“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 of the work 168 6.2 Experimental 169 6.2.1 Pre-formulation studies 169 6.2.2 Preparation of NLCs 171 6.2.3 Characterization of NLCs 171 6.2.4 In vivo pharmacokinetic studies 172 6.2.5 Neuroprotection effects of the NLC formulations on Alzheimer’s

disease model induced by Aβ 25–35 174 6.2.6 Statistical analysis 176 6.3 Results and discussion 176 6.3.1 Pre-formulation studies 176 6.3.2 RP-HPLC chromatographic method 181 6.3.3 Preparation and characterization of NLCs 184 6.3.4 Brain distribution of NLCs 189 6.3.5 Neuroprotection effects of the NLC formulations on Alzheimer’s

disease model induced by Aβ 25–35 193 6.4 Conclusion 196 6.5 References 196

Chapter 7: Conclusions 199 Appendix 201 Publications 203

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List of Figures

Figure Title Page No. No. 1.1 Factors responsible for skin aging. 5 1.2 Aβ-associated OS and neurotoxicity in AD brain. 10 1.3 How skin cells respond to OS. 13 1.4 Comparison of antioxidant in conventional delivery (CD) and in 21 NDDS. 1.5 Different types of lipid nanoparticles. 24 1.6 Difference between SLN and NLC particle matrix structure. 26 1.7 Types of NLCs formed. 32 1.8 Skin (cutis) drawing illustrating the main layers that structure the 36 largest human organ, accounting more than 10% of the body mass. 1.9 Film formation associated to the occlusion effect. 38 1.10 Possible mechanisms for skin permeation enhancement of drugs or 39 active ingredients from NLCs. 1.11 Exchange phenomena between the lipophilic oil phase of emulsions 40 (left), and the lipophilic bilayer and aqueous core of liposomes (middle) and in case of NLCs with solid matrix, these exchange phenomena and degradation are practically eliminated or at least distinctly slowed down/minimised (right). 1.12 Pathways for brain targeting after intranasal administration. 42 1.13 General organization of the olfactory region. 44 4.1 Polarized light microscopic images featuring A. Idebenone crystals. 92 B. Binary mixture of WE 85 and Capmul MCM (7:1), and C. Ternary mixture of IDB, WE 85 and Capmul MCM (1.25:7:1). 4.2 Crystallographic investigations. A. Differential scanning 94 calorimetry (DSC) scans. B. X-ray diffractions patterns of Idebenone (IDB), Witepsol E 85 (WE 85), a binary mixture of WE 85 and Capmul MCM (7:1), and a ternary mass mixture of IDB, WE 85 and Capmul MCM (1.25:7:1).

VI

4.3 Main effects plot for S/N ratios of (A) particle size and (B) 99 entrapment efficiency. 4.4 Transmission electron microscopy (TEM) images. A. IDB-NLCs 101 stabilized with TegoCare 450, magnification 55,000 ×. B. NR-IDB- NLCs stabilized with TegoCare, after zoom-in, original magnification 55,000 ×. Arrow indicates a thin layer encircling the particle. 4.5 In vitro release profile of IDB from IDB-NLCs and IDB-PD. 102 4.6 A Comparison of the IDB levels from IDB-NLCs and IDB-PD. 103 4.7 Viability of HaCaT cells. A. After 12, 24 and 48 h of incubation 105 with different concentrations of IDB-PD (1, 2, 4, 6, 8 and 10 µM/0.1mL). B. After 24 h of incubation with placebo-NLCs and IDB-NLCs (equivalent to 1, 2, 4, 6, 8 and 10 µM/0.1mL of IDB). 4.8 Photoprotective effect of IDB-PD, placebo-NLCs and IDB-NLCs 107 (equivalent to 1, 2, 4, 6, 8 and 10 µM/0.1mL of IDB) against 55 µW/cm2 UVB (200 mJ/cm 2 for 1 h)-mediated phototoxicity in HaCaT cells. 4.9 Quantitative (spectrophotometric) intracellular uptake of IDB from 109 IDB-NLCs and IDB-PD (equivalent to 2 µg/mL of IDB) by HaCaT cells at 37°C. 4.10 Confocal images (Qualitative) featuring the time dependent (1, 2, 3, 110 6, 12 and 24 h) intracellular uptake of NR-IDB-NLCs by HaCaT cells at 37°C. 4.11 Photoprotective effect of placebo-NLCs and IDB-NLCs (equivalent 113 to 6 µM/0.1ml of IDB) against 55 µW/cm 2 UVB (200 mJ/cm 2 for 1 h)-mediated oxidative stress in HaCaT cells. 5A.1 Cell proliferation rate in UVB irradiated HaCaT cells. 124 5A.2 Effect of increasing concentration of (A) CS-11; (B) LS-11; (C) 126 MS-11 and (D) CPZ on cell viability of HaCaT cells. 5B.1 UV spectra of IDB, RSV and PBN, in the range 290 – 400 nm, at 138 10 M concentration.

VII

5B.2 Viability of HaCaT cells. (A) Cytotoxicity assay, after 24 h of 140 incubation with different concentrations of IDB, RSV and PBN. (B) Photoprotective effect of IDB, RSV and PBN against UVB (200 mJ/cm 2 for 1 h) -mediated phototoxicity in HaCaT cells. 5B.3 Mechanism of action of PBN 141 5B.4 Miscibility of the selected Gelucire 50/13 and Labrafil M 1944 CS 143 at various ratios: (1) 9:1, (2) 8:2, (3) 7:3, (4) pure Gelucire 50/13, (5) 6:4 and (6) 5:5. 5B.5 DSC thermograms: (a) RSV, (b) PBN, (c) Gelucire 50/13, (d) G 144 50/13 + Labrafil M 1944 CS at 9:1 ratio, (e) RSV + G 50/13 + Labrafil M 1944 CS at 0.5:9:1 ratio, (f) PBN + G 50/13 + Labrafil M 1944 CS at 0.5:9:1 ratio and (g) RSV+PBN + G 50/13 + Labrafil M 1944 CS 1:9:1 ratio. 5B.6 FT-IR spectra of (a) RSV pure drug, (b) PBN pure drug and (c) 146 RSV+PBN mixture. 5B.7 Main effects plot for S/N ratios of (a) particle size (B) % 152 entrapment efficiency of RSV and (c) % entrapment efficiency of PBN. 5B.8 Transmission electron microscopy (TEM) image of combination- 157 NLCs. 5B.9 Release profiles of resveratrol pure-drug (RSV-PD), PBN pure- 158 drug (PBN-PD) and, resveratrol (RSV) and PBN (PBN) from combination-NLCs. 5B.10 A Comparison of the resveratrol pure-drug (RSV-PD), PBN pure- 159 drug (PBN-PD) and, resveratrol (RSV) and PBN (PBN) from combination-NLCs 5B.11 Protective effect of combination-NLCs, placebo-NLCs RSV-NLCs 163 and PBN-NLCs against 55 W/cm 2 UVB (200 mJ/cm 2 for 1 h)- mediated phototoxicity in HaCaT cells. 5B.12 Photoprotective effect of different NLCs against 55 W/cm 2 UVB 164 (200 mJ/cm 2 for 1 h)-mediated oxidative stress in HaCaT cells. 6.1 Steps involved in lipid phase screening. 179

VIII

6.2 DSC thermograms: (a) RSV, (b) PBN, (c) Gelucire 44/14 (G40/14), 180 (d) G 44/14 +Labrafil M 1944 CS at 9:1 ratio, (e) RSV + G 44/14 + Labrafil M 1944 CS at 0.5:9:1 ratio, (f) PBN + G 44/14 + Labrafil M 1944 CS at 0.5:9:1 ratio and (g) RSV+PBN + G 44/14 + Labrafil M 1944 CS 1:9:1 ratio. 6.3 HPLC chromatogram of simultaneous determination of RSV and 181 PBN in mobile phase. 6.4 HPLC chromatogram of extracted blank brain. 182 6.5 HPLC chromatogram of RSV in brain tissue samples. 182 6.6 HPLC chromatogram of PBN in brain tissue samples. 182 6.7 HPLC chromatogram of brain spiked with RSV, PBN and 183 carbamazepine (CBZ). 6.8 Images depicting different NLCs on the day of production. 185 6.9 ESEM image of combination-NLCs. 187 6.10 Release profiles of PBN-PD, RSV-PD, PBN and RSV from 188 combination-NLCs in PBS pH 6. 6.11 Concentration (in brain) vs. time curves of different samples after 190 intranasal administration of A) Resveratrol pure drug (RSV-PD) and Resveratrol-NLCs (RSV-NLCs) and B) PBN pure drug (PBN- PD) and PBN-NLCs. 6.12 Concentration (in brain) vs. time curves of combination samples 191 after intranasal administration of Resveratrol (RSV) from combo- PD, RSV from combo-NLCs, PBN form combo-PD and PBN from combo-NLCs.

6.13 Aβ25-35 (2 µL bilaterally injected slowly into hippocampus using 194 stereotaxic coordinates)-induced memory impairment and neuroprotective effect of RSV-NLCs, PBN-NLCs, combination- NLCs and test or standard drug in in-vivo behavioral tests (Y- maze).

IX

List of Tables

Table Title Page No. No. 1.1. The list of key ROS with their half-life. 3 1.2. List of single exogenous antioxidants with photoprotective or 16 damage protective effects. 1.3. List of combination exogenous antioxidants with photoprotective 17 or damage protective effects. 1.4 List of exogenous antioxidant molecules as therapeutic agents for 18 AD management. 1.5 List of lipids used in preparation of NLCs. 28 1.6 List of surfactants used in preparation of NLCs. 29 1.7 List of miscellaneous agents used in preparation of NLCs. 30 1.8 Mechanism involved in lipid nanoparticles formation by various 33 production techniques. 1.9 Overview of NLC-based dermal cosmetic products currently on 37 the market. 1.10 Key benefits offered by nasal drug delivery system. 41 1.11 Number of articles published by different research groups between 47 years 2010 to 2015. 1.12 List of relevant articles published on “NLCs in topical delivery of 47 antioxidants” and “NLCs in direct nose to brain delivery of antioxidants”, with outcome of the study and references. 1.13 List of relevant patents published on “NLCs in topical delivery of 50 antioxidants” and “NLCs in brain targeting”, with findings and references. 4.1 Solubility of idebenone in different solid and liquid lipids. 91

X

4.2 Variables, levels and observed responses (dependent variables) of 96 Taguchi L9 (33) orthogonal array. The coded values (1), (2) and (3) correspond, respectively, to 30, 60, and 90 for sonication amplitude (A), 1, 2, and 4 for sonication time (B), and 0.5, 1.0, and 2.0 for surfactant concentration (C). 4.3 S/N ratios for particle size and entrapment efficiency. 97 4.4 Mean S/N response for particle size and standard deviation. 98 4.5 Mean S/N response for entrapment efficiency and standard 98 deviation. 4.6 Final formulation compositions of the optimized IDB-NLCs. 100 4.7 Physicochemical characteristics of different NLCs. 100 4.8 Stability study (3 months, at varying temperature) data of IDB- 104 NLCs.

5A.1 IC 50 values of acylglutamate surfactants and CPZ on HaCaT cells. 125 5A.2 PIF mean and MPE mean values of acylglutamate surfactants and 127 CPZ on HaCaT cells. 5B.1 Solubility of individual RSV, PBN and their combination in 142 different solid and liquid lipids. 5B.2 Different control factors and levels. 147 1 3 5B.3 The Taguchi’s L 18 (6 × 3 ) standard OA with actual experimental 149 levels and coded levels (in parentheses) of parameters. 5B.4 Experimental and S/N ratio results for particle size and % EE of 151 RSV and PBN. 5B.5 Final formulation compositions of the investigated NLCs. 153 5B.6 ANOVA for S/N ratio of particle size and % EE of RSV and PBN. 154 5B.7 Response table for particle size and % EE of RSV and PBN. 155 5B.8 Results of the prediction and confirmation experiment for particle 156 size and % EE. 5B.9 Stability study (90 days, at varying temperature) data of 160 combination-NLCs. 5B.10 In vitro Cell uptake studies of RSV-PD, PBN-PD, and RSV and 161 PBN from combination-NLCs in HaCaT cells.

XI

6.1 Groups and treatments. 174 6.2 Solubility studies of RSV and PBN in Gelucire 44/14, Labrafil M 177 1944 CS, and at their 9:1 combination. 6.3 Clarity of emulsions produced by different surfactants. 180 6.4 Regression analysis of calibration curves for individual RSV and 183 PBN, and simultaneous estimation of RSV and PBN in plasma and brain tissue over the specified concentration rate. 6.5 Composition of lipid nanoparticles (Batch size 10 mL) 184 6.6 Physicochemical characteristics of prepared NLCs. 185 6.7 Stability study (90 days, at varying temperature) data of 189 combination-NLCs. 6.8 Pharmacokinetic parameters in rat brain for RSV and PBN 192 following intranasal administration of pure drug solutions and single antioxidant loaded-NLCs (single dose). 6.9 Pharmacokinetic parameters in rat brain for RSV and PBN 193 following intranasal administration of pure drug solutions and combination-NLCs (single dose). 6.10 The AChE activity in brains of AD rats treated with different 195 formulations.

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Chapter 1

Introduction

This chapter is an introduction to the thesis and gives a brief overview of oxidative stress and its role in photoaging and in Alzheimer’s disease, novel antioxidant delivery approaches to overcome the oxidative stress and colloidal carriers with more emphasis on lipid nanoparticles (particularly NLCs). The detailed description of NLCs composition, models of drug incorporated, production and characterization techniques, properties, advantages, role of NLCs in topical skin delivery of antioxidants and in direct nose to brain delivery of antioxidants have been discussed. A part of the chapter also provide the review of literature comprised of research papers, review articles and previous patents, on the preparation of variety of NLCs and their applications in topical skin and intranasal delivery of antioxidants and various other drugs.

Chapter 1: Introduction

1. Introduction

1.1 Background In recent past, antioxidants have gained tremendous importance because of their potential as prophylactic and therapeutic agents in numerous diseases. The discovery of the role of free radicals in premature skin-aging, neurodegenerative diseases, cancer, diabetes, cardiovascular diseases and autoimmune diseases has led to a medical revolution that is promising a new paradigm of healthcare. Although only few antioxidants have made their way into various pharmacopoeia listings, immeasurable research is being carried out worldwide on these agents, and majority of them have been proven pharmacologically active. Traditionally, herbal medicine with antioxidant properties have been used for diverse causes and epidemiological data indicates worldwide acceptance and use of these agents. In current scenario, the main antioxidant component from these herbal sources are extracted, purified and tested for their potential use in aforementioned diseases. Currently, the worldwide market for antioxidants is growing at sizable rate and is expected to increase at rapid pace in the years to come (i.e. compound annual growth rate (CAGR) of 4% by 2018), because of growing awareness about health, as a result more number of consumers are selecting products containing antioxidants [1].

1.2 Oxidative stress The oxidation and reduction reactions in biological systems (redox reactions) represent the basis for numerous biochemical mechanisms of metabolic changes [2]. In biological systems, instead of using the terms reducing and oxidant agent, it is more frequent to use the denominations of antioxidant and pro-oxidant, respectively [3]. A reducing agent, or antioxidant, is a substance which donates electrons, whereas an oxidant, or pro-oxidant agent, is a substance that accepts electrons. Cells are constantly exposed to oxidants from both physiological processes, such as mitochondrial respiration [4] and pathophysiological conditions such as inflammation, foreign compound metabolism, and radiation among others [5]. Oxidative stress (OS) constitutes a unifying mechanism of injury of many types of disease processes. This alteration is encountered when there is an imbalance between the production of reactive oxygen species (ROS) and the ability of the biological system to readily detoxify these reactive intermediates or easily repair the

1 Chapter 1: Introduction resulting damage. ROS are a family of highly reactive species that can be beneficial, as they are used by the immune system as a way to attack and kill pathogens. Nevertheless, when these species are found in excess they might cause cell damage either directly or working as intermediates in diverse signaling pathways.

1.2.1 Generation of ROS The generation of ROS is a physiological and normal attribute of any kind of aerobic life. In mammalian, under physiological conditions, cells metabolize approximately 95% of the oxygen (O2) to water, without formation of any toxic intermediates. Water if formed according to the following tetravalent reaction:

O2 + 4H+ + 4e– → 2H2O The first impressions about oxygen as an element were made by the Swedish researcher C.W. Scheele in the 18th century. However, it was only in 20th century when it was demonstrated what Scheele himself had already anticipated that O2 in its pure state at high pressure and concentration is toxic for animals, and herein for several life forms. The later was followed by new interesting discoveries, generating the controversy called until these days as “the oxygen paradox”. Several investigations from the last thirty years were needed to agree that, in normal conditions, a minimal 5% of O2 is metabolized through univalent reduction, following four different reactions or stages: •– Reaction 1: O2 + e → O2 (superoxide anion) •– Reaction 2: O2 + e → H2O2 (hydrogen peroxide)

Reaction 3: H2O2 + e → •OH (hydroxyl radical)

Reaction 4: •OH + e → H2O (water)

Indeed, the final product is still H2O. However, through these four reactions •– three highly toxic species are formed, two of them being free radicals: O2 and •OH.

H2O2 is still a highly reactive compound, but not a radical in strict sense. Table 1.1 provides the list of key ROS with their reported half-life (sec). The four stages model was the first to be discovered, and in fact it explains in general terms the mitochondrial generation of ROS in normal cellular metabolism. The intermediates do not leave the complex before the process is finished, but in some pathophysiological conditions ROS can leave the respiratory burst. In mammalian cells ROS might be formed through different pathways, either •– enzymatically or non-enzymatically. For instance, the generation of O2 , as well as

2 Chapter 1: Introduction other ROS, requires cell activation involving alteration of the cell membrane structure what in turn activates the generation of lipid peroxidation product molecules [6].

Table 1.1: The list of key ROS with their half-life [7].

ROS Half-life (sec) Singlet oxygen 10-6 Hydrogen peroxide Long Superoxide anion Long Hydroxyl radical 10-9 Alkoxy radical 10-6 Alkyl peroxy radical 1-10 Nitric oxide 1-10 Peroxynitrite Long Ozone Short

1.2.2 Pathophysiological conditions In pathophysiological conditions, the main sources of ROS include the mitochondrial respiratory electron transport chain, xanthine oxidase (XO) activation through ischemia–reperfusion, the respiratory burst associated with neutrophil activation, and arachidonic acid (AA) metabolism. Activated neutrophils produce

O2•– as a cytotoxic agent as part of the respiratory burst via the action of membrane- bound nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase on O2.

Neutrophils also synthesize nitric oxide (NO) that can react with O2•– to produce peroxynitrite anion (ONOO-), a powerful oxidant, which may decompose to form •OH. Additionally, in ischemia-reperfusion XO catalyzes the formation of uric acid with the co-production of O2•–. The enhanced O2•– released results in the recruitment and activation of neutrophils and their adherence to endothelial cells, which in turn stimulates the formation of XO in the endothelium, with further O2•– production as a positive feedback model pathway. As mentioned previously, low concentrations of ROS have physiological functions that are essential in cells, such as mitochondrial respiration, prostaglandin production pathways, host defense and sometimes ROS also act as the stimulating

3 Chapter 1: Introduction agent for biochemical processes within the cell [8]. Moreover, NO plays an important role in antagonizing the vasoconstrictor effects of Angiotensin II (Ang-II), endothelins and ROS [9]. However, ROS have well known involvement in common- shared pathophysiological models causing cell damage, either directly or through behaving as intermediates in diverse signaling pathways, including DNA damage, protein oxidation and lipid peroxidation [10]. Oxidative damage to the mitochondrial membrane can also occur, resulting in membrane depolarization and the uncoupling of oxidative phosphorylation (OXPHOS), with altered cellular respiration [11]. This can ultimately lead to mitochondrial damage, with release of cytochrome c, activation of caspases and apoptosis [12]. These consequences of oxidative stress construct the molecular basis in the development of premature skin-aging, neurodegenerative diseases, cancer, diabetes, cardiovascular diseases and autoimmune diseases. Since, the thesis focuses on loading of antioxidant (one or more) into lipid nanoparticles and their effects in combating photoaging (Ultraviolet irradiation induced skin-aging) and Alzheimer’s disease (amyloid-beta (Aβ) peptide induced degeneration of neurons), the role of OS in the pathogenesis of aforementioned diseases is discussed in some detail here.

1.2.3 Role of OS in photoaging It is well known that skin aging is caused by both intrinsic and extrinsic factors, all leading to reduced structural integrity and loss of physiological function (Fig. 1.1) [13]. Typical intrinsic factors for skin aging are ‘mitochondrial’ ROS that lead to decreased replicative ability of skin cells (keratinocytes, fibroblasts and melanocytes) and increased degradation of extracellular matrix (ECM) [14].

4 Chapter 1: Introduction

Figure 1.1: Factors responsible for skin aging [15].

1.2.3.1 Extrinsic aging Extrinsic aging is caused by environmental oxidative factors, like ultraviolet radiation (UVR) [16], cigarette smoke [17] or other pollution factors. Epidemiological studies worldwide revealed that UV exposure and tobacco smoke both independently caused largest percentage of environmentally accelerated skin aging [18, 19]. These factors add to intrinsic aging. Exposure to UVR is the key factor of extrinsic skin aging, also referred as to photoaging. The degeneration of the skin by UVR is a cumulative process and the rate of degeneration depends on the frequency, duration and intensity of solar exposure and the natural protection by skin pigmentation [20]. Photoaging accounts for as much as 80% of facial aging [21]. Photoaged skin is characterized by deep wrinkling, loss of elasticity, dryness, laxity, rough-textured appearance, teleangiectasies and pigmentation disorders and its appearance is quite noticeable from mostly intrinsically aged skin [22]. The extremity of photoaging depends on the skin type, being more notable in fair skinned people (skin types I and II) and less noticeable in people with skin type III or higher [23]. Thus, the extremity of photoaging mainly depends on the cumulative dose of UV exposure received and on the pigmentation ranking of the skin.

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1.2.3.2 UV-mediated ‘OS’ drives photoaging UVR emitted by the sun that reaches the earth’s surface has a wavelength range from 290 to 400 nm and is divided into three wavelength ranges: UVC (270–

290), UVB (290–320) and UVA (320–400) [24, 14]. The rise in UVR on earth’s surface due to stratospheric ozone depletion constitutes a serious environmental threat to the skin increasing its risk of photooxidative damage by ROS. Though ROS are part of normal regulatory circuits and the cellular redox state is tightly controlled by antioxidants, increased ROS load and loss of cellular redox homeostasis can promote carcinogenesis and photoaging. Loss of cellular redox homeostasis is causally linked to UV-mediated ROS substantially compromising the enzymatic and nonenzymatic antioxidant defense of the skin, thus tilting the balance towards a pro-oxidant state. The resulting OS causes damage to cellular components and changes the pattern of gene expression finally leading to skin pathologies such as nonmelanoma and melanoma skin cancers, phototoxicity and photoaging. The absorption of UVB photons by DNA and RNA bases and subsequent structural changes, generation of ROS following irradiation with UVB requires the absorption of photons by endogenous photosensitizer molecules. The excited photosensitizer subsequently reacts with oxygen, resulting in the generation of ROS •– 1 •– 1 including the O2 and singlet oxygen ( O2). O2 and O2 are also produced by neutrophils that are increased in photodamaged skin and contribute to the overall •– pro-oxidant state. Superoxide dismutase (SOD) converts O2 to H2O2. H2O2 is able to cross cell membranes easily and in conjunction with transitional Fe (II) drives the generation of the highly toxic •OH which can initiate lipid peroxidation of cellular membranes with the generation of carbonyls and to date poorly understood consequences [25].

1.2.3.3 UVA or UVB? Significant UVB radiation reaches our skin with the sun in overhead position (at noon) and accounts for several types of skin damage. UVB is mostly responsible for the development of sunburn. A substantial part of UVB is mostly absorbed in the stratum corneum, but attenuated UVB radiation intensity also reaches the viable epidermal cells [26], causing biological damage.

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UVA is not directly related with sunburn because UVA is roughly 1000 times less effective than UVB in creating sunburn [27]. Therefore, UVB but not UVA, is related with skin photodamage and photoaging for a long time. At same UV irradiation doses, skin exposure to UVB has much more biological impact on the skin than exposure to UVA. The radiation intensity (W/m2) of both UVB and UVA depends on various factors, like the solar zenith angle, reflection by the soil [28], elevation and the presence of clouds or dust particles in the sky that filter or scatter the radiation [29]. The irradiation dose (J/m2) is the product of the radiation intensity multiplied by the exposure time, and predominantly determines the UV-induced damage to the skin [14].

1.2.4 Role of OS in Alzheimer’s disease The brain utilizes about 20% of respired oxygen, even though it represents only 5% of the body weight. Free radicals are generated in the brain during normal intake of oxygen, during infection, and during normal oxidative metabolism of certain substrates. Oxidative damage is highly relevant in the brain because it: (1) is a post-mitotic tissue with a high energy demand; (2) is exposed to high oxygen concentrations, utilizing about one-fifth of the oxygen consumed by the body; (3) contains relatively poor concentrations of antioxidants and related enzymes; (4) is rich in polyunsaturated fatty acids (PUFA) and catecholamines that are prone to oxidation; and (5) is enriched in Iron, which accumulates in the brain as a function of age and can be a potent catalyst for oxidative species formation [30]. All of these factors with Aβ peptide, especially in the early phase of disease process, Aβ peptide could enter the mitochondria where it would increase the generation of ROS and induce OS [31]. Alzheimer’s disease (AD) is the most frequent neurodegenerative disorder associated with the onset of dementia in the elderly. It’s the most common form of dementia in adults over age 65, and the sixth leading cause of death worldwide, currently affecting nearly 36 million people, and is predicted to be double every 2 decades to at least 81 million by 2040. Worldwide, the cost of AD and dementia is estimated to be $605 billion, which is equivalent to 1% of the entire world’s gross domestic product. [32]

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Considerable data have been accumulated indicating that the brain in AD is under increased OS and this may have a role in the pathogenesis of neuron degeneration and death in this disorder. The following evidence strongly supports the role of oxidative stress in AD [6]: 1. A number of studies show that Aβ peptide and hyperphosphorylated forms of tau protein are capable of generating free radicals oxidative stress. 2. Increased brain Iron (Fe), Aluminium (Al), and Mercury (Hg) found in AD is capable of stimulating radical species. 3. Increased lipid peroxidation and decreased polyunsaturated fatty acids (PUFA) in the AD neurons, with a concomitant increase of 4-hydroxynonenal, an aldehyde product of lipid peroxidation, is found in AD ventricular fluid. 4. Increased protein and DNA oxidation is a characteristic of an AD patient brain. 5. Diminished energy metabolism and decreased cytochrome c oxidase levels are observed in AD brains. 6. Advanced glycation end products (AGE), malondialdehyde, carbonyls, peroxynitrite, heme oxygenase-1 and SOD-1 are found in neurofibrillary tangles and AGE, heme oxygenase-1, SOD-1 in senile plaques (SP) of AD brains.

1.2.4.1 Aβ a hallmark of AD pathogenesis? Aβ-peptide generated by sequential proteolytic cleavage of Aβ-protein precursor protein (APP), a large transmembrane glycoprotein that is initially cleaved by the β-site APP cleaving enzyme 1 (BACE1) and subsequently by membrane bound proteolytic enzymes, called secretases in the transmembrane domain [33-35]. β- and γ-secretase, are the two main proteases that cleave APP at the amino and carboxyl-terminus of the Aβ peptide, respectively and are hence, directly responsible for Aβ peptide generation. It has been suggested that Aβ peptides might have a toxic effect, and impair synaptic plasticity long before its SP formation and deposition [36]. Aβ appears to promote neuronal death, at least in part by the generation of oxidative stress. Indeed, this process seems to be dependent of the predominant β- sheet conformation. It has been proposed that soluble Aβ might be the initial event that triggers the neurodegenerative cascade. However, whether OS is cause or consequence of the amyloid deposition is still in dispute [37].

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Senile plaques can be differentiated into two histological forms; a) diffuse plaques of amorphous extracellular neurite lacking, deposits of Aβ or b) neuritic plaques, composed of extracellular deposits of insoluble Aβ surrounded by dystrophic neurites, activated astrocytes and microglia. Recent studies suggest that Aβ can impair synaptic plasticity through mechanisms that might contribute to cognitive decline in AD. Evidence is mounting that Aβ oligomers can mediate these effects, possibly accounting for why plaque number is such a poor predictor of cognitive impairment. The deposit of SP is a frequent finding in the elder, not always linked to cognitive decline. Early studies suggested that there was a direct relation between the deposit of SP and cognitive decline [38]. Interestingly, there is a direct link between the function of cholinergic neurons, the cerebrovascular system and amyloid pathology. Acetylcholine is a potent vasodilator affecting cerebral blood flow [39]. Amyloid deposits are also found around cortical vessels where they promote Aβ deposition that consequently contributes to brain hypoperfusion or vice versa [40]. Also, endothelial dysfunction of cells participating in the BBB may contribute to inadequate supply of nutrients to the brain. Indeed, increased hypertension, leading to augmented BBB permeability in cortex and hippocampus has been directly associated with Aβ deposit in murine models [41]. Current evidence also supports a role for cholinergic innervation in the non-amyloidogenic maturation of APP, whereas amyloidogenic related peptides depress the activity of cholinergic neurons. These cholinergic neurons innervate the hippocampus and neocortex, being central to the loss of cognitive function in AD. It has been shown that Aβ peptides may have acute detrimental effects on acetylcholine synthesis and release and are neurotoxic on the long term [40]. For a long time researchers have been trying to elucidate If Aβ and Tau modifications are related, or represent parallel mechanisms in the pathogenesis of AD. There’s evidence to support that Aβ toxicity can induce Tau phosphorylation and production of 17 kD Tau fragments [42]. Moreover, there’s evidence that aggregated soluble Aβ peptide and not fibrils per se are necessary for OS and neurotoxicity associated with Aβ peptide. Given the centrality of Aβ to the pathogenesis of AD, and the notable OS available in AD brain, an Aβ-associated OS model for neurodegeneration in AD provides a framework that unites these observations (Fig. 1.2).

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Figure 1.2: Aβ-associated OS and neurotoxicity in AD brain [43].

1.2.4.2 Aβ and mitochondria There is significant evidence that links mitochondrial oxidative damage to AD development and progression. Several in vitro studies suggest mitochondrial involvement in AD pathology. For example, Aβ peptides require functional mitochondria to induce cell toxicity [44]. Casley et al. 2002, [45, 46] have shown that Aβ significantly reduces states 3 and 4 of mitochondrial respiration. Aβ also inhibits the activities of cytochrome oxidase, α-ketoglutarate dehydrogenase, and pyruvate dehydrogenase. Kim et al. [47] have shown that the addition of Aβ to isolated mitochondria from rat brain directly induces the release of cytochrome c and the swelling of mitochondria. These researchers have also suggested that in AD, Aβ may accumulate intracellularly due to abnormal APP processing and that the

10 Chapter 1: Introduction intracellular Aβ may exert neurotoxicity by interacting with mitochondria, resulting in oxidative damage and apoptosis. It is considered that free radicals in AD are generated in the mitochondrial matrix, in both sides of the inner membrane of mitochondria and in the outer mitochondrial membrane. The mechanism underlying Aβ-induced induction of free radicals and the exact site of induction (complex 1 or 3, or the TCA cycle) are not yet well established and are currently under investigation in many laboratories. In summary, there is good evidence that Aβ can develop neurotoxic effects under certain conditions. Independent of the particular cell death pathways that are induced by Aβ aggregates, OS appears to be a major player at various stages.

1.3 Antioxidative strategy to lower OS In 21st century, demand for intake of antioxidant food or dietary antioxidant increasing with the hope that they keep body healthy and free from diseases [48, 49]. The potential beneficial effects of antioxidants in protecting against disease have been well established. It is increasingly thought that antioxidants may play a vital role in helping to defend against OS and damage induced by free radicals. Therefore, dietary components with antioxidant properties are important for the protection against OS injury to the body. Antioxidants can be defined as “any substance (man-made or natural, enzymatic or non-enzymatic) that, when available at small concentrations in contrast with that of an oxidizable substrate (a reagent which undergo oxidation) significantly delays or inhibits oxidation of that substrate” [50]. In body antioxidants (exogenous, endogenous or both) do their job (against OS) at three different levels (1) prevention – keeping formation of reactive species to minimum, (2) interception – scavenging reactive species either by using catalytic and non-catalytic molecules and (3) repair – repairing damaged target molecules [51] and therefore it is logical to assume that they are useful in OS-related disease. Moreover, antioxidants may function as immune modulators and can be used for prophylaxis or therapy of certain diseases along with the mainstream therapy. Supplements of exogenous antioxidants can act directly to quench the free radical or free radical reactions, prevent lipid peroxidation and also boost the endogenous antioxidant system and hence deliver the prophylactic or therapeutic activity. Many novel approaches and significant findings have come to light in the last few years.

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The natural food, spices and medicinal plants are rich sources of antioxidants and can be prime source to treat such diseases. Several antioxidants like epigallocatechin-3-O-gallate, lycopene, ellagic acid, coenzyme Q10 (CoQ10), indole- 3-carbinol, resveratrol, genistein, spin trap, quercetin, vitamin C and vitamin E have been found to be pharmacologically active as prophylactic and therapeutic agents. Therefore targeting OS or boosting the endogenous levels of antioxidants by the use of antioxidants and or is likely to have beneficial outcome in the management of several disorders [1, 52].

1.3.1 Antioxidants in prevention of photoaging As the outermost barrier of the body, the skin is directly exposed to a pro- oxidative environment (UVR and air pollutants) [53]. These external inducers of oxidative attack lead to the generation of ROS and other free radicals. To counteract the harmful effects of ROS, the skin is equipped with antioxidant systems consisting of a variety of primary (preventive, e.g. vitamin C) and secondary (interceptive, e.g. vitamin E) antioxidants forming an ‘antioxidant network’. Human skin contains the lipophilic antioxidants vitamin E (tocopherols and tocotrienols), CoQ10 and carotenoids, as well as the hydrophilic antioxidants vitamin C (ascorbate), uric acid (urate) and glutathione (GSH) [53]. The fig. 1.3 summarizes how human skin cells respond to OS. Generally, higher antioxidant concentrations were found in the epidermis as compared to the dermis. Some antioxidants are also present in the stratum corneum. α-Tocopherol was shown to be the predominant antioxidant in human stratum corneum, whereas the outermost layers seem to contain lower amounts than the layers in closer proximity to the nucleated epidermis [54]. Particularly, the antioxidants contained in the stratum corneum were demonstrated to be susceptible to UVR. For example, a single suberythemal dose of UVR depleted human stratum corneum α-tocopherol by almost half, while dermal and epidermal α- tocopherol were only depleted at much higher doses [54]. The high susceptibility of stratum corneum vitamin E to UVR may be, at least in part, due to a lack of co- antioxidants in this outermost skin layer. CoQ10, the most abundant ubiquinone found in human skin, was undetectable in human stratum corneum.

