“Lipid Nanoparticles for Improved Delivery of Antioxidants” A THESIS SUBMITTED TO BHARATI VIDYAPEETH DEEMED UNIVERSITY, PUNE FOR AWARD OF DEGREE OF DOCTOR OF PHILOSOPHY IN PHARMACEUTICS UNDER THE FACULTY OF PHARMACEUTICAL SCIENCES SUBMITTED BY MR. ABHAY KYADARKUNTE (M. Pharmacy) UNDER THE GUIDANCE OF PROF. VARSHA POKHARKAR RESEARCH CENTRE POONA COLLEGE OF PHARMACY BHARATI VIDYAPEETH DEEMED UNIVERSITY, ERANDWANE, PUNE - 411038. OCTOBER 2015 CERTIFICATE This is to certify that the work incorporated in the thesis entitled “Lipid Nanoparticles for Improved Delivery of Antioxidants” for the degree of ‘Doctor of Philosophy’ in the subject of Pharmaceutics under the faculty of Pharmaceutical Sciences has been carried out by Mr. Abhay Kyadarkunte in the Department of Pharmaceutics, at Bharati Vidyapeeth Deemed University’s Poona College of Pharmacy, Pune during the period from October 2012 to October 2015 under the guidance of Prof. Varsha Pokharkar, HOD and Vice-Principal, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune. Place: Pune Principal Date: Prof. K. R. Mahadik Professor and Principal, Poona College of Pharmacy Bharati Vidyapeeth Deemed University, Pune. CERTIFICATION BY GUIDE This is to certify that the work incorporated in the thesis entitled “Lipid Nanoparticles for Improved Delivery of Antioxidants” submitted by Mr. Abhay Kyadarkunte for the degree of ‘Doctor of Philosophy’ in the subject of Pharmaceutics under the faculty of Pharmaceutical Sciences has been carried out in the Department of Pharmaceutics, Bharati Vidyapeeth Deemed University’s Poona College of Pharmacy, Pune during the period from October 2012 to October 2015, under my direct supervision. Place: Pune Prof. Varsha Pokharkar Date: HOD and Vice-Principal Poona College of Pharmacy Bharati Vidyapeeth Deemed University, Pune. DECLARATION BY THE CANDIDATE I hereby declare that the thesis entitled “Lipid Nanoparticles for Improved Delivery of Antioxidants” submitted by me to the Bharati Vidyapeeth Deemed University, Pune for the degree of Doctor of Philosophy (Ph.D.) in Pharmaceutics under the faculty of Pharmaceutical Sciences is original piece of work carried out by me under the supervision of Prof. Varsha Pokharkar HOD and Vice-Principal, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune. I further declare that it has not been submitted to this or any other University or Institution for the award of any Degree or Diploma. I also confirm that all the material which I have borrowed from other sources and incorporated in this thesis is duly acknowledged. If any material is not duly acknowledged and found incorporated in this thesis, it is entirely my responsibility. I am fully aware of the implications of any such act which might have been committed by me advertently or inadvertently. Place: Pune Mr. Abhay Kyadarkunte Date: Research student Acknowledgement Many people deserve my sincere acknowledgements, without them this thesis could not have been written. My first thanks goes to our Honorable Vice-Chancellor, Dr. Shivajirao Kadam for his encouragement and providing excellent facilities. I am also grateful to Dr. K. R. Mahadik, Principal, Poona College of Pharmacy, for providing constant support and fabulous infrastructure. I wish to express my sincere gratitude to my advisor Prof. Varsha Pokharkar for her supervision, advice and guidance throughout the past three years. Above all, she provided me encouragement and support in a number of ways. I will be forever grateful for her patience and generosity in letting me navigate my own path through my PhD work, I did not take the shortest path but I learned more than I ever thought possible. The financial support from University Grants Commission (UGC), India, must be as well acknowledged and thus all the members belonging to it. I am thankful to Dr. S. L. Bodhankar, for supporting my animal studies in Department of Pharmacology, Poona College of Pharmacy. I express my sincere thanks to Dr. A. P. Pawar, Dr. S. R. Dhaneshwar, Prof. G. N. Zambre, Dr. R. N. Kamble, Dr. V. M. Kulkarni, Dr. Bhosale, Dr. J. R. Rao, Dr. S. S. Dhaneshwar, Dr. Kolhe, Dr. Purohit, and Dr. Bothiraja for their constant support and encouragement during my research work and throughout the PhD tenure. I extend my sincere thanks to Mr. S. S. Potdar, Mr. B. D. Khade, Mr. D. J. Joshi, Mr. Mandke, Arjun, Mr. Patil and all non-teaching staff, office staff and others, who have helped me directly or indirectly during my Ph.D. thesis. I would also like to thank Dr. Milind Patole, Mr. Bhimashankar Utge and cell line repository team, National Centre for Cell Science (NCCS), India for their friendly support during cell culture activities. I would like to thank to the people who always helped me and supported me, colleagues