Prof. Kennedy Erlwanger Dr. Pierre Kondiah, Dr. Thashree Marimuthu, Dr. Ravindra Badhe, Dr. Dharmesh Chejara, Dr. Mostafa Mabrouk, Dr. Mershen Govender, Sunaina Indermun, Greta Mbitsi-ibouily, Fatema Mia, Mukaza Eliphaz, Khuphukile Madida, Karmani Muruga, Margaret Siyawamwaya, Poornima Ramburrun, Bibi Choonara, Mduduzi Sithole, Zikhona Hayiyana, Pride Mothobi, Pariksha Kondiah. I thank you all for your supports. I thank the technical staff of the Department of Pharmacy and Pharmacology, Mr. Sello Ramarumo, Ms. Nompumelelo Damane, Mr. Kleinbooi (Mkhaya) Mohlabi, Mr. Bafana Themba and Phumzile Madodo. I also thank the staff of the CAS at Wits, Sr. Mary-Ann Costello, Patrick Selahle, Sr. Ammelia Rammekwa, Kershneee Chetty, Lorrraine Setimo and Nico Douths. viii DEDICATION This work is dedicated to my late father, Simon Babatunde Akilo. ix ANIMAL ETHICS DECLARATION I, Olufemi David Akilo confirm that the study entitled “In vivo animal studies to assess the performance of an intranasal drug delivery device in a rabbit model” received the approval from the Animal Ethics Committee of the University of the Witwatersrand with ethics clearance number 2015/05/C (see Appendix D) x TABLE OF CONTENTS DECLARATION........................................................................................................................i PUBLICATIONS.......................................................................................................................ii RESEARCH OUTPUTS AT CONFERENCE PROCEEDINGS......................................iii PATENT FILED........................................................................................................................v ABSTRACT.............................................................................................................................vi ACKNOWLEDGEMENTS.......................................................................................................vii DEDICATION.........................................................................................................................viii ANIMAL ETHICS DECLARATION….......................................................................................ix TABLE OF CONTENTS……………...………………………………………………………………x LIST OF FIGURES.............................................................................................................xxviii LIST OF TABLES................................................................................................................xxxv LIST OF EQUATIONS…………………………………………………………………..……...xxxviii LIST OF ABBREVIATIONS……………………………………………………………………..xxxix xi CHAPTER 1 INTRODUCTION 1. CHAPTER 1......................................................................................................... 1 1.1. BACKGROUND TO THIS STUDY ................................................................ 1 1.2. RATIONALE AND MOTIVATION FOR THIS STUDY ................................... 4 1.3. AIM AND OBJECTIVES ................................................................................ 7 1.4. NOVELTY OF THIS WORK .......................................................................... 8 1.5. OVERVIEW OF THIS THESIS ...................................................................... 9 1.6. CONCLUDING REMARKS ......................................................................... 11 1.7. REFERENCES ............................................................................................ 11 xii CHAPTER 2 A REVIEW OF NOSE-TO-BRAIN NEUROTHERAPEUTIC INTERVENTIONS 2. CHAPTER 2....................................................................................................... 14 2.1. INTRODUCTION ......................................................................................... 14 2.2. BIOLOGICAL BARRIERS AS AN IMPEDIMENT TO CNS DRUG DELIVERY.. .......................................................................................................... 15 2.2.1. Blood-Brain Barrier ............................................................................... 15 2.2.2. Blood-Cerebrospinal Fluid Barrier ........................................................ 16 2.2.3. Blood-Tumor Barrier ............................................................................. 16 2.3. NASAL ANATOMY AS AN AID TO CNS DRUG DELIVERY ...................... 17 2.3.1. The Olfactory Pathway as an Opportunity for CNS Drug Delivery ........ 22 2.3.2. Proposed Pathways of Therapeutic Agents Introduced into the Nasal Cavity………… .................................................................................................. 23 2.3.3. Mechanism of Direct Nasal Mucosa to Brain Delivery via the Olfactory Pathway ............................................................................................................. 24 2.4. EVIDENCE OF NOSE-TO-BRAIN DRUG DELIVERY ................................ 26 2.4.1. Delivery of RNA, Enzymes, Insulin and Protein .................................... 26 2.4.2. Delivery of Drug-Loaded Nanoparticles ................................................ 28 2.4.3. Microemulsion Formulations ................................................................. 31 2.4.4. Liposomes as Delivery Vehicles ........................................................... 33 2.4.5. Direct Drug Application into the Nasal Cavity ....................................... 35 2.5. CONVENTIONAL NASAL DRUG DELIVERY DOSAGE FORMS ............... 37 xiii 2.5.1. Nasal Drops .......................................................................................... 37 2.5.2. Nasal Sprays ........................................................................................ 37 2.5.3. Nasal Gels ............................................................................................ 38 2.5.4. Nasal Powders ..................................................................................... 40 2.6. THE USE OF SPECIALIZED INTRANASAL DRUG DELIVERY SYSTEMS……………. .......................................................................................... 40 2.6.1. Liposomes ............................................................................................ 41 2.6.2. Microspheres ........................................................................................ 41 2.6.3. Nanoparticles ........................................................................................ 42 2.6.4. Microemulsions ..................................................................................... 43 2.6.5. Nanoemulsions ..................................................................................... 45 2.6.6. Iontophoretic Delivery ........................................................................... 46 2.7. CHALLENGES AND FUTURE TRENDS OF DIRECT NOSE-TO-BRAIN DELIVERY ............................................................................................................ 46 2.8. CONCLUDING REMARKS ......................................................................... 49 2.9. REFERENCES ............................................................................................ 50 xiv CHAPTER 3 AN IN VITRO EVALUATION OF A CARMUSTINE-LOADED NANO-CO-PLEX FOR POTENTIAL MAGNETIC-TARGETED INTRANASAL DELIVERY TO THE BRAIN 3. CHAPTER 3....................................................................................................... 67 3.1. INTRODUCTION ......................................................................................... 67 3.2. MATERIALS AND METHODS .................................................................... 69 3.2.1. Materials ............................................................................................... 69 3.2.2. Preparation of the Iron Oxide Nanoparticles ......................................... 70 3.2.3. Preparation of the Nano-co-Plex .......................................................... 70 3.2.4. Determination of Structural Integrity of the BCNU-loaded Nano-co- Plex……….. ....................................................................................................... 72 3.2.4.1. Fourier Transform Infrared spectroscopy analysis of the BCNU- loaded Nano-co-Plex ...................................................................................... 72 3.2.4.2. Nuclear Magnetic Resonance analysis of the Nano-co-Plex .......... 72 3.2.5. Thermal Stability and Quantitative Analysis of the Nano-co-Plex ......... 72 3.2.6. Size and Stability Analysis of the BCNU-Loaded Nano-co-Plex ........... 72 3.2.7. Determination of Size and Morphology of BCNU-Loaded Nano-co- Plex…………. .................................................................................................... 73 3.2.8. Determination of Magnetization Properties of the BCNU-Loaded Nano- co-Plex……….. .................................................................................................. 73 3.2.9. Analysis of the Crystalline Structural Properties of BCNU-Loaded Nano- co-Plex……….. .................................................................................................. 73 xv 3.2.10. Establishment and Confirmation of the Interaction Profile Employing Computational Simulations ................................................................................ 73 3.2.11. Determination of the Loading Capacity and Efficiency of BCNU within the Nano-Co-Plex .............................................................................................. 74 3.2.12. Determination of the In Vitro Drug Release Profile of BCNU released from the BCNU-Loaded Nano-co-Plex ............................................................... 75 3.2.13. Cytotoxicity Analysis, Cellular Uptake and Internalization of the BCNU- Loaded Nano-co-Plex ........................................................................................ 75 3.2.13.1. Cytotoxicity studies via 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide assay on the conventional BCNU and BCNU-loaded Nano-co-Plex .................................................................................................. 75 3.2.13.2. Cellular uptake and internalization studies of the BCNU-loaded Nano-co-Plex .................................................................................................. 77 3.3. RESULTS AND DISCUSSION .................................................................... 78 3.3.1. Synthesis of the BCNU-Loaded Nano-co-Plex ..................................... 