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Highly reduced Resting cell Generally reduced environment (high concentration of GSH, ascorbate, etc). Some moreoxidized microenvironments e.g. endoplasm reticulum

Mild oxidative stress

Proliferation stimulated rise in Ca++ and increased phosphorylation

Greater oxidative stress Transcription factor activation adaptive Oxidative damage level rises; release of response transition metal ions that catalyze free-radical Increasing levels of reactions is an early stage in oxidative damage. protective systems Some may bind to DNA to damage it by site- (e.g.chaperones, specific ·OH formation. Rise in free Ca++ antioxidant enzyme, HO- occur due to oxidative damage to the ion 1, Ferritin to sequester transporter that normally keep it low iron) render cell more resistant to subsequent insults. Cell cycle halts to allow repair of DNA damage

More oxidative damage Mitochondrial damage by ROS and by excessive free Ca++ can be mitochondrial permeability transition and/or cytochrome c release. Excessive DNA damage (via p53) halts the cell cycle or initiates apoptosis.

Greater oxidative stress

Severe oxidative damage Mitochondrial damage or excessive damage (via) p53 often initiates apoptosis Apoptosis halted, survival badly-damaged cells or necrotic cell death, the latter Intense oxidative releasing metal ions and stress other toxins to spread injury to surrounding cells Highly oxidized Shut-down of caspases by oxidation of their active site – SH groups

Figure 1.3: How skin cells respond to OS [7].

Additionally, ascorbate, the major hydrophilic co-antioxidant that is capable of recycling photooxidized α -tocopherol [55], seems to be present only at very low levels in human stratum corneum. Consequently, direct depletion of α -tocopherol and formation of its radical may also affect other endogenous antioxidant pools. In

13 Chapter 1: Introduction addition to direct depletion by UVR, skin α -tocopherol levels may be consumed as a consequence of its chain-breaking antioxidant action. Also, the hydrophilic antioxidants were shown to be sensitive to UVR. However, ascorbic and uric acid were less susceptible to UVR than α -tocopherol or CoQ10 as was shown using cultured human skin equivalents [56]. Further, epidermal GSH levels in hairless mice were depleted by 40% within minutes after UVB exposure but returned to normal levels after half an hour [57]. Moreover, skin contains enzymatic antioxidants such as catalase, superoxide dismutase, GSH peroxidase and GSSG reductase, which were also shown to be susceptible to UVR [53].

1.3.1.1 Topical application of a single antioxidant Apart from using chemical and/or physical sunscreens to diminish the intensity of UVR reaching the skin, supplementation of the skin with antioxidants and thereby strengthening its antioxidative capacity is an emerging approach in limiting ROS-induced skin damage [53]. Topical application of antioxidants, such as vitamin E, provides an efficient means of increasing antioxidant tissue levels in human epidermis and dermis [58]. As the most susceptible skin layer for UVR- and ozone-induced depletion of cutaneous antioxidants, the stratum corneum may particularly benefit from an increased antioxidant capacity due to topical supplementation.

1.3.1.2 Topical application of antioxidant combinations The cutaneous antioxidant system is complex and far from being completely understood. As mentioned above, the system is interlinked and operates as an ‘antioxidant network’ [53]. α -Tocopherol is readily regenerated from its radical at the expense of reductants like ascorbate. Ascorbate itself can be regenerated by GSH. Thus, an enhanced photoprotective effect may be obtained by applying appropriate combinations of antioxidants. As was shown in a human study, the co- application of vitamin E and vitamin C resulted in a much more pronounced photoprotective effect as compared to the application of one single antioxidant alone in the identical vehicle [59]. The list of single and mixture of exogenous antioxidants with photoprotective or damage protective effects are listed in Table 1.2 and 1.3, respectively [60]

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1.3.2 Antioxidants in prevention of AD Of the therapeutic approaches to AD, one of the most thought of, yet underestimated, is the role of antioxidants. The brain is acutely influenced and maintained by our diet. Approximately 60% of this organ’s dry weight is composed of lipids [61]. The aging cellular membrane is characterized by higher levels of cholesterol, decreased cholesterol turnover and decreased levels of PUFA. This may be related to poor uptake of PUFA over the blood brain barrier (BBB), decreased incorporation into the membrane and/or reduced enzymatic activity [62]. Fats obtained from our diet directly affect the composition and structure of cell membranes [63]. Obtaining data from the Washington Heights-Inwood Columbia Aging Project (WHICAP), a total of 2,258 community-based non demented individuals were evaluated regarding the following of a mediterranean diet consisting of a higher intake of vitamin C, vitamin E, flavonoids, unsaturated fatty acids, fish, higher levels of vitamin B12 and folate, modest to moderate ethanol intake, and lower total fat consumption. Adherence to mediterranean diet was associated with a significant reduction in the risk for AD [64]. Even though epidemiological studies have reported conflicting data, the evidence to date supports a contribution of antioxidants in the risk, and particularly prevention of AD. Increasing evidence reveals that dietary components with antioxidant properties are far from being a mere energy substrate, rather, stimulating neuronal plasticity, and ameliorating ongoing neurodegenerative processes [65]. Table 1.4 provides the list of exogenous antioxidant molecules as therapeutic agents for AD management [66].

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Table 1.2: List of single exogenous antioxidants with photoprotective or damage protective effects.

Antioxidants Outcome of the study Ref. Ascorbic acid Ascorbic acid found to be a photoprotectant when applied 67 to mice and pig skin before exposure to UVR. Ascorbic acid led to a significant and remarkable reduction 68 of the UVB-induced damage. Vitamin E UV-induced vitamin E depletion. 69 The interaction of vitamin E with the eicosanoid system 70 resulted in an anti-inflammatory effect and thereby complemented the photoprotective effects of other antioxidants in the skin. Vitamin E has skin barrier-stabilizing properties. 71 Lycopene Lycopene contributed to life-long protection against 72 harmful UV radiation. Carotenoids Carotenoids are efficient in photoprotection, scavenging 73 (carotene and singlet oxygen, and peroxyl radicals. β-carotene) Dietary β-carotene has effect on wrinkles and elasticity, 74 procollagen gene expression, and UV-induced DNA

damage in human skin.

CoQ10 CoQ10 protects against oxidative stress-induced cell death. 75

CoQ10 was shown to reduce UVA-induced MMPs in cultured human dermal fibroblasts. 76 GSH GSH a photoprotective agent in skin cells. 57 Resveratrol Application of resveratrol to the skin of hairless mice 77 effectively prevented the UVB-induced increase in skin thickness and the development of the skin edema. Resveratrol proved to be a photoprotective agent and 78 reduced cell death in UVB damaged skins. Green tea Topical treatment or oral consumption of green tea 79 polyphenols (GTP) inhibited chemical carcinogen- or UV radiation-induced skin carcinogenesis.

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Antioxidants Outcome of the study Ref. Green tea or Oral administration of green tea or caffeine in amounts 80 caffeine equivalent to three or five cups of coffee per day to UVB- exposed mice increased levels of p53, slowed cell cycling, and increased apoptotic sun burn cells in the epidermis. Sylimarin Silymarin strongly prevents both photocarcinogenesis and 81 skin tumor promotion in mice. Genistein Antioxidant and anticarcinogenic effects on skin. 82 Cocoa Dietary flavanols from cocoa contributed to endogenous 83 photoprotection, improve dermal blood circulation, and affect cosmetically relevant skin surface and hydration variables. Ferulic acid FAEE give a sun protection factor (SPF) similar to that of 84 ethyl ester benzymidazilate, a filter permitted in European Union

(FAEE) (EU).

Table 1.3: List of combination exogenous antioxidants with photoprotective or damage protective effects.

Antioxidant mixture Outcome of the study Ref.

Ferulic acid, Vitamin C Provided meaningful synergistic protection 85 and E against oxidative stress in skin, photoaging & skin cancer. Caffeic and ferulic acids Successfully employed as topical protective 86 agents against UV radiation-induced skin damage Oral vitamin E and beta- Reduced UVR-induced OS in human Skin. 87 carotene supplementation Carotenoids and Scavenging reactive oxygen species generated 88 tocopherols during photooxidative stress. β-carotene, lutein, and UV irradiation induced intensity of erythema was 89 lycopene diminished. Tomato extract Reduction in erythema formation following UV 90 irradiation.

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Antioxidant mixture Outcome of the study Ref. Quercetin, hesperetin and Protective agents in certain skin diseases caused, 91 naringenin initiated, or exacerbated by sunlight irradiation. α-Tocopherol and Minimal Erythemal Dose (MED) increased 92 ascorbate markedly after intake of the combination of α - tocopherol and ascorbate. Vitamin C, vitamin E, Vitamin C, vitamin E, and carnosic acid showed 93 lycopene, photoprotective potential human dermal beta-carotene, the fibroblasts exposed to UVA. rosemary polyphenol, and carnosic acid Lycopene, β-carotene, α- Many parameters of the epidermal defense 94 tocopherol, and selenium against UV-induced damage were significantly improved. β-Carotene, lycopene, Significant increase of melanin concentrations in 95 tocopherol, and ascorbic skin was found. acid Carotenoids (β-carotene A selective protection of the skin against 96 and lycopene), vitamins irradiation was confirmed. C and E, selenium, and proanthocyanidins

Table 1.4: List of exogenous antioxidant molecules as therapeutic agents for AD management

Antioxidants Outcomes in AD treatment Ref. Resveratrol Promoted clearance of Aβ-peptides. 97 Inhibited Aβ oligomeric cytotoxicity 98 Neuroprotective effects against Aβ-induced 99 neurotoxicity.

Inhibited Aβ-induced neuronal apoptosis. 100

N-acetyl-L- Increased GSH levels. 101 cysteine Reduced protein oxidation.

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Antioxidants Outcomes in AD treatment Ref.

N-acetyl-L- Modulation of Ab processing and production via 102 cysteine inhibition of g-secretase. 103 Idebenone Protected hippocampal neurons against Aβ-peptide- 104 induced neurotoxicity in rat primary cultures.

Vitamin E Reduced lipid peroxidation and Aβ deposition in the 105 brain.

Vitamin E supplementation reduced the in vitro oxidation 106 of cerebrospinal fluid (CSF) lipoproteins.

Ferulic acid Reduced Aβ and IL-1b levels. 107 Reduced brain parenchymal and cerebral vascular Aβ 108 deposits as well as abundance of various Aβ species

decreased Aβ production and reduced amyloidogenic APP proteolysis.

Vitamin D Regulates levels of GSH. 109 Improved phagocytosis of Aβ-peptides and inhibited 110 fibrillar Aβ-induced apoptosis.

Melatonin Prevented Aβ-induced increases in intracellular Ca2+ and 111 lipid peroxidation. 112 Increased SOD, catalase and GSH levels. CoQ10 Prevented a decrease in mitochondrial transmembrane 113 potential and reduced mitochondrial ROS generation. Attenuated Aβ overproduction and intracellular Aβ 114 deposits in AD cells and mouse models. Reduced astrogliosis, synaptic loss and caspase activation 115 in AD mouse models. MitoQ Extended lifespan, delayed Aβ-induced paralysis, 116 ameliorated depletion of the mitochondrial lipid cardiolipin and protected complexes IV and I of the ETC in Caenorhabditis elegans treated with Aβ

Lipoic acid Ameliorated cognitive processes and declines in AD 117 human brain and mouse models. 118 Decreased mitochondrial oxidative stress and damage

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1.4 Antioxidant delivery approaches

1.4.1 Conventional delivery Conventional dosage forms such as tablets, capsules and liquid orals through the most acceptable oral route are easy to formulate and are inexpensive, but these dosage forms are experiencing serious disadvantages in the delivery of antioxidants. At times, these dosage forms lead to loss of efficacy of the active agent which may be due to various reasons like poor bioavailability, first pass effect or instability of the active agent in GI tract (Fig. 1.4). Currently, there are lots of antioxidant products on market which have been formulated into these conventional dosage forms, with vitamins leading the group. Vitamins have been formulated mainly into tablets and capsules. Generally, these agents were found in combinations rather than individual products. Antioxidants like idebenone, resveratrol, PBN, quercetin, lycopene, ellagic acid and CoQ10 are difficult to deliver by these conventional dosage forms because of poor water solubility, lack of targeting to cell organelle or part of body, insufficient bioavailability (due to first-pass metabolism), fluctuating plasma levels and undesirable side effects are the main and common problems. Many of these are not formulated in the pure forms; instead the plant extracts of these agents are generally formulated and marketed. These are rarely formulated as liquid orals considering their instability in the solution form. Addressing these problems with the help of conventional dosage forms is difficult and help of novel drug delivery systems (NDDS; colloidal carriers) is a must to maximize the potential roles of antioxidants in prophylaxis and therapy. Different types of strategies were implicated by different groups to achieve successful delivery of these antioxidants [1]

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Figure 1.4: Comparison of antioxidant in conventional delivery (CD) and in NDDS. Antioxidant in CD has to go through various phases like liberation, absorption, distribution, metabolism, elimination and response (LADMER). The problems associated with CD at any stage in LADMER can be overcome by implication of NDDS [1].

1.4.2 Colloidal carriers Colloidal carriers are promising novel antioxidant delivery systems because they fulfil the requirements mentioned above and depicted in Fig 1.4. Nanosized carriers are treated as hopeful means to increase the solubility, surface area, absorption and therefore the bioavailability / permeability of poorly water-soluble active ingredients belonging to the classes II and IV in the biopharmaceutical classification system (BCS) [119]. The common characteristic of all colloidal carriers is the submicron particle size (50-1000 nm). Nanometric carriers vary in terms of ingredients, rigidity, stability, release properties and ability to incorporate materials with different solubilities. There is no universal nanometric carrier system that can be employed for all applications, although some are more versatile than others. The choice of

21 Chapter 1: Introduction which carrier system to use depends on the characteristics (size, solubility, charge, etc.) of the antioxidants to be incorporated, safety and efficiency of the antioxidant- carrier complex, as well as the intended application and route of administration of the complex to the body. Moreover, optimal formulations have to be chosen carefully for each antioxidant, according to the features of the nanometric carriers. With respect to manufacturing such complexes, possibility of mass production with minimum consumption of material, solvents, equipment and time should seriously be considered [120]

1.4.2.1 Overview of colloidal carrier systems Colloidal carrier system can be broadly categorized as polymer, lipid and surfactant-based systems or a combination of these. Typical examples for carrier systems are micro and nanoemulsion, liposomes, niosomes, nanocapsules, nanosponges, solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs) etc. Examples of combined carrier systems are micelles and nanoparticles that can be prepared from polymer-surfactant mixtures and polymer-lipid conjugates respectively. The polymeric nanoparticles have their own disadvantages such as use of toxicologically harmful reactive cross-linkers and carcinogenic monomers in the production, complete removal of these constituents is difficult [121] and slow degradation of the polymer results in its accumulation and may produce toxic metabolites [122], hence restricting their applications as a drug delivery system. Lipid-based colloidal carriers (particularly liposomes, SLNs and NLCs) have been introduced to overcome these toxicological issues exhibited by polymeric nanoparticulate systems and hence prominent research has been carried out over the past few decades on lipidic systems. Although liposomes have been the hallmark of lipid-based colloidal carriers for site- specific delivery of drugs, there are, however, some drawbacks are associated with them such as storage stability difficulties, Incorporation of a drug into the phospholipid bilayer can decrease the carrier stability, rapid degradation by the gastrointestinal pH or by intestinal enzymes and the bile salts if taken orally, burst release of drug, limitations associated with large scale manufacturing and numerous to say [123].

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All of these points make liposomal colloidal carrier not optimal as a pharmaceutical drug carrier system. Hence, nanoparticles based on lipid components other than phsopholipids such as SLNs and NLCs (commonly known as lipid nanoparticles) are alternative approaches for safe and efficient delivery of drugs. Also, it is expected that these lipid nanoparticles may allow for higher control over drug release and delivery of therapeutics, which may not efficiently load in to liposomes [124].

1.5 Lipid nanoparticles

1.5.1 History and scope The first lipid particles were produced by the research group of Speiser, the father of the nanoparticles, in Zurich [125]. High-speed stirring was applied for the production of an o/w emulsion between a melted lipid phase and a hot aqueous surfactant solution. The obtained emulsion was then cooled and the inner lipid phase formed solid particles. However, in general, the use of stirring techniques has some drawbacks, such as the relatively broad size distribution of particles and the fact that relatively high concentrations of surfactant molecules are usually required to obtain a mean particle diameter in the nanometer range. The product developed by Speiser was called “lipid nanopellets” and was intended for oral delivery. The patent obtained by Speiser was not followed up by the owner Rentschler, and patent protection no longer exists in a number of countries [126]. A similar process was developed and patented by Domb, who prepared particle dispersions applying a sonication procedure. The product developed by Domb was called “lipospheres” [127], and they also found no broad application in pharmaceutical products. In 1991, the patent application of the first generation of lipid nanoparticles— SLNs—was submitted describing the nanoparticle production by high-pressure homogenization (HPH) (128) and also via microemulsion technique [129]. Since then use of lipid nanoparticles as drug carriers has been greatly exploited ever since

(e.g. Cutanova Cream nano Repair Q10, NanoLipid Restore CLR, IOPE SuperVital, etc.). Currently more than 20 research groups are working on lipid nanoparticles world wide, estimated by the published articles. This proves the increasing interest in the field of lipid nanoparticles. Lipid nanoparticles have been investigated for

23 Chapter 1: Introduction various pharmaceutical applications like parenteral [130, 131], pulmonary [132, 133] oral [134, 135], topical [136, 137], ocular [138, 139] administration.

1.5.2 What exactly are lipid nanoparticles? Lipid nanoparticles in solid state are derived from o/w emulsions, simply replacing the liquid lipid (oil) by a solid lipid (SLNs; Fig. 1.5a), mixing appropriate ratios of solid lipid and liquid lipid (NLCs; Fig. 1.5b), conjugation of drug with lipid molecule and transformed into a more lipophilic and insoluble molecule (Lipid-drug conjugate, LDC; Fig. 1.5c), and use of hydrophobic polymer as a core material and lipid/lipid shells (polymer-lipid hybrid nanoparticles, PLNs; Fig. 1.5d). A schematic diagram depicting different types of lipid nanoparticles encapsulating different kinds of drugs is shown in Fig. 1.5.

Figure 1.5: Different types of lipid nanoparticles [140].

Both SLNs and NLCs provide a suitable environment for entrapment of lipophilic drugs because of their hydrophobic core. This is important, as nearly 4 out of 10 drug candidates are lipophilic in nature [141] In this thesis, the efforts were made to entrap lipophilic drugs and not the hydrophilic one; hence, the LDCs and PLNs (suitable drug carriers for hydrophilic drugs) detailed composition, models of drugs incorporated, different production and characterization techniques are not mentioned / discussed in this thesis.

1.5.2.1 Solid lipid nanoparticles SLNs were developed at the beginning of the 1990s and are often referred to as the first generation of lipid nanoparticles. As mentioned SLNs are derived from o/w emulsions by replacing liquid lipids with a lipid matrix that is solid (from 0.1 % (w/w) to 30 % (w/w)) at body temperature and stabilized by the use of surfactants

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(preferably 0.5 % (w/w) to 5 % (w/w)) with particle size ranging 50 - 1000 nm. [142].

1.5.2.2 Nanostructured lipid carriers Evolved from the SLNs, NLCs are second generation of lipid nanoparticle technology; the particles are prepared using binary mixture of a solid lipid and a spatially distinct liquid lipid (preferably in a ratio of 70:30 up to a ratio of 99.9:0.1). The overall solid content of NLCs could be increased as high as 95 %. [143]. Due to NLCs binary mixture as a main composition they do not form a perfect crystal. The imperfections present in the solid matrix accommodate the drugs either as molecules or as amorphous crystals [144]. NLCs were developed to overcome the shortcomings associated with the SLNs discussed in following section and are depicted in Fig. 1.6 [145]

1.5.3 Major shortcomings associated with SLNs

1.5.3.1 Very low pay-load for a number of drugs Sufficiently high drug-load is achieved only when a less ordered solid lipid matrix is available to accommodate the drug. The presence of drug can be found in between the lipid layers or fatty acid chains. Some amount can also be found in imperfections. Due to conversion of lipid matrix into perfect β modification, very limited amount of drug gets accommodated into the lipid matrix [146].

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Figure 1.6: Difference between SLNs and NLCs particle matrix structure [145].

1.5.3.2 Drug expulsion during storage SLNs are prepared using blend of solid lipids only. After preparation, the particles get crystallize partially in higher energy modifications viz. α and β’. During storage these high energy modifications get transformed into lower energy modifications which are more ordered and generally called as β modification. This more ordered conversion leads to reduction in crystal lattice imperfections thereby leading to expulsion of drug from lipid matrix [147]

1.5.3.3 High water content of SLNs dispersions The lipid concentration of 0.1 to 30 % w/w is the optimum concentration for formation of SLNs as above 30 % concentration, formation of bicoherent creams takes place. The corresponding water content of 99.9 to 70 % potentially creates problem during incorporation of SLNs dispersion into cream, thus creating a need to reduce the water content of SLNs [148].

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1.6 NLCs

1.6.1 Compositions NLCs are typically composed of lipids (solid and liquid), surfactant(s), co- surfactant (optional) and active ingredients (typically drugs). The lipids used in the production of lipid nanoparticles are physiological lipids. Based on their structural diversity, lipids used in the production are broadly categorized into fatty acids, fatty esters, fatty alcohols, triglycerides or partial glycerides. A few researchers have also reported the use of waxes as solid lipid in the preparation of lipid nanoparticles [149]

1.6.1.1 Lipids The lipid, itself, is the main ingredient of lipid nanoparticles that influence their drug loading capacity, their stability and the sustained release behavior of the formulations. Most of these lipids, with the notable exception of cetyl palmitate, are approved as generally-recognised-as-safe (GRAS) and are physiologically well- tolerated. Selection of appropriate lipids is essential prior to their use in preparation of lipid nanoparticle dispersions. Although there are no specific guidelines, empirical values, such as the solubility of drug in the lipid have been proposed as suitable criteria for selection of an appropriate lipid [150]. Examples of the lipids (including cationic lipids) which have been used in the preparation of NLCs are listed in Table 1.5.

1.6.1.2 Surfactants Surfactants (also known as surface-active agents or emulsifiers) form the other critical component of the lipid nanoparticle formulation. Surfactants are amphipathic molecules that possess a hydrophilic moiety (polar) and a lipophilic moiety (non-polar), which together form the typical head and the tail of surfactants. At low concentrations, surfactants adsorb onto the surface of a system or interface. They reduce the surface or interfacial free energy and consequently reduce the surface or interfacial tension between the two phases. [151,152]. The relative and effective proportions of these two moieties are reflected in their hydrophilic lipophilic balance (HLB) value.

27 Chapter 1: Introduction

Table 1.5: List of lipids used in preparation of NLCs [151].

Fatty lipids Waxes Dodecanoic acid Cetyl palmitate Myristic acid Carnauba wax Palmitic acid Beeswax Stearic acid Hydrogenated coco-glycerides Monoglycerides Liquid lipids Glyceryl monostearate Soya bean oil Glyceryl hydroxystearate Oleic acid Glyceryl behenate Medium chain triglycerides (MCT)/caprylic- Diglycerides and capric triglycerides Glyceryl palmitostearate α-tocopherol/Vitamin E Glyceryl dibehenate Squalene Hydroxy octaco sanylhydroxystearate Isopropyl myristate Triglycerides Cationic lipids Caprylate triglyceride Stearylamine (SA) Caprate triglyceride Benzalkonium chloride (alkyl dimethyl benzyl Glyceryl tristearate/Tristearin ammonium chloride, BA) Glyceryl trilaurate/Trilaurin Cetrimide (tetradecyl trimethyl ammonium Glyceryl trimyristate/Trimyristin bromide, CTAB) Glyceryl tripalmitate/Tripalmitin Cetyl pyridinium chloride (hexadecyl Glyceryl tribehenate/Tribehenin pyridinium chloride, CPC) Dimethyl dioctadecyl ammonium bromide (DDAB) N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium chloride (DOTAP)

Surfactants used in the preparation of lipid nanoparticle preparations play two quite distinct and important roles • Surfactants disperse the lipid melt in the aqueous phase during the production process • Surfactants stabilize the lipid nanoparticles in dispersions after cooling [151].

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Surfactants can be broadly categorized into three classes based on their charge: ionic, non-ionic and amphoteric. Table 1.6 lists a few surfactants from each class used in the preparation and stabilization of lipid nanoparticles. Amongst the ionic surfactants, cationic surfactants are more toxic than anionic or amphoteric surfactants. Therefore, the surfactants arranged in the decreasing order of toxicity are: cationic > anionic > non-ionic > amphoteric [151].

Table 1.6: List of surfactants used in preparation of NLCs [151].

Surfactants Ionic surfactants Non-ionic surfactants Sodium cholate Tween 20 Sodium glycocholate Tween 80 Sodium taurocholate Span 20 Sodium taurodeoxycholate Span 80 Sodium oleate Span 85 Sodium dodecyl sulphate Tyloxapol Amphoteric surfactants Poloxamer 188 Egg phosphatidylcholine (Lipoid E PC S) Poloxamer 407 Soy phosphatidylcholine (Lipoid S 100, Lipoid S PC) Poloxamine 908 Hydrogenated egg phosphatidylcholine (Lipoid E PC- Brij78 3) Tego care 450 Hydrogenated soy phosphatidylcholine (Lipoid S PC- Solutol HS15 3, Co-surfactants Phospholipon 80 H, Phospholipon 90 H) Butanol Egg phospholipid (Lipoid E 80, Lipoid E 80 S) Butyric acid Soy phospholipid (Lipoid S 75)

1.6.1.3 Other agents Apart from lipids and surfactants, lipid nanoparticle formulations can also contain a number of other ingredients including counter-ions and surface modifiers. Table 1.7 lists some of the counter-ions and surface-modifiers used in lipid nanoparticle preparation [151].

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Table 1.7: List of miscellaneous agents used in preparation of NLCs [151].

Counterions Surface modifiers Organic salts Dipalmitoyl-phosphatidyl-ethanolamine conjugated

Mono-octyl phosphate with polyethylene glycol 2000 (DPPE-PEG2000) Mono-hexadecyl phosphate Distearoyl-phosphatidyl-ethanolamine-N-

Mono-decyl phosphate poly(ethylene glycol) 2000 (DSPE-PEG2000)

Sodium hexadecyl phosphate Stearic acid-PEG 2000 (SA-PEG2000) Ionic polymers α-methoxy-PEG 2000-carboxylic acid-α-lipoamino

Dextran sulphate sodium salt acids (mPEG2000-C-LAA18) α-methoxy-PEG 5000-carboxylic acid-α-lipoamino

acids (mPEG5000-C-LAA18)

1.6.2 Models of drugs incorporated in NLCs Different models have been described in the literature for how active molecules can be incorporated into NLCs [153]. The type of NLCs depends on various factors such as the ration between the active ingredient and lipid content, chemical and physical nature of the active ingredient and lipid, the solubility of actives in the melted lipid, nature and concentration of surfactants, type of production (hot vs. cold HPH), and the production temperature [153], As the preparation cools, the lipid phase will solidify, and depending on the lipid to active ratio and their melting point, three different scenarios become possible. Fig. 1.7 provides a schematic representation of type of NLCs formed. Type I: If the ratio is ideal, the lipid and active will precipitate together and create a nanoparticle with a constant concentration as a function of depth from the particle surface. Another reason is the melting point of lipid and drug. When the melting point of lipid and melting point of drug are similar then the corresponding drug incorporation is of solid solution or homogenous matrix type (i.e. Type I). Type II: A second scenario occurs when there is relatively too much lipid; in such Type II nanoparticles, the lipid precipitates first, thus lowering the lipid content of the mixture until the ideal lipid to active ratio is reached and a constant content of active precipitates within the lipid. When the melting point of drug is lower than that of the melting point of the drug then the morphology that is obtained is drug- enriched shell (i.e. Type II). Finally

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Type III: if there is relatively too much active, the active will precipitate first, lowering the active content until the ideal lipid to active ratio is reached and a constant content of active precipitates with the lipid. Moreover, higher the melting point of the drug than the lipid, the drug will get incorporated in the core and Such Type III nanoparticles will be known as active-enriched on the inside. Actives having higher water solubilities tend to create Type II NLCs. If the solubility of the active in water increases at elevated water temperatures, a reasonable to substantial amount of active will escape to the water phase. When the temperature is subsequently lowered—and with it the solubility of the active in the water phase, the active ingredient will partition back into the partially formed nanoparticles. Therefore, the outer layer will be active enriched on the outside, i.e., a Type II NLCs [153]. The NLCs type is important because it determines the release characteristics of the particle. Type I NLCs (Fig. 1.7) give a sustained, constant and relatively long release of active (drug is released by diffusion and/or by degradation of the lipid matrix). Type II NLCs deliver a relatively short, burst release of active. Finally, Type III NLCs provide a slow release [153].

1.6.3 Production techniques Several approaches for the preparation of lipid nanoparticle dispersions have been reported since these carriers were first described in early 1990s [129, 154, 155, 146]. The preparation technique has a significant role in the performance of the colloidal formulation. The choice of preparation technique for lipid nanoparticle dispersions may be influenced by: .

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Figure 1.7: Types of NLCs formed [153, 154].

• Physicochemical properties of the drug to be incorporated • Stability of the drug to be incorporated • Desired particle characteristics of the lipid nanoparticle dispersion • Stability of the lipid nanoparticle dispersion • Availability of the production equipment A brief description of a variety of production techniques is discussed here. Table 1.8 gives a brief outline of the mechanisms involved in lipid nanoparticle formation by various production techniques.

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Table 1.8: Mechanism involved in lipid nanoparticles formation by various production techniques [156].

Production technique Mechanism of particle formation High pressure High mechanical shear due to strong turbulent eddies homogenization Lowering of pressure across the valves of homogenizers Strong cavitation forces Solvent evaporation Lipid crystallization due to solvent evaporation in an anti-solvent Double emulsion Lipid crystallization due to solidification of emulsion Solvent diffusion Lipid crystallization due to diffusion of solvent from internal organic phase to external aqueous phase Solvent injection (or Lipid crystallization due to rapid diffusion of solvent displacement) from internal organic phase to external aqueous phase. Phase inversion Spontaneous inversion of o/w emulsion to w/o emulsion temperature due to thermal treatment (subsequent heating cooling technique cycles) Lipid crystallization as a result of emulsion breakage due to irreversible shock induced by rapid cooling. Supercritical fluid Parallel processes of supercritical fluid extraction extraction of (diffusion) of organic solvent from emulsions and lipid emulsions dissolution. Expansion of organic phase; leads to lipid crystallization

1.6.4 Characterization techniques NLCs are known to present three features—a colloidal (or smaller) particle size, solid nature and lipid matrix. These features are theorized to impart controlled drug release, biocompatibility and improved drug dissolution to the colloidal carriers. This section introduces a number of methods commonly applied for characterization of NLCs dispersions [148, 157, and 158].

1.6.4.1 Particle size The two major techniques that are used for measuring the particle size of lipid nanoparticles are photon correlation spectroscopy (PCS) and laser diffraction

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(LD). The principle of PCS is based on light scattering. In colloidal dispersions, colloidal sized particles undergo random “Brownian” motion due to collisions happening with the thermally driven molecules of the liquid. This movement of these particles creates fluctuations of the intensity of the scattered light, which is measured by the PCS. The measurement size range is from few nanometers to about 3 μm. LD measures the particle size by measuring the intensity of light scattered (Fraunhofer scattering) as a laser beam passes through a dispersed particulate sample. The gathered data is then analyzed to calculate the size of the particles that created the scattering pattern. The measurement size range is 0.02 - 2000 μm. The reports suggest that PCS is sensitive and highly accurate method but it is highly advised to perform particle size analysis by PCS and LD simultaneously. Various electron microscopy techniques such as scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) are very helpful in determining the particle size, particle size distribution and morphology of lipid nanoparticles. SEM and TEM disclose valuable information about particle size and shape. However sufficient attention should be paid towards the sample preparation techniques in all the three types of microscopy as they may influence the nanoparticle shape.

1.6.4.2 Polydispersity index Considering the polydispersed nature of colloidal particles, the measurement of polydispersity index (PDI) is the important requirement. PCS is used for the measurement of PDI. PDI value of 0 to 0.5 indicates the dispersion is monodisperse and homogenous and value above 0.5 indicates nonhomogenity and polydispersity. PDI value of less than 0.3 is accepted as the optimum value.

1.6.4.3 Surface charge Another most important factor determining stability of colloidal dispersion is surface charge (zeta potential). The high values of zeta potential (more than ± 30 mV) indicate less particle aggregation and more electric repulsion between charged particles. This rule cannot be applied for system which contains steric stabilizers, because the adsorption of steric stabilizer will decrease the zeta potential due to the shift in the shear plane of the particle [159].

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1.6.4.4 Degree of crystallinity and lipid modification Drug incorporation or encapsulation efficiency and release rate are the two important parameters that are governed by degree of crystallinity and lipid modification. The thermodynamic stability and lipid packing density are reported to increase and drug incorporation rates decrease in the following order: supercooled melt < α-modification < β-modification < β’-modification. Due to the small size of the particles and the presence of emulsifiers, lipid crystallization and modification changes might be highly retarded. Differential scanning calorimetry (DSC) and X- ray diffractometry (XRD) are the two useful techniques used to investigate the nature of lipid present in the nanoparticles. DSC uses the fact that different lipid modifications possess different melting points and melting enthalpies. XRD helps to assess the length of the long and short spacings of the lipid lattice. Measurement of dispersion crystallinity is the best option as solvent removal process leads to modification changes. The low sensitivity and long measurement times of conventional X-ray sources are the two disadvantages of XRD technique. Synchrotron irradiation helps to overcome these problems but it has limited accessibility. Other useful tools to investigate structural properties of lipids include infrared and Raman spectroscopy [160].

1.7 Role of NLCs in topical delivery of antioxidants Three anatomical locations might be targeted from topical antioxidant delivery namely the skin itself, the deeper tissues (e.g., muscles) for regional delivery, and the systemic circulation (transdermal delivery). Since, the treatments of skin diseases by dermal application of antioxidants have several advantages among those e.g., minimal systemic effects, the potential drug targeting of skin areas and cutaneous layers. The primary challenge for developing a successful nanotechnological product for dermal administration of cosmetic actives relies on the complex structure of the skin barrier [161]. The challenge is proportional to potential opportunity.