and “food-club” friends Dr. Leenata, Dr. Arpana, Sameer, Dr. Ganesh, Dr. Sharvil, Dr. Vividha, Dr. Deepak, Dr. Ashwin, Akhil, Dr. Ashwin mali, Dr. Arvind, Dr. Hemant, Dr. Suyog, Dr. Abhijeet, Priyanka, Shaivee, Mithila, Prachi, Saba and Megha. I spent a great time with them and will never forget it. I thank and dedicate this thesis to all my family members for their love, strength, and everlasting patience. Most importantly, Special thanks to my wife, Keerti, and our two son’s Anish and Shivansh for their encouragement and support throughout the years. Table of contents List of figures VI List of tables X Chapter 1: Introduction 1. Introduction 1 1.1 Background 1 1.2 Oxidative stress 1 1.2.1 Generation of ROS 2 1.2.2 Pathophysiological conditions 3 1.2.3 Role of OS in photoaging 4 1.2.4 Role of OS in Alzheimer’s disease 7 1.3 Antioxidative strategy to lower OS 11 1.3.1 Antioxidants in prevention of photoaging 12 1.3.2 Antioxidants in prevention of AD 15 1.4 Antioxidant delivery approaches 20 1.4.1 Conventional delivery 20 1.4.2 Colloidal carrier 21 1.5 Lipid nanoparticles 23 1.5.1 History and scope 23 1.5.2 What exactly are lipid nanoparticles? 24 1.5.3 Major shortcomings associated with SLNs 25 1.6 NLCs 27 1.6.1 Compositions 27 1.6.2 Models of drugs incorporated in NLCs 30 1.6.3 Production techniques 31 1.6.4 Characterization techniques 33 1.7 Role of NLCs in topical delivery of antioxidants 35 1.7.1 Benefits of NLCs in topical delivery of antioxidants 38 1.8 Role of NLCs in intranasal (direct nose to brain) delivery of antioxidants 40 1.8.1 Intranasal (direct nose to brain) pathway 41 I 1.8.2 Strategies to enhance i.n. (direct nose to brain) drug delivery 45 1.9 Literature survey 46 1.10 References 51 Chapter 2: Genesis, specific aims and objectives of work 2.1 Genesis 61 2.2 Specific aims 62 2.3 Objectives 62 2.4 References 64 Chapter 3: Materials, instruments and software’s 3.1 Materials 65 3.1.1 Antioxidants 65 3.1.2 Excipients 65 3.1.3 Chemicals and reagents 66 3.1.4 Cell culture requirements 66 3.1.5 Miscellaneous 67 3.2 Instruments 67 3.3 Software’s 68 3.4 Antioxidants 68 3.4.1 Idebenone 68 3.4.2 Resveratrol 70 3.4.3 Phenyl butyl nitrone 71 3.5 Excipients 72 3.5.1 Solid lipids 72 3.5.2 Liquid lipids 74 3.5.3 Surfactants 75 3.6 References 77 Chapter 4: Photoprotection aspects of topically administered IDB loaded NLCs (IDB-NLCs) 4.1 Genesis and outline of the work 78 4.2 Experimental 79 II 4.2.1 Lipid phase screening 79 4.2.2 Surfactant selection 80 4.2.3 Crystallographic investigations 81 4.2.4 Optimization of IDB-NLCs preparation 81 4.2.5 Preparation of IDB-NLCs 81 4.2.6. Characterization of IDB-NLCs 82 4.2.7 Cell culture study 85 4.2.8 Statistical analysis 90 4.3 Results and discussion 90 4.3.1 Selection of the lipid phase composition 90 4.3.2 Selection of surfactant 93 4.3.3 Optimization of IDB-NLC preparation 95 4.3.4 TEM investigations 100 4.3.5 In vitro IDB release studies 101 4.3.6 Occlusion and ex-vivo drug penetration studies 102 4.3.7 Stability investigations 103 4.3.8 Cell viability and photoprotective studies 104 4.3.9 Cell uptake studies 108 4.3.10 Oxidative stress 111 4.4 Conclusions 113 4.5 References 114 Chapter 5A: Cytotoxicity and phototoxicity assessment of acylglutamate surfactants 5A.1 Genesis and outline of the work 118 5A.2 Experimental 119 5A.2.1 Surfactants tested 119 5A.2.2 Cell culture 120 5A.2.3 UVB-irradiation and cell viability 120 5A.2.4 Surfactant treatment 121 5A.2.5 MTS assay 121 5A.2.6 Photoirritation evaluation 121 5A.2.7 Data analysis 122 III 5A.2.8. Statistical analysis 122 5A.3 Results and discussion 122 5A.3.1 UVB dose optimization 123 5A.3.2 Cytotoxicity and phototoxicity of commercial acylglutamates 124 5A.4 Conclusion 128 5A.5 References 128 Chapter 5B: Photoprotection aspects of topically administered RSV and PBN loaded NLCs (Combination-NLCs) 5B.1 Genesis and outline of the work 130 5B.2 Experimental 131 5B.2.1 Screening and selection of antioxidant combination 131 5B.2.2 Pre-formulation studies 133 5B.2.3 Preparation of NLCs 135 5B.2.4 Optimization of NLCs 135 5B.2.5 Characterization of NLCs 135 5B.2.6 Cell culture study 136 5B.2.7 Statistical analysis 137 5B.3 Results and discussion 137 5B.3.1 Screening and selection of antioxidant combination 137 5B.3.2 Pre-formulation studies 141 5B.3.3 Optimization of the NLCs preparation 145 5B.3.4 TEM investigations 156 5B.3.5 In vitro drug release studies 156 5B.3.6 In vitro occlusion and ex-vivo drug penetration studies 158 5B.3.7 Stability investigations 160 5B.3.8 Cell uptake studies 161 5B.3.9 Photoprotective effects of NLCs 162 5B.4 Conclusion 164 5B.5 References 165 IV Chapter 6: Neuroprotection aspects of intranasally administered RSV and PBN loaded NLCs (Combination-NLCs) 6.1 Genesis and outline