78 3.3.2. Assessment of the Structural Modifications of the BCNU-Loaded Nano- co-Plex……… .................................................................................................... 80 3.3.2.1. Structural modification assessment via Fourier Transform Infrared spectroscopy .................................................................................................. 80 3.3.2.2. Structural modification assessment via Nuclear Magnetic Resonance analysis ....................................................................................... 82 3.3.3. Morphology and Particle Size of the BCNU-Loaded Nano-co-Plex ...... 83 3.3.4. Size Distribution and Zeta Potential Analysis of the BCNU-Loaded Nano-co-Plex ..................................................................................................... 85 3.3.5. Assessment of the Crystalline Structural Properties of the BCNU- Loaded Nano-co-Plex ........................................................................................ 87 xvi 3.3.6. Assessment of the Thermal Stability and Quantitative Analysis of the Nano-co-Plex ..................................................................................................... 88 3.3.7. Assessment of Attraction Sensitivity of the BCNU-Loaded Nano-co-Plex to an External Magnetic Field ............................................................................ 90 3.3.8. BCNU Loading into the Nano-co-Plex .................................................. 92 3.3.9. Assessment of the In Vitro Drug Release Profile of BCNU from the Nano-co-Plex ..................................................................................................... 93 3.3.10. Establishment and Confirmation of the Interaction Profile Employing Computational Simulations ................................................................................ 95 3.3.11. Assessment of the Cytotoxicity and Cellular Uptake of the BCNU- Loaded Nano-co-Plex ........................................................................................ 96 3.3.11.1. Cytotoxicity studies of the BCNU-loaded Nano-co-Plex ................ 96 3.3.11.2. Cellular uptake and internalization of the BCNU-loaded Nano-co- Plex………………… ....................................................................................... 98 3.4. CONCLUDING REMARKS ......................................................................... 99 3.5. REFERENCES .......................................................................................... 100 xvii CHAPTER 4 PREPARATION, PHYSICOCHEMICAL AND PHYSICOMECHANICAL CHARACTERIZATION OF AN OPTIMIZED NANOGEL COMPOSITE 4. CHAPTER 4..................................................................................................... 106 4.1. INTRODUCTION ....................................................................................... 106 4.2. MATERIALS AND METHODS .................................................................. 108 4.2.1. Materials ............................................................................................. 108 4.2.2. Preparation of the Thermosensitive Electro-Responsive Mucogel ..... 108 4.2.3. Preparation of Carmustine-Loaded Nano-co-Plex .............................. 108 4.2.4. Incorporation of the Carmustine-Loaded Nano-co-Plex into the Thermosensitive Electro-Responsive Mucogel ................................................ 109 4.2.5. Optimization Studies Employing a Box-Behnken Experimental Design for the Nanogel Composite .............................................................................. 109 4.2.6. Determination of Structural Modification and Chemical Interaction of the Nanogel Composite ......................................................................................... 110 4.2.7. Determination of the Mucoadhesive Behavior of the Nanogel Composite………… ......................................................................................... 111 4.2.8. Determination of Gelation Temperature of the Nanogel Composite ... 111 4.2.9. Determination of the Electrical Conductivity of the 15 Nanogel Composite Formulations .................................................................................. 112 4.2.10. In vitro Determination of the Release Profiles of the 15 Nanogel Composite Formulations upon Application of an Electrical Stimulation ........... 112 xviii 4.2.10.1. Quantitative analysis of the BCNU-loaded Nano-co-Plex released from the Nanogel Composite ...................................................................... 113 4.2.11. Ex Vivo Evaluation of Mucoadhesive Strength of the Nanogel Composite ....................................................................................................... 114 4.2.12. Evaluation of the Thermal Behavior of the Optimized Nanogel Composite ....................................................................................................... 115 4.2.13. Determination of Surface Morphology and Porosity of the Optimized Nanogel Composite ......................................................................................... 115 4.2.13.1. Scanning Electron Microscopy .................................................... 115 4.2.13.2. Determination of the porosity profile of the Nanogel Composite . 116 4.2.14. Rheological Analysis of the Optimized Nanogel Composite ............ 117 4.2.15. Evaluation of the Electro-activity of the Optimized Nanogel Composite……… ............................................................................................. 118 4.2.16. In Vitro Drug Release Analysis from the Optimized Nanogel Composite on the Application of an Electrical Stimulation ............................... 119 4.2.16.1. Quantitative determination of the BCNU-loaded Nano-co-Plex released from the optimized Nanogel Composite ......................................... 119 4.3. RESULTS AND DISCUSSION .................................................................. 119 4.3.1. Preparation of the Thermosensitive Electro-Responsive Mucogel and the Nanogel Composite ................................................................................... 119 4.3.2. Evaluation of Box–Behnken experimental design optimization ........... 120 4.3.3. Assessment of the Mucoadhesive Behavior of the Nanogel Composite………….. ....................................................................................... 124 4.3.4. Assessment of the Gelation Temperature .......................................... 126 xix 4.3.5. Electro-Responsiveness of the Nanogel Composite via Electric Conductivity Assessment ................................................................................. 127 4.3.6. In Vitro Assessment of Release Behavior of the 15 Nanogel Composite Formulations on Application of Electrical Stimulation ...................................... 128 4.3.7. Ex vivo Mucoadhesion Analysis ......................................................... 131 4.3.8. Assessment of the Structural Modification of the Optimized Nanogel Composite ....................................................................................................... 133 4.3.8.1. Assessment of the structural modification employing Fourier Transmission Infrared spectroscopy ............................................................. 133 4.3.8.2. Assessment of structural integrity by XRD spectroscopy ............. 136 4.3.9. Evaluation of the Thermal Stability of the Optimized Nanogel Composite…. ................................................................................................... 137 4.3.10. Assessment of the Surface Morphology and Porosity of the Nanogel Composite ....................................................................................................... 139 4.3.11. Assessment of Gelation Temperature of the Optimized Nanogel Composite ....................................................................................................... 145 4.3.12. Assessment of Rheological Properties of the Nanogel Composite.. 145 4.3.12.1. Shear viscosity ............................................................................ 145 4.3.12.2. Yield stress .................................................................................. 146 4.3.12.3. Oscillation stress sweep .............................................................. 147 4.3.12.4. Frequency sweep ........................................................................ 148 4.3.12.5. Creep recovery test ..................................................................... 150 4.3.12.6. Temperature ramp analysis ......................................................... 151 xx 4.3.13. Assessment of the Electro-active Properties of the Nanogel Composite Employing Cyclic Voltammetric Analysis ....................................... 153 4.3.14. Assessment of the In Vitro Drug Release Analysis of the Optimized Nanogel Composite on Application of Electrical Stimulation ............................ 154 4.4. CONCLUDING REMARKS ....................................................................... 157 4.5. REFERENCES .......................................................................................... 157 xxi CHAPTER 5 EX VIVO EVALUATION OF AN IN SITU NANOGEL COMPOSITE 5. CHAPTER 5..................................................................................................... 161 5.1. INTRODUCTION ....................................................................................... 161 5.2. MATERIALS AND METHODS .................................................................. 162 5.2.1. Materials ............................................................................................. 162 5.2.2. Preparation of the Nanogel Composite ............................................... 162 5.2.3. Ex Vivo Evaluation of the Nanogel Composite ................................... 162 5.2.3.1. Preparation of the rabbit nasal tissue for permeation studies ...... 162 5.2.4. Ex Vivo Evaluation of Mucoadhesive Strength of the Nanogel Composite.. ..................................................................................................... 163 5.2.5. Determination of the Permeation Capability of the BCNU-Loaded Nano- co-Plex across Rabbit Nasal Tissue ................................................................ 163 5.2.5.1. Quantitative analysis of the BCNU-loaded Nano-co-Plex permeated through the rabbit nasal epithelial tissue from the Nanogel Composite ........ 165 5.2.5.2. Evaluation of BCNU-loaded Nano-co-Plex permeability .............. 