Fig. 1.8 illustrates the complexity of the skin consisting of several layers and structures which are organized to represent an excellent biological barrier. Skin anatomy refers to the structure of the skin, which consists of two principal parts: the outer, thinner portion which is called the epidermis and the inner, thicker portion

35 Chapter 1: Introduction which is known as the dermis. Anatomically, the skin consists of the following basic layers: the stratum corneum (nonviable epidermis), viable epidermis, dermis and subcutaneous tissues. In addition to these structures, there are also several associated appendages: hair follicles, sweat glands, apocrine glands, and nails. The stratum corneum is considered to act as the main barrier for the exchange of substances between the body and the environment because of its composition and structure. Therefore it became the real challenge on active agent (in this case antioxidants) delivery into and through the skin.

Figure 1.8: Skin (cutis) drawing illustrating the main layers that structure the largest human organ, accounting more than 10 % of the body mass. Upper layer: Epidermal layer (Epidermis); Intermediate layer: Dermal layer (Dermis); Bottom layer: Hypodermal layer (Hypodermis) [162].

Due to NLCs unique size (which ensures a close contact to the stratum corneum and can increase the amount of drug penetration into the skin) and composition-dependent properties, NLCs pose the ability to penetrate through skin barrier, hence providing release of their contents and drug targeting. Moreover, NLCs increases chemical stability of the incorporated antioxidant and are safe carriers which can be produced easily on large scale. [163-166]. Many aspects of NLCs that are advantageous for dermal application of cosmetic products have been reported, e.g. occlusive properties, increase in skin hydration, modified release profile, increase of skin penetration associated with a targeting effect and avoidance of systemic uptake, etc. These positive features of

36 Chapter 1: Introduction lipid nanoparticles led to the market introduction of a number of cosmetic products (Table 1.9).

Table 1.9: Overview of NLC-based dermal cosmetic products currently on the market [167].

Product name Main active ingredients

Cutanova Cream NanoRepair Q10 Q10, polypeptide, Hibiscus extract, ginger extract, Ketosugar

Intensive Serum NanoRepair Q10 Q10, polypeptide, Mafane extract

Cutanova Cream NanoVital Q10 Q10, TiO2, polypeptide ursolic acid, oleanolic acid, sunflower extract SURMER Crème Legère Coconut oil, Monoi Tiare Tahiti®, Nano-Protection pseudopeptide, milk extract from coconut, wild ginger, Noni extract SURMER Masque Crème Coconut oil, Monoi Tiare Tahiti®, milk Nano-Hydratant extract from cocosnut, wild ginger, pseudopeptide.

NanoLipid Restore Coenzyme Q10, ω-3 and ω-6 unsaturated fatty acids

NanoLipid Q10 Coenzyme Q10 NanoLipid Basic unloaded NCL, effect of lipids and particles

NLC Deep Effect Eye Serum Coenzyme Q10, highly active oligosaccharides

NLC Deep Effect Repair Cream Q10, TiO2, highly active oligosaccharides NLC Deep Effect

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1.7.1 Benefits of NLCs in topical delivery of antioxidants

1.7.1.1 Increase of skin occlusion Skin occlusion is related to lipid film formation of NLCs on the skin which may result in increased hydration effect [168] (Fig. 1.9). Any increase of the hydration effect results in a facilitation to access the natural pathways through the skin permeability barrier [169]. For cosmetic applications it is important that the cosmetic active is not systemically absorbed, but it is crucial a certain penetration into the skin for the desired effect to take place. It was reported by Wissing et al. [170] that the highest occlusion efficiency will be reached by using low temperature melting lipids, highly crystalline and very small particles. Nanoparticles have been found to be 15-folds more occlusive than microparticles [171].

Figure 1.9: Film formation associated to the occlusion effect [162].

1.7.1.2 Increase of skin hydration and elasticity The reduction of transepidermal water loss (TEWL) caused by occlusion leads to an increase in skin hydration after dermal application NLCs or formulations containing them. A significant higher increase in skin hydration was found by Müller et al. for NLCs containing cream compared to conventional cream [172].

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1.7.1.3 Enhancement of skin permeation and drug targeting The stratum corneum in healthy skin has typically a water content of 20 % and provides relatively an effective barrier against percutaneous absorption of exogenous substances. Skin hydration after applying NLCs leads to a reduction of corneocytes packing and an increase in the size of the corneocytes gaps. This will facilitate the percutanious absorption and drug penetration to the deeper skin layers (Fig. 1.10) [173, 174].

Figure 1.10: Possible mechanisms for skin permeation enhancement of drugs or active ingredients from NLCs [175].

1.7.1.4 Enhancement of chemical stability of chemically labile antioxidants Enhancement of chemical stability after incorporation into NLCs was proven for many cosmetic actives, e.g. Idebenone [176] and ascorbyl palmitate [177]. This is primarily due to the solid state nature of NLCs, where very low exchange between the solid particle phase and the external water phase (Fig. 1.11).

1.7.1.5 Enhancement in skin photoprotection Enhancement of skin photoprotection after incorporation into NLCs was proven for many cosmetic actives, e.g. CoQ10 [179], and combination of rice bran and raspberry seed-oil [180]. Significant increase in SPF up to about 50 was reported after the encapsulation of titanium dioxide into NLCs. Encapsulation of antioxidant and inorganic sunscreens into NLCs is therefore a promising approach to obtain well tolerable sunscreens with high SPF.

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Figure: 1.11: Exchange phenomena between the lipophilic oil phase of emulsions (left), and the lipophilic bilayer and aqueous core of liposomes (middle) and in case of NLCs with solid matrix, these exchange phenomena and degradation are practically eliminated or at least distinctly slowed down/minimised (right) [178].

1.8 Role of NLCs in intranasal (direct nose to brain) delivery of antioxidants All organisms with a well-developed central nervous system (CNS) have a BBB. In all mammals the BBB is created by the endothelial cells forming the capillaries of the brain and spinal cord microvasculature. The combined surface area of these microvessels constitutes by far the largest surface area for blood–brain exchange. This surface area, depending on the anatomical region, is between 150 and 200 cm2/g of tissue giving a total area for exchange in the brain of 12–18 m2 for the average human adult [181, 182]. A BBB is required because the CNS needs to maintain an extremely stable internal fluid environment surrounding the neurons. This stability of the internal environment of the brain is an absolute requirement for reliable synaptic communication between nerve cells [182]. The BBB plays a key role in maintaining brain function by allowing selective access to essential nutrients and signaling molecules from the vascular compartment, and restricting the entry of xenobiotics. The protective function of the BBB, however, becomes a major obstacle to the treatment of many devastating diseases of the CNS such as neurodegenerative disorders (Parkinson’s disease and AD). Thus, there is a need to develop delivery technologies (particularly noninvasive) that are capable of transporting drugs across the BBB efficiently [183]. In recent years, compared to other drug delivery system, direct transport of drugs from the nose to the brain come to light as an effective and noninvasive means (Table 1.10) to circumvent the BBB for a variety of therapeutic molecules [184].

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Table 1.10: Key benefits offered by nasal drug delivery system [185].

Key benefits Nasal Oral Parenteral Higher drug levels Yes No Yes Rapid onset Yes No Yes Pain at the site of administration No No Yes Self administration Yes Yes No BBB bypass Yes No No Patient compliance High High Low Hepatic first-pass metabolism No Yes No Drug degradation Low High No

1.8.1 Intranasal (direct nose to brain) pathway There are 3 distinct functional areas (Fig. 1.12) in the nasal cavity, the vestibular, olfactory and respiratory zones. The vestibular area (approx. 0.6 cm2) serves as a first barrier against airborne particles with low vascularization comprised of stratified squamous and keratinized epithelial cells with nasal hairs. The olfactory area (approx. 15 cm2) enables olfactory perception and is highly vascularized. The respiratory area (approx. 130 cm2) serves with its mucus layer produced by highly specialized cells as an efficient air- cleansing system [186]. Due to the rich vascularization, the olfactory and in particular the respiratory zone serve as an efficient absorption surface for topically applied drugs. The olfactory zone with its vicinity to the cerebrospinal fluid and direct nervous interface to the brain has attracted research interest for possible nose- to- brain delivery. Since, the nasal epithelium (olfactory and respiratory) represents the most likely sites of absorption for drugs administered intranasally (i.n), we highlighted the pathways and mechanisms by which a drug travels from nasal epithelium to various regions of the CNS. There are at least three sequential steps necessary to carry the drug to the CNS after i.n. administration includes:

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Figure 1.12: Pathways for brain targeting after intranasal administration [187].

(1) Transport across the epithelial ‘barriers’ (olfactory or respiratory) in the nasal passages. (2) Transport from the nasal mucosa to sites of brain entry near the pial brain surface in the cranial compartment (i.e. entry points of peripheral olfactory or trigeminal nerve-associated components comprising the delivery pathways) and (3) Transport from these initial brain entry sites to other sites within the CNS.

1.8.1.1 Transport across the epithelial ‘barriers’ Transport across the olfactory or respiratory epithelia may occur by intracellular or extracellular pathways. Intracellular pathways across the olfactory epithelium include endocytosis into olfactory sensory neurons (OSN) and subsequent intraneuronal transport to the olfactory bulb or transcytosis (i.e. transcellular transport) across sustentacular cells to the lamina propria as shown in Figure 1.13. OSN have the ability to endocytose certain viruses (e.g. herpes) as well as large molecules such as horseradish peroxidase (HRP) from the nasal passages and then transport them intracellularly along the axon in the anterograde direction towards the olfactory bulb [188-193]. Intracellular pathways across the respiratory epithelium potentially include endocytosis into peripheral trigeminal nerve processes

42 Chapter 1: Introduction located near the epithelial surface and subsequent intracellular transport to the brainstem or transcytosis across other cells of the respiratory epithelium to the lamina propria (Fig. 1.13) for relationships viruses and bacteria may also be transmitted to the CNS along trigeminal nerve components within the nasal passages [194, 195]. Extracellular transport pathways across either the olfactory or respiratory epithelia primarily include paracellular diffusion to the underlying lamina propria (Fig. 1.13)

1.8.1.2 Transport from the nasal mucosa to sites of brain entry Transport from the nasal mucosa to brain entry points at the level of the olfactory bulbs or brainstem may in theory occur via intracellular pathways (endocytosis and intraneuronal transport within OSN or trigeminal ganglion cells) or extracellular pathways (diffusion or convection within perineural, perivascular or lymphatic channels associated with olfactory and trigeminal nerve bundles extending from the lamina propria to the brain). The possible fates of substances reaching the extracellular environment of the lamina propria are numerous and include: (1) absorption into blood vessels and entry into the general circulation; (2) absorption into lymphatic vessels draining to the deep cervical lymph nodes of the neck; (3) extracellular diffusion or convection in compartments associated with nerve bundles, particularly perineural or perivascular spaces, with subsequent entry into the cranial compartment (Fig. 1.13).

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Figure 1.13: General organization of the olfactory region [196].

Once a substance crosses the nasal epithelium into the lamina propria, it may be absorbed by the rich supply of nasal blood vessels and enter the systemic circulation. Drugs absorbed into the systemic circulation would then have to cross the BBB or blood–CSF barriers to reach the CNS. In the nasal submucosa, substances that aren't absorbed into the bloodstream may drain to the deep cervical lymph nodes through nasal lymphatic vessels in the lamina propria [197,198]. Substances which have crossed the nasal epithelium to reach the lamina propria but escape local absorption into the blood stream and drainage within nasal lymphatics to the deep cervical lymph nodes may be available to enter the CNS. Finally, the perineural spaces of cranial nerves such as the olfactory and trigeminal nerves appear to allow communication with the CSF of the

44 Chapter 1: Introduction subarachnoid space for some substances, providing a potential route for molecules to reach the CNS from the nasal cavity [199].

1.8.1.3 Transport from brain entry sites to other CNS areas Final distribution from drug entry points at the level of the olfactory bulb and brainstem to other areas of the CNS may be envisioned to occur either by intracellular transport (i.e. transfer and uptake to second order neurons synapsing with peripheral OSN or trigeminal ganglion cells) or extracellular transport [160] e.g. widespread distribution via convective transport within the cerebral perivascular spaces, local diffusion at the entry points and local diffusion from perivascular spaces into the parenchyma).

1.8.2 Strategies to enhance i.n. (direct nose to brain) drug delivery Lipid nanoparticles are promising strategy for drug delivery to the brain, due to its rapid uptake by the brain, bioacceptability and biodegradability. Many drug molecules like Rivastigmine [200], Ondansetron hydrochloride [201], Artemether [202], Iloperidone [203], Idebenone [204], Duloxetine (205), and Basic fibroblast growth factor [206] were delivered by NLCs as a direct nose to brain transport system. Among the various strategies surface engineering of lipid nanoparticles is one of the excellent approaches to manage drug delivery properties of formulations by interaction of surface coating with a biological system. The various surface modifications that are being reported include, 1) Chitosan surface modification 2) PEG surface modification 3) Lectin surface modification 4) Peptide surface modification 5) Lipid surface modification

Lipid surface modification: Since, this strategy is related with our hypothesis; the strategy is briefly discussed here. Patel et al. [206] reported the formulation and evaluation of risperidone-loaded lipid nanoparticles for brain targeting and their findings substantiate the existence of a direct nose to brain route for nanoparticles

45 Chapter 1: Introduction administered to the nasal cavity. However, modification of the lipid matrix of lipid- based nanoparticle formulations may improve the formulation characteristics. Recently, Pardeshi et al. [207] prepared surface-engineered lipid nanoparticles for brain targeting. They assessed the influence of stearylamine, a surface charge modifier, on particle size, z-potential and stability of lipid nanoparticles. Stearylamine induces positive charge on the surface of nanoparticles and also contributes to increased stability. Stearylamine serves as an electrostatic stabilizer by maintaining zeta potential of fabricated system to the optimum. Also, ropinirole- loaded surface modified lipid nanoparticles had shown enhanced therapeutic efficacy in terms of ability to reduce tremors in a tremor induced rat model of Parkinson’s disease (PD), when compared with marketed tablet formulation of the same drug. Thus, lipid surface modification may contribute to the development of products with improved formulation and stability characteristics, which would serve as a promising approach for brain targeting via intranasal administration of neurotherapeutics.

1.9 Literature survey As a result of intensive work of different research groups, the NLCs have been investigated as delivery systems for antioxidants with respect to production, characterization and application. The increasing interest in the field of these lipidic structures is demonstrated in Table 1.11 by appropriate keyword (“-”) search in

PubMed. After the removal of irrelevant articles, a total of 10 (research article only) were considered for full-text review and are listed in Table 1.12. Moreover, a total of 4 relevant patents published on “NLCs in topical delivery of antioxidants” and “NLCs in brain targeting”, with findings and references are listed in Table 1.13.

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Table 1.11: Number of articles published by different research groups between years 2010 to 2015.

Keyword* Results NLCs in topical delivery of antioxidants “Nanostructured lipid carriers” 354 “Nanostructured lipid carriers” and “Topical” 70 “Nanostructured lipid carriers” and “Skin” and “Dermal” 19 “Nanostructured lipid carriers” and “Antioxidant” 13 Total 456 NLCs in direct nose to brain delivery of antioxidants “Nanostructured lipid carriers” and “Nose to brain” 06 “Nanostructured lipid carriers” and “Brain” and “Antioxidant” 02 Total 08 * In title and abstract only

Table 1.12: List of relevant articles published on “NLCs in topical delivery of antioxidants” and “NLCs in direct nose to brain delivery of antioxidants”, with outcome of the study and references.

Research group Out come of the study Ref NLCs in topical delivery of antioxidants Charoenputtakun Developed and evaluated dermal delivery of all-trans- 208 et al. retinoic acids (ATRA). Results indicated that the physicochemical characteristics of terpene composited lipid nanoparticles influenced skin permeability. NLCs significantly protected ATRA from photodegradation and were non-toxic to normal human foreskin fibroblast cells in vitro. CLSM image analysis showed higher epidermal permeation for terpene composited SLNs and NLCs. Hence SLNs and NLCs can be potentially used as dermal drug delivery carriers for ATRA.

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Research group Out come of the study Ref Tichota et al. Developed a topical formulation of argan oil NLCs to 209 improve skin hydration and the entrapment of the NLCs in the hydrogel net did not affect their colloidal sizes. Additionally, it was observed that this formulation precipitated an increase in skin hydration of healthy volunteers. Therefore, concluded that NLCs using argan oil as the liquid lipid is a promising strategy, since a synergistic effect on the skin hydration was obtained. Gokce et al. RSV was loaded into SLNs and NLCs for dermal 210 applications. When the two systems were compared, NLCs penetrated deeper into the skin. RSV-loaded NLCs with smaller particle size and higher drug loading appears to be superior to SLNs for dermal applications. Mitri et al. Lutein incorporated into nanocarriers [SLNs, NLCs 211 and a nanoemulsion (NE)] for dermal delivery with particle size in the range of 150-350 nm. Release profiles were biphasic (lipid nanoparticles) or triphasic (NE). The nanocarriers were able to protect lutein against UV degradation. Based on size, stability and release/permeation data, the lipid nanoparticles were found to potential dermal nanocarriers for lutein. Okonogi et al. Lycopene was loaded into NLCs using high pressure 212 homogenization (HPH). The particle size, size distribution, and zeta potential of lycopene-loaded NLCs stored at different temperatures did not change in time, demonstrated an excellent colloidal stability of the systems. Chemical stability data indicated that the utilization of NLC increased the stability of lycopene. In conclusion, NLCs are attractive systems for the cutaneous delivery of lycopene.

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Research group Out come of the study Ref NLCs in topical delivery of antioxidants

Keck et al. Developed coenzyme Q10 (Q10) loaded ultra-small NLC 213 (usNLCs) with a particle size below 50 nm. usNLCs showed a higher release, a higher antioxidant capacity,

and a better skin penetration for Q10. Hence, usNLC might represent a novel and promising carrier system for the improved delivery of lipophilic actives.

Nanjwade et al. Developed coenzyme Q10 (Q10) loaded NLCs. Results 214

indicated that Q10-loaded NLCs has more antioxidant activity than that of solution form and it can be used to reduce the oxidative stress and to increase the antioxidant enzyme activity in many neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease etc. Li et al. Idebenone loaded NLCs, NE or oil solution were 215 specifically evaluated for ex vivo permeation and stability studies. Results obtained demonstrated that NLCs improved the chemical stability of IDB and enhanced the skin permeation much better than NE and oil solution. Thus, suggesting that NLCs containing Idebenone would be of potential use for skin care as an alternative to topical formulation. NLCs in direct nose to brain delivery of antioxidants Madane et al. Developed a stable NLCs system as a carrier for 216 curcumin (CRM). Increased cytotoxicity of CRM- NLCs than that of CRM to astrocytoma-glioblastoma cell line (U373MG) in the cancer cell lines was observed. Moreover, biodistribution studies showed higher drug concentration in brain after intranasal administration of NLCs than pure drug solution. Hence, suggesting NLCs are promising drug delivery system for brain cancer therapy.

49 Chapter 1: Introduction

Research group Out come of the study Ref Mandpe et al. Developed and evaluated the outcome of combining 217 iloperidone with idebenone in the form of brain- targeted NLCs. The combination NLCs demonstrated good targeting potential. Finally, concluded that combining iloperidone with idebenone and converting into NLCs has contributed in effectively reducing oxidative stress in the brain and helped in reversing the catalepsy.

Table 1.13: List of relevant patents published on “NLCs in topical delivery of antioxidants” and “NLCs in brain targeting”, with findings and references.

Patent no. Patent/application Findings Ref title NLCs in topical delivery of antioxidants US20100104522 Composition for The invention relates to the Stable 218 A1 (application) skin external use preparations of NLCs containing containing omega- omega-3 fatty acid as an effective 3 fatty acid ingredient in preventing skin aging, moisturizing skin, improving skin roughness, restoring or strengthing skin barrier function, improving skin elasticity, recovering or preventing skin damage induced by UV CN103860389 A NLCs loaded with The invention relates to topical 219 (application) phenylethyl antioxidant (phenylethyl resorcinol) resorcinol, loaded-NLCs for skin whitening preparation effect, wherein preparation method thereof & methods, composition, and a skin- cosmetic care product or a cosmetic containing same. containing the NLCs.

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Patent no. Patent/application Findings Ref title CN1278682 C Nanometer The invention describes topical 220 (grant) preparation of preparation of natural vitamin E natural vitamin E loaded NLCs, wherein preparation process and composition of NLCs such as lipid materials (solid and liquid lipids) and emulsifier mixtures are disclosed. NLCs in brain targeting US Nanostructured This invention relates to 221 20100247619 Lipid nanoparticles consisting of riluzole A1 (application) Carriers trapped in lipids, Containing and their use to prepare medicinal Riluzole And products for the treatment of Pharmaceutical Amyotrophic Lateral Sclerosis and Formulations Multiple Sclerosis. Containing Said Particles

1.10 References

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183) Elena V. Batrakova and Alexander V. Kabanov. Strategies to Overcome the Blood–Brain Barrier. Elka Touitou, Brian W. Barry (eds). Enhancement in drug delivery, 1st ed. FL, USA: CRC Press; 2007. pp. 593-614. 184) Pardeshi CV, Belgamwar VS. Direct nose to brain drug delivery via integrated nerve pathways by passing the blood-brain barrier: an excellent platform for brain targeting. Expert Opin Drug Deliv. 2013; 10:957-72. 185) Dhakar RC. Nasal drug delivery: success through integrated device development. J. Drug Deliv. Ther. 2011; 1:2-7. 186) Pires A, Fortuna A, Alves G, Falcão A. Intranasal drug delivery: how, why and what for? J Pharm Pharm Sci. 2009; 12:288-311. 187) Bitter C, Suter-Zimmermann K, Surber C. Nasal drug delivery in humans. Curr Probl Dermatol. 2011; 40:20-35. 188) Thorne RG, Emory CR, Ala TA, Frey WH 2nd. Quantitative analysis of the olfactory pathway for drug delivery to the brain. Brain Res. 1995; 692:278-82. 189) Doty RL. The olfactory vector hypothesis of neurodegenerative disease: is it viable? Ann Neurol. 2008; 63:7-15. 190) Kristensson K, Olsson Y. Uptake of exogenous proteins in mouse olfactory cells. Acta Neuropathol. 1971; 19:145-54. 191) Broadwell RD, Balin BJ. Endocytic and exocytic pathways of the neuronal secretory process and trans-synaptic transfer of wheat germ agglutinin-horseradish peroxidase in vivo. J Comp Neurol. 1985; 242:632-50. 192) Baker H, Spencer RF. Transneuronal transport of peroxidase-conjugated wheat germ agglutinin (WGA-HRP) from the olfactory epithelium to the brain of the adult rat. Exp Brain Res. 1986; 63:461- 73. 193) Kristensson K. Microbes' roadmap to neurons. Nat Rev Neurosci. 2011; 12:345-57. 194) Deatly AM, Haase AT, Fewster PH, Lewis E, Ball MJ. Human herpes virus infections and Alzheimer's disease. Neuropathol Appl Neurobiol. 1990; 16:213-23. 195) Jin Y, Dons L, Kristensson K, Rottenberg ME. Neural route of cerebral Listeria monocytogenes murine infection: role of immune response mechanisms in controlling bacterial neuroinvasion. Infect Immun. 2001; 69:1093-100. 196) Lochhead JJ, Thorne RG Intranasal delivery of biologics to the central nervous system. Adv Drug Deliv Rev. 2012; 64:614-28. 197) Yoffey JM, Drinker CK. The lymphatic pathway from the nose and pharynx: the absorption of dyes. J. Exp. Med. 1938; 68: 629–640. 198) Yoffey JM, Sullivan ER, Drinker CK. The lymphatic pathway from the nose and pharynx: the absorption of certain proteins. J. Exp. Med. 1938; 68:941–947. 199) M.W.B. Bradbury, H.F. Cserr. Drainage of cerebral interstitial fluid and of cerebrospinal fluid into lymphatics, in: M.G. Johnston (Ed.), Experimental Biology of the Lymphatic Circulation, Elsevier, Amsterdam and New York, 1985, 355–391. 200) Wavikar PR, Vavia PR. Rivastigmine-loaded in situ gelling nanostructured lipid carriers for nose to brain delivery. J Liposome Res. 2014; Sep 9:1-9. 201) Devkar TB, Tekade AR, Khandelwal KR. Surface engineered nanostructured lipid carriers for efficient nose to brain delivery of ondansetron HCl using Delonix regia gum as a natural mucoadhesive polymer. Colloids Surf B Biointerfaces. 2014; 122:143-50. 202) Jain K, Sood S, Gowthamarajan K. Optimization of artemether-loaded NLC for intranasal delivery using central composite design. Drug Deliv. 2014; 1-15. 203) Mandpe L, Kyadarkunte A, Pokharkar V. Assessment of novel iloperidone- and idebenone- loaded nanostructured lipid carriers: brain targeting efficiency and neuroprotective potential. Ther Deliv. 2013; 4:1365-83. 204) Alam MI, Baboota S, Ahuja A, Ali M, Ali J, Sahni JK. Intranasal infusion of nanostructured lipid carriers (NLC) containing CNS acting drug and estimation in brain and blood. Drug Deliv. 2013; 20:247-51. 205) Zhao YZ, Li X, Lu CT, Lin M, Chen LJ, Xiang Q, Zhang M, Jin RR, Jiang X, Shen XT, Li XK, Cai J. Gelatin nanostructured lipid carriers-mediated intranasal delivery of basic fibroblast growth factor enhances functional recovery in hemiparkinsonian rats. Nanomedicine. 2014; 10:755-64. 206) Patel S, Chavhan S, Soni H, Babbar AK, Mathur R, Mishra AK, Sawant K. Brain targeting of risperidone-loaded solid lipid nanoparticles by intranasal route. J Drug Target. 2011; 19:468-74. 207) Pardeshi CV, Rajput PV, Belgamwar VS, Tekade AR, Surana SJ. Novel surface modified solid lipid nanoparticles as intranasal carriers for ropinirole hydrochloride: application of factorial design approach. Drug Deliv. 2013; 20:47-56.

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60 Chapter 2

Genesis, specific aims and objective of work

This chapter reports the main obstacles that hinder the clinical application of antioxidant and importance of nanotechnology particularly colloidal carriers systems as a drug delivery system. The specific aim and objectives of the present investigation are described in this chapter which highlights our strategies to deliver antioxidant effectively and efficiously via topical skin and intranasal delivery.

Chapter 2: Genesis, Specific aims and Objectives of Work

2.1 Genesis Antioxidants obtained from various natural sources are one of the most widely used entities to treat human diseases (particularly augmented by oxidative stress, directly or indirectly) and they have been used since ancient times. Today, the use of natural as well as synthetic antioxidants is seen in a range of applications from appearance-enhancing cosmetics to life-threatening diseases like Alzheimer’s. The main obstacles that hinder the clinical application of these antioxidant compounds are: 1. Poor water solubility 2. Poor bioavailability and short half-life 3. Instability in gastrointestinal tract (if taken orally) 4. Limited dose regimen Conventional antioxidant dosage forms (Solid and liquid-oral dosage forms such as tablets, capsules and liquid orals), have limited success due to: 1. Lack of controlled release 2. Lack of targeting (inability to cross membrane barriers of skin and brain) 3. Fluctuating blood plasma levels and rapid clearance 4. Undesirable side effects Thus, the nanotechnology might be the answer to many of the inherent problems associated with antioxidants and conventional dosage forms. In the past 30 years, the emergence of nanotechnology had a ground breaking impact on drug delivery, therapeutics, diagnostic and imaging. In recent years, we have seen a paradigm change in delivery methods of antioxidants for the treatment and prevention of various diseases. One of the most important and useful methods is the utilization of nanotechnology as an efficient delivery system. The chemical structure, relatively safe history, and easy surface modulation of nanoparticles make them ideal carriers to deliver a multiple drug load to the desired site [1-3]. Colloidal systems (particle size ranging 50 – 1000 nm), particularly nanostructured lipid nanoparticles (NLCs) are in the current limelight of interest due to controlled release action [4], higher loading capacity [4], enhancement in chemical stability [5], outstanding potential to target physiological sites, organs, tissues, or cells where the pharmacological activity of a drug moiety is required. For e.g. penetration into skin [6], overcoming membrane barriers particularly in the nose [7] and drug transport over blood brain barrier (BBB) [8].

61 Chapter 2: Genesis, Specific aims and Objectives of Work

In conclusion, the combination of antioxidants and NLCs might have the potential to treat different oxidative stress-related diseases, and thereby overall has the potential to increase the life expectancy and quality of life.

2.2 Specific aims The aim of this research work was “to improve the topical and intranasal delivery of antioxidants (i.e. idebenone (IDB), resveratrol (RSV) and phenyl butyl nitrone (PBN)) via loading into NLCs”. In addition to the design and investigation of the feasibility of process to obtain topical and intranasal NLCs, photoprotection and neuroprotection aspects of NLCs were also assessed. Towards this goal, the following primary objectives were set.

2.3 Objectives Part 1 (Chapter 4, P. 78): Photoprotection aspects of topically administered IDB loaded NLCs (IDB-NLCs) 1) To prepare IDB-NLCs using a melt emulsification ultrasound homogenization technique with appropriate excipient (solid lipid, liquid lipid and surfactants) considered acceptable in topical formulations. 2) To investigate the effect of various process parameters (sonication amplitude and sonication time) and formulation parameter (surfactant concentration) on formulation characteristics (particle size and entrapment efficiency) using Taguchi robust orthogonal design. 3) To study and compare the in vitro occlusion test, in vitro release characteristics and ex vivo drug diffusion/penetration studies of optimized IDB-NLCs with pure IDB (suspension in PBS; IDB-PD) and NLCs without drug (placebo-NLCs). 4) To carry out stability studies of optimized IDB-NLCs stored at different temperature and humidity conditions. 5) To evaluate the in vitro cytotoxicity and biocompatibility of IDB-PD, placebo-NLCs and optimized IDB-NLCs on human keratinocytes cells (HaCaT). 6) To investigate the photoprotective effect (using intracellular ROS scavenging activity and mitochondrial membrane potential assay) of IDB-PD, placebo- NLCs and optimized IDB-NLCs on HaCaT cell line against UVB radiation.

62 Chapter 2: Genesis, Specific aims and Objectives of Work

7) To study the quantitative (using spectrophotometry) and qualitative (using Confocal Laser Scanning Microscope) cell uptake of optimized IDB-NLCs and Nile Red loaded IDB-NLCs (NR-IDB-NLCs).

Part 2 (Chapter 5A and 5B, P. 118 and 130): Photoprotection aspects of topically administered RSV and PBN loaded NLCs (Combination-NLCs) 1) To assess cytotoxicity and phototoxicity of various acylglutamate surfactants (sodium cocoyl glutamate, sodium lauroyl glutamate and sodium myristoyl glutamate) using HaCaT cell line and pick most suitable surfactant for the preparation and stabilization of combination-NLCs 2) To review, screen and select appropriate antioxidant combination based on various chemical and cell-based assays. 3) To carry out preformulation studies such as lipid phase screening, surfactant selection, crystallographic investigations and antioxidant interaction studies. 4) To prepare and optimize combination-NLCs using ultrasonication homogenization method and mixed-level orthogonal array design. 5) To investigate the influence of various process parameters (sonication amplitude and sonication time) and formulation parameters (lipid and surfactant concentration) on important formulation characteristics like particle size, polydispersity index, surface charge and entrapment efficiency. 6) To characterize optimized combination-NLCs with respect to surface morphology, in vitro drug release and occlusion test, ex vivo drug diffusion / penetration studies and in vitro cellular uptake studies. 7) To carry out stability studies of optimized combination-NLCs stored at different temperature and humidity conditions. 8) To perform, investigate and compare in vitro cytotoxicity, biocompatibility and photoprotective effect of optimized combination-NLCs against pure RSV, pure PBN (suspension in PBS; RSV-PD and PBN-PD) and placebo- NLCs on HaCaT cells. 9) To confirm the photoprotective effect of optimized combination-NLCs using intracellular ROS scavenging activity.

63 Chapter 2: Genesis, Specific aims and Objectives of Work

Part 3 (Chapter 6, P. 168): Neuroprotection aspects of intranasally administered RSV and PBN loaded NLCs (Combination-NLCs) 1) To perform essential preformulation studies for the development of combination-NLCs such as lipid phase screening, surfactant selection, XRD, DSC and simultaneous HPLC estimation of RSV and PBN. 2) To prepare and optimize combination-NLCs via ultrasonication homogenization method. 3) To investigate the effect of sonication, lipids and surfactant parameters on NLCs particle size, polydispersity index, zeta potential, entrapment efficiency and drug release study. 4) To carry out stability studies of optimized combination-NLCs stored at different temperature and humidity conditions. 5) To study the in vivo pharmacokinetic performance of optimized combination-NLCs and compare it with individual PD as well as placebo- NLCs through intranasal route. 6) To assess the neuroprotection effect of the optimized combination-NLCs on

Alzheimer’s disease rat model induced by A β 25-35 .

2.4 References 1) Bawarski WE, Chidlowsky E, Bharali DJ, Mousa SA. Emerging nanopharmaceuticals. Nanomedicine. 2008;4:273-82. 2) Chan WC. Bionanotechnology progress and advances. Biol Blood Marrow Transplant. 2006; 12:87-91. 3) Salata O. Applications of nanoparticles in biology and medicine. J Nanobiotechnology. 2004;2: 3 4) Souto EB, Wissing SA, Barbosa CM, Müller RH. Development of a controlled release formulation based on SLN and NLC for topical clotrimazole delivery. Int J Pharm. 2004; 278:71–77. 5) Li B, Ge ZQ. Nanostructured lipid carriers improve skin permeation and chemical stability of idebenone. AAPS PharmSciTech. 2012; 13:276-83. 6) 213) Keck CM, Baisaeng N, Durand P, Prost M, Meinke MC, Müller RH. Oil-enriched, ultra- small nanostructured lipid carriers (usNLC): a novel delivery system based on flip-flop structure. Int J Pharm. 2014; 477:227-35 7) Devkar TB, Tekade AR, Khandelwal KR. Surface engineered nanostructured lipid carriers for efficient nose to brain delivery of ondansetron HCl using Delonix regia gum as a natural mucoadhesive polymer. Colloids Surf B Biointerfaces. 2014; 122:143-50. 8) Mandpe L, Kyadarkunte A, Pokharkar V. Assessment of novel iloperidone- and idebenone-loaded nanostructured lipid carriers: brain targeting efficiency and neuroprotective potential. Ther Deliv. 2013; 4:1365-83.

64 Chapter 3

Materials, instruments and software’s

This chapter includes a list of all the materials and instruments used for characterizations purpose. Brief information about all the antioxidants (idebenone, resveratrol and PBN) and excipients (solid and liquid lipids, surfactants) used in the formulation development of NLCs are mentioned in this chapter.

Chapter 3: Materials, Instruments and Software’s

3.1 Materials Supplier 3.1.1 Antioxidants - Idebenone Purchased from Xi’an Biosunny Biological Technology Co., Ltd., China - Resveratrol Gift sample from Bachem AG, Bubendorf, Switzerland. - PBN Purchased from GeroNova Research, Inc., CA, USA. 3.1.2 Excipients 3.1.2.1 Solid lipids - Witepsol E 85, Witepsol H 175, Gift sample from Sasol Germany Witepsol H 35, Dynasan 116, GmbH, Witten Germany Dynasan 118 and Sofigen 767 - Gelucire 50/13, Gelucire 40/14, Gift sample from Gattefosse, France Precirol ATO 5 and Compritol 888 ATO - Stearic acid Purchased from Sigma Aldrich, India. 3.1.2.2 Liquid lipids / Oils - Capmul 908 P, Capmul PG12, Gift sample from Abitech Captex 300, Caproyl 90, Captex 200 P, Corporations, Wisconsin, USA. Captex 355, Captex 300, Captex 500 P, and Capmul MCM - Miglyol 840 and Miglyol 812 Gift sample from Sasol GmbH, Witten Germany. - Olive oil and Crodamol GTCC Gift sample from Croda Inc, USA. - Phosal 53 MCT Gift sample from Lipoid, USA. - Jojoba oil and Rose hip seed oil Gift sample from N. V. Organics, India. 3.1.2.3 Surfactants - TegoCare 450 Gift sample from Evonik Goldschmidt GmbH, Essen Germany.

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Materials (cont..) - Amisoft CS 11, Amisoft LS 11 and Gift sample from Western Amisoft MS 11 Commercial Corporation, India. - Human serum albumin Purchased from Reliance life sciences, India. 3.1.3 Chemicals and reagents - Methanol-HPLC grade, Anesthetic Purchased from Merck, India. ether and Glacial acetic acid - Absolute ethanol 99.9 % Purchased from Changshu Yangyuan Chemical Co., Ltd. China. - Poloxamer 188 Gift sample from BASF Corporation, Germany. - Phosphate-buffered saline, Nile red, Purchased from Sigma, MO, USA Chlorpromazine, Dimethyl sulfoxide and Paraformaldehyde 3.1.4 Cell culture requirements - HaCaT cell line Acquired from NCCS, India. - L-glutamine, Fetal bovine serum Purchased from Gibco® life (FBS), technologies, NY, USA. Trypsin, Penicillin 10,000 units/mL, Streptomycin 10,000 g/mL and Dulbecco’s-Modified Eagle’s Medium (DMEM) high glucose. - 75 cm 2 tissue culture flask, 96 and 24- Procured from BD Falcon, NJ, USA. well flat-bottom polystyrene tissue culture plate. - DCF-DA, Rhodamine-123 and Purchased from Sigma, MO, USA. Amyloid β-protein fragment 25-35 - MTS reagent Purchased from Promega, WI, USA. - Mounting medium Gift sample from Ibidi GmbH, Germany. - Sterile glass coverslip (12mm, 1#) Purchased from Blue star, India. Materials (cont..)

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- 0.22 µm sterile syringe filters Purchased from Merck Millipore, India 3.1.5 Miscellaneous - 0.45 and 0.22 µm hydrophilic syringe Purchased from Axiva Sichem filters Biotech, New Delhi, India. - Cellophane dialysis membrane Purchased from Hi-media Labs, India (MWCO 12000 Da )

3.2 Instruments • Particle size and zeta potential analyzer 90 Plus: Brookhaven Instruments Corporation., USA. • Differnential Scanning Calorimetry: Metler, Toledo GmbH, Switzerland. • Electronic balance: Contech electronic balance, India • pH meter: Toshniwal Process Instruments Ltd • Ultrasonic processor: Sonics and Materials Inc, Newton, CT, USA. • Magnetic stirrer: Remi scientific, Mumbai, India • Malvern Mastersizer 2000SM: Malvern Instruments, Malvern,U.K • UV-Visible spectrophotometer: Jasco V-530, Japan • Bath Sonicator: Toshniwal Process Instruments Ltd, Mumbai • Hot air oven: Kumar Industries, India • Shaker water bath CS-26a: Classic scientific, Thane, India • Centrifuge mahcine: Allegra 64R, Beckman Coulter, USA • FT-IR: Jasco 5300, Japan • X-Ray Diffractometer: PW 1729 Philips, The Netherlands • Hot plate: Meta-lab, Mumbai, India • Stability chamber: Thermolab, Scientific Equipment Pvt. Ltd., Mumbai, India • Transmission electron microscope: Technai G2 F30 S-TWIN Instrument, The Netherlands • FluoView FV10i inverted CLSM system: Olympus, Tokyo, Japan. • Flow cytometer FACSCanto II : Becton Dickinson, CA, USA.

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• Polarized light microscope Eclipse E600 POL: Nikon Corporation, Tokyo, Japan. • Cell incubator model 3193: Forma Scientific Inc, USA. • UV Crosslinker system: UVP, Upland, CA, USA • Lyophilizer, FreeZone Plus 2.5 L: Labconco Corp, MO, USA. • Microplate reader (Model 680): BIO-RAD Laboratories, CA, USA. • Incubator: Thermo Electron Corporation, USA • HPLC plus intelligent HPLC pump with autosampler, model Jasco PU 2080: Jasco, Mumbai, India • Tissue homogenizer: Remi scientific, Mumbai, India • Stereotaxic apparatus:

3.3 Software’s • Phototox, Ver 2.0 • Image-Pro Premier, Ver 9.1 • Kaluza, Ver 1.2 • GraphPad Prism, Ver 6.0 • Minitab, Ver 17.0 • Origin, Ver 8.0 • Kinetica, Ver 5.0

3.4 Antioxidants 3.4.1 Idebenone General description: Chemically, IDB is an organic compound of the quinone family. It is also promoted commercially as a synthetic analog of ubiquinone

(coenzyme Q10 ; CoQ 10) and was initially patented by Takeda Chemical Industries, Osaka, Japan. In 1986 it was introduced to the market in Japan where it was initially suggested to aid in AD [1]. Currently, investigated for benefit in the treatment of various mitochondrial and neuromuscular diseases which are associated with respiratory chain dysfunction [2]. IDB differs in structure from CoQ 10 by means of a shorter carbon side chain as it possess an alkyl chain with a terminal polar hydroxyl group whereas CoQ 10 contains ten isoprenoid repeats [3]. This means IDB has a lower molecular weight (approximately 60 % smaller) subsequently increasing its solubility, characteristics of which should assist in the penetration of IDB in the skin in comparison to CoQ 10 [4]. 68

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Appearance: Yellow-orange crystalline powder CAS No: 58186-27-9 IUPAC: 2-(10-hydroxydecyl)-5,6-dimethoxy-3-methylcyclohexa-2,5-diene-1,4- dione Chemical structure: [5]

Molecular formula : C 19 H30 O5 Molecular weight : 338.439 g·mol -1 Solubility: Soluble in DMSO (>25 mg/mL), ethanol (>25 mg/mL), methanol, and chloroform. Insoluble in water. pKa : 15.19 Density (tapped) : 1.09 g/cm 3 Melting point : 52-55 °C Storage : Store in closed container at room temperature. Mechanism of action : In cellular and tissue models, IDB acts as a transporter in the electron transport chain of mitochondria and thus increases the production of adenosine triphosphate (ATP) which is the main energy source for cells, and also inhibits lipoperoxide formation. A positive effect on the energy household of mitochondria has also been observed in animal models. Pharmacokinetics : After absorption, IDB is rapidly metabolized by first-pass metabolism and shows dose-proportional pharmacokinetics in healthy subjects in daily doses up to 2250 mg. IDB is well absorbed from the gut but undergoes excessive first pass metabolism in the liver, so that less than 1 % reach the circulation. This rate can be improved with special formulations (suspensions) of IDB and by administering it together with fat food; but even taking these measures bioavailability still seems to be considerably less than 14 % in humans. More than

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99 % of the circulating drugs are bound to plasma proteins. IDB metabolites include glucuronides and sulfates, which are mainly (~ 80%) excreted via the urine [6].

3.4.2 Resveratrol General description: Chemically, RSV (3,5,4 '-trihydroxy-stilbene) is a polyphenolic phytoalexin that is generated in response to environmental stress in Japanese plant Polygonum cuspidatum . It is also found in grape skins, peanuts and red wine, as well as host of nonedible plants [7, 8], and is produced preferentially in response to fungal infections as seen in mature vine berries [9]. RSV exists in cis- and trans -isomers, where later is more abundant and biologically active. However, trans-isomers get converted in to cis- when not stored properly (exposed to white light) and if it is protected from high pH. trans -isomer is reported to be stable for 4 weeks in absence of light [10,11]. RSV has been reported to have numerous biological effects, including antioxidant activity. Appearance: White to off-white crystalline powder. CAS No: 501-36-0 IUPAC: 5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol Chemical structure: [12]

Molecular formula: C14 H12 O3 Molecular weight: 228.24 g·mol -1 Solubility: Soluble in DMSO (16 mg/mL), ethanol (50 mg/mL), methanol, and chloroform. Water solubility 0.03 mg/mL. pKa (strongest acidic): 8.99 pKa (strongest basic): − 5.7 Density (tapped) : 1.4±0.1 g/cm 3 Melting point: 271.57 °C Storage : Store in closed container at − 20°C, avoid direct exposure to white light. 70

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Mechanism of action: The exact mechanism of RSV apparent effects on life extension (anti-aging) is not yet clear, but it is believed that it is both a free radical scavenger and a potent antioxidant due to its ability to promote the activities of a variety of antioxidant enzymes [13]. RSV also exhibits neuroprotective activity in AD model by inhibiting A β protein aggregation and modulates intracellular effectors involved in neuronal cell survival/death [14]. Pharmacokinetics: Oral absorption of resveratrol is high ( ≈ 75%) and occurs primarily through transepithelial diffusion [15], However, its oral bioavailability is very poor ( < 1%), mainly due to metabolism in the lower part of GI tract and extensive hepatic gluconuridation and sulfation. Hence, only 5 ng/mL of unchanged RSV was detected in blood after 25 mg oral dose.

3.4.3 Phenyl butyl nitrone General description: PBN otherwise known as “spin trap” or “intelligent” antioxidants, are the compounds that stabilize or trap free radicals, hence reducing the cascade effect on other molecules. Appearance: White to off-white crystalline powder Chemical structure: [16]

CAS No: 33-7624-7 IUPAC: 5-nitrosopentylbenzene

Molecular formula: C11 H15 NO Molecular weight: 177.25 g·mol -1 Solubility: In most solvents such as methanol, ethanol and dichloromethane. Slightly soluble in water. Density (bulk) : 1.0 ± 0.1g/cm 3 Melting point : 72.96 °C Storage : Store in closed container at − 20°C, protect from light.

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Mechanism of action: Conventional antioxidants, the compound destructively act upon the free radicals by chemically reacting with them to convert ROS into water, whereas, “spin trap” “traps” the ROS and convert them into harmless oxygen and then transport them back into the electron transport chain of cellular respiration. “Spin trap” are the only antioxidants which can differentiate between the good oxygen molecules and harmful ones [17, 18]. Pharmacokinetics: In a reported study when PBN administered intraperitoneally (i.p.) the concentration of PBN in plasma peaked at 15 min, whereas the maximum concentration in all the major organs (brain, heart and kidney) tested occurred at 30 min. PBN was detected in urine for as long as 24 h after injection. In another study an in vivo microdialysis study in rats indicated that PBN distributes preferentially to the brain; about 15-fold better than the water-soluble PBN analog [19,20].

3.5 Excipients 3.5.1 Solid lipids 3.5.1.1 Witepsol E85 [21]. CAS no: 91744-42-2 Regulatory information: Product conforms to EP and USP. Chemical description/INCI name : Hard fat, PhEur: Adeps solidus, USP: Hard Fat and hydrogenated coco glycerides. General description : They are white, odourless hard fats in pastill shape which comprises of glycerides of plant origin. It is a mixture of 5 % mono-, 29 % di-, and

66 % triglycerides esters of fatty acid C 8 – C18 , with hydroxyl value (mg KOH/g) in the range of 5-15. Melting point : 46°C Administration route: Topical, oral and or rectal/vaginal routes

3.5.1.2 Gelucire 50/13 [22]. CAS no: 9011-21-6 Regulatory information: Product conforms to EP, USP/NF. Chemical description: Stearoyl macrogol-32 glycerides EP / Stearoyl polyoxyl-32 glycerides INCI name: Hydrogenated palm oil PEG-32 esters Form : Waxy solid pellets 72

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Colour : Creamy white Odour : Faint

General description: It is a mixture of caprylic acid C 8 ( < 3.0%), capric acid C 10

(<3.0 %), lauric acid C 12 ( < 5.0 %), myristic acid C 14 ( < 5.0 %), palmitic acid C 16

(40 to 50 %), stearic acid C 18 (48 to 58 %), with hydroxyl value (mg KOH/g) in the range of 36 - 56. Hydrophilic-Lipophilic Balance (HLB): 13 Melting point: 50° C Administration route : Oral, topical and/or rectal/vaginal routes. Storage : Keep the product in its original packaging sealed tightly, protect from light and moisture.

3.5.1.3 Gelucire 44/14 [23]. CAS no: 57107-95-6 Regulatory information: Product conforms to EP, USP/NF. Chemical description: Lauroyl macrogol-32 glycerides EP / Lauroyl polyoxyl-32 glycerides NF. INCI name: Hydrogenated coconut PEG-32 esters Form : Waxy solid Colour : White Odour : Faint

General description: It is a mixture of caprylic acid C 8 ( < 15.0 %), capric acid C 10

(< 12.0 %), lauric acid C 12 ( < 30 to 50 %), myristic acid C 14 ( < 5 to 25 %), palmitic acid C 16 (4 to 25 %), stearic acid C 18 (5 to 35 %), with hydroxyl value (mg KOH/g) in the range of 36 - 56. HLB: 14 Melting point: 44° C Administration route : Oral, topical and/or rectal/vaginal routes. Storage : Keep the product in its original packaging sealed tightly, protect from light and moisture.

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3.5.2 Liquid lipids 3.5.2.1 Capmul MCM [24]. CAS no: 91744-32-0, 26402-22-2 and 26402-26-6 Regulatory information: Product conforms to USP/NF. Ingredients: Caprylic/capric mono- & diglycerides Chemical name: Glyceryl caprylate/caprate Synonyms: Glycerol monocaprylocaprate; Glycerides C8-10 mono-, di-, tri-; Medium chain mono- & diglycerides; Glyceryl mono- & dicaprylo/caprate; Monodiglycerides of caprylic/capric acid Appearance: Liquid (at 25 °C) Specific gravity: 0.97 – 1.02 General description: Capmul products are mono-, di- and triglyceride emulsifiers prepared through the glycerolysis of select fats and oils. They are lipophilic, insoluble in water and soluble in oils at elevated temperatures. They are used to produce stable emulsions and to modify viscosity. Caprylic and capric mono- diglyceride esters function as very effective carriers and solubilizers of active compounds. Storage : Store in tight, light-resistant containers. Store in a dry location at ambient temperature.

3.5.2.2 Labrafil M 1944 CS [25]. CAS no: 69071-70-1 Regulatory information: Product conforms to USP/NF and EP. Chemical description: Oleoyl macrogol-6 glycerides EP / Oleoyl polyoxyl-6 glycerides NF INCI name: Apricot Kernel Oil PEG-6 Esters Form : Liquid at 40°C, colourless Odour : Faint

General description: It is a mixture of palmitic acid C 16 (4 to 9 %), stearic acid C 18

(6 %), oleic acid C 18:1 (58 - 80 %), linoleic acid C 18:2 (15 - 35 %), linolenic acid C 18:3

(2 %), arachidic acid C 20 (2 %), eicosenoic acid C 20:1 (2 %), with hydroxyl value (mg KOH/g) in the range of 45 - 65. HLB: 9 Administration route : Oral and topical. 74

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Storage : Keep the product in its original packaging sealed tightly, protect from light and moisture

3.5.3 Surfactants 3.5.3.1 TegoCare 450 [26]. CAS no: 68936-95-8 and 9009-32-9 Regulatory information: Only for cosmetics use, product conforms to USP and listed in European Cosmetic Directive 76-768 / EEC. INCI name: Polyglyceryl-3-Methylglucose Distearate Form: Pellets Colour: Ivory HLB: 12 General description: TegoCare 450 is a non-ionic, PEG-free emulsifier based on natural renewable raw materials. TegoCare 450 is an emulsifier for the formulation of O/W creams and lotions providing a high compatibility with active ingredients. TegoCare 450 forms stable emulsions with all common oils and fats used for skin care products, including polar oils. Storage: Keep the product in its original packaging sealed tightly, protect from light and moisture.

3.5.3.2 Amisoft CS 11 [27, 28]. CAS no: 68187-32-6 INCI name: Sodium Cocoyl Glutamate Chemical name: Sodium N-Cocoyl-L –Glutamate Average Mol. Wt: 359 Appearance: White, fine powder General description: Amisoft series is derived from L-glutamic acid (an amino acid), natural fatty acids and is anionic in nature. It is extremely hypoallergenic and well suited as a mild cleansing agent. The pH level of the agents is similar to that of the skin, therefore, they are very mild to the skin. They vary with different fatty acid chain length and available form.

Fatty acid chain length: C 8-14 mixture 86.9 % Storage: Keep the product in its original packaging sealed tightly.

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3.5.3.3 Amisoft LS 11 [27, 28]. CAS no: 29923-31-7 INCI name: Sodium Lauroyl Glutamate Chemical name: Sodium N-Lauroyl-L –Glutamate Average Mol. Wt: 356 Form: Fine powder Colour: White General description: Same as CS 11

Fatty acid chain length: C12 97.3 % Storage: Keep the product in its original packaging sealed tightly.

3.5.3.4 Amisoft MS 11 [27, 28]. CAS no: 38517-37-2 INCI name: Sodium Myristoyl Glutamate Chemical name: Sodium N-Myristoyl-L –Glutamate Average Mol. Wt: 384 Form: Fine powder Colour: White General description: Same as CS 11

Fatty acid chain length: C14 97.8 % Storage: Keep the product in its original packaging sealed tightly.

3.5.3.5 Human serum albumin [29]. CAS no: 9048-49-1

Protein chemical formula: C2936 H4624 N786 O889 S41 Protein average weight: 66472.2000 Form: Yellowish liquid General description: Human serum albumin (HSA) is an abundant multifunctional non-glycosylated, negatively charged plasma protein, with drug carrier properties, emulsification properties, ligand-binding and transport properties, antioxidant functions, and enzymatic activities [30]. In addition, albumin is a multi-functional excipient due to its intrinsic physiochemical and biochemical properties which makes it an ideal excipient in various drug delivery platforms [31]. Half-life: 20 days 76

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Storage: Keep the product in cool place, preferably at 8 °C in freeze.

3.6 References:

1) Geromel V, Darin N, Chrétien D, Bénit P, DeLonlay P, Rötig A, Munnich A, Rustin P. Coenzyme Q(10) and idebenone in the therapy of respiratory chain diseases: rationale and comparative benefits. Mol Genet Metab. 2002; 77:21-30. 2) Carbone C, Pignatello R, Musumeci T, Puglisi G. Chemical and technological delivery systems for idebenone: a review of literature production. Expert Opin Drug Deliv. 2012; 9:1377-92. 3) Haefeli RH, Erb M, Gemperli AC, Robay D, Courdier Fruh I, Anklin C, Dallmann R, Gueven N. NQO1-dependent redox cycling of idebenone: effects on cellular redox potential and energy levels. PLoS One. 2011; 31:e17963. 4) McDaniel DH 1, Neudecker BA, DiNardo JC, Lewis JA 2nd, Maibach HI. Clinical efficacy assessment in photodamaged skin of 0.5% and 1.0% idebenone. J Cosmet Dermatol. 2005: 4:167-73. 5) http://www.chemspider.com/Chemical-Structure.3558.html (accessed 01.11.2014) 6) Becker C, Bray-French K, Drewe J. Pharmacokinetic evaluation of idebenone. Expert Opin Drug Metab Toxicol. 2010; 6:1437-44. 7) Neves AR 1, Lucio M, Lima JL, Reis S. Resveratrol in medicinal chemistry: a critical review of its pharmacokinetics, drug-delivery, and membrane interactions. Curr Med Chem. 2012; 19:1663-81. 8) Guo W 1, Li A, Jia Z, Yuan Y, Dai H, Li H. Transferrin modified PEG-PLA-resveratrol conjugates: in vitro and in vivo studies for glioma. Eur J Pharmacol. 2013; 718:41-7. 9) Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CW, Fong HH, Farnsworth NR, Kinghorn AD, Mehta RG, Moon RC, Pezzuto JM. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science. 1997; 275:218-20. 10) Lopez-Hernandez J, Paseiro-Losado P, Sanches-Silva AT, Lage-Yusty MA. Study of the changes of trans-resveratrol caused by ultraviolet light and the determination of trans- and cis-resveratrol in Spanish white wines. Eur. Food Res. Technol. 2007; 225: 789–796. 11) Trela BC, Waterhouse AL. Resveratrol: isomeric molar absorptivities and stabilities. J. Agric. Food Chem. 1996; 44:1253–1257. 12) http://www.chemspider.com/Chemical-Structure.392875.html (accessed 01.11.2014) 13) de la Lastra CA, Villegas I. Resveratrol as an antioxidant and pro-oxidant agent: mechanisms and clinical implications. Biochem Soc Trans. 2007; (Pt 5):1156-60. 14) Bastianetto S, Ménard C, Quirion R. Neuroprotective action of resveratrol. Biochim Biophys Acta. 2015; 1852:1195-1201. 15) W alle, T., 2011. Bioavailability of resveratrol. Ann. N. Y. Acad. Sci. 1215, 9–15. 16) http://www.chemspider.com/Chemical-Structure.554348.html?rid=7a33fa61-27e6-4247-9a2f- a533ea64df9a (accessed 03.11.2014) 17) http://www.beautymagonline.com/beauty-articles-4/1112-spin-traps-2 (accessed 06.11.2014) 18) http://geronova.com/pbn-overview/ (accessed 06.11.2014) 19) Yashige K. Pharmacologic Properties of Phenyl N-tert-Butylnitrone. Antioxid Redox Signal. 1999; 1:481-499. 20) Cheng HY, Liu T, Feuerstein G, Barone FC. Distribution of spin-trapping compounds in rat blood and brain: in vivo microdialysis determination. Free Radic Biol Med. 1993; 14:243-50. 21) www.cremeroleo.de/de-wAssets/docs/.../02-broschuere-witepsol.pdf (accessed 05.11.2014) 22) http://www.gattefosse.com/en/applications/gelucire-5013.html (accessed 07.11.2014) 23) http://www.gattefosse.com/en/applications/gelucire-4414.html (accessed 03.11.2014) 24) http://www.abiteccorp.com/product-lines/capmul (accessed 03.12.2014) 25) http://www.gattefosse.com/node.php?articleid=10 (accessed 08.12.2014) 26)http://www.centroaloe.it/index.php?page=shop.getfile&file_id=633&product_id=2395&option=c om_virtuemart&Itemid=90 (accessed 15.12.2014) 27) http://www.ajichem.com/products/product-list.aspx (accessed 04.12.2014) 28) http://www.ajichem.com/en/products/anionic-surfactants.aspx (accessed 04.12.2014) 29) http://www.drugbank.ca/drugs/db00062 (accessed 06.01.2015) 30) Quinlan GJ, Martin GS, Evans TW. Albumin: biochemical properties and therapeutic potential. Hepatology. 2005; 41:1211-1219 31) http://www.europlat.org/albumin-as-a-multifunctional-excipient-a-paradigm-shift-in-drug- development-and-delivery.htm (accessed 06.01.2015)

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Photoprotection aspects of topically administered IDB-NLCs

This chapter deals with the cellular interactions and photoprotective effects of idebenone (IDB; an antioxidant) - loaded nanostructured lipid carriers (NLCs) stabilized using PEG-free surfactant (TegoCare 450). The two-step preformulation 3 strategy followed by three-level, three-variable, L9 (3 ) Taguchi robust orthogonal design was employed to optimize IDB-NLCs. Results of this study showed that NLCs markedly improved biocompatibility of IDB under normal as well as stress conditions. Quantitative and qualitative cell uptake studies demonstrated a significant uptake of IDB–NLCs. Hence, exerted improved photoprotective effects.

Chapter 4: Photoprotection aspects of IDB-NLCs

4.1 Genesis and outline of the work Skin being a largest organ of the human body is constantly exposed to the sun’s harmful Ultraviolet (UV) radiation and hence deserves extra care and protection. UV radiation emitted by the sun is divided into three wavelength ranges: UVC (270-290), UVB (290-320) and UVA (320-400) [1]. Though UVB constitute only 4-5% of UV radiation is thought to be blameworthy for most of the UV- produced detrimental effects (erythema, oedema, pigment darkening and thickening of epidermis and dermis) on the skin and is one of the key etiological agent in the production of ROS [2-4]. ROS induce direct cellular injury, premature skin aging (photoaging) and skin cancer [5]. Fortunately, human skin possesses a wide range of endogenous enzymatic and nonenzymatic antioxidants that works in a coordinated manner to neutralize and restore ROS balance. In spite of these inbred defenses, increased oxidative stress can vanquish the skin’s antioxidant defense machinery. Hence, topical supplementation of exogenous antioxidants may help neutralize and restore ROS balance, and prevents skin aging phenomenon and IDB is one such antioxidant [6-8]. IDB [2-(10-hydroxydecyl)-5,6-dimethoxy-3-methyl-2,5-cyclohexadiene-1,4- dione] is a synthetic derivative of CoQ10 , with a shorter carbon side chain, and increased solubility and skin penetration compared to CoQ 10 [9]. Owing to antioxidant activity, IDB has been researched extensively as a means for treating number of mitochondrial and neuromuscular diseases associated with respiratory chain dysfunction [10-12]. Recently, Santhera pharmaceuticals Raxone ® (IDB 150 mg, in the treatment of Leber hereditary optic neuropathy) received temporary authorization (ATU) for use granted by the French National Agency for Medicines and Health Products Safety (ANSM) [13]. Regarding the cosmetic applications, IDB has shown the highest oxidative protection capacity when compared to dl-alpha tocopherol, kinetin, CoQ 10 , L-ascorbic acid, and dl-alpha lipoic acid [14]. Hence, the cosmetic industry has shown keen interest in IDB which lead to the development of anti-ageing products Prevage® MD (IDB 1.0%) and TRUE Youth Revealing Complex® (0.5%) by Allergan Inc, USA and TRUE cosmetics, USA, respectively. However, it has been reported that these IDB products offered little or no skin photoprotection [15], this effect could be attributed to IDB chemical instability upon UV exposure [6]. Moreover, to exert improved and sustained photoprotective

78 Chapter 4: Photoprotection aspects of IDB-NLCs effects in skin, IDB need to cargo into efficient topical drug delivery system, and NLCs is one such option. NLCs represent the second generation of lipid nanoparticle technology, composed of spacially very different biocompatible/biodegradable solid lipid and liquid lipid (oils), and exhibits mean particle size in the submicron range, ranging from 50 to 1000 nm [16]. For the last two decades NLCs has become increasingly interesting for researchers around the globe owing to their potential to overcome the challenges associated with the topical drug delivery system [17]. The key advantages associated with NLCs compared to “old school” colloidal systems (liposomes and nanoemulsion) are enhancement of chemical stability of chemically labile compound’s [18-20], improved skin photoprotection [21], characteristic sustain drug release pattern [22], improved benefit/risk ratio [23], enhanced drug loading and limited drug expulsion during storage [24], and finally cosmetic acceptability [25]. In this context, solid lipid nanoparticles (SLNs) and NLCs have lately been utilized efficiently for the topical delivery of IDB owing to its aforementioned characteristic property compared to CoQ 10 . However, majority of these studies remained, more or less, limited to targeting of IDB into the upper layers of the skin [26], antioxidant activity of IDB [27], improvement of IDB chemical stability [28], and IDB release from carrier system [29], but no attention has been paid to their interaction with HaCaT cells in terms of viability and uptake, and assessment of photoprotective effects, still remains to be explored. Therefore, the specific aim of the present study was to prepare, optimize IDB-NLCs using two-step preformulation strategy and orthogonal design, and to appraise cellular interactions (cell viability and uptake) and photoprotective effects of IDB, placebo-NLCs (without adding the IDB) and the IDB-NLCs against UVB-mediated oxidative stress in HaCaT cells. Since, UVB affects the outermost layer of the skin, the epidermis, and keratinocytes are the most copious cell type constituting almost 80-95 % of epidermal cells [30], we therefore chose HaCaT cells as keratinocyte cell model.

4.2 Experimental 4.2.1 Lipid phase screening Selection of solid lipid was initiated by transferring 50 mg of IDB to the glass vial containing 1 g of solid lipid. Next, the vial content was then melted at

79 Chapter 4: Photoprotection aspects of IDB-NLCs

80°C and vortexed for 2 min. The resulting mixture was then visually observed for the solubility of IDB. Following dissolution of the initial amount of IDB in the molten lipid, the IDB was added in steps of 50 mg until IDB crystals failed to dissolve in the molten lipid. The saturation solubility of IDB in variety of liquid lipids was determined by transferring an approximately 0.5 g of IDB to the amber-colored glass vial containing 1 g of liquid lipid, vortexed for 2 min and equilibrated at room temperature (to avoid IDB photo-degradation) for 24 h using shaker water bath. After 24 h, the resulting mixture was centrifuged at 10,000 rpm for 15 min. The clear supernatant was filtered through 0.45 µm hydrophilic AXIVA nylon syringe filters. A 100 µL of aliquot of the filtrate diluted to 3 mL with analytical grade absolute ethanol 99.9 % and the resulting solution was quantified spectrophotometrically at 278 nm. The miscibility of the selected solid lipid and liquid lipid (binary mixture) that dissolved maximum amount of IDB was mixed in three main ratios such as, 7:1, 7:2 and 7:3. In next step, a small quantity of binary mixture was smeared on Whatman filter paper and observed for any oil expulsion on filter paper. Additionally, to get information about recrystallization of IDB from selected binary mixture ratio, the samples were scrutinized using polarized light microscopy at 40x objective. Only the best binary mixture ratio which did not show any oil expulsion on filter paper and which did not show any IDB crystals was selected for the preparation of IDB-NLCs.

4.2.2 Surfactant selection 4.2.2.1 Photosafety assessment of surfactant The initial surfactant selection was carried out by performing a basic UV/VIS spectral analysis (a key indicator of photosafety) and determining molar extinction coefficient (MEC or ɛ) of surfactant [31]. Briefly, surfactant was dissolved in double distilled water (DDW) at final concentrations of 5 µM, 10 µM and 20 µM. UV/VIS absorption spectra were recorded (specifically from 290-320 nm) on UV/VIS spectrophotometer. A standard rectangular spectrophotometer quartz cell with 10 mm path length was utilized (Starna; Optiglass Ltd., Hainault, UK). For each peak, MEC was calculated: ɛ = A/ (c × l ), A: absorbance, c: concentration of surfactant, l: path length.

80 Chapter 4: Photoprotection aspects of IDB-NLCs

4.2.3 Crystallographic investigations Differential scanning calorimetry (DSC) measurements were conducted to get information about the melting behavior of IDB, Witepsol E 85 (WE 85), binary mixture of WE 85 and Capmul MCM (7:1) and ternary mass mixture of IDB, WE 85 and Capmul MCM (1.25:7:1). DSC measurements were carried out using a METLER DSC 821e module controlled by STAR e software. Briefly, 6 mg sample was weighed directly and hermetically sealed into a standard 40 µL aluminium pan, before heating under nitrogen flow (20 mL/min) at a scanning rate of 10 °C/min, over the temperature range of 0 to 150 °C. An empty aluminium pan was used as reference. X-Ray diffraction (XRD) patterns were captured using a X-ray diffractometer. Samples were irradiated with monochromatized Cu-Kα radiation ( λ= 1.54056 Å) and analyzed over 2 Theta (θ) range of 5-50°. The voltage and current used were 30 kV and 30 mA respectively.

4.2.4 Optimization of IDB-NLCs preparation After appropriate selection of solid to liquid lipid ratio (7:1), the optimum process parameters (sonication amplitude and time) and surfactant concentration required for the preparation of IDB-NLCs were optimized using Taguchi robust orthogonal design.

4.2.5 Preparation of IDB-NLCs IDB-NLCs were prepared by a melt-emulsification-ultrasonication method as described elsewhere [32], with some minor changes. Briefly, the weighted quantities of Witepsol E 85 (solid lipid, m. p. 48 °C), Capmul MCM (liquid lipid) and TegoCare 450 (surfactant, m. p. 62 °C) were melted together and cyclo-mixed for 1 min. The obtained lipid-surfactant mixture was quickly added in hot DDW (maintained at 70 °C) under ultrasonication (amplitude 90 %., 4 min., 30 sec “on”, 5sec “off” cycle) using Ultrasonic processor. The resulting dispersions were allowed to cool down to room temperature and initial particle size and zeta potential were recorded. Placebo-NLCs and NR-IDB-NLCs (with nile red (NR) in lipid-surfactant mixture) were prepared the same way as the IDB-NLCs. Final compositions of the investigated NLC are depicted in Table 4.6.

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4.2.6. Characterization of IDB-NLCs 4.2.6.1 Particle size analysis and zeta potential determination The mean particle size (z-ave), and the polydispersity index (PI, related to the width of size distributions) of IDB-NLCs was determined by particle size analyzer (DLS) at a fixed scattering angle of 90 °C and a temperature of 25 °C ± 1 °C. 50 µL of each sample was diluted with a 3 mL of DDW in order to obtain suitable scattering intensity before DLS analysis. 3 DLS measurements were performed on each sample. The zeta potential ( ζ) of IDB-NLCs was determined by zeta potential analyzer after switching the instrument from DLS to the Electrophoretic Light Scattering (ELS) mode. 50 µL of each sample was diluted with a 3 mL of DDW with conductivity adjusted to 50 µS/cm using 0.9 % (w/v) sodium chloride solution and a pH ~ 5.5. Three DLS measurements (at 25 °C ± 1°C) were performed on each sample.

4.2.6.2 Entrapment efficiency and drug content The % entrapment efficiency (% EE) of IDB in the NLC was determined by centrifugation method (measuring the concentration of free IDB in the aqueous phase of the NLC dispersion), as described elsewhere [32], with some minor changes. Briefly, 1 mL of NLC dispersion (equivalent to 5 mg of IDB) was measured and filled in centrifuge tubes. The samples were centrifuged at 25,000 rpm for 30 min at 25 °C ± 1°C, in order to settle prepared nanoparticles. The clear supernatant was filtered through 0.45 µm syringe filters. A 100 µL of aliquot of the filtrate diluted to 3 mL with ethanol and the resulting solution (free IDB) was quantified spectrophotometrically at 278 nm. The % EE was calculated using the following equation: Total amount of IDB - Amount of free IDB % EE = × 100 Total amount of IDB Drug content of prepared IDB-NLCs was determined by rupturing lipid nanoparticles into suitable solvent. For that, a specific volume (50 µL) of IDB-NLCs dispersion was suitably diluted (3 mL) with ethanol. The resulting solution sonicated in a bath-type sonicator for 5 min, and subsequently vortexed for 5 min. In next step, the solution was filtered through 0.45 µm syringe filter and the total drug content was quantified spectrophotometrically at 278 nm.