165 5.2.5.3. Evaluation of the effect of magnetic field on cytotoxicity of the BCNU-loaded Nano-co-Plex on human glioblastoma A170 cells ................. 166 5.3. RESULTS AND DISCUSSION .................................................................. 167 5.3.1. Ex Vivo Evaluation of Mucoadhesive Strength of the Optimized Nanogel Composite ....................................................................................................... 167 5.3.2. Calibration Curve of Iron ..................................................................... 168 xxii 5.3.3. Assessment of the Permeation of the BCNU-Loaded Nano-co-Plex across the Rabbit Nasal Tissue ....................................................................... 169 5.3.4. Assessment of the Magnetic Field Influence on the Cytotoxicity of BCNU-Loaded Nano-co-Plex on Human Glioblastoma A170 Cells ................. 172 5.4. CONCLUDING REMARKS ....................................................................... 173 5.5. REFERENCES .......................................................................................... 174 xxiii CHAPTER 6 IN VIVO EVALUATION OF THE OPTIMIZED NANOGEL COMPOSITE 6. CHAPTER 6..................................................................................................... 176 6.1. INTRODUCTION ....................................................................................... 176 6.2. MATERIALS AND METHODS .................................................................. 177 6.2.1. Materials ............................................................................................. 177 6.2.2. Animal Ethics Clearance..................................................................... 178 6.2.3. Preparation and Sterilization of Nanogel Composite .......................... 178 6.2.4. In Vivo Experimental Studies .............................................................. 178 6.2.4.1. Pilot studies for determining the workability and suitability of the application of electric stimulation and magnetic field. ................................... 178 6.2.4.2. In vivo experimental design and procedure for the studies .......... 181 6.2.5. Quantitative Chromatographic Determination of BCNU in Plasma, CSF and the Brain Tissue of Rabbit ........................................................................ 187 6.2.5.1. Development of a method for sample analysis employing Ultra Performance Liquid Chromatography ........................................................... 188 6.2.5.2. Extraction of BCNU from rabbit plasma and CSF for UPLC analysis……… .............................................................................................. 188 6.2.5.3. Validation of liquid-liquid extraction procedure ............................. 189 6.2.5.4. Preparation of calibration curve ................................................... 190 6.2.5.5. Determination of BCNU-NCP in the brain tissue employing Field Emission Electron Probe Micro Analyzer (FE EPMA) .................................. 191 xxiv 6.2.5.6. Histopathological Evaluation of the Nasal Mucosa of the Rabbits 191 6.3. RESULTS AND DISCUSSION .................................................................. 191 6.3.1. Pilot Study to Assess the Effect of Electric Stimulation, Magnetic Headband and Fluid Sampling on the Rabbits ................................................ 191 6.3.2. Drug Extraction, UPLC Method Validation and Assay Procedure ....... 193 6.3.3. Concentration of BCNU in the blood, CSF and brain tissue of the rabbit model………….. ............................................................................................... 196 6.3.4. Assessment of BCNU-NCP in the Brain Tissue Employing Field Emission Electron Probe Micro Analyzer (FE EPMA) ...................................... 205 6.3.5. Histopathological Evaluation of the Nasal Epithelia of the Rabbit ....... 206 6.4. CONCLUDING REMARKS ....................................................................... 208 xxv CHAPTER 7 RECOMMENDATIONS AND FUTURE OUTLOOK 7. CHAPTER 7..................................................................................................... 211 7.1. RECOMMENDATIONS ............................................................................. 211 7.2. FUTURE OUTLOOK ................................................................................. 213 7.2.1. Hypothesis: Lactoferrin-Galantamine Proteo-Alkaloid Conjugate for The Management of Alzheimer‟s Disease .............................................................. 214 7.2.1.1. Introduction .................................................................................. 214 7.2.1.2. The hypothesis ............................................................................. 216 7.2.1.3. Evaluation of the hypothesis ........................................................ 216 7.2.1.4. Galantamine and Alzheimer‟s intervention ................................... 217 7.2.1.5. Lactoferrin and metal chelation .................................................... 218 7.2.1.6. Lactoferrin-Galantamine Proteo-Alkaloid conjugate ..................... 219 7.2.2. Concluding Remarks .......................................................................... 222 7.3. REFERENCES .......................................................................................... 222 xxvi APPENDICES 8. APPENDICES.................................................................................................. 228 8.1. APPENDIX A ............................................................................................. 228 8.1.1. Research Publications ........................................................................ 228 8.1.1.1. Book chapter ................................................................................ 228 8.1.1.2. Research paper ........................................................................... 229 8.2. APPENDIX B ............................................................................................. 230 8.2.1. Abstracts of Research Outputs at Conference Proceedings............... 230 8.2.1.1. School of Therapeutic Sciences Research Day, University of the Witwatersrand, Johannesburg, South Africa, September 10, 2013, Poster presentation). ............................................................................................... 230 8.2.1.2. 6th Cross-Faculty Graduate Symposium, University of the Witwatersrand, Johannesburg, South Africa, September 17, 2014, (Poster presentation). ............................................................................................... 231 8.2.1.3. International Society of Biomedical Polymers and Polymeric Biomaterials, Orlando, Florida USA, 8th-10th July, 2015, (Poster presentation). 232 8.2.1.4. International Society of Biomedical Polymers and Polymeric Biomaterials, Orlando, Florida USA, 8th-10th July, 2015, (Podium presentatation). ............................................................................................ 233 8.2.1.5. Faculty of Health Sciences Research Day, University of the Witwatersrand, Johannesburg, South Africa, September 8-9, 2015. (Podium Presentation). ............................................................................................... 234 xxvii 8.2.1.6. Academy of Pharmaceutical Sciences of South Africa, Johannesburg, South Africa, September 17-19, 2015, (Podium Presentation). 235 8.2.1.7. 7th Cross-Faculty Graduate Symposium, University of the Witwatersrand, Johannesburg, South Africa, March 2-3, 2016, (Podium presentation). ............................................................................................... 236 xxviii LIST OF FIGURES Figure 1.1: Schematic conceptualization of design criteria for release of drugs using an external electric stimulation and a magnetic field .................................................. 6 Figure 2.1: Lateral wall of the nasal cavity, and cross-sections through the internal ostium, the middle of the nasal cavity, and the choanae. Hatched area in the upper figure, olfactory region. NV, nasal vestibule, IT, inferior turbinate and orifice of the nasolacrimal duct; MT, middle turbinate and orifices of frontal sinus, anterior ethmoidal sinuses and maxillary sinus; ST, superior turbinate and orifices of posterior ethmoidal sinuses. Source (Mygind and Dahl, 1998). ............................... 19 Figure 2.2: Illustrations of the various types of cells in the olfactory region. Reproduced from (Mistry et al., 2009). ..................................................................... 23 Figure 2.3: Possible transport pathways of a compound introduced into the nasal cavity. Adapted from (Graff and Pollack, 2005; Illum, 2004, 2003, 2000). ............... 24 Figure 2.4: Showing fluorescently labeled siRNA distribution in the olfactory bulb of rat. Scale= 30µm: ONB=olfactory nerve bundle, OM=olfactory mucosa, CP=cribiform plate, ONL=olfactory nerve layer, GL=glomerular layer. Adapted from (Renner et al., 2012b). ..................................................................................................................... 28 Figure 2.5: Distribution and retention of Cy5.5-Lf in the brain following intranasal administration. Adapted from (Liu et al., 2013). ........................................................ 29 Figure 2.6: SEM image showing surface morphology of optimized bromocriptine loaded Chitosan nanoparticles. Adapted from (Shadab et al., 2012). ...................... 30 Figure 2.7: Dynamics of MPEG–PCL–Tat complex in brain tissue following intranasal and intravenous administration (Kanazawa et al., 2013). ......................................... 31 Figure 2.8: Environmental scanning electron microscopy (ESEM) micrographs of unloaded (A) and UDCA–AZT loaded (B) SLMs based on tristearin (Dalpiaz et al., 2014). ....................................................................................................................... 33 xxix Figure 2.9: TEM image of GH loaded flexible liposomes adapted from (W. Li et al., 2012) ........................................................................................................................ 35 Figure 3.1: Schematic of the synthesis of BCNU-loaded Nano-co-Plex. .................. 80 Figure 3.2: FTIR spectra of a) synthesized magnetite, b) native PVA, c) native PEI, d) native FITC, e) BCNU, f) NCP-3 and g) BCNU-NCP-3. ....................................... 