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4.2.6.3 Transmission electron microscopy (TEM) The shape and surface morphology of IDB-NLC was examined by TEM at an acceleration voltage of 200 kV. The sample was prepared by placing a drop of IDB-NLCs dispersion on a copper grid coated with carbon film. The grid was allowed to air-dry at room temperature, before visualization of the sample using TEM. Staining agent (such as osmium tetroxide) was not used in current TEM examination.

4.2.6.4 In vitro occlusion test The occlusivity (occlusion factor F) of IDB-NLCs and IDB-PD (pure drug suspension in PBS) was performed as described previously [33]. Briefly, 100 mL capacity glass beaker was filled with 50 g of DDW, covered with Whatman ® filter paper grade 6 (cellulose filters, 9 cm, circle, cutoff size: 3 m) and sealed with water- resistant adhesive. Approximately 200 mg of IDB-NLCs and IDB-PD sample was evenly distributed with a spatula on the filter surface (18.8 cm 2). A visible film formation on top of the filter paper was observed during the experiment. In the next step, the beaker was immediately stored in stability chamber at 32 °C ± 1°C (the skin temperature), 60 % ± 5 % relative humidity (RH) for 48 h in order to simulate the temperature of the skin surface. The weight of the water present in the beaker was carefully weighed on an analytical balance at 6, 12, 24 and 48 h. The glass beaker covered with filter paper but without applied sample served as a reference. Each experiment was performed in triplicate ( n = 3). F was calculated using the following equation:  A − B  F =  ×100  A 

Where, A is water loss without sample (reference) and B is water loss with sample. An F value of 0 (zero) means no occlusive effect compared to the reference, whereas F value of 100 means maximum occlusive factor.

4.2.6.5 In vitro IDB release studies In vitro IDB release studies ( n = 3) from NLCs and IDB-PD were carried out as described previously [26], with some minor changes. The jacketed Franz-type diffusion cells with a diffusion area 0.636 cm 2 and a receptor compartment of 5 mL

83 Chapter 4: Photoprotection aspects of IDB-NLCs were used in the current study. Initially, a cellophane dialysis membrane (MW cut- off 12000 Da, Hi-media Labs, India) was equilibrated in receptor solution (water/ethanol; 50:50, v/v) for 24 h. After 24 h, the membrane was installed in between the donor and receptor compartment and 200 µL of IDB-NLCs and IDB- PD (equivalent to 1 mg of IDB) was gently placed on the donor compartment under non-occlusion conditions. The receptor solution was continuously stirred with a magnetic bead and thermostated at 37 °C ± 1°C. By maintaining the release medium at 37°C, a temperature of 32°C at the membrane surface was assured, thus mimicking the human skin conditions. The experiments were carried out over a period of 24 h and at fixed time intervals (1, 2, 4, 6, 8, 12, 16 and 24 h), 200 µL aliquot of the receptor solution was withdrawn and immediately replaced with equal volume of fresh medium (pre-equilibrated at 37°C ). The % IDB release was quantified spectrophotometrically at 278 nm with suitable dilutions.

4.2.6.6 Ex vivo drug diffusion studies Ex vivo drug diffusion studies ( n = 3) of IDB-NLCs and IDB-PD were performed in jacketed Franz-type diffusion cells, in similar conditions of the In vitro IDB release studies (section 4.2.6.5), but using newborn porcine skin model (as a surrogate for human skin, provided by a local slaughterhouse) [34], installed in between the donor and receptor compartment, with stratum corneum (SC) side facing the donor compartment. A 5 % Poloxamer 188 aqueous solution as a receptor medium has been used since the water/ethanol (50:50, v/v) solution could damage the barrier integrity of animal skin [35]. The studies were performed for 12 h according to the clinical application time [36]. The % IDB permeated in receptor compartment was quantified spectrophotometrically at 278 nm with suitable dilution. Initially, the skin was gently cleaned and subcutaneous fat was carefully removed, and excised into 3 × 3 cm 2 samples, wrapped in an aluminium foil and stored (less than a week) at −80°C until used. Before the experiments, the frozen skin samples were thawed, and pre-equilibrated by placing in PBS pH 7.4 for 24 h at 4 °C. At the end of the experiment (12 h), the diffusion cell was dismantled and % IDB remained on the skin was determined. The skin surface was washed three times with receptor solution. Those washings were filtered through 0.22 µm syringe filters

84 Chapter 4: Photoprotection aspects of IDB-NLCs and quantified spectrophotometrically at 278 nm with suitable dilution. % IDB deposited in skin was estimated after washing skin with a PBS pH 7.4. In the next step, skin was cut into small pieces and 5 mL of ethanol was added to the test tube and IDB present in the skin was extracted using tissue homogenizer for 2 min at 5,000 rpm, followed by bath sonication for 15 min. later, a skin homogenate was centrifuged (10,000 rpm, for 10 min. at 4 °C), and the supernatants were filtered through 0.22 µm syringe filters and quantified spectrophotometrically at 278 nm with suitable dilution.

4.2.6.7 Stability studies The IDB-NLCs formulation was subjected to stability study for over a period of 90 days according to International Conference on Hormonisation (ICH) guidelines. 10 mL of IDB-NLC dispersion was transferred in each of several airtight amber-colored glass vials, stored at different temperature and humidity conditions such as 25°C ± 2 °C/60 % ± 5% RH and at 40 °C ± 2 °C/75 % ± 5 % RH in a stability chamber. Particle size, encapsulation efficiency, zeta potential and polydispersity index of the samples were monitored at regular interval (Initial, 7 days, 15 days, 30 days and 90 days).

4.2.7 Cell culture study 4.2.7.1 HaCaT culture Early-passage (P 09) HaCaT keratinocytes, a spontaneously transformed human epithelial cell line, acquired from National Centre for Cell Sciences (NCCS, Pune, India) was grown as adherent cultures into 75 cm 2 culture flasks using, DMEM high glucose (4.5 g/L) as base medium, supplemented with 10 % heat- inactivated FBS, 2mM L-glutamine, antibiotics (penicillin 10,000 U/mL and streptomycin 10,000 g/mL) and routinely maintained at 37 °C with 5% CO 2, 95 % air in a humidified incubator. When cell line reached the exponential phase of growth (nearly 70 - 80 %), the old culture media were carefully removed from the flask and the cells were briefly rinsed with 10 ml of PBS pH 7.4, harvested with 0.25 % trypsin and expanded by subculturing into three (3) 75cm 2 flasks. Across all experiments, cells were used between 9 th and 12 th passage.

85 Chapter 4: Photoprotection aspects of IDB-NLCs

4.2.7.2 Keratinocyte treatment with IDB, placebo-NLCs and IDB-NLCs samples Initially, IDB (stock, 10 mM) was dissolved in DMSO and stored at − 20°C until use. Working concentrations (1, 2, 4, 6, 8 and 10 µM/0.1 mL) of IDB-PD were prepared by serially diluting the stock in freshly prepared culture medium. Similarly, placebo-NLCs, and IDB-NLCs were diluted in culture medium as required. HaCaT keratinocyte were seeded 100 µL/well from a density of 2 × 10 5/mL, into 96-well flat bottom tissue culture plate and the cells were arbitrarily divided into two major groups, such as negative control group (untreated) and compound group (working concentrations of IDB-PD). In next step, the plate was placed in an incubator and cells were allowed to attach overnight. Next day, cell media from overnight incubation were carefully removed from the plate and cells were briefly rinsed with 100 µL/well of PBS pH 7.4. Immediately cells were treated with working concentrations of IDB-PD (as above), followed by incubation for 12, 24 and 48 h and cell viability was quantified by MTS assay. In a separate experiment, the biocompatibility of placebo-NLCs and IDB- NLCs samples (formulation group) were determined in HaCaT cells. Briefly, cells were treated with formulation equivalent to 1, 2, 4, 6, 8 and 10 µM/0.1mL of IDB, followed by incubation for 24 h and cell viability was quantified by MTS assay. Later, to determine the photoprotective effect of IDB-PD, placebo-NLCs and IDB-NLCs on HaCaT cells against UVB radiation, cells were pretreated (24 h, 37°C) with IDB-PD and formulations equivalent to 1, 2, 4, 6, 8 and 10 µM/0.1mL of IDB. Next day, cells were irradiated with 200 mJ/cm2 of UVB for 1 h. After UVB irradiation, cells were replenished with a fresh cell culture medium and incubated for 24 h and cell viability was quantified by MTS assay. Untreated and non-irradiated cells were served as a negative control, whereas untreated and irradiated cells were served as positive control. For this experiment, UVB dose 200 mJ/cm 2 was chosen based on our previous results [37].

4.2.7.3 UV irradiation system The CL1000M UV Crosslinker system consist of five UVB tubes (8 W), was used to deliver an energy spectrum of UVB radiation (midrange, peak intensity 302 nm). To prevent UVB light absorption by the cell culture medium, the medium was

86 Chapter 4: Photoprotection aspects of IDB-NLCs removed just before irradiation and replenished with a thin layer of PBS to cover the cells. A 200 mJ/cm 2 of UVB corresponds to 15 min – 4 h of solar irradiation [38], a common period of time for out door exposure.

4.2.7.4 MTS assay Cytotoxicity was quantified using a ready-to-use The CellTiter 96 ® Aqueous One Solution of, 3-(4,5-dimethylthiazol-2-yl) -5-(3-carboxymethoxyphenyl) -2-(4- sulfophenyl) -2H-tetrazolium, inner salt (MTS) and is often described as a ‘one-step’ MTT assay. This colorimetric assay is fast, convenient, easy-to-use in comparison with 3-(4,5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT). MTS is bioreduced by cells into a colored formazan product which is soluble in tissue culture media. An increase or decrease in cell number results in a concomitant change in the amount of formazan formed, indicating the degree of cytotoxicity caused by investigated samples with or without UVB irradiation. After 24 h of incubation, 20 µL MTS solution (according to a protocol proposed by the manufacturer) was added directly into each well of 96-well assay plate containing investigated samples followed by additional 3 h of incubation. Finally, the cell viability was quantified by recording the formazan absorbance at 490 nm using a Bio-Rad microplate reader. Since the mean absorbance (OD) directly correlated with the number of viable cells, the mean OD of negative control cells was set to 100 % viability.

4.2.7.5 Cellular uptake of IDB-NLCs and NR-IDB-NLCs Both quantitative (using spectrophotometry) and qualitative (using Confocal Laser Scanning Microscope; CLSM) in vitro cell uptake studies were performed in HaCaT cells. For quantitative determination of IDB-PD and IDB-NLCs uptake by HaCaT cells, the cells were seeded (50,000 cells/1mL/well) into 24-well flat bottom tissue culture plate and the cells were arbitrarily divided into two major groups, such as IDB-PD group and IDB-NLCs group. In next step, the plate was placed in an incubator and cells were allowed to attach overnight. Next day, cell media from overnight incubation were carefully removed from the plate and cells were briefly rinsed with 1mL/well of PBS pH 7.4. Immediately cells were treated with IDB-PD and IDB-NLCs (equivalent to 6 µM/1mL of IDB; i.e. 2 µg/mL), followed by incubation for 1, 2, 3, 6, 12 and 24 h at 37 °C in CO 2 incubator. At each time point,

87 Chapter 4: Photoprotection aspects of IDB-NLCs medium (1mL) was carefully withdrawn from each well of 24-well plate. The cells were permeabilized by three cycles of freezing and thawing ( − 80 °C, F-T). Both permeabilized cells and recovered medium were lyophilized ( − 50 °C, 0.120 mbar, 24 h) and suspended in a 3 mL of ethanol followed by sonication for 1 min for complete dissolution of IDB. Later, the cell lysate was centrifuged at 5,000 rpm for 5 min and the clear supernatant was filtered through a 0.22 µm syringe filter. The amount of IDB in cells and media was quantified spectrophotometrically at 278 nm. The time-dependent intracellular uptake of NR-IDB-NLCs (qualitative) by HaCaT cells was studied using CLSM. For this experiment, HaCaT cells were grown in 24-well flat bottom tissue culture plate. Cell numbers were adjusted in order to get 20,000 cells in each well on sterile glass coverslip. Cells were treated with NR-IDB-NLCs (equivalent to 16ng/1mL of NR and 6 µM/1mL of IDB) and incubated at 37°C in CO 2 incubator for predetermined time intervals (as above). At each time point, the medium was replaced from one set of cells and cells were washed two times with 1mL/well of PBS pH 7.4 and cells were then fixed with 4 % paraformaldehyde in PBS pH 7.4 for 10 min (at room temperature) followed by washing three times with PBS. Next, the coverslips were carefully removed from culture wells using fine-point stainless steel tweezers and coverslips were mounted onto microscopic slides using Ibidi mounting medium. Fluorescence imaging of HaCaT cells was performed using the FluoView FV10i inverted CLSM system. The preset filter options were used to record the NR signal in the red-narrow channel (excitation, 599 nm; emission, 600 nm) and images were acquired with a 60x oil immersion objective lens with additional x2 digital zoom at 1024 × 1024-pixel resolution. Laser intensity were kept fixed (below 10 %) throughout the experiment. Furthermore, the integrated optical density (IOD) (red fluorescence) of minimum 6 confocal images (obtained from respective time points) of two experiments was analyzed by using Media Cybernetics' (Rockville, MD, USA) Image-Pro Premier 9.1 image analysis software.

4.2.7.6 Intracellular ROS scavenging activity 2′,7 ′-dichlorofluorescin diacetate, commonly known as DCF-DA, a cell- permeant fluorogenic probe was used to detect intracellular ROS levels in HaCaT cells (DCF-DA assay). Cells were seeded (20,000 cells/100 µL/well) into 96-well flat bottom tissue culture plate and the cells were arbitrarily divided into three major

88 Chapter 4: Photoprotection aspects of IDB-NLCs groups, such as negative control group (untreated and non-irradiated), positive control group (untreated but irradiated cells) and formulation group (treated and irradiated cells). In next step, the plate was placed in an incubator and cells were allowed to attach overnight. Then, cell media from overnight incubation were carefully removed from the plate and cells were briefly rinsed with 100 µL/well of PBS pH 7.4. Immediately cells were treated with placebo-NLC and IDB-NLCs (formulation equivalent to 6 µM/0.1mL of IDB), followed by incubation for 24 h at

37 °C in CO 2 incubator. After treatment, cell media from overnight incubation were carefully removed from the plate and cells were briefly rinsed twice with 100 µL/well of PBS pH 7.4. Then cells were irradiated with 200 mJ/cm 2 of UVB for 1 h, followed by further incubation for 24 h. Following incubation, the cells were incubated with 25 µM DCF-DA at 37 °C for 30 min. Later, the dye solution was removed, the cells were briefly rinsed with 100 µL/well of PBS pH 7.4 and cells were gently scraped off and resuspended in PBS pH 7.4 for analysis. Fluorescence emitted by DCF-DA was detected using a flow cytometer in FL-1 channel (535 nm). The data acquisition and analysis were performed using Kaluza software version 1.2. The level of intracellular ROS was expressed as median fluorescence intensity (MFI).

4.2.7.7 Mitochondrial membrane potential assay Rhodamine-123, a cell-permeant, lipophilic cationic, fluorogenic probe was used to detect UVB radiation-mediated loss of mitochondrial membrane potential (Ψ m) in HaCaT cells [39]. Cells were seeded (20,000 cells/100 µL/well) into 96- well flat bottom tissue culture plate and the cells were arbitrarily divided into three major groups, such as negative control group (untreated and non-irradiated), positive control group (untreated but irradiated cells) and formulation group (treated and irradiated cells). In next step, the plate was placed in an incubator and cells were allowed to attach overnight. Then, cell media from overnight incubation were carefully removed from the plate and cells were briefly rinsed with 100 µL/well of PBS pH 7.4. Immediately cells were treated with placebo-NLCs and IDB-NLCs (formulation equivalent to 6 µM/0.1mL), followed by incubation for 24 h at 37 °C in

CO 2 incubator. After treatment, cell media from overnight incubation were carefully removed from the plate and cells were briefly rinsed twice with 100 µL/well of PBS pH 7.4. Then cells were irradiated with 200 mJ/cm 2 of UVB for 1 h, followed by

89 Chapter 4: Photoprotection aspects of IDB-NLCs further incubation for 24 h. Following incubation, the cells were incubated with 25 µM Rhodamine-123 at 37 °C for 20 min. Later, the dye solution was removed, the cells were briefly rinsed with 100 µL/well of PBS pH 7.4 and cells were gently scraped off and resuspended in PBS pH 7.4 for analysis. Fluorescence emitted by Rhodamine-123 was detected using a flow cytometer FACSCanto II in FL-1 channel (535 nm). The data acquisition and analysis were performed using Kaluza software version 1.2. The loss of Ψ m was expressed as MFI.

4.2.8 Statistical analysis Statistical data analysis was performed using the demo version of GraphPad Prism 6.0 Software. p < 0.05 considered statistically significant. Taguchi design of experiments was employed using the demo version of MINITAB 17 software.

4.3 Results and discussion Initially a two-step preformulation strategy was adopted before the actual optimization applied to the IDB-NLCs preparation. The first step comprising preliminary lipid phase screening studies such as the ability of lipids to dissolve IDB and miscibility of the selected binary mixture ratio, and the second step was the selection of surfactant, based on the photosafety assessment of surfactant and literature review.

4.3.1 Selection of the lipid phase composition Choosing an appropriate lipid phase composition could increase % EE of IDB and slow the release rate of IDB from NLCs. Hence, the solubility of IDB in several different types of solid and liquid lipids was investigated and presented in Table 4.1. These data indicate that Witepsol E 85 (a mixture of 5 % mono-, 29 % di-

, and 66 % triglycerides esters of fatty acid C 8 - C18 ; melting point 46 °C) exhibited relatively high solubility value for IDB (more than 0.5 %, w/w), in comparison with Precirol ATO 5 (a mixture of approximately 8 – 22 % mono-, 40 – 60 % di-, and 25 – 35 % triglycerides of palmitic acid and stearic acid; melting point 52 °C). This observed difference was mainly attributed to the presence of emulsifier in Witepsol E 85 [40]. In addition, Witepsol E 85 could provide ‘drug-enriched core’ type NLCs (when a melting point of drug is more than the melting point of lipid used) [41], when compared to Precirol ATO 5. After the selection of Witepsol E 85 as ideal

90 Chapter 4: Photoprotection aspects of IDB-NLCs solid lipid, it was compulsory to select an appropriate liquid lipid for the preparation of NLCs. According to the results (Table 4.1), Capmul MCM (a mixture of mono- and diglycerides; mainly caprylic acid; max. 90 %) exhibited the highest solubilizing potential for IDB, i.e. more than 0.5 % (w/w) and hence Capmul MCM was selected as a liquid lipid for the preparation of IDB-NLCs.

Table 4.1: Solubility of idebenone in different solid and liquid lipids.

Lipids Idebenone solubility (%, w/w) 0.1 0.2 0.3 0.4 0.5 Solid lipids Witepsol E 85 + + + + +* Dynasan 116 − − − − − Precirol ATO 5 + + + − − Stearic acid − − − − − Compritol 888 ATO − − − − − Liquid lipids Captex 355 + + − − − Captex 300 + + − − − Captex 500 P + + + − − Phosal 53 MCT + + + − − Capmul MCM + + + + +* Miglyol 840 + + + + − Olive oil + − − − − Jojoba oil − − − − − Miglyol 812 + + − − − Rose hip seed oil − − − − −

Data are expressed as mean ± SD, n = 3. − Indicates insoluble (presence of idebenone crystals); + indicates soluble (absence of idebenone crystals); * indicates idebenone soluble more than 0.5 %.

Since, the Witepsol E 85 and Capmul MCM are two principal components of NLCs; they were mixed in different ratios to establish the ideal composition of a binary mixture for the preparation of NLCs. Binary mixtures that are miscible and manifested melting points higher than 40 °C were considered appropriate for use [42].

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The physical appearance of selected solid and liquid lipid at 7:1 ratio revealed no oil expulsion and remained solid at 32 °C (the skin temperature), in comparison with 7:2 and 7:3 ratios. In addition, the melting temperature of 7:1 ratio (assessed by DSC, Fig. 4.2 A) was well above (44.54 °C) the cut-off line (40 °C). On a further addition of liquid lipid, a soft mixture with expulsion of liquid lipid was observed on filter paper. Also, melting point depression occurred which was below the cut-off line, i.e. 39.35 °C, 37.71 °C for 7:2 and 7:3 ratio, respectively. To reconfirm the suitability and get information about the crystallization of IDB (0.5%, w/w) from 7:1 binary mixture ratio, we further evaluated this mixture by polarizing light microscopy. According to the results (Fig. 4.1 C), IDB crystals were not observed, which means binary mixture ratio 7:1 was able to dissolve 0.5 % (w/w) IDB, and has finally confirmed their suitability and overcome the prerequisite to obtain a sufficient drug payload, entrapment efficiency, and sustained release profile describe later on [43].

Figure 4.1: Polarized light microscopic images featuring A. Idebenone crystals. B. Binary mixture of WE 85 and Capmul MCM (7:1), and C. Ternary mixture of IDB, WE 85 and Capmul MCM (1.25:7:1).

The DSC thermograms and XRD patterns of IDB, Witepsol E 85, binary mixture of Witepsol E 85 and Capmul MCM (7:1) and a ternary mass mixture of IDB, Witepsol and Capmul MCM (1.25:7:1) are depicted in Fig. 4.2. The DSC thermogram of IDB and pure lipid (Fig. 4.2 A) showed a sharp melting endotherm at 54.01 °C and 48.10 °C, respectively, indicating the crystalline nature of the drug and lipid. Next, binary mixtures and a ternary mass mixture with the same formulation composition as NLCs were heated to 70 °C and maintained at that temperature for 10-15 min to simulate production state of NLCs. The bulk mixture showed widening of the melting peak and temperature was lower (44.54 °C) compared to pure lipid,

92 Chapter 4: Photoprotection aspects of IDB-NLCs which indicate that liquid lipid Capmul MCM was completely dissolved in pure lipid Witepsol E 85. Ternary mass mixture thermogram did not show a melting peak for IDB (Fig. 4.2 A), which indicate that the IDB was dissolved in the binary mixture and is in an amorphous state. In addition, the melting point of the ternary mass mixture was lower (43.78 °C) than that of binary mixture, but higher than 40°C (Fig. 4.2 A), which is prerequisite for topical application of lipid nanoparticles [44]. The XRD pattern of IDB (Fig. 4.2 B) showed four sharp and distinct diffraction peaks at 2 θ equals 12.7°, 14.3°, 22.5°, and 24.6°. On the contrary, IDB peaks disappeared from ternary mass mixture (Fig. 4.2 B), indicating the crystalline IDB was dissolved and embedded (amorphization) in binary mixtures of Witepsol E 85 and Capmul MCM at 7:1 ratio. The diffraction curve of Witepsol E 85 (Fig. 4.2 B) presented some sharp and distinct peak in the range of 20° - 22°. Compared to the Witepsol E 85 (Fig. 4.2 B), the peak intensities of binary and ternary mixture are weaker, indicating incorporation of liquid lipid to the solid lipid leads to formation of imperfect crystal model (fewer ordered crystals with many imperfections). According to these results, many authors have observed the same feature using NLCs [45,46].

4.3.2 Selection of surfactant Since, a surfactant prevents particle agglomeration of lipid nanoparticles, their nature and concentration plays a crucial role in particle size reduction and stabilization of lipid nanoparticles [47]. The exposure of cell-membrane to non-ionic surfactant, such as Tween 80 and Cremophor EL leads to a reduction in intracellular GSH level, which is responsible for part of the oxidative stress that causes damage to cells [48, 49]. Hence, with the aim to prepare lipid nanoparticles, that protects keratinocyte cells from UVB-mediated oxidative stress, the non-ionic, PEG-free surfactant based on natural renewable raw materials, i.e., TegoCare 450 was selected. Moreover, their potential applications in topical lipid nanoparticles are well reported [50, 51].

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Figure 4.2: Crystallographic investigations. A. Differential scanning calorimetry (DSC) scans. B. X-ray diffractions patterns of Idebenone (IDB), Witepsol E 85 (WE 85), a binary mixture of WE 85 and Capmul MCM (7:1), and a ternary mass mixture of IDB, WE 85 and Capmul MCM (1.25:7:1).

To this end, we evaluated the photosafety of TegoCare 450 and the results of 1 1 this study showed MEC value < 50 M¯ cm ¯ in UVB region. According to the 1 1 literature [31], chemicals with a MEC value < 1000 M ¯ cm ¯ were less of a phototoxic risk. Hence, TegoCare 450 was finally selected for the stabilization of IDB-NLCs.

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4.3.3 Optimization of IDB-NLC preparation The optimum level for the preparation of IDB-NLCs were selected using a 3 three-level, three-variable, L9 (3 ) Taguchi orthogonal array design. For that, based on our experience on the nanoparticles, three key independent variables and levels were pinpointed. These included process variables such as sonication amplitude and time, and formulation variables such as surfactant concentration. The choice is thus based on the fact that, these variables are known to exhibit a significant impact on z- ave and EE of the NLCs [52]. IDB 0.5 % (w/w) and solid to liquid lipid (7:1) ratio kept constant during the optimization process. Usually, for three-level with three- variable design (i.e. complete factorial design), 33 = 27 experiments should be conducted. As a “simple”, fast, and economical method, Taguchi design, the standard orthogonal array, namely L 9 was used and yielded a total of 9 experiments

(i.e. formulations; F). The designed L 9 is an array of 9 formulations with the particular combination of levels, where, row determines the levels of variables, used to run each individual formulation and column is assigned to every variable or a combination of variables to study the interactions. The structure of Taguchi orthogonal robust design and observed responses (z-ave and %EE) of each formulation are shown in Table 4.2. It is seen that NLC with z-ave between ~ 150 and 390 nm ranges and with EE values between ~ 94 and

99 % has been produced. However, % EE seems unchanged ( > 94%) across 9 formulations. This suggests that selected lipid phase composition had profound effect (owing to the imperfections) on % EE (Table 4.2), as anticipated during lipid phase screening. F9 showed the lowest particle size and PDI (Table 4.2), and highest % EE values, when compared to all other formulations. Slight increase in ζ value was observed in case of F3, F5 and F7, when compared to all other formulations (Table 4.2), which is mainly attributed to the surfactant nature. TegoCare 450 when dispersed in water, the OH group available in the sugar part of the molecule gains a negative charge [53, 54]. Hence, an increase in surfactant concentration from 0.5 % to 2 % resulted in slight increase in ζ values.

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Table 4.2: Variables, levels and observed responses (dependent variables) of 3 Taguchi L 9 (3 ) orthogonal array. The coded values (1), (2) and (3) correspond, respectively, to 30, 60, and 90 for sonication amplitude (A), 1, 2, and 4 for sonication time (B), and 0.5, 1.0, and 2.0 for surfactant concentration (C).

F Independent variables Dependent variables A B C Particle size PDI ζζζ (mV) ± EE (%) ± (%) (min) (%) (z-ave) (nm) S.D. S.D. ± S.D. 1 30 (1) 1 (1) 0.5 (1) 391.8±14.4 0.741 −31.70±2.27 96.04±3.11 2 30 (1) 2 (2) 1.0 (2) 328.0±9.24 0.719 −33.47±0.99 95.28±3.74 3 30 (1) 4 (3) 2.0 (3) 312.0±7.82 0.680 −36.06±1.25 94.98±2.61 4 60 (2) 1 (1) 1.0 (2) 247.5±10.14 0.573 −34.17±2.0 95.37±0.96

5 60 (2) 2 (2) 2.0 (3) 282.5±8.91 0.492 −36.12±0.83 95.61±1.69 6 60 (2) 4 (3) 0.5 (1) 226.3±5.28 0.406 −32.23±0.52 96.29±5.28

7 90 (3) 1 (1) 2.0 (3) 218.2±11.09 0.395 −36.40±0.81 95.16±2.49 8 90 (3) 2 (2) 0.5 (1) 207.6±9.13 0.358 −31.20±1.12 95.82±1.72

9 90 (3) 4 (3) 1.0 (2) 151.2±4.75 0.289 −35.57±0.68 98.73±0.83

Results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate. F indicates formulation; EE indicates entrapment efficiency .

In present study, the S/N ratio was selected according to the criterion “smaller is better” and “larger is better”, to minimize and maximize the particle size and entrapment efficiency, respectively, and hence they were calculated using following formula [55].

(y2+ y 2 + y 2 + ...) Smaller is better: S/N ratio (dB) = − 10 log 1 2 3 n

Where, yi is particle size and standard deviation, n is the number of measurement in each formulation ( n = 3).

(1/y2+ 1/ y 2 + 1/ y 2 + ...) Larger is better: S/N ratio (dB) = − 10 log 1 2 3 n

Where, yi is entrapment efficiency and standard deviation, n is the number of measurement in each formulation ( n = 3).

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Table 4.3 shows the S/N (signal-to-noise) ratios (dB) for particle size and entrapment efficiency, calculated using earlier mentioned formula. Then, for the selection of optimum level of each variable from the obtained data, the mean S/N ratios are further categorized in Table 4.4 and 4.5. It can be noticed from Table 4.3, 4.4, 4.5, and Fig. 4.3 A and B, level 3 of sonication amplitude, and sonication time and level 2 of surfactant concentration showed the maximum S/N ratio for particle size and entrapment efficiency. The maximum is the S/N ratio; the smaller is the variance of particle size and entrapment efficiency around the desired value. Hence, the optimum levels for the particle size and entrapment efficiency are A3B3C2 .

Table 4.3: S/N ratios for particle size and entrapment efficiency

F Particle size (nm) Entrapment efficiency (%) Mean Standard Mean Standard deviation deviation Data S/N Data S/N ratio Data S/N Data S/N ratio (±) (dB) ratio (±) ratio (dB) (dB) (dB) 1 391.8 −51.86 14.40 −23.16 96.04 39.64 3.11 9.85 2 328.0 −50.31 9.24 −19.31 95.28 39.58 3.74 11.45 3 312.0 −49.88 7.82 −17.86 94.98 39.55 2.61 8.33 4 247.5 −47.87 10.14 −20.12 95.37 39.58 0.96 −0.35 5 282.5 −49.02 8.91 −18.99 95.61 39.61 1.69 4.55 6 226.3 −47.09 5.28 −14.45 96.29 39.67 5.28 14.45 7 218.2 −46.77 11.09 −20.89 95.16 39.56 2.49 7.92 8 207.6 −46.34 9.13 −19.20 95.82 39.62 1.72 4.71 9 151.2 −43.59 4.75 −13.53 98.73 39.88 0.83 −1.61

Results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate.

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Table 4.4: Mean S/N response for particle size and standard deviation

Variables Mean S/N ratio (dB) for Mean S/N ratio (dB) for particle size standard deviation Level 1 Level 2 Level 3 Level 1 Level 2 Level 3 A) Sonication −50.69 −48.00 −45.57 −20.11 −17.86 −17.88 amplitude B) Sonication −48.84 −48.56 −46.86 −21.40 −19.17 −15.28 time C) Surfactant −48.43 −47.26 −48.56 −18.94 −17.66 −19.25 concentration

Table 4.5: Mean S/N response for entrapment efficiency and standard deviation Variables Mean S/N ratio for Mean S/N ratio for standard entrapment efficiency deviation Level 1 Level 2 Level 3 Level 1 Level 2 Level 3 A) Sonication 39.59 39.62 39.70 9.88 6.21 3.67 amplitude B) Sonication 39.60 39.61 39.70 5.80 6.90 7.05 time C) Surfactant 39.65 39.69 39.58 9.67 3.16 6.93 concentration

Considering the results shown in Table 4.2, 4.4 and 4.5, F9 was selected as optimized IDB-NLCs batch (Table 4.6). NR was then included into batch F9 as a fluorescent probe to investigate a qualitative intracellular uptake of NR-IDB-NLCs. Analysis of placebo-NLCs and NR-IDB-NLCs (Table 4.7) revealed no significant change in the z-ave, PDI and ζ, when compared to the IDB-NLCs indicating the robustness of the optimized NLC system. In addition, the stability of placebo-NLCs, IDB-NLCs and NR-IDB-NLCs were analyzed by incubating these NLC systems for 24 h in cell culture medium (DMEM with 10 % FBS). The obtained results revealed a slight increase in the mean particle size of NLCs (Table 4.7) indicating no aggregations of NLCs (as evidenced in NR-IDB-NLCs TEM investigation; Fig. 4.4 B).

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A]

B]

Figure 4.3: Main effects plot for S/N ratios of (A) particle size and (B) entrapment efficiency

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Table 4.6: Final formulation compositions of the optimized IDB-NLCs

Ingredients Quantity [(%) w/w] Placebo-NLCs IDB-NLCs NR-IDB-NLCs Idebenone - 0.5 0.5 Witepsol E 85 2.8 2.8 2.8 Capmul MCM 0.4 0.4 0.4 Nile red - - 0.004 TegoCare 450 1.0 1.0 1.0 Double distilled water ≈ 95.8 ≈ 95.3 ≈ 95.3

Table 4.7: Physicochemical characteristics of different NLCs.

Sample z-ave (nm) ± PDI ζζζ (mV) ± S.D. SD Placebo-NLCs 147.5 ± 2.91 0.272 −34.18 ± 0.18 Placebo-NLCs in medium at 24 h 161.27 ± 1.01 0.281 - IDB-NLCs in medium at 24 h 159.59 ± 2.05 0.321 - NR-IDB-NLCs 157.11 ± 0.75 0.309 −34.19 ± 0.24 NR-IDB-NLCs in medium at 24 h 168.2 ± 1.29 0.326 -

Results are expressed as mean ± SD, n = 3.

4.3.4 TEM investigations The TEM image of IDB-NLCs stabilized with TegoCare 450 demonstrated ideal spherical lipid nanoparticles with particle sizes between ~ 140 and 146 nm range (Fig. 4.4 A). Note also that the width of the particles is in line with the z-ave measurement by DLS (151.2 ± 4.75 nm). Moreover, no IDB crystals (free drug) were observed at or near the surface of the nanoparticles during the investigation. The absence of free drug mainly attributed to the higher entrapment efficiency feature of the optimized NLCs system. At higher magnification TEM clearly revealed the presence of a thin layer encircling the particle (Fig. 4.4 B) indicating formation of ‘drug-enrich core’ type NLCs. This type of NLCs is primarily

100 Chapter 4: Photoprotection aspects of IDB-NLCs attributed to the higher melting point of IDB (54.01 °C) when compared to Witepsol E 85 (48.10 °C), this finding corroborates our previous study [53].