82 Figure 3.3: 1H NMR spectra of (a) PVA, (b) PEI and (c) Polyplex. The red circle and arrow indicating the new NH-CH2 bond formed by the conjugation of PEI and PVA at peak 3.14 ppm. ........................................................................................................ 83 Figure 3.4: TEM image of a) Uncoated magnetite, b), c) and d) BCNU-loaded Nano- co-Plex (BCNU-NCP-1, BCNU- NCP-2 and BCNU- NCP-3 respectively). The blue box in a) indicates uncoated particle while the red boxes in b), c) and d) indicate coated particles. ....................................................................................................... 85 Figure 3.5: Zeta potential of a) synthesized magnetite b) Nano-co-Plex (NCP-3) and c) BCNU-loaded Nano-co-Plex (BCNU-NCP-3). ...................................................... 87 Figure 3.6: XRD spectra of uncoated Magnetite and Nano-co-Plex (NCP-1, NCP-2 and NCP-3). ............................................................................................................. 88 Figure 3.7: TGA thermograms of a) uncoated magnetite and Nano-co-Plex b) NCP- 1, c) NCP-2 and d) NCP-3. ....................................................................................... 89 Figure 3.8: Magnetization curve of the synthesized magnetite and that of the Nano- co-Plex (NCP-1, NCP-2 and NCP-3) measured at 25°C showing the hysteresis loop. ................................................................................................................................. 91 Figure 3.9: BCNU loading profile of Nano-co-Plex (NCP-1, NCP-2 and NCP-3). ..... 93 Figure 3.10: In vitro release profile of BCNU-loaded Nano-co-Plex (NCP-1, NCP-2 and NCP-3). ............................................................................................................. 94 Figure 3.11: Computational simulations of interaction profile of a) PVA and PEI, b) FITC and PEI and c) BCNU and PEI. The color codes for various elements are as xxx follows: Carbon=cyan, Nitrogen=blue, Oxygen=red, Sulphur=yellow and Hydrogen=white. ...................................................................................................... 96 Figure 3.12: Cell viability of human glioblastoma A172 cell line treated with BCNU- NCP-1, BCNU-NCP-2, BCNU-NCP-3 and BCNU incubated at 37°C for 72 hours. .. 98 Figure 3.13: Fluorescent microscopy analysis of human glioblastoma A172 cell line treated with BCNU-NCP-1, BCNU-NCP-2 and BCNU-NCP-3 incubated at 37°C for 2 hours without and with application of magnet. .......................................................... 99 Figure 4.1: A schematic diagram depicting the electro-stimulation in vitro release experimental setup. ................................................................................................ 113 Figure 4.2: Residual plots of (a) average release per cycle, (b) conductivity and (c) mucoadhesion. ....................................................................................................... 122 Figure 4.3: Response surface plots showing the effects of F127, CS and PANI on (a) electro-responsive drug release, (b) conductivity and (c) mucoadhesion. .............. 123 Figure 4.4: Response Optimization output plot by desirability function with the values of independent variables and predicted values of the responses of Nanogel Composite. ............................................................................................................. 124 Figure 4.5: Percentage of mucin adsorbed by the 15 formulations. ....................... 125 Figure 4.6: Electrical conductivity of the 15 formulations of the Nanogel Composite ............................................................................................................................... 128 Figure 4.7: BCNU-NCP release profiles in simulated nasal fluid (pH 6.0) for Formulations 1-15. ................................................................................................. 130 Figure 4.8: Tensile detachment measurement displaying Maximum force of adhesion of the Nanogel Composite formulations from rabbit nasal mucosal tissue wetted with simulated nasal buffer at pH 6.0 (SD≤ 0.015; N=5). ............................................... 132 Figure 4.9: The total work of adhesion for removing Nanogel Composite from rabbit nasal mucosal tissue wetted with simulated nasal buffer at pH 6.0 (SD≤ 0.00997; N=5). ...................................................................................................................... 132 xxxi Figure 4.10: FTIR spectra of a) HPMC, b) PANI, c) CS and d) F127. .................... 133 Figure 4.11: FTIR spectra of a) F127, b) TERM and c) Nanogel Composite .......... 136 Figure 4.12: XRD spectra of the Nanogel Composite (black), F127 (red), CS (green), PANI (blue) and HPMC (purple). ............................................................................ 137 Figure 4.13: TGA thermograms of a) Nanogel Composite, b) F127, c) CS, d) HPMC and f) PANI. ............................................................................................................ 138 Figure 4.14: DSC thermogram of Nanogel Composite (black), F127 (red), CS (green), PANI (blue) and HPMC (purple). .............................................................. 139 Figure 4.15: SEM images of native a) F127, b) CS, c) HPMC and d) PANI. Magnification=740x. ............................................................................................... 141 Figure 4.16: SEM images of a) TERM b) cross section view of TERM, c) Nanogel Composite before application of electrical stimulation and d) Nanogel Composite after application of electrical stimulation and 5 released cycle. Magnification=740x. ............................................................................................................................... 142 Figure 4.17: Isotherm linear plots of a) TERM, b) Nanogel Composite before application of electrical stimulation and c) Nanogel Composite after application of electrical stimulation. (N2 adsorption in red and desorption in burgundy color). ..... 143 Figure 4.18: Test tube inverted method for the determination of gelation temperature, a) before gelation at room temperature and b) immediately after gelation at 29±1°C. ............................................................................................................................... 145 Figure 4.19: Rheogram of viscosity as a function of shear rate of Nanogel Composite and F127. ............................................................................................................... 146 Figure 4.20: Rheogram of Yield stress of Nanogel Composite and F127............... 147 Figure 4.21: Rheogram of oscillation stress sweep of Nanogel Composite and F127. ............................................................................................................................... 148 Figure 4.22: Frequency sweep rheogram of Nanogel Composite and F127. ......... 149 xxxii Figure 4.23: Rheogram of creep recovery test for the Nanogel Composite and F127. ............................................................................................................................... 150 Figure 4.24: Rheogram of temperature ramp of Nanogel Composite and F127. .... 151 Figure 4.25: Rheogram showing G‟G” plot against temperature for a) F127 and b) Nanogel Composite ................................................................................................ 152 Figure 4.26: Voltammogram of a) PANI and b) Nanogel Composite ...................... 154 Figure 4.27: The three forms of PANI showing their transition and reversible states ............................................................................................................................... 154 Figure 4.28: BCNU-NCP release profile in simulated nasal fluid (pH 6.0). ............. 156 Figure 4.29: In vitro BCNU release profile from the BCNU-NCP for a period of 6 hours ...................................................................................................................... 156 Figure 5.1: Schematic diagram of side-by-side diffusion cell. ................................. 165 Figure 5.2: Calibration curve of iron ....................................................................... 169 Figure 5.3: Permeation curve showing the cumulative amount of BCNU-NCP Permeated across the nasal epithelial tissue of rabbit at 34±1°C in a diffusion cell. Data are expressed as mean ± SD (N=3). ............................................................. 170 Figure 5.4: effect on toxicity of human glioblastoma A172 cell line showing the control treatment, treated with NCP, BCNU, BCNU-NCP and BCNU-NCP (with magnetic field) incubated at 37°C for 72 hours. ..................................................... 173 Figure 6.1: Digital images of a) application of electric stimulus on the nose and b) Magnetic Headband fitted on the head of the rabbit. .............................................. 180 Figure 6.2: A digital image depicting the conditions under which the rabbits were housed in a vivarium according to ARVO Resolution guidelines. ........................... 181 Figure 6.3: Schematic diagrams showing the outline summary of the in vivo animal studies. ................................................................................................................... 183 xxxiii Figure 6.4: Digital images of the procedure for withdrawing CSF from the rabbit showing a) shaving of the back of the neck of rabbit, b) shaved area, c) insertion of needle directed towards the nose of the rabbit and d) the withdrawal of CSF with the aid of 1mL syringe). ................................................................................................ 184 Figure 6.5: Photographic evidence of (a) Housing of the rabbits (b) preparation for surgery with anesthetization (c) intravenous dosing (d) intranasal dosing e) application of ES (f) blood withdrawal (g) CSF withdrawal (h) administration of oxygen after dosing and sampling (i) rabbit recovering from sedation. .................. 187 Figure 6.6: Typical chromatogram displaying a) the spiking plasma separation of the Gal, b) BCNU and c) the separation of both Gal and BCNU after plasma extraction. ............................................................................................................................... 193 Figure 6.7: Typical chromatogram depicting a) the spiking CSF separation of the Gal, b) BCNU and c) the separation of both Gal and BCNU after CSF extraction. ........ 195 Figure 6.8: Calibration curve of BCNU at 230nm. ................................................. 196 Figure 6.9: Mean BCNU concentration-time profile in plasma after IV injection of the conventional BCNU, IN administration of Nanogel Composite without application of ES/MFand IN administration of Nanogel Composite with the application of ES/MF (SD≤ 0.053; N=5 at each time point). ..................................................................... 197 Figure 6.10: Mean BCNU concentration–time profile in CSF after IV injection of conventional BCNU, IN administration of Nanogel Composite without application of ES and IN administration of Nanogel Composite with the application of ES and MF at 1mg/kg dose in rabbits. (SD≤ 0.051; N=5 at each time point). ............................... 199 Figure 6.11: Percentage of BCNU released in CSF following IN administration. (SD≤ 0.051; N=5 at each time point). .............................................................................. 200 Figure 6.12: Mean BCNU concentration–time profile in Brain after IV injection of the conventional BCNU, IN administration of Nanogel Composite without application of ES and IN administration Nanogel Composite with the application of ES and MF at 1mg/kg dose in rabbits. (SD≤ 0.05; N=5 at each time point). ................................. 201 xxxiv Figure 6.13: BCNU concentration in the Plasma, CSF and Brain tissue after IV injection of conventional BCNU. ............................................................................. 203 Figure 6.14: BCNU concentration in the Plasma, CSF and Brain tissue after IN administration of Nanogel Composite with application of ES and MF. ................... 204 Figure 6.15: BCNU concentration in the plasma, CSF and brain tissue after IN administration of Nanogel Composite without application of ES and MF. .............. 204 Figure 6.16: Comparison of the concentration of BCNU in CSF and brain following IV of conventional BCNU and IN administration of Nanogel Composite with application of ES and MF. ........................................................................................................ 205 Figure 6.17: FE EPMA spectra of brain tissue of rabbit after IN administration of Nanogel Composite for Fe detection. ..................................................................... 206 Figure 6.18: Histological evaluation of nasal epithelia tissue samples from rabbits showing light digital microscopic images of the H&E stained slides of a) normal nasal epithelia tissue, b) placebo sample, c) control group sample and d) experimental group sample. Magnification is x10. ....................................................................... 208 Figure 7.1: Molecular structure of Galantamine ..................................................... 213 Figure 7.2: Schematic diagram of the mechanism of release of Galantamine and absorption of iron by Lf-Gal conjugate. ................................................................... 221 Figure 7.3: Schematic diagram demonstrating the potential neuroprotective- neurotherapeutic interventional mechanism of the lactoferrin-galantamine conjugate. ............................................................................................................................... 221 xxxv LIST OF TABLES Table 2.1: Advantages and Limitation of Intranasal Drug Administration. ................ 21 Table 2.2: Review of recent development in nasal sprays for delivery of therapeutic agents. ..................................................................................................................... 38 Table 2.3: Review of recent development in nasal gel for delivery of therapeutic agents ...................................................................................................................... 39 Table 2.4: Review of recent development in nasal powder for delivery of therapeutic agents. ..................................................................................................................... 40 Table 2.5: Review of recent development in nasal liposomes for delivery of therapeutic agents. ................................................................................................... 41 Table 2.6: Review of recent development in microspheres for nasal delivery of therapeutic agents. ................................................................................................... 42 Table 2.7: Summary of recent development in nanoparticles for intranasal delivery of therapeutic agents. ................................................................................................... 44 Table 2.8: Summary of recent development in microemulsion for intranasal delivery of therapeutic agents. ............................................................................................... 45 Table 2.9: Review of recent development in nanoemulsion for nasal delivery of therapeutic agents. ................................................................................................... 46 Table 3.1: Formulation percentage ratio of conjugated polymers used .................... 71 Table 3.2: DPBS, NCP, BCNU-NCP and BCNU contents distribution in each plate 76 Table 3.3: Percentage weight loss of the synthesized magnetite and the various formulations. ............................................................................................................. 89 Table 4.1: Lower and upper limits of the variables employed for the Box- Behnken design. .................................................................................................................... 109 xxxvi Table 4.2: Series of formulations statistically generated by the Box–Behnken design. ............................................................................................................................... 110 Table 4.3: X-ray diffraction parameters .................................................................. 111 Table 4.4: The parameter settings for the mucoadhesion analysis of the Nanogel Composite. ............................................................................................................. 115 Table 4.5: Porositometry parameters employed in the measurement of TERM/Nanogel Composite ..................................................................................... 116 Table 4.6: The parameter settings for the rheological analysis of Nanogel Composite and F127. ............................................................................................................... 118 Table 4.7: Formulations and their corresponding responses .................................. 121 Table 4.8: Gelation temperature of the 15 formulations ......................................... 126 Table 4.9: FTIR spectra of HPMC, PANI, CS and F127 showing their major characteristic native bands. .................................................................................... 134 Table 4.10: Results of the porosity analysis of the TERM and the Nanogel Composite .............................................................................................................. 144 Table 5.1: Assessment procedures used in the permeation studies ...................... 165 Table 5.2: Distribution of DPBS, NCP, BCNU-NCP and BCNU contents in individual plate. ...................................................................................................................... 167 Table 5.3: Flux, Permeability coefficient and Enhancement ratio for permeation of BCNU-NCP through the nasal tissue of New Zealand white rabbit. ....................... 172 Table 6.1: Details of drugs, anesthetics, analgesics and other medicinal or experimental substances used, including the doses and routes of administration . 178 Table 6.2: Weight of the three rabbits on day of arrival and a day prior to procedure. ............................................................................................................................... 179 xxxvii Table 6.3: UPLC parameters employed for the determination of BCNU concentration ............................................................................................................................... 188 xxxviii LIST OF EQUATIONS Equation 3.1 ............................................................................................................. 77 Equation 4.1 ........................................................................................................... 116 Equation 4.2 ........................................................................................................... 117 Equation 4.3 ........................................................................................................... 117 Equation 5.1 ........................................................................................................... 166 Equation 5.2 ........................................................................................................... 166 Equation 5.3 ........................................................................................................... 166 Equation 6.1 ........................................................................................................... 