Figure 4.4: Transmission electron microscopy (TEM) images. A. IDB-NLCs stabilized with TegoCare 450, magnification 55,000 ×. B. NR-IDB-NLCs stabilized with TegoCare, after zoom-in, original magnification 55,000 ×. Arrow indicates a thin layer encircling the particle.

4.3.5 In vitro IDB release studies IDB-NLCs under investigation exhibited slow and sustained release profile when compared to IDB-PD (~ 95 % IDB release at 6 h) (Fig. 4.5). A similar sort of release profile was reported employing Witepsol E 85 as a lipid matrix [40]. The sustained release of IDB without a burst release from IDB-NLCs was primarily attributed to their characteristic ‘drug-enriched core model’ (IDB was present within the core of the nanoparticles, as a result longer diffusion distance δ for the IDB) feature as evidenced in TEM investigations (Fig. 4.4 A and B). Polymorphic transitions of the lipid matrix (IDB-NLCs dispersion turns slowly into a semisolid gel due to non-occlusion conditions) could also be the reason for a sustained release of IDB from NLCs [26]. Note also that there is a good agreement between the selected binary mixture lipids (with melting point above 40 °C) and sustained release of the IDB from an optimized NLCs system. This type of release profile would allow maintain a certain level of IDB in the skin over a prolonged period of time (discussed in section 4.3.6).

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Figure 4.5: In vitro release profile of IDB from IDB-NLCs and IDB-PD. Each data point is the mean ± S.D. from three independent experiments, each performed in triplicate.

4.3.6 Occlusion and ex-vivo drug penetration studies In the current investigation, in vitro occlusion test was carried out with the objective to determine their effect on penetration profile of IDB-NLCs and IDB-PD. Generally, lipid nanoparticles with particle size less than 200 nm applied onto the skin forms a hydrophobic monolayer, which subsequently leads to an occlusive effect. This in turn reduces the water loss from skin (increased skin hydration), and hence, penetration of lipophilic active ingredient is enhanced [56-58]. The same holds true for IDB-NLCs, and yielded occlusion factor 29.02 ± 2.91 (vs 7.34 ± 1.74 of IDB-PD) and demonstrated a higher skin deposition (77.09 %) at 12 h when compared to 25.01 % of IDB from IDB-PD sample (Fig. 4.6). Over 3-fold increase in IDB skin deposition from IDB-NLCs was primarily attributed to their nanometer- sized particles with narrow size distribution (Table 4.2), solid state nature of lipid matrix (as evidenced in DSC studies; Fig. 4.2 A), and sustained release profile (Fig. 4.5). The results (at 12 h) of these experiments indicate that the occlusion effect of NLCs boosted the penetration of IDB into the skin and no IDB was detected in receptor compartment from IDB-NLCs when compared to IDB-PD (11.47 % IDB), because lipid nanoparticles like NLC can make closer contact with superficial

102 Chapter 4: Photoprotection aspects of IDB-NLCs junctions of corneocytes clusters and furrows present between corneocytes islands and favor’s accumulation for several hours [59, 60], hence allowing sustained IDB release when compare to IDB-PD.

Figure 4.6: A Comparison of the IDB levels from IDB-NLCs and IDB-PD. Bars represent mean ± S.D. from three independent experiments, each performed in triplicate.

4.3.7 Stability investigations The physical stability of IDB-NLCs was evaluated by measuring the mean particle size, the polydispersity index, the zeta potential and the % EE after 7, 15, 30, 60, and 90 days of storage at 25 °C ± 2 °C/60 % ± 5 % RH and at 40 °C ± 2 °C/75 % ± 5 % RH. The results are displayed in Table 4.8. The IDB-NLCs revealed no significant ( p > 0.05) changes in the mean particle size, the polydispersity index and, the zeta potential during 90 days of storage at different temperatures. Moreover, instabilities such as gelation, phase separation and aggregation are not observed, indicating a good physical stability of the optimized NLCs system. The % EE remained in the range of 95.65 to 99.82 during 90 days of storage, hence providing evidence that IDB is efficiently encapsulated in Witepsol E 85 and Capmul MCM lipid matrix.

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Table 4.8: Stability study (3 months, at varying temperature) data of IDB-NLCs.

Time (days) Particle size (z- PDI ζζζ (mV) ± EE (%) ± ave) (nm) ± S.D. S.D. S.D. 25°C±2°C/60% ±5% RH Initial 151.2 ± 4.75 0.289 −35.57 ± 0.68 98.73±0.83 7 days 153.0 ± 1.89 0.295 −32.81 ± 0.27 97.05±1.28 15 days 151.91 ± 2.31 0.294 −33.98 ± 1.04 99.82±3.16 30 days 155 ± 5.20 0.291 −36.47 ± 0.70 95.65±4.21 90 days 155.13 ± 5.19 0.296 −31.38 ± 1.29 96.07±3.84 40°C ±2°C/75% ±5% RH 7 days 154.29 ± 4.46 0.294 −34.93 ± 0.80 96.54±4.38 15 days 155.47 ± 2.86 0.293 −31.32 ± 0.54 98.02±1.15 30 days 153 ± 0.28 0.296 −30 ± 1.04 97.10 ±3.08 90 days 156.3 ± 4.71 0.301 −33 ± 0.40 96.52±1.06

Results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate.

4.3.8 Cell viability and photoprotective studies To investigate the potential photoprotective effects of the IDB-PD, placebo- NLCs and IDB-NLCs on HaCaT cells, first, the effect of different concentrations of IDB-PD on cell viability was quantified by MTS assay right away after predetermined incubation times (12, 24 and 72 h) (Fig. 4.7 A), and later, the biocompatibility of placebo-NLCs and IDB-NLCs was determined (Fig. 4.7 B). IDB-PD results (Fig. 4.7 A) showed a concentration and time dependent decrease in cell viability of HaCaT cells, with highest for 10 µM (29.38 % and 19.76 %) and lowest for 1 µM dose (98.05 % and 98.02 %) after 12 and 24 h incubation, respectively, when compared to control cells. The observed results could be due to the pro-oxidant property of IDB; however, these properties seem to be rather minute and are not sufficient to question its good safety records [61].

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Figure 4.7: Viability of HaCaT cells. A. After 12, 24 and 48 h of incubation with different concentrations of IDB-PD (1, 2, 4, 6, 8 and 10 µM/0.1mL). B. After 24 h of incubation with placebo-NLCs and IDB-NLCs (equivalent to 1, 2, 4, 6, 8 and 10 µM/0.1mL of IDB). Results are represented as percentage of cell viability compared to negative control group set on 100%. Bars represent mean ± S.D. from three independent experiments, each performed in triplicate. *** p < 0.001, **p < 0.01, ns (non significant) vs. Control (One-Way ANOVA test, Dunnett’s Multiple comparison Test); °°° p < 0.001 vs. 12 h, NS vs. 24 h, °° p < 0.01 vs. placebo-NLCs (One-Way ANOVA test, Tukey’s Multiple Comparison Test).

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Extending the incubation time from 24 to 48 h did not cause any significant decrease in HaCaT cell viability ( p > 0.05) (Fig. 4.7 A), hence the biocompatibility of placebo-NLCs and IDB-NLCs was determined after 24 h of incubation and the obtained results revealed significant increase in cell viability ( p < 0.05) at all concentration tested, when compared to the IDB-PD concentrations (Fig. 4.7 A and B) indicating incorporation of IDB into NLC eliminates its cytotoxicity, primarily believed to be due to sustained release profile of IDB-NLCs (as evidenced in release studies; Fig. 4.5), thus avoiding instantaneous and huge intracellular concentration in cells, when compared to the IDB-PD. At 10 µM placebo-NLCs and IDB-NLCs increased cell viability by 4 and nearly 5-fold, respectively, when compared to 10 µM IDB-PD (24 h) (Fig. 4.7 A and B). Additionally, at 6, 8, and 10 µM of IDB-NLCs exhibited a significant increase in cell viability ( p < 0.01) when compared to respective placebo-NLCs concentrations (Fig. 4.7 A and B), indicating an exceptional biocompatibility of optimized IDB- NLCs system. Though the results of placebo-NLCs at all the concentrations (except 1 and 2 µM) showed a slight cytotoxicity when compared to control cells, their cell viability did not drop below 80 %. Since the present study not intended to deal with the biochemical process by which solid lipid cause cell apoptosis, the slight cytotoxicity could possibly be attributed to the solid lipid we used for the preparation of placebo-NLCs [62]. However, according to OECD guideline 439 [63], an irritant substance or a mixture is predicted if they decrease cell viability below defined threshold levels (< 50%) when compared to negative controls for a 15 - 60 min exposition time. In the present study, cells were exposed to placebo-NLCs for 24 h with cell viability well above the threshold level. Thus, placebo-NLCs can be considered as non-irritant. Since it has been reported that interactions of NLCs with cells and biomolecules should be frequently appraised for understanding their compatibility and toxicity, not only under normal conditions, but also under any chemical or physical stress, which these delivery might be subjected to during their employment [64]. Hence, we investigated the photoprotective effects of IDB-PD, placebo-NLCs and IDB-NLCs (at different concentration) on HaCaT cells against 55 µW/cm 2 of UVB radiation (200 mJ/cm 2 for 1 h). The obtained results (Fig. 4.8) revealed that positive control cells significantly reduced the cell viability by 46.01 % ( p < 0.001), when compared to negative control cells, indicating that the irradiation system and

106 Chapter 4: Photoprotection aspects of IDB-NLCs method used in this study were appropriate [37]. The photoprotection offered by IDB is displayed in Fig. 4.8. The results showed that IDB at 1, 2, and 4 µM significantly increased cell viability ( p < 0.001), when compared to positive control cells, indicating that the IDB at 1, 2, and 4 µM provided UV photoprotection. On the contrary, decrease in cell viability was observed at higher concentration of IDB (8 and 10 µM) and no significant increase or decrease in cell viability was observed in case of IDB at 6 µM.

Figure 4.8: Photoprotective effect of IDB-PD, placebo-NLCs and IDB-NLCs (equivalent to 1, 2, 4, 6, 8 and 10 µM/0.1mL of IDB) against 55 µW/cm 2 UVB (200 mJ/cm 2 for 1 h)-mediated phototoxicity in HaCaT cells. Results are represented as percentage of cell viability compared to negative control group set on 100%. Bars represent mean ± S.D. from three independent experiments, each performed in triplicate. *** p < 0.001 vs negative control; °°° p < 0.001, ns (non significant) vs positive control; $$ p < 0.01, $$$ p < 0.001 vs IDB-PD and vs positive control (One- Way ANOVA test, Tukey’s Multiple Comparison Test).

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Regarding the photoprotection offered by NLCs (Fig. 4.8), it is shown that both placebo-NLCs and IDB-NLCs at 6 µM significantly increased cell viability ( p < 0.05) (2-fold increase in case of IDB-NLC), when compared to IDB-PD and positive control cells, indicating the photoprotective effect of carrier (NLCs) and IDB, primarily attributed to: (1) IDB, known to scavenge ROS generated by UVB light (discussed in more detail in section 4.3.10.1), (2) lipids in the NLCs, as lipids are known to play an important role in cell structure and metabolism, and (3) synergistic effect of IDB and NLCs. No significant increase in cell viability was observed in case of placebo- NLCs and IDB-NLCs at 8 and 10 µM ( p > 0.05), when compare to positive control cells, possibly indicating no photoprotection and pro-oxidant effect at high dose, respectively. Since, placebo-NLCs and IDB-NLCs at 6 µM demonstrated more than 80 % and ~ 100 % cell viability, respectively, under UVB conditions; hence, this dose was used in all further experiments.

4.3.9 Cell uptake studies Over 3-fold increase in IDB skin deposition from IDB-NLCs, when compared to IDB-PD, prompted us to probe whether NLCs improve a cellular uptake of IDB or not. Hence, we performed both quantitative (using spectrophotometry) and qualitative (using CLSM) in vitro cell uptake studies in HaCaT cell line. First, a quantitative estimation of the IDB (µg/mL), intracellular and extracellular (medium) from IDB-NLCs and IDB-PD was determined and results are depicted in Fig. 4.9. The obtained results revealed that the concentration of IDB in cells was highest at 6 h (1.756 µg/mL) and 2 h (1.793 µg/mL) from IDB-PD and IDB-NLCs, respectively, indicating the faster uptake (3 times faster) of NLCs by HaCaT cells, when compare to IDB-PD. At the end of the experiment (24 h), the concentration of IDB in cells was significantly higher in case of IDB-NLCs (1.413 µg/mL), when compared to IDB-PD (0.462 µg/mL), indicating a sustained release of IDB from IDB-NLCs. These significant differences in the uptake can be well correlated with the results of ex vivo penetration (Fig. 4.6) and in vitro drug release studies (Fig. 4.5), respectively. To further confirm this observation (faster uptake and sustained release of IDB from NLCs), a time-dependent intracellular uptake of NR-IDB-NLCs by

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HaCaT cells was studied using CLSM and results are illustrated in Fig. 4.10. The obtained results revealed that there is no uncertainty that the fluorescence is intracellular in origin (localized brighter spots) and a significant uptake was evident at 2 h.

Figure 4.9: Quantitative (spectrophotometric) intracellular uptake of IDB from IDB-NLCs and IDB-PD (equivalent to 2 µg/mL of IDB) by HaCaT cells at 37 °C. In all cases (except 6 h of IDB-PD and 2 h of IDB-NLCs), the IDB values in cells and medium from IDB-NLCs were statistically significant from the IDB-PD ( p < 0.01) (the unpaired, two-tailed Student t-test). Bars represent mean ± S.D. from three independent experiments, each performed in triplicate.

Not all time-point samples showed distinct localized brighter spots, as a result the number of spot also varied between samples. The presence of localized brighter spots can indicate NLCs in vesicle after endocytosis. The IOD of the red fluorescence was significantly higher ( p < 0.05) at 2 h time-point, when compared to 1 h (28 vs 12), and it decreased over time (26, 25, 23 and 18 for 3, 6, 12 and 24 h respectively), indicating the sustained NR release from NLCs and that NR might extracellularly transferred (as evidenced same pattern in quantitative cell uptake study, where concentration of IDB in medium was increased after 2 h; Fig. 4.9),

109 Chapter 4: Photoprotection aspects of IDB-NLCs hence resulting in decreased IOD values over time. However, note also that IOD at 24 h was higher when compared to 1 h (18 vs 12), suggesting retention of encapsulated NR in NLCs.

Figure 4.10: Confocal images ( Qualitative) featuring the time dependent (1, 2, 3, 6, 12 and 24 h) intracellular uptake of NR-IDB-NLCs by HaCaT cells at 37 °C. FV10- ASW 2.0.0.7 imaging software (Olympus) was used for imaging.

These results, together with those obtained from quantitative cell uptake studies; seem to suggest that NLC successfully improved the cellular uptake of IDB and this effect was primarily attributed to number of reasons. The NLCs under investigation had mean particle size well below 200 nm (151.2 ± 4.75 nm for IDB- NLC and 157.11 ± 0.75 nm for NR-IDB-NLCs) with narrow size distribution and a negative zeta potential (−35.57 ± 0.68 for IDB-NLC and − 34.19 ± 0.24 for NR- IDB-NLCs), moreover, NLCs were spherical in shape and no aggregation occurred (as evidenced during TEM investigation; Fig. 4.4 A and B) when they were incubated with a cell culture medium at 37 °C with 5 % CO 2, 95 % air in a humidified incubator for 24 h (Table 4.7). According to the literature, nanoparticle structure and surface properties (shape, size, charge and surface modifications) are crucial in dictating cellular fate [65]. Literature also indicates that nanoparticles

110 Chapter 4: Photoprotection aspects of IDB-NLCs were taken up by HaCaT cells in a size-dependent manner (preferably below 200 nm) and predominantly clathrin-mediated endocytosis was proposed to be the major pathway for the uptake of particles with size below 200 nm [66, 67]. Spherical nanoparticles were fast internalized (5 times faster) when compared to rod-like nanoparticles via endocytosis [68]. It is also reported that positively charged nanoparticles are easily adsorbed on negatively charged areas of cell membrane and later facilitate their internalization, provided there should be no aggregation of nanoparticles when incubated with a cell culture medium, because positively charged nanoparticles are tend to aggregate, leading to lower internalization [66]. Moreover, a cell membrane possesses slight negative charge areas when compared to positively charged areas [69]. Finally, we can claim that spherical, negatively charged NLCs with mean particle size below 200 nm without aggregation could fast take up by HaCaT cells via clathrin-mediated endocytosis.

4.3.10 Oxidative stress After having discussed the importance of preformulation strategy and their influence on a number of aspects of IDB-NLCs including cellular uptake. We are now able to investigate whether IDB-NLCs can protect HaCaT cells against UVB (200 mJ/cm 2 for 1 h)-mediated oxidative stress.

4.3.10.1 Intracellular ROS scavenging activity Fig. 4.11 A depicts the number of cells against fluorimetric intensity. The peaks are the results of ROS produced by UVB irradiation inside the cells, and this production is expressed in fluorescence. Therefore, the lower the fluorescence, the higher the ROS scavenging activity of test samples. The MFI of all test samples, as obtained from flow cytometric analysis, discerned the following order: Negative control cells (43.46) < IDB-NLCs + UVB (51.40) < placebo-NLCs + UVB (171.04) < positive control cells (246.53). As expected, the positive control cells exhibited over 5-fold higher fluorescent intensity, when compared to negative control cells, a significant difference ( p < 0.001) owing to UVB-mediated ROS production without a NLC or any antioxidant treatment, indicating a single dose of UVB (200 mJ/cm 2 for 1 h) was more than sufficient to generate the ROS inside the cells. On the contrary, IDB-NLCs showed no-significant increase in MFI after

111 Chapter 4: Photoprotection aspects of IDB-NLCs exposure to UVB at a given fluence, indicating less ROS production. This activity primarily attributed to two major contributing factors: 1) NLCs as a drug carrier improved the cellular uptake of IDB (as evident in the precedent section) and 2) a well-known antioxidant and photoprotective properties of IDB [70, 14]. It is well known that excessive production of ROS or reduction in the endogenous antioxidants to neutralize them results in oxidative stress which in turn causes damage to intracellular lipids, proteins and DNA [71]. IDB, a key cell membrane antioxidant and essential constituent of electron transport chain (ETC) in mitochondria, acts by inhibiting the lipid peroxidation and thus protects cell membrane and mitochondria membrane integrity from oxidative stress [72, 73]. Regarding the placebo-NLCs + UVB (Fig. 4.11 A), it is seen that it exhibited significantly lower MFI, when compared to positive control cells ( p < 0.001), indicating the placebo-NLCs under UVB irradiation provided photoprotection but they in spite of that compromised over 3-fold increase in ROS production, when compared to IDB-NLCs + UVB, indicating loading of IDB in NLCs system boosted HaCaT cell’s protection against oxidative stress.

4.3.10.2 Mitochondrial membrane potential Since, ROS can induce rapid disruption of inner Ψ m and subsequent impairment of oxidative phosphorylation (OXPHOS) [74]. We evaluated the effect of UVB irradiation on HaCaT cell’s mitochondrial function; assessed by extent of disruption of Ψ m and protection provided by test samples. Fig. 4.11 B depicts the number of cells against fluorimetric intensity. The peaks are the results of disruption of Ψ m produced by UVB irradiation inside the cells, and this disruption is expressed in fluorescence. Therefore, the lower the fluoresence, higher Ψ m (healthy cells) activity of test samples. The MFI of all test samples, as obtained from flow cytometric analysis, discerned the following order: Negative control cells (51.35) < IDB-NLCs + UVB (58.16) < placebo-NLCs + UVB (175.74) < positive control cells (293.38). Consistent with intracellular ROS results, the IDB-NLCs + UVB did not reveal any toxic effect on cells, rather, exhibited over 3-fold and 5-fold increase in protection of Ψ m, when compared to placebo-NLCs + UVB and positive control cells, respectively, and is primarily ascribed to synergistic effect of IDB and to NLCs aspects (as evident in the course of the investigation). Similar

112 Chapter 4: Photoprotection aspects of IDB-NLCs results (ROS scavenging activity and protection of Ψ m) have been reported in recent past by several researchers [64, 21].

Figure 4.11: Photoprotective effect of placebo-NLCs and IDB-NLCs (equivalent to 6 µM/0.1mL of IDB) against 55 µW/cm 2 UVB (200 mJ/cm 2 for 1 h)-mediated oxidative stress in HaCaT cells. A and B represents flow cytometric analysis of intracellular ROS and mitochondrial membrane potential ( Ψ m) disruption levels in HaCaT cells, respectively.

4.4 Conclusions In the present study, we show that meticulously executed two-step preformulation study followed by Taguchi robust orthogonal design as “simple” and “economical” optimization process was capable of improving IDB-NLCs key physicochemical aspects. Regarding cellular interactions, NLCs markedly improved biocompatibility of IDB and efficiently transported it across the cell membrane and that intracellular uptake mechanism was clathrin-mediated endocytosis. The antioxidant potency of the IDB is well known and in the present investigation IDB and placebo-NLCs prompted HaCaT cell photoprotection in a dose-dependent manner, but the loading of IDB into NLCs system further boosted to thwart the oxidative stress induced by UVB irradiation. Overall, suggesting that NLC has ability to interact with skin components (owing to their nano-size and high occlusive

113 Chapter 4: Photoprotection aspects of IDB-NLCs effect) and subsequently deliver IDB in a sustained release manner that in turn improved the cellular uptake and exerted photoprotective effects. Since, NLCs proved to be a suitable topical delivery system for IDB, future research could emphasize on loading of two or more antioxidants in NLC system (combination strategy) to treat premature skin aging and skin cancer caused by overexposure to UV radiation.

4.5 References 1) Mancebo SE, Hu JY, Wang SQ. Sunscreens: a review of health benefits, regulations, and controversies. Dermatol. Clin. 2014; 32:427-438. 2) Afaq F. Natural agents: cellular and molecular mechanisms of photoprotection. Arch. Biochem. Biophys. 2011; 508:144-151. 3) Nichols JA and Katiyar SK. Skin photoprotection by natural polyphenols: anti- inflammatory, antioxidant and DNA repair mechanisms. Arch. Dermatol. Res. 2010; 302: 71-83. 4) Lautenschlager S, Wulf HC, Pittelkow MR. Photoprotection. Lancet 2007; 370:528-537. 5) Gonzalez S, Gilaberte Y, Philips N, Juarranz A. Current Trends in photoprotection - A New Generation of Oral photoprotection. Open Dermatol. J. 2011; 5:6-14. 6) Chen L, Hu JY, Wang SQ. The role of antioxidants in photoprotection: a critical review. J. Am. Acad. Dermatol. 2012; 67:1013-1024. 7) Pandel, R.; Poljšak, B.; Godic, A.; Dahmane, R. Skin photoaging and the role of antioxidants in its prevention. ISRN Dermatol. 2013, 930164. 8) Dinardo JC, Lewis JA, Neudecker BA, Maibach HI. Antioxidants compared in a new protocol to measure protective capacity against oxidative stress-part II. J. Am. Acad. Dermatol. 2004; 50:P30. 9) McDaniel DH, Neudecker BA, DiNardo JC, Lewis JA, Maibach HI. Clinical efficacy assessment in photodamaged skin of 0.5% and 1.0% idebenone. J. Cosmet. Dermatol. 2005; 4:167-173. 10) Klopstock T, Metz G, Yu-Wai-Man P, Büchner B, Gallenmüller C, Bailie M, Nwali N, Griffiths PG, von Livonius B, Reznicek L, Rouleau J, Coppard N, Meier T,Chinnery PF. Persistence of the treatment effect of idebenone in Leber's hereditary optic neuropathy. Brain. 2013;136:e230. 11) Parkinson MH, Schulz JB, Giunti P. Co-enzyme Q10 and idebenone use in Friedreich's ataxia. J. Neurochem. 2013; 126:125-141. 12) Giorgio V, Petronilli V, Ghelli A, Carelli V, Rugolo M, Lenaz G, Bernardi P. The effects of idebenone on mitochondrial bioenergetics. Biochim. Biophys. Acta. 2012; 1817:363-369. 13) News release ATU: http://hugin.info/137261/R/1755914/592922.pdf (accessed 12.07.2014). 14) McDaniel DH, Neudecker BA, DiNardo JC, Lewis JA. Maibach, H. I. Idebenone: a new antioxidant - Part I. Relative assessment of oxidative stress protection capacity compared to commonly known antioxidants. J. Cosmet. Dermatol. 2005; 4:10-17. 15) Tournas JA, Lin FH, Burch JA, Selim MA, Monteiro-Riviere NA, Zielinski JE, Pinnell SR. Ubiquinone, idebenone and kinetin provide ineffective photoprotection to skin when compared to a topical antioxidant combination of vitamins C and E with ferulic acid. J. Invest. Dermatol. 2006; 126:1185-1187. 16) Pardeike J, Hommoss A, Müller RH. Lipid nanoparticles (SLN, NLC) in cosmetic and pharmaceutical dermal products. Int. J. Pharm. 2009; 366:170-184. 17) Müller RH, Radtke M, Wissing SA. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) in cosmetic and dermatological preparations. Adv. Drug Delivery Rev. 2002; 54:S131-155. 18) Mitri K, Shegokar R, Gohla S, Anselmi C, Müller RH. Lipid nanocarriers for dermal delivery of lutein: preparation, characterization, stability and performance. Int. J. Pharm. 2011; 414:267-275. 19) Hung LC, Basri M, Tejo BA, Ismail R, Nang HL, Abu Hassan H, May CY. An improved method for the preparations of nanostructured lipid carriers containing heat-sensitive bioactives. Colloids Surf. B. Biointerfaces. 2011; 87:180-186. 20) Fan H, Liu G, Huang Y, Li Y, Xia Q. Development of a nanostructured lipid carrier formulation for increasing photo-stability and water solubility of phenylethyl resorcinol. Appl. Surf. Sci. 2014; 288:193-200. 21) Yue Y, Zhou H, Liu G, Li Y, Yan Z, Duan M. The advantages of a novel CoQ10 delivery system in skin photo-protection. Int. J. Pharm. 2010; 392:57-63.

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22) Wang J, Xia Q. Alpha-lipoic acid-loaded nanostructured lipid carrier: sustained release and biocompatibility to HaCaT cells in vitro. Drug Delivery 2014; 21:328-341. 23) Schäfer-Korting M, Mehnert W, Korting HC. Lipid nanoparticles for improved topical application of drugs for skin diseases. Adv. Drug Delivery Rev. 2007; 59:427-443. 24) Martins S, Sarmento B, Ferreira DC, Souto EB. Lipid-based colloidal carriers for peptide and protein delivery-liposomes versus lipid nanoparticles. Int. J. Nanomed. 2007; 2:595-607. 25) Souto EB, Müller RH. Cosmetic features and applications of lipid nanoparticles (SLN, NLC). Int. J. Cosmet. Sci. 2008 ; 30:157-165. 26) Montenegro L, Sinico C, Castangia I, Carbone C, Puglisi G. Idebenone-loaded solid lipid nanoparticles for drug delivery to the skin: in vitro evaluation. Int. J. Pharm. 2012; 434:169-174. 27) Leonardi A, Crasci L, Panico A, Pignatello R. Antioxidant activity of idebenone-loaded neutral and cationic solid-lipid nanoparticles. Pharm. Dev. Technol. [Online early access]. Published online: May 6, 2014. http://informahealthcare.com/doi/abs/10.3109/10837450.2014.915572 (assessed Aug 6, 2014). 28) Li B, Ge ZQ. Nanostructured lipid carriers improve skin permeation and chemical stability of idebenone. AAPS PharmSciTech. 2012; 13:276-283. 29) Sarpietro MG, Accolla ML, Puglisi G, Castelli F, Montenegro L. Idebenone loaded solid lipid nanoparticles: Calorimetric studies on surfactant and drug loading effects. Int. J. Pharm. 2014; 471:69-74. 30) Lorencini M, Brohem CA, Dieamant GC, Zanchin NI, Maibach HI. Active ingredients against human epidermal aging. Ageing Res. Rev. 2014; 15:100-115. 31) Henry B, Foti C, Alsante K. Can light absorption and photostability data be used to assess the photosafety risks in patients for a new drugmolecule? J. Photochem. Photobiol. B. 2009; 96:57-62. 32) Mandpe L, Kyadarkunte A, Pokharkar V. Assessment of novel iloperidone- and idebenone- loaded nanostructured lipid carriers: brain targeting efficiency and neuroprotective potential. Ther. Delivery 2013; 11:1365-1383. 33) de Vringer T. Topical preparation containing a suspension of solid lipid nanoparticles. Eur. Pat. 91200664, 1992. 34) Cilurzo F, Minghetti P, Sinico C. Newborn pig skin as model membrane in in vitro drug permeation studies: a technical note. AAPS PharmSciTech. 2007; 8:E94. 35) Friend DR. In vitro skin permeation techniques. J. Controlled Release. 1992; 18:235-248. 36) Vaghasiya H, Kumar A, Sawant K. Development of solid lipid nanoparticles based controlled release system for topical delivery of terbinafine hydrochloride. Eur. J. Pharm. Sci. 2013; 49:311- 322. 37) Kyadarkunte A, Patole M, Pokharkar V. In Vitro cytotoxicity and phototoxicity assessment of acylglutamate surfactants using a human keratinocyte cell line. Cosmetics 2014; 1:159-170. 38) Kudish AI, Lyubansky V, Evseev EG, Ianetz A. Statistical analysis and inter-comparison of the solar UVB, UVA and global radiation for Beer Sheva and Neve Zohar (Dead Sea), Israel. Theor. Appl. Climatol. 2005; 80:1-15. 39) Wang CB, Huang MQ, Tao GL, Yu GY, Han ZW, Yang ZH, Wang YJ. Polypeptide from Chlamys farreri protects HaCaT cells from UVB-induced apoptosis. Chem. Biol. Interact. 2004; 147:119-127. 40) Vivek K, Reddy H, Murthy RS. Investigations of the effect of the lipid matrix on drug entrapment, in vitro release, and physical stability of olanzapine-loaded solid lipid nanoparticles. AAPS PharmSciTech. 2007; 8:E83. 41) Wiechers JW, Solutions JW, Souto EB. Delivering Actives via Solid Lipid Nanoparticles and Nanostructured Lipid Carriers: part I. Cosmetics & Toiletries® magazine. [Online] 2011. http://www.cosmeticsandtoiletries.com/formulating/function/delivery/premium-Delivering-Actives- via-Solid-Lipid-Nanoparticles-and-Nanostructured-Lipid-Carriers-Part-I-208736461.html (accessed July 12, 2014). 42) Kasongo KW, Pardeike J, Müller RH, Walker RB. Selection and characterization of suitable lipid excipients for use in the manufacture of didanosine loaded solid lipid nanoparticles and nanostructured lipid carriers. J. Pharm. Sci. 2011; 100:5185-5196. 43) Müller RH, Mäder K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drug delivery - a review of the state of the art. Eur. J. Pharm. Biopharm. 2000; 50:161-177. 44) Teeranachaideekul V, Souto EB, Junyaprasert VB, Müller RH. Cetyl palmitate-based NLC for topical delivery of Coenzyme Q(10) - development, physicochemical characterization and in vitro release studies. Eur. J. Pharm. Biopharm. 2007; 67:141-148. 45) Jia LJ, Zhang DR, Li ZY, Feng FF, Wang YC, Dai WT, Duan CX, Zhang Q. Preparation and characterization of silybin-loaded nanostructured lipid carriers. Drug Delivery 2010; 17:11-18.

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46) Pardeike J, Weber S, Haber T, Wagner J, Zarfl HP, Plank H, Zimmer A. Development of an itraconazole-loaded nanostructured lipid carrier (NLC) formulation for pulmonary application. Int. J. Pharm. 2011; 419:329-338. 47) Uner M, Wissing SA, Yener G, Müller RH. Influence of surfactants on the physical stability of solid lipid nanoparticle (SLN) formulations. Pharmazie 2004; 59:331-332 48) Hirama S, Tatsuishi T, Iwase K, Nakao H, Umebayashi C, Nishizaki Y, Kobayashi M, Ishida S, Okano Y, Oyama Y. Flow-cytometric analysis on adverse effects of polysorbate 80 in rat thymocytes. Toxicology 2004; 199:137-143. 49) Tatsuishi T, Oyama Y, Iwase K, Yamaguchi JY, Kobayashi M, Nishimura Y, Kanada A, Hirama S. Polysorbate 80 increases the susceptibility to oxidative stress in rat thymocytes. Toxicology 2005; 207:7-14. 50) Farboud ES, Nasrollahi SA, Tabbakhi Z. Novel formulation and evaluation of a Q10-loaded solid lipid nanoparticle cream: in vitro and in vivo studies. Int. J. Nanomedicine. 2011; 6:611-617. 51) Junyaprasert VB, Teeranachaideekul V, Souto EB, Boonme P, Müller RH. Q10-loaded NLC versus nanoemulsions: stability, rheology and in vitro skin permeation. Int. J. Pharm. 2009; 377:207- 214. 52) Das S, Ng WK, Tan RB. Sucrose ester stabilized solid lipid nanoparticles and nanostructured lipid carriers. I. Effect of formulation variables on the physicochemical properties, drug release and stability of clotrimazole-loaded nanoparticles. Nanotechnology 2014; 25:105101. 53) Mandpe L, and Pokharkar V. Targeted Brain Delivery of Iloperidone Nanostructured Lipid Carriers Following Intranasal Administration: In Vivo Pharmacokinetics and Brain Distribution Studies. J. Nanopharmaceutics Drug Delivery 2013; 1:212-225. 54) Hommoss A. Nanostructured lipid carriers (NLC) in dermal and personal care formulations. Ph.D. Dissertation, The Freie Universität Berlin, Berlin, 2009. 55) Taguchi G. Introduction to Quality Engineering; Asian Productivity Organization: Tokyo, 1990. 56) Wissing SA and Müller RH. Cosmetic applications for solid lipid nanoparticles (SLN). Int. J. Pharm. 2003; 254:65-68. 57) Wissing S, Lippacher A, Müller RH. Investigations on the occlusive properties of solid lipid nanoparticles (SLN). J. Cosmet. Sci. 2001; 52:313-324. 58) Hafeez F and Maibach H. Occlusion effect on in vivo percutaneous penetration of chemicals in man and monkey: partition coefficient effects. Skin Pharmacol. Physiol. 2013; 26:85-91. 59) Alvarez-Román R, Naik A, Kalia YN, Guy RH, Fessi H. Skin penetration and distribution of polymeric nanoparticles. J. Controlled Release. 2004; 99:53-62. 60) Cevc G. Lipid vesicles and other colloids as drug carriers on the skin. Adv. Drug Delivery. Rev. 2004; 56:675-711. 61) Haefeli RH. Molecular effects of idebenone. Ph.D. dissertation, University of Basel, Basel, 2012. 62) Weyenberg W, Filev P, Van den Plas D, Vandervoort J, De Smet K, Sollie P, Ludwig A. Cytotoxicity of submicron emulsions and solid lipid nanoparticles for dermal application. Int. J. Pharm. 2007; 337:291-298. 63) OECD 439. OECD guidelines for the testing of chemicals: In vitro skin irritation-reconstructed human epidermal test method. OECD: Paris, July 2013. 64) Brugè F, Damiani E, Puglia C, Offerta A, Armeni T, Littarru GP, Tiano, L. Nanostructured lipid carriers loaded with CoQ10: effect on human dermal fibroblasts under normal and UVA-mediated oxidative conditions. Int. J. Pharm. 2013; 455:348-356. 65) Ma N, Ma C, Li C, Wang T, Tang Y, Wang H, Moul X, Chen Z, Hel N. Influence of nanoparticle shape, size, and surface functionalization on cellular uptake. J. Nanosci. Nanotechnol. 2013; 13:6485- 6498. 66) Rancan F, Gao Q, Graf C, Troppens S, Hadam S, Hackbarth S, Kembuan C, Blume-Peytavi U, Rühl E, Lademann J, Vogt A. Skin penetration and cellular uptake of amorphous silica nanoparticles with variable size, surface functionalization, and colloidal stability. ACS Nano 2012; 6:6829-6842. 67) Hillaireau H and Couvreur P. Nanocarriers' entry into the cell: relevance to drug delivery. Cell. Mol Life. Sci. 2009; 66:2873-2896. 68) Rybak-Smith MJ, Tripisciano C, Borowiak-Palen E, Lamprecht C, Sim RB. Effect of functionalization of carbon nanotubes with psychosine on complement activation and protein adsorption. J. Biomed. Nanotechnol. 2011; 7:830-839. 69) Jin H, Heller DA, Sharma R, Strano MS. Size-dependent cellular uptake and expulsion of single- walled carbon nanotubes: single particle tracking and a generic uptake model for nanoparticles. ACS Nano 2009; 3:149-158. 70) Mordente A, Martorana GE, Minotti G, Giardina B. Antioxidant properties of 2,3-dimethoxy-5- methyl-6-(10-hydroxydecyl)-1,4-benzoquinone (idebenone). Chem. Res. Toxicol. 1998; 11:54-63.