190 xxxix LIST OF ABBREVIATIONS AESC BBB BET BHN BCNU BCNU-NCP CAS CSF CV ES F127 FE EPMA FTIR FITC Gal HG HPLC HPMC IN IS MF NCP NMR Animal Ethics Screening Committee Blood-Brain Barrier Brunauer-Emmett-Teller Brinell Hardness Number Carmustine Carmustine-loaded Nano-co-Plex Central Animal Service Cerebrospinal Fluid Cyclic Voltammetry Electric Stimulation Pluronic F127 Field Emission Electron Probe Micro Analyzer Fourier Transform Infrared Spectroscopy FIuorecein isothiocyanate Galantamine Human Glioblastoma High Performance Liquid Chromatographic hydroxypropyl methylcellulose Intranasal Internal Standard Magnetic Field Nano-co-Plex Nuclear Magnetic Resonance xl PANI PD PEI PVA SEM SQUID TEM TERM TGA UPLC UV WITS XRD Polyaniline Potential Difference Polyethyleneimine Polyvinyl alcohol Scanning Electron Microscopy Superconducting Quantum Interference Device Transmission Electron Microscopy Thermosensitive Electro-Responsive Mucogel Thermogravimetric analysis Ultra Performance Liquid Chromatographic Ultraviolet-visible Witwatersrand University X-ray Diffractometry 1 CHAPTER 1 INTRODUCTION 1.1. BACKGROUND TO THIS STUDY Delivery of drugs to the brain has been fraught with issues of low bioavailability. This is due to the impervious nature of the Blood-Brain-Barrier (BBB). The BBB consists of an endothelial membrane separating the systemic circulation and central interstitial fluid (Pardridge, 1999). The vasculature serving the Central Nervous System (CNS) has capillaries with tight junctions preventing permeation of drugs. The BBB also comprises a double layered structure called the arachnoid membrane, which acts as a barrier between the blood and Cerebrospinal Fluid (CSF) (Vyas et al., 2005a). These characteristics of the BBB restrict the movement of molecules from the general circulation to the brain and spinal cord (Lerner et al., 2004). The BBB usually restricts the passage of hydrophilic substances from the blood to the brain but allows certain lipophilic substances to pass through to the brain (Sharma et al., 2015). The development of therapies to treat brain diseases has long been frustrated by the failure of systemically administered drugs to permeate the brain. The delivery mechanism through the BBB and the physicochemical properties of the molecule of the drug are factors that must be considered when designing drug delivery systems for brain targeting. The systemic delivery of drugs through the oral, intravenous and the transdermal routes requires drug to pass through the blood circulation in order to reach the brain, and it is fraught with the challenge of passing through the BBB. The delivery of drugs using intranasal delivery has been explored by a number of investigators as it is purportedly a more reliable method for drug delivery to the brain than the systemic administration (Serralheiro et al., 2015; Mistry et al., 2015; Nasr, 2015; Serralheiro et al., 2014). This is due to its ability to bypass the BBB and directly propel the drugs through the olfactory lobe to the brain which enhances their bioavailability. Many therapeutic agents such as peptides and proteins which are poorly absorbed orally and extensively metabolized in the gastrointestinal tract 2 (GIT) and the liver have been found to be ideally delivered through the nasal route. Advantages of intranasal delivery over other routes of delivery include; 1) non- invasiveness, 2) affords direct delivery of drug to brain by circumventing theBBB, 3) targeted delivery to the brain is possible, 4) rapid delivery, low toxicity and side effects, 5) by-passes first-pass metabolism, and 6) provides a larger surface area for drug delivery. Comparative studies of the nasal route and other routes of administration such as intravenous, oral and transdermal have been explored (Lalatsa et al., 2014; Kumar et al., 2008a; Westin et al., 2005). It was observed that the nasal route was more efficient in delivering a wide range of therapeutic compounds including peptides and proteins in the presence of permeation enhancers resulting in higher bioavailability (Costantino et al., 2007; Türker et al., 2004). The drug uptake into the brain from the nasal mucosa mainly occurs via two different pathways. One is the systemic pathway by which some of the drugs administered into the nasal cavity are absorbed into the systemic circulation via the blood-rich vessels and capillaries of the nasal mucosa. The other is the olfactory pathway by which the drug travels from the nasal cavity directly to CSF and brain tissue (Mistry et al., 2009; Seju et al., 2011; Shah et al., 2015; Vyas et al., 2005b). Almost all research in intranasal drug delivery have employed the mechanism of passive diffusion that relies mainly on a combination of proper drug instillation deep in the nasal cavity, gravity, and the properties of the neuro-epithelium to gain access to the CNS. However, a substantial quantity of drugs goes through the systemic pathway thereby reducing the quantity via the olfactory pathway (Kumar et al., 2008a). Such phenomenon is a limitation in the intranasal delivery of drugs directly to the brain as this reduces the bioavailability of the drugs delivered to the brain. Furthermore, there has been no controlled way in which the therapeutics get delivered to the brain when they are introduced into the nasal cavity. Furthermore, drugs meant for direct nose-to-brain delivery should have a protective embodiment that can entrap/encapsulate the therapeutic substances whilst in the nasal cavity. There is therefore a need for the drugs to be protected whilst in the nasal cavity and be delivered into the brain in a controlled manner in conjuction with a „driving 3 force‟ that can propel the drugs directly to the brain after being released in the nasal mucosa. The use of electroactive polymers as a protective embodiment as well as in release of therapeutics when external electric stimulus is applied may be very useful. The use of magnetic nanoparticles in fabricating the drug delivery systems, which on application of magnetic field may act as a „driving force‟, has the potential of guiding and propelling the drugs to the brain via the olfactory pathway and therefore bypassing the systemic pathway. This will improve the bioavailability of drugs in the CNS. It is envisaged that the application of an external electric and magnetic fields may cause controlled release and uptake of drugs through the olfactory pathway thereby circumventing the BBB thus enhancing the delivery and subsequent distribution of drug within the brain. Learner and coworkers (2004) investigated a method to increase intranasal delivery and drug distribution within the brain using iontophoresis, to actively drive drug movement across an electric field to the brain. In their method, electrodes containing a drug reservoir were applied to the nasal neuro-epithelium of the rabbit with a return electrode placed at the back of the rabbit‟s head. Upon the application of a relatively small sustained current, charged drug molecules were driven from the nasal cavity into the brain. This method actually allows the passage of more drugs to the brain compared to other methods. However, the method is cumbersome and invasive since electrodes have to be inserted deep into the nasal cavity and patience non-compliance is envisaged. Therefore, a drug delivery device administered via the intranasal route which applies electric and magnetic fields, and is not cumbersome and invasive, for targeted brain delivery is paramount for the management of CNS diseases in order to enhance the bioavailability of drug in the brain. Hence this study was undertaken to explore the use of electro-active polymers to formulate an electro-responsive gel and containing drug-loaded magnetic nanoparticles which in the presence of an external electric stimulus, control the release of drug and the presence of external magnetic field enhances rapid transportation of the drug-loaded nanoparticles to the brain and CNS via the olfactory pathway. 4 1.2. RATIONALE AND MOTIVATION FOR THIS STUDY This study proposed to develop an electro-magneto-responsive polymeric drug delivery system by exploring the use electro-active polymers which are polymers that respond to external electric stimulations. These polymers include polyaniline (PANI), polypyrrole (PPy), ethylene vinyl acetate (EVA), polyethylene, polylactic acid (PLA), polyglycolic acid (PGA), polylactic-co-glycolic acid (PLGA), and polythiophene which could be employed in preparing a drug delivery system for direct delivery of therapeutics to the brain through the nasal mucosa. A number of these polymers were explored in designing the intranasal drug delivery system described herein. The applications of these polymers are mostly in the production of artificial muscles. On application of an electric field these polymers are able to swell and/or contract. Such physicochemical changes are converted into mechanical work in a muscle actuator. This concept was employed in designing a drug delivery system that can deliver therapeutic drugs directly to the brain through the nasal cavity. Magnetic nanoparticles were fabricated by employing substances and compounds which possess the capacity to respond to a magnetic field. Such substances include iron and gadolinium compounds. These compounds were employed to develop the magnetic nanoparticles using techniques such as co-precipitation in an alkaline medium. The magnetic nanoparticles were coated with biodegradable and biocompatible polymers such as polyvinyl alcohol (PVA), poly ethylene glycol (PEG), polyethyleneimine (PEI) and loaded with the therapeutic agents. Due to the magnetic property of the nanoparticles, it is anticipated that the nanoparticles would move in the direction of the magnetic field which would be positioned on the patient‟s head in the form of a headband for a nose-to-brain delivery. The target size of the nanoparticles was less than 200nm. The driving force which is the magnetic field would create a „pulling‟ effect on the nanoparticles attracting them towards the brain. By so doing it was envisaged that the nanoparticles would bypass the systemic pathway for uptake of drugs into the brain through the olfactory pathway thereby circumventing the BBB since most of the nanoparticles would migrate rapidly to the brain through the olfactory pathway. When the 5 nanoparticles reach the brain, the drug would be released by dissolution and diffusion into the cells thereby evading the BBB. The magnetic nanoparticles would be subsequently incorporated into a gel. The gel was fabricated employing thermosensitive and mucoadhesive polymers in addition to the electroactive polymers, this was to enable the gel to be liquid at room temperature and then gel at physiological temperature for ease of administration. This gel would possess mucoadhesive capabilities in order to provide a long residence time in the nasal mucosa. In developing the electro- responsive gel, the electroactive polymer would be incorporated or blended homogenously with the other polymers such as chitosan and hydroxypropyl methylcellulose using techniques such as polymer blending, copolymerization or crosslinking to generate an inter-penetrating network that would erode degrade or dissociate in the presence of an electric field. When the gel erodes, degrades or dissociates, the drug-loaded nanoparticles embedded within the gel would be released and in the presence of external magnetic field, the drug-loaded nanoparticles would migrate rapidly to the brain through the olfactory pathway with the aid of external magnetic field. The gel was employed to control the release of the drug-loaded nanoparticles for 24 hours as