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71) Linder J. Antioxidants: Crucial Additions to Dermal Photoprotection. Cosmet. Dermatol. 2010; 23:40-44. 72) Suno M and Nagaoka A. Inhibition of lipid peroxidation by a novel compound, idebenone (CV- 2619). Jpn. J. Pharmacol. 1984; 35:196-198. 73) Zs –Nagy I. Chemistry, toxicology, pharmacology and pharmacokinetics of idebenone: a review. Arch. Gerontol. Geriatr. 1990; 11:177-186. 74) Piao MJ, Lee NH, Chae S, Hyun JW. Eckol inhibits ultraviolet B-induced cell damage in human keratinocytes via a decrease in oxidative stress. Biol. Pharm. Bull. 2012; 35:873-880.

117 Chapter 5A

Cytotoxicity and phototoxicity assessment of acylglutamate surfactants

This particular chapter deals with In vitro cytotoxicity and phototoxicity of three acylglutamate surfactants such as sodium cocoyl glutamate (Amisoft CS-11), sodium lauroyl glutamate (Amisoft LS-11) and sodium myristoyl glutamate (Amisoft MS-11) using HaCaT as in vitro cell culture model. All the acylglutamate surfactants tested on HaCaT cells demonstrated significantly less cytotoxicity (+ UVB / − UVB) and no phototoxic potential was observed in any of the acylglutamate surfactants, when compared with the positive control chlorpromazine. Based on biocompatible properties and low cytotoxicity profile, the LS-11 would be the most suitable surfactant for the synthesis and stabilization of lipid nanoparticles.

Chapter 5A: Cytotoxicity and Phototoxicity of Acylglutamate Surfactants

5A.1 Genesis and outline of the work In cosmeceuticals, surfactant has several applications, such as cleansing, foaming wetting, thickening, solubilizing, and conditioning. In addition to these applications, surfactant is also used for the synthesis and stabilization of lipid nanoparticles for improved delivery of cosmeceutical actives [1–3]. Cosmeceutical products were frequently designed in order to retain the agents on the skin in spite of rinsing, and the recurrent use of phototoxic surfactant containing cosmeceuticals to the skin would inevitably increase risk. Moreover, the disposal of these products can adversely affect the aquatic environment, their biocompatibility and biodegradability become virtually as important as their functional performance to the consumer. Hence, there is a predilection for utilizing biocompatible and biodegradable surfactants for cosmeceutical formulations [4]. Surfactant of this type can be achieved by designing molecules that mimic natural amphiphilic structures (N α-acyl amino acids, phopholipids and alkyl-glycosides) [5–7]. Surfactant molecules from renewable crude substance (such as amino acids) that mimic natural lipoamino acids are one of the preferred choices for cosmeceutical formulations. The importance of amino acid as crude substance for the synthesis of surfactant came into existence immediately after they were discovered in 1930 [8]. Acylglutamate surfactants are remarkably superior to conventional surfactants in characteristic features. They have an excellent skin moisturization effect without a taut feeling, ability to improve conventional surfactant mildness, and usable even by patients suffering from skin disease [9–12]. Until 11 March 2013, experimentation of finished cosmetic products and ingredients was prohibited in the EU (European Union), but the 7th amendment (Directive 2003/15/EC [13]) to the cosmetic directive (Directive 76/768/EEC [13]) has now placed a Europe-wide prohibition on the sale of cosmetics if they have been experimented on animals anywhere on the globe. The main aim of the new directive is to safeguard and enhance the welfare of animals for experimental and research purposes by supporting the development of alternative methods, as well as to firmly anchor the principle of “3Rs”, to replace, reduce and refine the use of animals [14]. As a result, Over the past two decades, many different types of cell and tissue culture based in vitro models have been developed, not only in response to the need to find ethical alternatives to the animal-based tests, but also because many in vitro models allow one to obtain more relevant, objective test results [15]. 118

Chapter 5A: Cytotoxicity and Phototoxicity of Acylglutamate Surfactants

In the current study we chose a human keratinocyte (HaCaT) cell line, as keratinocytes represent the major cell type in the epidermis and in vivo keratinocytes are biological relevant targets for surfactants once they pass through the stratum corneum. Furthermore, this cell line provides a virtually endless supply of look-alike cells, assuring immense reproducibility across-laboratory and is of greater human relevance to epidermal-induced irritation, than animal derived fibroblast cells [16,17]. Earlier studies reported that HaCaT cells are a promising screening tool for predicting the toxic and irritation potential of amino acid-based surfactants, anionic surfactants, certain phytochemicals, and lipid nanocapsules [18–21]. Phototoxicity is of growing concern in dermatology due to stratospheric ozone depletion resulting in an increasing penetration of ultraviolet (UV) radiation. Particularly, ultraviolet B (UVB; mid wave, 290–320 nm) irradiation is thought to be responsible for more of the UV-induced adverse effects in the skin and serves as an important etiologic factor causing inflammatory skin damages, oxidative stress, DNA damage, cellular, as well as tissue injuries, cell death, skin cancer, and premature skin aging [22,23]. On that account, the ultimate goal of the current study was to elucidate the effects of the fatty acid chain length of acylglutamate surfactants on their cytotoxicity and the UVB-induced phototoxicity using the HaCaT cell line. Ultimately, it was foreseen that the results from this study generate the first set of data which would allow us to (a) categorize acylglutamte surfactants into non-phototoxic, probable phototoxic and phototoxic, (b) pick the most suitable surfactant for the synthesis and stabilization of lipid nanoparticles.

5A.2 Experimental

5A.2.1 Surfactants tested Commercially available anionic amino acid-based, weakly acidic surfactants (Amisoft ®, Acylglutamte salt), namely sodium cocoyl glutamate (CS-11), sodium lauroyl glutamate (LS-11) and sodium myristoyl glutamate (MS-11) were tested. Each acylglutamate surfactant solution was prepared by dissolving them in serum- free DMEM supplemented with 2 mM L-glutamine and antibiotic mixture. The CPZ stock solution was prepared in DMSO and further dilutions by serum free DMEM.

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5A.2.2 Cell culture Early-passage HaCaT cell line was acquired from National Centre for Cell Sciences (NCCS, Pune, India) was grown as adherent cultures into 75 cm 2 culture flasks using, DMEM high glucose (4.5 g/L) as base medium, supplemented with 10

% heat-inactivated FBS, 2mM L-glutamine, antibiotics (penicillin 10,000 units/mL and streptomycin 10,000 g/mL) and maintained at 37 °C with 5 % CO 2, 95 % air in a humidified incubator. When cell line reached nearly 80 % confluences, the culture media were removed from the flask and the cells were briefly rinsed with 10 mL of PBS pH 7.4, harvested with 0.25 % trypsin and was then used for further experiments.

5A.2.3 UVB-irradiation and cell viability For cell viability experiment, cells were seeded 100 L/well from a density of 2 × 10 5 cells/mL, into 96-well tissue culture plate and then allowed to attach overnight in an incubator. After 24 h, cells were exposed to UVB light (13, 27, 41, 55, 83, 111, and 138 W/cm 2, which corresponds exactly to 50, 100, 150, 200, 300, 400 and 500 mJ/cm 2) without plastic lid. The W/cm 2 doses were achieved after 60 min of irradiation time using CL1000M UV Crosslinker (UVP, Upland, CA, USA). The irradiance was measured using a UV dosimeter (UVX Digital Radiometer, UVX-31 sensor, UVP). To prevent UVB light absorption by the cell culture medium, the medium was removed just prior to irradiation and replenished with a thin layer of PBS to cover the cells [24]. After the UVB irradiation, cells were replenished with fresh cell culture medium and incubated for 24 h. Later, cell viability was assessed by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) assay (Section 5A.2.5). Non-irradiated cells were served as control. No quantifiable rise in temperature was recorded during the irradiation. For colony formation assay, cells were seeded (2 × 10 5 cells/mL), into 24 well-tissue culture plate, allowed to attach overnight and later irradiated. After 240 h (cell media was changed every 48 h), cells were fixed with 3.7 % formaldehyde (in PBS) and stained with crystal violet (0.05 % in PBS). Excess crystal violet stain was rinsed off by PBS (twice), and air-dried.

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Chapter 5A: Cytotoxicity and Phototoxicity of Acylglutamate Surfactants

5A.2.4 Surfactant treatment Cells were seeded 100 L/well from a density of 2 × 10 5 cells/mL, into the central 60 wells of 96 well-tissue culture plate. The peripheral wells of plate received 100 L of supplemented DMEM alone (no cells = blank). One column was used as negative control (cells in supplemented DMEM) and then allowed to attach overnight in an incubator. After 24 h of attachment, the culture media was decanted and cells were rinsed with 150 L of culture media used for incubation. The HaCaT cell line was exposed to 100 L of different concentrations of acylglutamate surfactants and CPZ solutions (1, 5, 10, 20, 40, 80, 160 and 320 g/mL, pH 7.2), followed by incubation for 24 h and cell viability was assessed by MTS assay.

5A.2.5 MTS assay Cytotoxicity was evaluated using a ready-to-use The CellTiter 96 ® Aqueous One Solution Cell Proliferation Assay (MTS) (Promega, Madison, WI). This colorimetric assay is fast, convenient, easy-to-use in comparison with MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). MTS is bioreduced by cells into a colored formazan product, which is soluble in tissue culture media [25, 26]. After 24 h of incubation, 20 L of MTS solution (according to a protocol proposed by the manufacturer) was added directly into each well of 96-well assay plate containing acylglutamate surfactants and CPZ solutions followed by additional 3 h of incubation. Finally, the cell viability was quantified by recording the formazan absorbance at 490 nm using a Bio-Rad microplate reader. Since the absorbance directly correlated with the number of viable cells, the percent viability was calculated from the absorbance.

5A.2.6 Photoirritation evaluation The HaCaT cell line was utilized as in vitro model cell system to predict the cutaneous photoirritation [17], determined by MTS assay. Briefly, two 96-well plates were prepared for each surfactant (section 5A.2.4). One plate (without plastic lid) was irradiated (+ UVB) at 200 mJ/cm 2 for 60 min using CL-1000M UV Crosslinker (UVP) and the other non-irradiated plate ( − UVB) was wrapped in foil and place into the crosslinker [27]. Measures were taken to prevent UVB light absorption by the cell culture medium (as described in Section 5A.2.3). After the

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UVB irradiation, cells were replenished with fresh cell culture medium and incubated for 24 h. Later, cell viability was assessed by MTS assay (section 5A.2.5).

5A.2.7 Data analysis The data obtained from cytotoxicity and photoirritation evaluation was fitted into two models, namely the Photo-Irritation Factor (PIF) and Mean Photo Effect (MPE) is commonly known as predictors for the photoirritation potential of a diverse set of surfactants. The PIF is calculated by dividing the IC 50 (the concentration of the surfactant by which the cell viability is reduced by 50 %) of −

UVB by the IC 50 of +UVB and MPE aims to overcome the obvious limitations in the application of the PIF, by comparing the value of the photo effect between two curves at arbitrary doses. The value obtained from two models allows categorize a surfactant into “no photoirritant” (PIF < 2 or MPE < 0.1), “probable photoirritant” (PIF > 2 and <5 or an MPE > 0.1 and <0.15) and “photoirritant” (PIF > 5 or an MPE

> 0.15) [28]. The IC 50 , PIF and MPE values were determined using software, Phototox (ver. 2.0) available from the Secretariat [29]. This program module performs a bootstrap resampling of the original concentration-response data, which results in a set of new computer-generated concentration-response data and can be regarded as equiprobable realizations of the “true” concentration-response data hidden in the experimental observations [30].

5A.2.8. Statistical analysis In present study, the series of experiments were performed at least in three independent examinations (same day) with six replicates for each surfactant and data were displayed as mean ± standard deviation (SD). All data compared by one-way ANOVA (analysis of variance) followed by tukey post hoc test (GraphPad Prism 6.0 Software, San Diego, CA, USA). p-value < 0.05 was regarded as statistical significant.

5A.3 Results and discussion The acylglutamate surfactants in all likelihood segregate into polar amino acids (hydrophilic moiety) and non polar fatty acids (hydrophobic moiety) in the presence of water. Since most of these amino acids and fatty acids are found in the foods, oral toxicity is not expected. Dermal toxicity as a result of dermal exposure is 122

Chapter 5A: Cytotoxicity and Phototoxicity of Acylglutamate Surfactants not expected to be different from oral exposure [31]. Cutaneous phototoxicity of acylglutamate surfactants is of concern, and the central focus of this paper. Cutaneous phototoxicity is an “acute” reaction caused by a single treatment with skin irritants, such as UV radiation and surfactant. In vivo , this reaction can be elicited in all subjects provided the suitable dose of radiation and concentration of surfactants. “Acute” include immediate as well as delayed (after 48 h) reactions. Conforming to the diverse classes and chemical structure of surfactants with toxic potential, it seems exceedingly possible that several pathways must be involved in cutaneous irritation. Generally, two different pathways (either alone or in combination) are associated with surfactants which can commence and regulate the cutaneous toxicity. First, by damaging the barrier function of the stratum corneum and second, by straightforward effects of surfactant on cutaneous cells [32].

5A.3.1 UVB dose optimization To examine the outcome of UVB on HaCaT cell proliferation rates, they were exposed to UVB at doses ranging from 50 to 500 mJ/cm 2 and later the viable cells were assessed by MTS assay and colony formation assay. As depicted in Fig. 5A.1A and 1B the viability rate of HaCaT was substantially decreased in dose- dependent manner ( p < 0.05) of UVB irradiation. Our data showed that 200 mJ/cm 2 UVB dose induced almost 50 % cell death compared to the control. The observed results are in accordance with previous results [33], which state that maintaining genomic integrity during cellular proliferation is essential for the continued viability of the organism. G2-M checkpoint makes certain that cells don’t start mitosis before they have a chance to repair damaged DNA after replication. Cells that have a defective G 2-M checkpoint enter mitosis before repairing their DNA. When cells bypass the normal restrictions on entrance into S phase imposed by DNA damage, the replication of damaged DNA can either result in cell death or an accumulation of genetic changes leading ultimately to cancer. UVB light is known to damage DNA 2 and therefore cells expose to single dose of 200 mJ/cm UVB causes G 2-M arrest. Moreover, this dose corresponds to 15 min – 4 h of solar irradiation [34], a common period of time for out door exposure. Hence, we selected this dose when performing photoirritation experiments examining the effect of acylglutamate surfactants.

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Figure 5A.1: Cell proliferation rate in UVB irradiated HaCaT cells. ( A) Viability assessed by MTS assay 24 h after UVB irradiation. The bar graph depicts the percent cell viability compared to control and represent the mean percentage ± S.D. (standard deviation) of three independent experiments. ( B) Colony formation assay. Representative photomicrographs of survival colonies after different doses of UVB, stained with crystal violet solution. Similar results were obtained in two independent experiments.

5A.3.2 Cytotoxicity and phototoxicity of commercial acylglutamates

The calculated IC 50 values for all acylglutamate surfactant on HaCaT cells with − UVB (blue points depict data and gray curve depicts statistical curve fitting by phototox software [30]) and + UVB (yellow points depict data and black curve depicts statistical curve fitting by phototox software) exposed group were higher (Fig.5A.2A–C and Table 5A.1) compared to the CPZ (+ UVB 1.054 ± 0.23, − UVB 16.00 ± 0.89,) (Fig.5A. 2D), also significant differences ( p < 0.05) were observed

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among the groups compared. The IC 50 values for acylglutamate surfactants were in the descending order: LS 11 > CS 11 > MS 11. The MS 11 showed highest cytotoxicity due to its larger fatty acid chain length (C 14 , 97.8 %) in comparison with shorter fatty acid chain length of LS 11 (C 12 , 97.3 %) and CS 11 (C 12 , 57.8 %). A possible explanation for this is that, as the alkyl side chain in the amino acid becomes larger, the sodium salts of acylglutamate cannot pack closely at water surfaces. As a result, their capacity to lower surface tension becomes less. On the contrary, the overall hydrophobicities of the molecules become larger [12,35]. Hence, causing greater amounts of surfactant adsorbed on the HaCaT cell membrane, leading to cellular disintegration, eventually cell death and the concomitant release of keratinocyte cytoplasm containing proinflammatory cytokine, IL-1α [36,37].

Table 5A.1: IC 50 values of acylglutamate surfactants and CPZ on HaCaT cells.

a Surfactant UVB light IC 50 ± SD ( µg/mL) Amisoft CS 11 − 43.48 ± 1.09 Amisoft CS 11 + 30.62 ± 2.72 Amisoft LS 11 − 69.97 ± 1.66 Amisoft LS 11 + 54.22 ± 1.81 Amisoft MS 11 − 26.48 ± 1.47 Amisoft MS 11 + 16.72 ± 0.92 CPZ − 16.00 ± 0.89 CPZ + 1.054 ± 0.23

aValues represent the mean ± standard deviation of three independent examinations, each performed with six replicates. “−” indicate UVB non-irradiated; “+” indicate 2 UVB irradiated at 200 mJ/cm for 60 min; IC 50 , the concentration of the surfactant by which the cell viability is reduced by 50 %; SD, standard deviation.

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Figure 5A.2: Effect of increasing concentration of ( A) CS-11; ( B) LS-11; ( C) MS- 11 and ( D) CPZ on cell viability of HaCaT cells. Yellow and blue points represent the original concentration-response data from + UVB and − UVB, respectively whereas, the black and gray curves depict statistical curve fitting by Phototox software. + UVB irradiated at 200 mJ/cm 2 for 60 min; − UVB non-irradiated.

Since, much more is known about the phototoxicity of CPZ and is one of the classic phototoxicants of the HaCaT cells [38]. Upon UV irradiation CPZ produces a variety of free radicals along with the corresponding cation radical, the neutral promazinyl radical, a chlorine radical and super-centered peroxy radical. The neutral promazinyl radical can react covalently with cell membrane, protein, and other macromolecules to yield antigens which could be responsible for the photoallergic response to CPZ resulting phototoxicity, both in vitro and in vivo [39]. Hence, CPZ was used as a positive control in the current study and as expected exhibited the

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Chapter 5A: Cytotoxicity and Phototoxicity of Acylglutamate Surfactants highest cytotoxic and phototoxic activity on HaCaT cell among all acylglutamate surfactant tested. Regarding the phototoxicity of acylglutamates, the results showed that all acylglutamate surfactant tested were non-phototoxic with + UVB, which is reflected in its low PIF mean and MPE values (PIF < 2 and MPE < 0.1) (Table 5A.2). Whereas, the resulting PIF mean and MPE values for CPZ tested on HaCaT cells were found to be 15.186 and 0.435, respectively, exhibiting the toxicity potential of a CPZ in the presence of UVB light (Table 5A.2). The non-phototoxic nature of all acylglutamate surfactant is due to the presence of amino acid (glutamic acid) in it and glutamic acid is one of the precursors for the synthesis of glutathione (GSH). GSH is the main protective mechanism against UVB radiation, as it is the only donor of hydrogen in H 2O2 neutralization thus preventing damage to important cellular components [40]. Thus, signify that higher the GSH level in HaCaT cell is expected to confer resistance to UVB irradiation [41].

Table 5A.2: PIF mean and MPE mean values of acylglutamate surfactants and CPZ on HaCaT cells. PIF Toxicity MPE Toxicity Phototoxic Surfactant mean a probability mean a probability potential Amisoft CS 11 1.292 0 0.019 0 No Amisoft LS 11 1.442 0 0.007 0 No Amisoft MS11 1.585 0 0.092 0.055 No CPZ 15.186 1.0 0.435 1.0 Yes a PIF and MPE values are averages over all bootstrap pairs.

Note that, PIF mean and MPE values of CPZ were in the range of the reference values (PIF > 14.4 and MPE 0.33 – 0.63) indicating that the irradiation system and method for measuring phototoxicity of acylglutamate surfactants were appropriate [28]. This was achieved because we used a wider concentration range of CPZ for both the + UVB and − UVB group.

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5A.4 Conclusion In the light of cytotoxicity and phototoxicity results, we could conclude that shorter the fatty acid chain length of acylglutamate surfactants, the milder it functions on HaCaT cell line and vice versa. In addition, both amino acid and fatty acid are valuable for the barrier effect of the skin cells. Considering their biocompatible properties and low cytotoxicity profile, the sodium lauroyl glutamate (Amisoft LS-11) would be the most suitable surfactant for the synthesis and stabilization of lipid nanoparticles for improved delivery of various cosmeceutical actives.

5A.5 References

1) Liu J, Hu W, Chen H, Ni Q, Xu H, Yang X. Isotretinoin-loaded solid lipid nanoparticles with skin targeting for topical delivery. Int. J. Pharm. 2007; 328:91–195. 2) Kovacevic A, Savic S, Vuleta G, Müller RH, Keck CM. Polyhydroxy surfactants for the formulation of lipid nanoparticles (SLN and NLC): Effects on size, physical stability and particle matrix structure. Int. J. Pharm. 2011; 406:163–172. 3) Mitri K, Shegokar R, Gohla S, Anselmi C, Müller RH. Lipid nanocarriers for dermal delivery of lutein: Preparation, characterization, stability and performance. Int. J. Pharm. 2011; 414:267–275. 4) Somasundaran P, Soma C, Puspendu D, Namita D, Somasundaran T. Contribution of surfactants to personal care products. In Surfactant in Personal Care Products and Decorative Cosmetics, 3rd ed.; Rhein, L.D., Olenick, A., Schlossman, M., Somasundaran, P., Eds.; CRC Press: Florida, FL, USA, 2007, pp. 121–135. 5) Infante MR, Molinero J, Erra P, Juliá MR, García Domínguez JJ, Robert M. The influence of steric configuration of some N α-lauroyl amino-acid derivatives on their antimicrobial activity. Fette Seifen Anstrichm. 1986; 88:108–110. 6) Okahata Y, Tanamachi S, Nagai M, Kunitake T. Synthetic bilayer membranes prepared from dialkyl amphiphiles with nonionic and zwitterionic head groups. J. Colloid Interface Sci. 1981; 82:401–417. 7) Kida T, Morishima N, Masuyama A, Nakatsuji Y. New cleavable surfactants derived from glucono-1,5-lactone. J. Am. Oil Chem. Soc. 1994; 71:705–710. 8) Heutrich W, Keppler H, Hintzmann K. Detergents, Wetting, Dispersing and Leveling Agents. German Patent 635522, 18 September 1936. 9) Amisoft ® CS 22. Amino Acid Based Anionic Surfactant. Available online: www.cosmesi.it/portals/7/documenti/amisoft%20CS-22_brochure.pdf (accessed on 15 April 2014). 10) Kawasaki Y, Quan D, Sakamoto K, Cooke R, Maibach HI. Influence of surfactant mixtures on intercellular lipid fluidity and skin barrier function. Skin Res.Technol. 1999; 5:96–101. 11) Lee CH, Kawasaki Y, Maibach H. Effect of surfactant mixtures on irritant contact dermatitis potential in man: Sodium lauroyl glutamate and sodium lauryl sulphate. Contact Dermat. 1994; 30:205–209. 12) Takehara M. Properties and applications of amino acid based surfactants. Colloids Surf. 1989; 38:149–167. 13) Cosmetic Directive. Available online: http://ec.europa.eu/consumers/archive/sectors/cosmetics/documents/directive/index_en.htm (accessed on 8 July 2014) 14) Full EU Ban on Animal Testing for Cosmetics Enters into Force. Available online: http://europa.eu/rapid/press-release_IP-13-210_en.htm (accessed on 15 April 2014). 15) Botham PA, Earl LK, Fentem JH, Roguet R, van de Sandt JJM. Alternative Methods for Skin Irritation Testing: The Current Status. Altern. Lab. Anim. 1998; 26: 195–211. 16) Wilhelm KP, Böttjer B, Siegers CP. Quantitative assessment of primary skin irritants in vitro in a cytotoxicity model: Comparison with in vivo human irritation tests. Br. J. Dermatol. 2001; 145:709– 715. 128

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17) Benavides T, Martínez V, Mitjans M, Infante MR, Moran C, Clapés P, Clothier R, Vinardell MP. Assessment of the potential irritation and photoirritation of novel amino acid-based surfactants by in vitro methods as alternative to the animal tests. Toxicology 2004; 201:87–93. 18) Sanchez L, Mitjans M, Infante MR, Vinardell MP. Potential irritation of lysine derivative surfactants by hemolysis and HaCaT cell viability. Toxicol. Lett. 2006; 161: 53–60. 19) Wilheilm KP, Samblebe M, Siegers CP. Quantitative in vitro assessment of N-alkyl sulphate- induced cytotoxicity in human keratinocytes (HaCaT): Comparison with in vivo human irritation tests. Br. J. Dermatol. 1994; 130:18–23. 20) Lohézic-Le Dévéhat F, Legouin B, Couteau C, Boustie J, Coiffard L. Lichenic extracts and metabolites as UV filters. J. Photochem. Photobiol. B 2013; 120:17–28. 21) Maupas C, Moulari B, Beduneau A, Lamprecht A, Pellequer Y. Surfactant dependent toxicity of lipid nanocapsules in HaCaT cells. Int. J. Pharm. 2011; 411:136–141. 22) Nichols JA, Katiyar SK. Skin photoprotection by natural polyphenols: Anti-inflammatory, antioxidant and DNA repair mechanisms. Arch. Dermatol. Res. 2010; 302:71–83. 23) Kim JK, Kim Y, Na KM, Surh YJ, Kim TY. [6]-Gingerol prevents UVB-induced ROS production and COX-2 expression in vitro and in vivo. Free Radic. Res. 2007; 41: 603–614. 24) Park K, Lee JH. Protective effects of resveratrol on UVB-irradiated HaCaT cells through attenuation of the caspase pathway. Oncol. Rep. 2008; 19:413–417. 25) Cory AH, Owen TC, Barltrop JA, Cory JG. Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture. Cancer Commun. 1991; 3: 207–212. 26) Yin JJ, Liu J, Ehrenshaft M, Roberts JE, Fu PP, Mason RP, Zhao B. Phototoxicity of nano titanium dioxides in HaCaT keratinocytes--generation of reactive oxygen species and cell damage. Toxicol. Appl. Pharm. 2012; 263:81–88. 27) Vinardell MP, Benavides T, Mitjans M, Infante MR, Clapés P, Clothier R. Comparative evaluation of cytotoxicity and phototoxicity of mono and diacylglycerol amino acid-based surfactants. Food Chem. Toxicol. 2008; 46:3837–3841. 28) Test No. 432: In vitro 3T3 NRU phototoxicity test. In OECD Guidelines for the Testing of Chemicals Section 4: Health Effects; Organisation for Economic Cooperation and Development (OECD) iLibrary: Paris, France, 2004; pp. 1–15. 29) OECD. Available online: http://www.oecd.org/document/55/0,2340,en_2649_34377_2349687_1_1_1_1,00.html (accessed on 1 April 2014). 30) Peters B, Holzhutter HG. In vitro phototoxicity testing: development and validation of new concentration response analysis software and biostatistical analyses related to the use of various prediction models. Altern. Lab. Anim. 2002; 30:415–432. 31) Safety Assessment of Amino Acid Alkyl Amides as Used in Cosmetics. Available online: www.cir-safety.org/sites/default/files/alkyl_amides_0.pdf (accessed on 4 April 2014) 32) Berardesca E, Distante F. The modulation of skin irritation. Contact Dermat. 1994; 31: 281–287. 33) Athar M, Kim AL, Ahmad N, Mukhtar H, Gautier J, Bickers DR. Mechanism of ultraviolet B- induced cell cycle arrest in G2/M phase in immortalized skin keratinocytes with defective p53. Biochem. Biophys. Res.Commun. 2000; 277:107–111. 34) Kudish AI, Lyubansky V, Evseev EG, Ianetz A. Statistical analysis and inter-comparison of the solar UVB, UVA and global radiation for Beer Sheva and Neve Zohar (Dead Sea), Israel. Theor. Appl. Climatol. 2005; 80:1–15. 35) Kanari M, Kawasaki Y, Sakamoto K. Acylglutamate as an anti-irritant for mild detergent system. J. Soc. Cosmet. Chem. Jpn. 1993; 27:498–505. 36) Osborne R, Perkins MA. An approach for development of alternative test methods based on mechanisms of skin irritation. Food Chem. Toxicol. 1994; 32:133–142. 37) De Brugerolle de Fraissinette A, Picarles V, Chibout S, Kolopp M, Medina J, Burtin P, Ebelin ME, Osborne S, Mayer FK, Spake A, et al. Predictivity of an in vitro model for acute and chronic skin irritation (SkinEthic) applied to the testing of topical vehicles. Cell Biol. Toxicol. 1999; 15:121– 135. 38) Suh YW. An Investigation of the Phototoxicity of Decabromodiphenyl Ether and Triclosan. Ph.D. Thesis, University of Iowa, Iowa City, IA, USA, 2010. 39) Chignell CF, Motten AG, Buettner GR. Photoinduced free radicals from chlorpromazine and related phenothiazines: Relationship to phenothiazine-induced photosensitization. Environ. Health Perspect. 1985; 64:103–110. 40) Afaq F, Mukhtar H. Effects of solar radiation on cutaneous detoxification pathways. J. Photochem. Photobiol. B 2001; 63:61–69. 41) Leccia MT, Richard MJ, Joanny-Crisci F, Beani JC. UV-A1 cytotoxicity and antioxidant defence in keratinocytes and fibroblasts. Eur. J. Dermatol. 1998; 8:478–482. 129

Chapter 5B

Photoprotection aspects of topically administered combination-NLCs

This chapter deals with feasibility of process to obtain RSV and PBN loaded NLCs (combination-NLCs) using amino acid-based surfactant (Amisoft LS-11). Combination-NLCs were formulated with the aim to reduce intracellular oxidative stress and provide maximum photoprotection to the skin. Optimized (using Taguchi’s mixed-level design) combination-NLCs demonstrated particle size in sub- micron range (< 200 nm), sustained release over a period of 24 h, due to the solid state nature of NLCs and high solubility of RSV and PBN in Gelucire 50/13 and Labrafil M 1944 CS binary mixture at 9:1 ratio. In vitro occlusion, skin deposition, stability studies, cellular uptake studies and photoprotection studies demonstrated superiority of combination-NLCs when compared to single antioxidant loaded NLCs

Chapter 5B: Photoprotection Aspects of Combination-NLCs

5B.1 Genesis and outline of the work Environmental factors such as UVR emitted by the sun and air pollution (primary as well as secondary air pollutant) generate OS in skin leading to skin cancer and photoaging changes. Endogenous antioxidants in skin interact to protect the tissue by neutralizing ROS [1], but most antioxidant protection largely depends on the average daily dietary intake and subsequent delivery to skin. Because the antioxidant tends to get destroyed or changed by oxidation during neutralization process, protection is often limited by the concentration of antioxidants remaining in the skin. [2]. Broad spectrum sunscreens are widely used to protect skin from sunlight but at optimal application only 55 % of the free radical generation is blocked [3]. Topical application of antioxidants when properly formulated for optimal percutaneous absorption, supplement skin’s antioxidant reservoir to provide additional protection. Moreover, since antioxidants do not work individually in the skin but work synergistically in an integrated and regulated way to protect skin against OS, Combination antioxidants (preferably of different mechanism of action) into drug carrier may be particularly efficacious to thwart UVB mediated OS [4]. To achieve this aim, it is necessary to develop topical nanoparticulate formulations that can hold two drugs and should provide photostability to these inherently unstable molecules. Of even more importance, this formulation should enter into the skin efficiently and release the drug in slow-sustained manner, so they can provide maximum protection against UVB mediated OS. RSV) and PBN were used as antioxidant models in current study. RSV (3,5,4 '-trihydroxy-stilbene) is a polyphenolic phytoalexin that is generated in response to environmental stress in Japanese plant Polygonum cuspidatum . It is also found in grape skins, peanuts and red wine, as well as host of nonedible plants [5, 6], and is produced preferentially in response to fungal infections as seen in mature vine berries [7]. Resveratrol exists in cis- and trans -isomers, where later is more abundant and biologically active. However, trans-isomers get converted in to cis- when not stored properly (exposed to white light) and if it is protected from high pH. trans -isomer is reported to be stable for 4 weeks in absence of light [8, 9]. RSV has been reported to have numerous biological effects, including antioxidant activity. Concerning its antioxidant activities, this polyphenols have the ability to scavenge free radicals, to inhibit lipid peroxidation and DNA damage as well as to improve

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Chapter 5B: Photoprotection Aspects of Combination-NLCs endogenous antioxidants defenses [10, 11]. However, the low water solubility, stability and therapeutic index of RSV make it unsuccessful in clinical therapy [12]. PBN (N-benzylidene-tert -butylamine N-oxide) otherwise known as “spin trap” or “intelligent” antioxidants, are the compounds that stabilize or trap free radicals, hence reducing the cascade effect on other molecules [13, 14]. Mechanisms other than spin-trapping have been investigated recently; these include 1) its antioxidant properties, 2) its action on important membrane enzymes including ion transport proteins and 3) its action as an anti-inflammatory agent [15]. In our two previous studies (chapter 4 and 5A), we proved that 1) NLCs are suitable topical delivery system for encapsulation single antioxidants that in turn improved the cellular uptake and exerted photoprotective effects and 2) amino acid-based surfactant (Amisoft LS-11) is biocompatible and exhibited low cytotoxicity profile among the other surfactant tested [16, 17]. In this light we decided to develop Amisoft LS-11 based topical NLC formulations containing RSV and PBN as single drug and in combination (combination-NLCs), and to evaluate the effect of co-encapsulation on the physico- chemical and photoprotective property of the NLCs against UVB-mediated phototoxicity and oxidative stress in HaCaT cells. This is the first study which explores the potential application of Aminsoft LS-11 as a stabilizer/emulsifier, feasibility of a process (optimized using Taguchi’s mixed-level design) to obtain stable combination-NLCs, and their capacity to improve the drug skin permeation and subsequent photoprotective effect when compare to pure-drug as well as individually loaded antioxidant into NLCs.