well as confine the drug-loaded nanoparticles in the nasal region to prevent premature clearance into the tracheal region as the patient breathes. The gel should be thermosensitive, and thermoreversible, possessed mucoadhesion capability and responded to an applied external electric field. The use of self- adhesive patch electrodes and a magnetic headband were employed to create an electric and magnetic field respectively. The electric field was required to dissociate or degrade the gel and transport the drug-loaded nanoparticles through the olfactory pathway to the brain. The self-adhesive patched electrode would be placed on the nose of the patient to generate an electric field creating an electric stimulus that dissociated or degraded the polymeric gel to release the drug-loaded nanoparticles. The magnetic headband would be able to generate magnetic fields thereby attracting the nanoparticles towards the brain for a nose-to-brain drug delivery. On reaching the brain, the nanoparticles were anticipated to circumvent 6 the BBB and then release the drugs in the brain cells. Figure 1.1 shows a schematic of the design. The nanoparticles were labelled with fluorescent markers such as fluorescein isothiocyanate (FITC) to aid imaging of the transport and bio- distribution of the nanoparticles. This drug delivery system may find applications in CNS diseases such as Alzheimer‟s and Parkinson‟s disease, schizophrenia, stroke, epilepsy, AIDS dementia Complex and brain tumor wherein drugs such as Galantamine, Levodopa, Clozapine, Alteplase, Clopidogrel, Zidovudine and Carmustine may be suitably incorporated into the nanoparticles. It is envisaged that the proposed drug delivery system, by circumventing BBB, will improve the bioavailability of CNS drugs thereby enhancing the pharmacological management of CNS diseases using an intranasal drug delivery system. Figure 1.1: Schematic conceptualization of design criteria for release of drugs using an external electric stimulation and a magnetic field 7 1.3. AIM AND OBJECTIVES The aim of this study was to design an intranasal drug delivery system that encompasses drug loaded magnetic nanoparticles which are incorporated into a thermosensitive electro-responsive mucoadhesive gel for controlled and targeted “nose-to-brain” delivery of therapeutic agents via the olfactory pathway with the aid of an external electric and magnetic fields for enhanced delivery. Figure 1 provides a schematic conceptualization of the design criteria for drug release using an electric and magnetic fields as examples of applied stimuli. In order to achieve this aim, the following objectives were fulfilled: 1. To undertake preliminary studies on the electro-active, mucoadhesive and thermosensitive polymers including modification of these polymers using techniques such as blending, copolymerization and crosslinking to assess their suitability for the development of a responsive gel. 2. To characterize the selected polymers employed in the formulation of the gel, using various techniques such as, Fourier Transform Infrared Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC), Thermogravimetric Analysis (TGA) and Scanning Electron Microscopy (SEM). 3. To fabricate stimuli-responsive polymeric nanostructures such as magnetic responsive nanoparticles via precipitation, chemical crosslinking, solvent evaporation/emulsification. The nanostructures were labelled using fluorescent markers such as fluorescein isothiocyanate (FITC), to aid imaging and detection of the bio-distribution of the nanostructures. 4. To incorporate a drug model such as carmustine, galantamine, levodopa, clozapine, alteplase, clopidogrel or zidovudine in the nanostructures for the treatment of brain tumors, Alzheimer‟s diseases (AD), Parkinson‟s, Schizophrenia, Stroke, epilepsy or AIDS dementia Complex, respectively. 8 5. To evaluate the physicochemical and electro-active properties of the nanogel using rheological and textural profiling analyses, as well as cyclic voltammetry in order to further characterize the nanogel. 6. To evaluate the physicochemical properties of the nanostructures using zetasize and zeta potential analyses, surface morphological characterization, drug loading/entrapment efficiency and conductivity analysis. 7. To assess in vitro drug release from the nanogel in the presence of various stimuli such as electric field and determine the optimum parameter required for the release and transport of nanostructures from the gel into the brain with the aid of an external magnetic field. 8. To undertake in vivo studies using New Zealand White rabbits to assess the performance of the novel intranasal drug delivery system, and evaluate the influence of an electric and magnetic stimuli. 1.4. NOVELTY OF THIS WORK The following points highlight the novelty of this research. 1. Design and formulation of novel nanoparticles employing polyvinyl alcohol (PVA), polyethyleneimine (PEI) and fluorescein isothiocyanate (FITC) referred to as Polyplex. 2. Coating of iron oxide nanoparticles with the Polyplex to form a Nano-co-Plex with the capability of significant drug loading and release. The drug-loaded Nano- co-Plex possesses the ability to respond to magnetic field. 3. Formulation of a thermosensitive electro-responsive mucogel (TERM) as an in situ intranasal drug delivery system that is liquid at room temperature and can form a gel at physiological temperature. The gel has the ability to control the release of therapeutic agents incorporated in it upon the application of external 9 electrical stimulus. The released therapeutic agents have the potential to be attracted to the brain via the olfactory pathway with the aid of an external magnetic stimulus. The electrical stimulus is in form of a self-adhesive electrode and the magnetic stimulus is in form of a magnetic headband. 4. The use of electrical stimulation on the nose in form of patched electrode to actuate controlled release of drug from the nasal mucosa. 5. The use of a magnet in form of a magnetic headband to enhance rapid migration of the drug-loaded nanoparticles to the brain after being released in the nasal cavity via the olfactory pathway. 1.5. OVERVIEW OF THIS THESIS Chapter 1 provides an introduction and background of the study, delineating the challenges experienced with delivering drug to the brain due to BBB. The use of intranasal drug delivery as alternative to systemic delivery was enumerated. The rationale and motivation for this research as well as the aim and objectives are outlined in this chapter. Chapter 2 provides a literature review, focusing on intranasal delivery of drugs for nose-to-brain delivery employing the olfactory route for the management of brain diseases. Techniques employed to fabricate and evaluate the intranasal drug delivery was enumerated. Chapter 3 provides a description of the synthesis of Nano-co-Plex by exploring the use of magnetic iron oxide nanoparticles and coated with Polyplex which was sythesized from PVA, PEI and FITC conjugate, loaded with carmustine (BCNU), an effective brain tumor chemotherapeutic agent. Detailed characterization of the Nano-co-Plex was carried out. In vitro drug release and cell studies were undertaken to determine the cytotoxicity and cellular uptake, as well as internalization of the drug-loaded Nano-co-Plex, in the absence and presence of a magnetic field. 10 Chapter 4 provides a description of formulating a thermosensitive electro- responsive mucogel (TERM) as a potential controlled intranasal drug delivery system, possessing mucoadhesive properties, which responds to an external electric field in releasing therapeutic agents. The carmustine-loaded Nano-co-Plex was incorporated into the gel to formulate the Nanogel Composite. A Design of Experiment (DoE) was extensively evaluated using a Box-Behnken Experimental Design (BBED). The BBED desirability function was employed to generate an optimized Nanogel Composite. Furthermore, this chapter deals with the physicochemical and physicomechanical characterization of the optimized Nanogel Composite. Chemical structure evaluation was undertaken using FTIR, XRD and NMR analyses. DSC and TGA were used to evaluate the thermal behaviour of the Nanogel Composite. SEM and porositometry analyses were undertaken to assess the surface morphology. Rheological evaluation was used to assess the viscoelastic properties and the gelation temperature of the Nanogel Composite. Cyclic voltammetry and conductivity analyses were employed to observe the electro-activity of the formulation. Texture profiling was undertaken to evaluate the mucoadhesive strength of the Nanogel Composite. In vitro studies were undertaken on the Nanogel Composite using an electrical stimulation to assess the release characteristics of the formulation. Chapter 5 provides the ex vivo evaluation of Nanogel Composite using an excised nasal epithelial tissue of New Zealand White rabbit. The ex vivo permeation studies were conducted to ensure the viability of the application of an electric stimulation on Nanogel Composite to release BCNU-loaded Nano-co-Plex. Furthermore, the effect of a magnetic field on the transportation of the BCNU- loaded Nano-co-Plex across the nasal epithelial tissue of rabbit was evaluated. This study represents a prototype investigation prior to in vivo studies. Chapter 6 details the in vivo performance of the optimized Nanogel Composite in comparison to the conventional drug available on the market. In vivo studies were carried out using New Zealand White rabbits. The effect of electric stimulation on the release behaviour of the nanoparticles and the effect of magnetic field on the 11 transport of the nanoparticles to the brain via the olfactory pathway were evaluated. Chapter 7 provides recommendations and future outlook of the study. A hypothesis based on “Proteo-Alkaloid” conjugation of Lactoferrin (Lf) and Galantamine (Gal) via the self-assembly functionality of Lf was postulated. The hypothesis explored the combination of these two compounds as one dosage form, taking into consideration their iron and free radical scavenging abilities. A dual neuroprotection and neurotherapeutic interventional approach was postulated. 1.6. CONCLUDING REMARKS The novel thermosensitive electro-responsive mucogel containing drug-loaded magnetic nanoparticles can be considered as an alternative therapy for circumventing the BBB. The novel development has the potential to increase bioavailability of drugs to the brain via a direct nose-to-brain delivery. It is therefore envisaged that this system is a promising technology for non-invasive treatment of brain diseases and disorders. 1.7. REFERENCES Costantino, H.R., Illum, L., Brandt, G., Johnson, P.H., Quay, S.C., 2007. Intranasal delivery: physicochemical and therapeutic aspects. Int. J. Pharm. 337, 1– 24. Kumar, M., Misra, A., Babbar, A., Mishra, A., Mishra, P., Pathak, K., 2008. Intranasal nanoemulsion based brain targeting drug delivery system of risperidone. Int. J. Pharm. 358, 285–291. Lalatsa, A., Schatzlein, A.G., Uchegbu, I.F., 2014. Strategies to Deliver Peptide Drugs to the Brain. Mol. Pharm. 11, 1081–1093. doi:10.1021/mp400680d 12 Lerner, E.N., van Zanten, E.H., Stewart, G.R., 2004. Enhanced delivery of octreotide to the brain via transnasal iontophoretic administration. J. Drug Target. 12, 273–280. Mistry, A., Stolnik, S., Illum, L., 2015. Nose-to-Brain Delivery: Investigation of the Transport of Nanoparticles with Different Surface Characteristics and Sizes in Excised Porcine Olfactory Epithelium. Mol. Pharm. 12, 2755–2766. Mistry, A., Stolnik, S., Illum, L., 2009. Nanoparticles for direct nose-to-brain delivery of drugs. Int. J. Pharm. 379, 146–157. Nasr, M., 2015. Development of an optimized hyaluronic acid-based lipidic nanoemulsion co-encapsulating two polyphenols for nose to brain delivery. Drug Deliv. 0, 1–9. Pardridge, W.M., 1999. Non-invasive drug delivery to the human brain using endogenous blood–brain barrier transport systems. Pharm. Sci. Technol. Today 2, 49–59. Seju, U., Kumar, A., Sawant, K.K., 2011. Development and evaluation of olanzapine-loaded PLGA nanoparticles for nose-to-brain delivery: In vitro and in vivo studies. Acta Biomater. 7, 4169–4176. Serralheiro, A., Alves, G., Fortuna, A., Falcão, A., 2015. Direct nose-to-brain delivery of lamotrigine following intranasal administration to mice. Int. J. Pharm. 490, 39–46. Serralheiro, A., Alves, G., Fortuna, A., Falcão, A., 2014. Intranasal administration of carbamazepine to mice: A direct delivery pathway for brain targeting. Eur. J. Pharm. Sci. 60, 32–39. Shah, B.M., Misra, M., Shishoo, C.J., Padh, H., 2015. Nose to brain microemulsion-based drug delivery system of rivastigmine: formulation and ex-vivo characterization. Drug Deliv. 22, 918–930. Sharma, U., Badyal, P.N., Gupta, S., 2015. Polymeric Nanoparticles Drug Delivery to Brain: A Review 2, 60–69. Türker, S., Onur, E., Ózer, Y., 2004. Nasal route and drug delivery systems. Pharm. World Sci. 26, 137–142. Vyas, T.K., Shahiwala, A., Marathe, S., Misra, A., 2005a. Intranasal Drug Delivery for Brain Targeting. Curr. Drug Deliv. 2, 165–175. 13 Vyas, T.K., Shahiwala, A., Marathe, S., Misra, A., 2005b. Intranasal Drug Delivery for Brain Targeting. Curr. Drug Deliv. 2, 165–175. Westin, U., Piras, E., Jansson, B., Bergström, U., Dahlin, M., Brittebo, E., Björk, E., 2005. Transfer of morphine along the olfactory pathway to the central nervous system after nasal administration to rodents. Eur. J. Pharm. Sci. 24, 565–573. 14 CHAPTER 2 A REVIEW OF NOSE-TO-BRAIN NEUROTHERAPEUTIC INTERVENTIONS 2.1. INTRODUCTION Delivery of drugs to the brain has been fraught with issues of low bioavailability (Krol, 2012). The central nervous system (CNS) which is made up of the brain and the spinal cord do not have adequate access to the blood compartment due to the Blood-Brain Barrier (BBB) and other barriers. CNS disorders have been recorded as the number one source of disability and account for more extended care and hospitalizations than the combination of most other diseases (Misra et al., 2003). For instance, over 36 million people in the world today have CNS related diseases and disorders, the numbers will continue to rise to about 66 million by 2030, and projected to be around 115 million by 2050 (Liu et al., 2013). Treating CNS disorders and ailments in general which include Alzheimer‟s disease, Parkinson‟s, Schizophrenia, Stroke, epilepsy, AIDS dementia Complex, brain tumor and Huntington disease is extremely challenging due to the presence of various obstacles that restrict passage of drug across the BBB for onward delivery to the brain (Radhika et al., 2011). The BBB which is both physiologically and anatomically a biological natural occurrence with no single scientific theory to explain all the events happening therein (Roth and Barlow, 1961) according to Roth and Barlow five decades ago, the challenges are still on even as of today. Researchers are therefore searching for the best methods to develop drug delivery systems and techniques to transfer pharmaceutical substances to the brain and CNS for the management of neurodegenerative disorders and delivery of drugs to the brain and the CNS in general having in mind the BBB obstacle. Lipophilic substances with molecular weight less than 600 Da are known to permeate the BBB, which implies that the more lipophilic the drug molecules the better the permeability of the drug, the use of prodrug will be an excellent approach in increasing the permeability, the prodrug on getting to the brain will be converted to the parent drug (Illum, 2000). Several 15 attempts and strategies such as disturbing the BBB (Alves, 2014), carrier mediated drug delivery osmotic BBB disruptions (Aryal et al., 2014), biochemical BBB disruption (Griep et al., 2013), drug manipulations including prodrugs, receptor-mediated drug delivery, chemical drug delivery, vector-mediated drug delivery (Lu et al., 2014), alternative routes such as intraventricular and intrathecal route, olfactory pathway, other techniques include injections, catheters, and pumps technique, implant of device directly to the brain, biodegradable polymer wafers, microspheres and nanoparticles techniques, have been explored (Misra et al., 2003). The use of intranasal route has been explored by various researchers and has been established to be a promising route of transferring therapeutic substances directly to the brain by employing the olfactory pathway through the nasal mucosa in order to bypass the BBB and its associated problems. 2.2. BIOLOGICAL BARRIERS AS AN IMPEDIMENT TO CNS DRUG DELIVERY There are biological barriers limiting the delivery of drugs to the CNS thereby contributing to the difficulties in treating CNS diseases and disorder. Due to these biological barriers, transcranial drug delivery has been used to overcome these barriers. This approach entails three types of delivery systems which are; intra- cerebroventricular injection, convection enhanced diffusion and intracerebral implantation. These methods however require an invasive approach through into the brain. Implantation of transcranial catheters has been confirmed to be connected to numerous complications and is known to be temporary (Bleier et al., 2013). These barriers are enumerated below. 2.2.1. Blood-Brain Barrier Knowledge about the BBB might assist researchers in designing better drugs or approaches in fabricating systems for delivering therapeutic agents to the brain. The BBB is a special membranous barrier that distinguishes the CNS from the systemic circulation (Begley, 1996; Misra et al., 2003; Schlosshauer and Steuer, 2002). The BBB is naturally designed to protect the brain from foreign organisms and deleterious chemicals in the blood, but allows the supply of necessary 16 nutrients to the brain for the well-being of the brain to maintain and regulate the microenvironment for accurate neuronal signaling (Abbott et al., 2010) and other functions. The protection of the Brain and the CNS is sorted by the barrier function of the brain capillary endothelial and the choroid epithelial cells. The blood capillaries of the brain are anatomically different from that of other tissues and organs. The blood capillaries consist of special cells known as endothelial cells; these cells are sealed with tight junctions with their membrane separating the systemic circulation and the central extracellular fluid of the brain. The vasculature serving the CNS also has capillaries with tight junctions. The arachnoid membrane which is a doubled layered structure covers the brain and constitutes a barrier between the blood and the CSF (Shawahna et al., 2013; Kuhnline Sloan et al., 2012; Nag et al., 2011; Kaur et al., 2008; Vyas et al., 2005a). Therefore, BBB is primarily a prominent obstruction in drug delivery to the brain. 2.2.2. Blood-Cerebrospinal Fluid Barrier The Blood Cerebrospinal Fluid Barrier (BCSFB) (Lam et al., 2012) is another barrier that exists, it inhibits the passage of drugs to the CNS during systemic administration (Chalbot et al., 2010; Yasuda et al., 2013). BCSFB is resides in the epithelium of the choroids plexus (Johanson et al., 2011), the arrangement of which limits the passage of therapeutic molecules into the CSF. The choroid plexus and the arachnoid membranes play a major role at the barriers between the blood and CSF (Chalbot et al., 2010; Misra et al., 2003). It is clear that the endothelium of the cerebral blood vessels and the epithelium of the choroid plexus prevent the penetration of large solutes directly into the brain and CSF. 2.2.3. Blood-Tumor Barrier Blood Tumor Barrier (BTB) is a barrier formation at the local site of the tumor cells that do not permit drugs from reaching brain tumors in therapeutic quantity to exterminate the tumor cells. A range of barriers that are physiologically oriented are found in solid tumors and they prevent drug delivery via the cardiovascular system. Solid tumor consists of neoplastic cells, drug delivery to these cells is hindered because uneven distribution of microvasculature throughout the tumor 17 interstitial resulting into drug delivery inconsistency (Misra et al., 2003). Apart from dealing with the BBB, there is also the problem of BTB. Many innovative methods have been used to improve drug delivery to the tumor cells, of which has been reviewed by Groothuis (2000). 2.3. NASAL ANATOMY AS AN AID TO CNS DRUG DELIVERY It is pertinent to understand the anatomy of the nasal cavity when discussing the intranasal administration, absorption and transporting of drugs to the brain and CNS. Figure 2.1 shows the Lateral wall of the nasal cavity. The nasal cavity is located within the skull; it spans from the base of the skull to the roof of the mouth. It is a midline structure that is divided into two equal haves by the nasal septum. Each half which consists of the roof and the floor as well as medial and lateral walls (formed by the b