5B.2 Experimental 5B.2.1 Screening and selection of antioxidant combination

5B.2.1.1 Chemical based assays 5B.2.1.1.1 UV measurements First, we determined the molar extinction coefficient (MEC, ɛ), briefly, a stock solution (10 mM) of each antioxidant (i.e. IDB, RSV and PBN) was prepared and diluted in absolute ethanol 99 % to get a final concentration of 10 M, and UV absorbance spectra were recorded on a spectrophotometer equipped with quartz cells of 10 mm light path. MEC was calculated: 131

Chapter 5B: Photoprotection Aspects of Combination-NLCs

ɛ = A/ (c × l ), A : absorbance, c: concentration of surfactant, l: path length. In the second method, we determine the UVA/UVB proportionality [18] and critical wavelength [19]. The UVA/UVB proportionality is nothing but the proportion of the average extinction in the UVA and UVB region. It is calculated by the following equation:

UVA/UVB =

Based on UVA/UVB proportion data, Boots and Chemist Ltd, defined five (05) different UVA Star Rating system: 1) a low UVA protection when proportion is

< 0.2 (“0” UVA Star Rating), 2) a moderate UVA protection with proportion between 0.21 – 0.40 (“1” UVA Star Rating), 3) a good protection with proportion between 0.41 – 0.6 (“2” UVA Star Rating), 4) a superior protection with a proportion between 0.61 – 0.8 (“3” UVA Star Rating) and 5) a maximum protection with a proportion > 0.8 (“4” UVA Star Rating) [18, 20]. The critical wavelength determination ( λc) [19], and can be defined as the wavelength where the integral of spectral absorbance curve reached 90 % of the integral from 290 - 400 nm. It is calculated by the following equation:

To classify the suncreens, a five-point scale was introduced [19], which is as follows: 1) λc < 325 nm (“0” Broad Spectrum Rating), 2) λc 325 – 335 nm (“1” Broad Spectrum Rating), 3) λc 335 – 350 nm (“2” Broad Spectrum Rating), 4) λc

350 – 370 nm (“3” Broad Spectrum Rating) and 5) λc > 370 nm (“4” Broad Spectrum Rating) [20].

5B.2.1.2 Cell based assays 5B.2.1.2.1 HaCaT culture HaCaT cells were cultured as previously described (section 4.2.7.1) [16]. Briefly, cells obtained from NCCS, Pune, India, were maintained at 37 °C with 5 %

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CO 2, 95 % air in a humidified incubator and were cultured in DMEM containing 10% FBS, L-glutamine and antibiotics.

5B.2.1.2.2 HaCaT treatment with IDB, RSV and PBN samples Stock solution, working concentrations and HaCaT treatment with each antioxidant (cytotoxicity and phototoxicity) were carried out as previously described (section 4.2.7.2) [16]. Briefly, in individual experiment, HaCaT cells were seeded 100 L/well from a density of 2 × 10 5/mL, into 96-well tissue culture plate. After 24 h, cells were treated with working concentrations of each antioxidant (i.e. IDB at 1, 2, 4, 6, 8 and 10 M/0.1 mL, both RSV and PBN at 5, 25, 50, 75, 100 and 200 M//0.1 mL, followed by incubation for 24 h and cell viability was quantified by MTS assay. To assess the photoprotective/antioxidative effect of each antioxidant, cells were pretreated (24 h, 37 °C) with working concentrations of each antioxidant (as above). Next day, cells were irradiated for 1 h with previously optimized UVB dose (200mJ/cm 2) (section 5A.2.3) [17], then incubated for another 24 h and cell viability was quantified by MTS assay. Untreated and non-irradiated cells were served as a negative control whereas untreated and irradiated cells were served as positive control. The detail of the UV irradiation system used in this study is described in section 4.2.7.3

5B.2.1.2.3 MTS assay To assess the effect of each antioxidant on HaCaT cell viability we used MTS assay as previously described (section 4.2.7.4) [16]. Briefly, after 24 h of incubation, 20 L MTS solution was directly added into each well plate containing different antioxidant samples followed by additional 3 h of incubation. Finally, the cell viability was quantified by recording the formazan absorbance at 490 nm using a microplate reader. Since the mean absorbance (OD) directly correlated with the number of viable cells, the mean OD of negative control cells was set to 100 % viability.

5B.2.2 Pre-formulation studies 5B.2.2.1 NLCs component screening and selection

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NLCs main components such as solid lipid, liquid lipid and its proper ratio was screened and selected as previously described (section 4.2.1) [16]. Briefly, 10 mg of RSV and PBN were added to 1 g of melted solid lipid or liquid lipid (melted 10 °C above their melting point) and vertexed for 2 min and then visually observed for the solubility. The addition of RSV and PBN was continued until it is saturated and the amount of RSV and PBN dissolved was reported directly. Solubility in liquid lipids was determined after separating the undissolved RSV and PBN by centrifugation. The amount of RSV and PBN dissolved was quantified spectrophotometrically at 306 nm and 293 nm, respectively. The miscibility of the selected solid lipid and liquid lipid (binary mixture) that dissolved maximum amount of RSV and PBN was mixed in five main ratios such as 9:1, 8:2, 7:3, 6:4 and 5:5. In next step, the solid mass of a binary mixture was placed on Whatman filter paper and observed for any oil expulsion on filter paper, during storage (40°C ± 2 °C/75 % ± 5% RH in a stability chamber). Only the best binary mixture ratio which did not show any oil expulsion was selected for the preparation of RSV loaded NLCs (RSV-NLCs), PBN loaded NLCs (PBN-NLCs) and RSV and PBN loaded NLCs (combination-NLCs). For the preparation of above mentioned NLCs “green-surfactants” such as amino-acid based surfactants otherwise known as “acylglutamates” were screened and “sodium lauroyl glutamate” was eventually selected based on our previous results (chapter 5A) [17].

5B.2.2.2 DSC studies The melting behavior of RSV, PBN, Gelucire 50/13 (G 50/13), binary mixture of G 50/13 and Labrafil M 1944 CS (LMC) at 9:1 ratio, ternary mass mixture containing drug and binary mixture (i.e. RSV, G 50/13 and LMC at 0.5:9:1 ratio; PBN, G 50/13 and LMC at 0.5:9:1 ratio; RSV+PBN, G 50/13 and LMC at 1:9:1 ratio) were studied using a DSC. Briefly, the ternary mixtures (6 - 8 mg) were tempered for 1 h at 70 °C to simulate production condition of NLCs. Samples were heated from 20 to 150 °C (except for RSV containing samples, heated from 20 to 300 °C). An empty pan was used as reference.

5B.2.2.3 Drug interaction studies

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Pure RSV, PBN and their mixture (2-3 mg sample mixed with dry potassium bromide) were subjected to Fourier transform infra-red (FTIR) spectroscopy studies by using Jasco FTIR-5300 in the region of 4000–400 cm –1.

5B.2.3 Preparation of NLCs RSV-NLCs, PBN-NLCs and combination-NLCs and placebo-NLCs (without drug) were prepared by a melt-emulsification-ultrasonication method as previously described (section 4.2.5) [16]. Briefly, the solid lipid (G 50/13, melting point 50 °C) was melted 10 °C above their melting point; to the above solution liquid lipid (LMC) was added and mixed together using cyclo-mixer (2 min). To the obtained mixture, a hot (60-65°C) aqueous surfactant solution (Amisoft LS 11) was quickly added and finally subjected to ultrasonication procedure (amplitude 90 %, 1 min, 30sec “on”, 5 sec “off” cycle). The resulting NLCs were allowed to cool down to room temperature and initial particle size and zeta potential were recorded.

5B.2.4 Optimization of NLCs NLCs were optimized using Taguchi’s experimental design with mixed-level factors. The experimental design with a 1 six-level factor and 3 three-level factors 1 3 i.e. L 18 (6 × 3 ) orthogonal array design. L and subscript 18 denote the Latin square and the number of experimental runs, respectively. Four researched factors were included: two formulation factors (surfactant concentration and lipid concentration) and two process parameters (sonication amplitude and sonication time). Since these factors known to exhibit a significant impact on z-ave and EE, they were included as dependent variables. The experimental results were then analyzed by the Minitab software version 17 to extract independently the main effects of these factors, followed by the analysis of variance (ANOVA) to find out which factors were statistically significant.

5B.2.5 Characterization of NLCs The prepared NLCs were characterized for their z-ave, PDI, zeta potential, entrapment efficiency and drug content. In addition to the above mentioned characterization, optimized NLCs, RSV-PD and PBN-PD (pure drug suspension in PBS) were evaluated for TEM, in vitro occlusion test, in vitro drug release studies,

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Chapter 5B: Photoprotection Aspects of Combination-NLCs ex vivo drug diffusion studies and stability studies (section 4.2.6.3 to 4.2.6.7). RSV and PBN were quantified spectrophotometrically at 306 and 293 nm, respectively.

5B.2.6 Cell culture study 5B.2.6.1 Cell viability and photoprotective effect of NLCs Working concentrations and HaCaT treatment with each NLC (cytotoxicity and phototoxicity) were carried out as previously described (section 4.2.7.2). Briefly, in an individual experiment, HaCaT cells were treated with placebo-NLCs, RSV-NLCs, PBN-NLCs and combination-NLCs (formulation equivalent to 75 M//0.1 mL for RSV; i.e. ≈ 1.8 g and 100 M/0.l mL for PBN; i.e. ≈ 1.8 g), followed by incubation for 24 h and cell viability was quantified by MTS assay. Regarding, the photoprotective effect of each NLCs (above mentioned), in an individual experiment, cells were pre-treated (24 h, 37 °C) with each NLCs. Next day, cells were irradiated for 1 h with previously optimized UVB dose (200mJ/cm 2) (section 5A.2.3) [17], and then incubated for 24 h and cell viability was quantified by MTS assay. Untreated and non-irradiated cells were served as a negative control, whereas untreated and irradiated cells were served as positive control. The UV irradiation system used in this study is described in section 4.2.7.3.

5B.2.6.2 Quantitative cellular uptake of NLCs For quantitative determination of RSV-PD, PBN-PD and combination-NLCs uptake by HaCaT cells we used method as previously described (section 4.2.7.5) [16]. Briefly, cells were treated with above mentioned NLCs and PD (equivalent to 75 M/1mL for RSV; i.e. ≈ 18 g and 100 M/mL for PBN; i.e. ≈ 18 g), followed by incubation for 2, 6 and 24 h at 37 °C in CO 2 incubator. At each time point, medium was carefully withdrawn from each well of 24-well plate and cells were permeabilized. Both permeabilized cells and recovered medium were lyophilized and suspended in ethanol followed by sonication. Later, the cell lysate was centrifuged and the clear supernatant was filtered. The amount of RSV and PBN in cells and media was quantified spectrophotometrically at 306 and 293 nm, respectively.

5B.2.6.3 Intracellular ROS scavenging activity

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Intracellular ROS level (DCF-DA assay) in HaCaT cells was determined as previously described (section 4.2.7.6) [16]. Briefly, in separate experiment, cells were treated with placebo-NLCs, RSV-NLCs, PBN-NLCs and combination-NLCs (equivalent to 75 M/0.1mL for RSV; i.e. ≈ 1.8 g and 100 M/0.1mL for PBN; i.e. ≈ 1.8 g) and exposed to UVB radiation (200 mJ/cm 2, 1 h), followed by further incubation for 24 h. The DCF-DA (25 M) was then added to detect UVB radiation- mediated intracellular ROS levels in HaCaT cells. Later, cells were incubated 37 °C for 30 min. Fluorescence emitted by DCF-DA and Rhodamine 123 was detected using a flow cytometer in FL-1 channel (535 nm). The data acquisition and analysis were performed using Kaluza software version 1.2. The level of intracellular ROS was expressed as MFI.

5B.2.7 Statistical analysis Statistical data analysis was performed using the demo version of GraphPad Prism 6.0 Software. p < 0.05 considered statistically significant.

5B.3 Results and discussion 5B.3.1 Screening and selection of antioxidant combination The comprehensive screening and selection of antioxidant combination is crucial step in the development of strategies to ensure their photoprotective /antioxidative properties. In order to ensure that, to this end, we performed various chemical-based and cell-based assays. In chemical-based assay, three parameters were used to classify the photoprotective effectiveness of an antioxidant. Fig.5B.1 depicts the UV spectra (in the range of 290 - 400 nm) of IDB, RSV and PBN. The IDB (has absorption maxima at 278 nm) showed very low absorbance value between 290 and 295 nm. Hence, IDB can not be regarded as efficient UVA or UVB filter. Both, RSV and PBN showed high specific absorbance values between 290 nm and 320 nm (UVB region). Two absorption maxima were observed for RSV at 306 nm ( ɛ RSV 306 = 0.20208) and 321 nm ( ɛ RSV 306 = 0.18769), while PBN showed single absorption maxima at 293 nm ( ɛ PBN 293 = 0.14169). The high absorptivity, particularly of RSV is due to the presence of multiple conjugate double bonds that permits a wider delocalization of the unpaired electron produced by the antioxidant action [20]. The high MEC value of RSV and PBN make these antioxidants good

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UV absorbers and as a consequence, they could be useful active component in sunscreen formulations, used in single or in combination [20].

Figure 5B.1: UV spectra of IDB, RSV and PBN, in the range 290 – 400 nm, at 10 M concentration.

In order to verify this possibility, we also determined their other photoprotective properties (UVA/UVB proportionality and critical wavelength). The obtained UVA/UVB proportionality data suggest that both RSV and PBN showed moderate (0.24, “1” UVA star rating) to low (0.15, “0” UVA star rating) UVA screen property, respectively. Hence, they cannot be considered as good UVA filters. Regarding critical wavelength, RSV ( λc = 340 nm) and PBN ( λc = 310 nm) are characterized by a rating value of 2 and 0, respectively, so indicating that they could be considered as a good UVA photoprotection antioxidants. Finally, we confirmed safety profile (cytotoxicity assay) and photoprotective effect (from UVB-mediated damage) of IDB, RSV and PBN on HaCaT cell line.

Fig.5B.2A and 2B, where IC 50 values of all the antioxidants investigated are reported. 138

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Higher IC 50 value (Fig.5B. 2A) of RSV (75 M, cell viability 53.71 ± 1.67) and PBN (100 M, cell viability 48.19 ± 3.19), which is almost 13-fold and nearly over 16-fold when compared to IDB (6 M, cell viability 46 ± 7.13) indicates biocompatibility and non-cytotoxic nature of RSV and PBN on HaCaT cells. Our data of RSV, in terms of biocompatibility, corroborate the findings of Baxter R.A [21], which report 17 fold greater antioxidant activity (using the oxygen radical absorbance capacity test, ORAC test) of RSV than IDB. This is the first study which shows the biocompatibility of PBN on HaCaT cells. Regarding the photoprotection offered by RSV (between 5 M and 75 M) and PBN (between 5 M and 100 M) (Fig.5B. 2B), the HaCaT cell viability increased significantly ( p < 0.001) when compared with positive control cells. Further, PBN at 100 M significantly ( p < 0.001) protected HaCaT cells when compared with IDB 6 M and RSV 75 M. Improved photoprotection offered by PBN is attributed to their unique mechanism of action. Conventional antioxidants like IDB and RSV act upon free radicals by chemically reacting with them to convert ROS into water, whereas, PBN “traps” the ROS and convert them into harmless oxygen and then transport them back into the electron transport chain of cellular respiration (Fig.5B.3) [22, 23]. IDB, despite of its superior oxidative protection capacity when compared to DL-alpha tocopherol, kinetin, CoQ 10 , L-ascorbic acid, and DL-alpha lipoic acid [24], in this study it didn’t show tendency to protect HaCaT cells when compared to RSV and PBN. In the light of all data, co-encapsulation of RSV and PBN into an efficient topical delivery system could assure a high protection over UVB and UVA region.

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Figure 5B.2: Viability of HaCaT cells. (A) Cytotoxicity assay, after 24 h of incubation with different concentrations of IDB, RSV and PBN. (B) Photoprotective effect of IDB, RSV and PBN against UVB (200 mJ/cm 2 for 1 h) -mediated phototoxicity in HaCaT cells. Results are represented as percentage of cell viability compared to negative control group set on 100 %. Bars represent mean ± S.D. from three independent experiments, each performed in triplicate. *** p < 0.001 vs. – control, ooo p < 0.001 and ns* vs. + control (One-Way ANOVA test, Dunnett’s Multiple comparison test); ** p < 0.01 and ns (non significant) vs. IDB 6 M, and $$$ p < 0.001 vs. IDB 6 M and RSV 75 M (One-Way ANOVA test, Tukey’s Multiple Comparison Test).

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Figure 5B.3: Mechanism of action of PBN

5B.3.2 Pre-formulation studies Before the experimental design, preliminary studies like solubility of RSV and PBN were carried out. DSC measurements and FT-IR studies were also carried out to get information about the melting behaviour between selected drug, solid lipid and liquid lipid at optimized ratio, and molecular interaction, respectively. To identify the solid lipid and liquid lipid that can dissolve the highest amount of RSV as well as PBN, lipid screening was done using various solid and liquid lipids that would be well suited for topical delivery. The various lipids screened and the amount of RSV and PBN dissolved in each lipid are illustrated in Table 5B.1. The highest amount (0.1 % each) of RSV and PBN was found to be dissolved in Gelucire 50/13 (solid lipid, a mixture of caprylic acid C 8 (< 3.0 %), capric acid C 10 (<3.0 %), lauric acid C 12 (< 5.0 %), myristic acid C 14 (< 5.0 %), palmitic acid C 16 (40 to 50 %), stearic acid C 18 (48 to 58 %), with hydroxyl value (mg KOH/g) in the range of 36 - 56) and Labrafil M 1944 CS (liquid lipid, a mixture of palmitic acid C 16 (4 to 9 %), stearic acid C 18 (6 %), oleic acid C 18:1 (58 – 80 %), linoleic acid C 18:2 (15 – 35 %), linolenic acid C 18:3 (2 %), arachidic acid C 20 (2 %), eicosenoic acid C 20:1 (2 %), with hydroxyl value (mg KOH/g) in the range of 45 - 65) (Table 5B.1). This is primarily due to higher content of hydroxyl value present in each lipid. In addition, Gelucire 50/13 has been used as solid lipid and also reported as stabilizer for the formulation of lipid nanoparticles [25, 26], hence Gelucire 50/13 and Labrafil M 1944 CS were selected as solid and liquid lipid, respectively [27, 28].

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Table 5B.1: Solubility of individual RSV, PBN and their combination in different solid and liquid lipids

Lipids RSV PBN RSV+ PBN 0.1 % w/w 0.1 % w/w 0.2 % w/w (total) Solid lipids Witepsol E 85 − − − Witepsol H 175 − − − Witepsol H 35 − − − Dynasan 118 − − − Dynasan 116 − − − Gelucire 50/13 +* +* +* Precirol ATO 5 − + − Stearic acid − + − Compritol 888 ATO − + − Liquid lipids Labrafac M 1944 CS + + + Captex 200 P − − − Captex 355 − − − Captex 300 − − − Captex 500 P − − − Phosal 53 MCT − − − Capmul MCM − − − Miglyol 840 − − − Miglyol 812 − − − Crodamol GTCC − − −

Data are expressed as mean ± SD, n = 3. “−” Indicates insoluble (presence of drug crystals); + indicates soluble (absence of drug crystals). * Indicates drug soluble more than 0.1 %.

It is a pre-requisite for the development of NLC that the solid and liquid lipids used to form technology are miscible at the specific concentrations to be used. In addition the solid lipid matrix formed using two lipid components should possess an onset melting point higher than 40°C in order to ensure that NLC remain in the 142

Chapter 5B: Photoprotection Aspects of Combination-NLCs solid state at both room and body temperatures [29-31]. Consequently, the second focus point of pre-formulation study was investigate the miscibility, at different concentrations of the solid lipid and liquid lipid determined in the solubility studies to have the best solubilizing potential for RSV as well as PBN. In this context, out of the various ratios studies, the physical appearance of Gelucire 50/13 and Labrafil M 1944 CS at 9:1 ratio revealed no oil expulsion and was remain solid (Fig.5B.4). Moreover, the melting temperature (assessed by DSC) of the said ratio was well above the skin temperature (32°C). On a further addition of liquid lipid, a soft mixture with expulsion of liquid lipid was observed on filter paper (Fig.5B.4).

Figure 5B.4: Miscibility of the selected Gelucire 50/13 and Labrafil M 1944 CS at various ratios: (1) 9:1, (2) 8:2, (3) 7:3, (4) pure Gelucire 50/13, (5) 6:4 and (6) 5:5.

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Figure 5B.5: DSC thermograms: (a) RSV, (b) PBN, (c) Gelucire 50/13, (d) G 50/13 + Labrafil M 1944 CS at 9:1 ratio, (e) RSV + G 50/13 + Labrafil M 1944 CS at 0.5:9:1 ratio, (f) PBN + G 50/13 + Labrafil M 1944 CS at 0.5:9:1 ratio and (g) RSV+PBN + G 50/13 + Labrafil M 1944 CS 1:9:1 ratio.

A DSC study was used to determine the melting behaviour of pure drug, individual lipid and the solid and liquid lipid blend (9:1). The obtained results were used to determine the best binary blend of a solid and liquid lipid for use in the

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Chapter 5B: Photoprotection Aspects of Combination-NLCs manufacture of the combination-NLCs. The DSC thermogram of drug (RSV and PBN) and pure lipid (Gelucire 50/13) (Fig.5B.5a-c) showed a sharp distinct melting endotherm at 271.57 °C, 72.96 °C and 48.61 °C, respectively, indicating the crystalline nature. Next, the DSC thermogram for lipid blend (binary mixture, Fig.5B.5d) and ternary mass mixture (Fig.5B.5e-g) with the same formulation composition as NLCs were reported. The binary mixture showed widening of the melting peak and temperature was lower (46.5 °C) compare to pure lipid, which indicates that liquid lipid Labrafil M 1944 CS was completely dissolved in pure solid lipid Gelucire 50/13. Further, ternary mixture did not show a melting peak for RSV and PBN, which clearly indicates that both the drug was dissolved in binary mixture and is in amorphous state. Moreover, the melting point of the ternary mixture was higher than 40 °C, which is pre-requisite for the development of topical NLCs [32]. To probe any molecular interaction between RSV and PBN upon their mixing, the FT-IR spectra of RSV+PBN mixture was recorded and compared with that of the pure drug form (Fig.5B.6). Pure RSV showed two typical strong absorption bands at 1589.06 cm -1 and 1383.68 cm -1 corresponding to C-C olefinic stretching and C-C stretching, respectively. The band at 966.18 cm -1 demonstrated the trans-form of RSV (Fig.5B.6a) [33]. The location of these characteristic bands, particularly trans-form of RSV did not alter much in RSV+PBN mixture (Fig.5B.6c). From these results we can conclude that no significant interaction between the individual drug and their mixture

5B.3.3 Optimization of the NLCs preparation The advantages and applications of liposomes, lipid nanoparticles (SLNs and NLCs), niosomes, polymeric nanoparticles, chitosan and PEGylated chitosan nanoparticles containing RSV or PBN have been well described in previous research [12, 34-37]. However, in many ways, this is the first study of its kind to show the feasibility of a process to obtain NLCs (stabilized by amino acid-based surfactant) containing the combination of RSV and PBN, two very distinct and largely researched antioxidants. Moreover, Taguchi’s design with mixed-level factors have been assigned and used for the first time to optimize combination-NLCs. .

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Figure 5B.6: FT-IR spectra of (a) RSV pure drug, (b) PBN pure drug and (c) RSV+PBN mixture (stored at 40°C ± 2 °C/75 % ± 5 % RH for 1 h in humidity chamber).

The experiments in current study were designed based on the orthogonal array technique. An orthogonal array is a fractional factorial design with pair wise balancing property. Using orthogonal array design the effects of multiple process variables and formulation variables on the performance characteristic (such as particle size and EE) can be estimated simultaneously while minimizing the number of test runs. As a result experimental cost, time and effort reduce. Using Taguchi’s

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Chapter 5B: Photoprotection Aspects of Combination-NLCs orthogonal array (OA), a total of 162 (6×3×3×3 for the full factorial design) sets of experiments are reduced to only 18 sets of experiments. In current investigation, the choice of surfactant concentration at 6 levels has been made because the amino acid-based surfactant (sodium lauroyl glutamate) is not reported to have any influence on nanoparticle; hence we sought to know whether sodium lauroyl glutamte has any impact on nanotechnological properties and physical stability of prepared combination NLCs, to probe the same we used wide range of concentrations (0, 0.25, 0.50, 1, 2 and 4%) and hence 6 levels. Regarding, the selection of 3 levels for remaining 3 factors was purely based on our experience on the lipid nanoparticles. 1 The control factors and their levels are shown in Table 5B.2, whereas L 18 (6 3 × 3 ) standard OA design as shown in Table 5B. 3 were employed for the present investigation. The numbers in each column indicate the levels of specific factors (A, B, C, and D). This array is most suitable to provide the minimum degrees of freedom (DOFs) as 11 [1 factor at 6 level (6 −1) = 5 and 3 factor at 3 level [3 × (3 -1)] = 6, total 6+5= 11] required for the experimental exploration.

Table 5B.2: Different control factors and levels

Code Control Unit Levels parameters 1 2 3 4 5 6 A Surfactant concentration % 0 0.25 0.50 1 2 4 B Lipid concentration 0.5 1 2 - - - C Sonication amplitude 30 60 90 - - - D Sonication time Sec 30 60 90 - - -

Tables 5B.4 represent the layout of the experimental design, obtained by

assigning the selected factors and their levels to appropriate columns of L 18 OA. This array has 18 rows and 4 columns and each row represents a experimental run condition while each column accommodates a specific formulation or process parameter. Then the experimental results are transformed into a signal-to- noise (S/N) ratio (Table 5B.4). In Taguchi method, the term “signal” represents the desirable effect for the output characteristic and the term “noise” stands for the undesirable

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Chapter 5B: Photoprotection Aspects of Combination-NLCs effect for the output characteristic, the S/N ratios are expressed on a decibel (dB) scale. S/N ratio measures the quality characteristics deviating from the desired values. There are three S/N ratios; smaller the better, nominal the best and larger the better, however the formula used to compute the S/N ratio depends on the objective function. A high value of S/N implies that the signal is much higher than the noise factors. A greater S/ N ratio corresponds to better quality characteristics (optimal level of the formulation or process parameters) [38, 39]. In the current study, particle size is a “Smaller the better S/N” and entrapment efficiency is a “larger the better S/N” type of characteristic, since the goal was to optimize NLCs with a small particle size and higher encapsulation. Quality characteristic likes smaller the better and larger the better is calculated based on the equations reported earlier (section 4.3.3). ANOVA is also performed to investigate which formulation or process parameter significantly affect the particle size and encapsulation efficiency of prepared combination NLCs. This is accomplished by separating the total variability of the S/N ratios, which is measured by the sum of the squared deviations (SS) from the total mean of the S/N ratio (MS), into contributions by each process parameter and the error. Equations for calculating the variance are well reported and can be found in [40]. Finally, an important step in Taguchi’s optimization technique is to verify the results using confirmation experiments, hence the confirmation experiment was conducted in present study using the optimal level of the design parameters and result is compared with that achieved from the regression model.

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1 3 Table 5B.3: The Taguchi’s L 18 (6 × 3 ) standard OA with actual experimental levels and coded levels (in parentheses) of parameters

Run Factor A Factor Factor C Factor D B 1 0 (1) 0.5 (1) 30 (1) 30 (1) 2 0 (1) 1 (2) 60 (2) 60 (2) 3 0 (1) 2 (3) 90 (3) 90 (3) 4 2 (0.25) 0.5 (1) 30 (1) 60 (2) 5 2 (0.25) 1 (2) 60 (2) 90 (3) 6 2 (0.25) 2 (3) 90 (3) 30 (1) 7 3 (0.50) 0.5 (1) 60 (2) 30 (1) 8 3 (0.50) 1 (2) 90 (3) 60 (2) 9 3 (0.50) 2 (3) 30 (1) 90 (3) 10 4 (1) 0.5 (1) 90 (3) 90 (3) 11 4 (1) 1 (2) 30 (1) 30 (1) 12 4 (1) 2 (3) 60 (2) 60 (2) 13 5 (2) 0.5 (1) 60 (2) 90 (3) 14 5 (2) 1 (2) 90 (3) 30 (1) 15 5 (2) 2 (3) 30 (1) 60 (2) 16 6 (4) 0.5 (1) 90 (3) 60 (2) 17 6 (4) 1 (2) 30 (1) 90 (3) 18 6 (4) 2 (3) 60 (2) 30 (1)

5B.3.3.1 Effect on particle size and entrapment efficiency

The experimental results and the S/N ratio values calculated for particle size and entrapment efficiency are listed in Table 5B.4 and the highest S/N ratio value for particle size ( − 41.00 dB; 112.3±5.31 nm) and entrapment efficiency (38.31 and 38.70 dB; 82.32±2.38 and 86.19±2.02 %) was found in experiment no.8. In Taguchi’s design the level of a parameter with the highest S/N ratio value is the optimal level. The average values of S/N ratios of the four control factors at each of the levels are shown in Fig.5B.7a-c, and from which the levels corresponding to the highest S/N ratio values are chosen for each parameter representing the optimum

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Chapter 5B: Photoprotection Aspects of Combination-NLCs condition. Here, the optimum condition is corresponding to the small particle size and high entrapment efficiency. It is clear from Fig.5B.7a-c that the optimum levels are: A 3 (surfactant concentration: 0.5%), B 2 (lipid concentration: 1 %), C 3 (sonication amplitude: 90 %) and D 3 (sonication time: 90 sec). In other words, based on the S/N ratio, the optimal conditions for producing combination-NLCs with small particle size and high entrapment efficiency are the A at level 3, B at level 2, C at level 3 and D at level 3. Final formulation compositions of the investigated NLCs are displayed in Table 5B.5. The increment of surfactant concentration between 1 % and 4 % resulted in significant increase in particle size and decrease in entrapment efficiency this may be due to the fact that when used in higher concentration surfactants tend to bring their solubilization effect. However, at 0.5 % LS 11 showed the sub-100 nm particle size with good encapsulation efficiency ( ≈ 82 to 86 %), indicating its potential emulsifying and stabilizing activity. In terms of particle size, all the experiments showed particle size down to the nanoscale, without micrometric particles (Table 5B.4). The absence of micrometric particles in the formulations containing RSV and/or PBN shows that they were not over concentrated. The ANOVA of the S/N data for particle size and entrapment efficiency are given in Table 5B.6. From the tables it is clear that surfactant concentration alone contributed ( ≈ 84 to 88 %) significantly to the both the mean and the variation in the particle size and entrapment efficiency, which is indicated by the higher values of sum of squares and lower p-values. The response table given in Table 5B.7 for S/N ratios shows the average of each response characteristic for each level of each factor. The tables include ranks based on Delta statistics, which compare the relative magnitude of effects. The Delta statistic is the highest minus the lowest average for each factor. Minitab assigns ranks based on Delta values; rank 1 to the highest Delta value, rank 2 to the second highest, and so on. The ranks and the delta values show that surfactant concentration has the greatest effect on particle size and entrapment efficiency, and is followed by sonication time (on particle size), sonication amplitude (on entrapment efficiency) and finally lipid concentration (on both particle size and entrapment efficiency).

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Table 5B.4: Experimental and S/N ratio results for particle size and % EE of RSV and PBN

Run Particle size (z-ave) Entrapment efficiency RSV PBN Experimental S/N Experimental S/N Experimental S/N result ratio result ratio result ratio (nm ± S.D) (dB) (% ± S.D) (dB) (% ± S.D) (dB) 1 821.4 ± 13.72 − 58.29 32.08 ± 2.84 30.12 38.01 ± 1.45 31.59 2 792.9 ± 17.24 − 57.98 38.94 ± 1.21 31.80 41.34 ± 4.11 32.32 3 753.1 ± 9.83 − 57.53 39.76 ± 4.02 31.98 45.61 ± 4.09 33.18 4 427.5 ± 10.69 − 52.61 47.10 ± 3.93 33.46 51.85 ± 2.17 34.29 5 347.9 ± 7.14 − 50.82 52.85 ± 5.92 34.46 61.76 ± 3.72 35.81 6 489.8 ± 8.43 − 53.80 58.62 ± 2.18 35.36 64.29 ± 5.04 36.16 7 250.7 ± 4.7 − 47.98 61.94 ± 4.87 35.83 64.62 ± 1.88 36.20 8 112.3 ± 5.31 − 41.00 82.32 ± 2.38 38.31 86.19 ± 2.02 38.70 9 202.4 ± 7.05 − 46.12 71.84 ± 3.55 37.12 70.14 ± 0.95 36.91 10 212.9 ± 9.11 − 46.56 68.57 ± 2.93 36.72 65.95 ± 1.64 36.38 11 372.3 ± 6.29 − 51.41 64.91 ± 5.21 36.24 60.07 ± 1.02 35.57 12 298.7 ± 5.98 − 49.50 63.07 ± 6.91 35.99 56.35 ± 7.52 35.01 13 447.0 ± 9.76 − 53.00 57.34 ± 5.37 35.16 54.26 ± 3.79 34.36 14 479.2 ± 4.83 − 53.61 51.81 ± 2.74 34.28 48.91 ± 4. 88 33.78 15 532.6 ± 6.14 − 54.52 42.06 ± 6.28 32.47 44.79 ± 6.20 33.02 16 531.1 ± 8.80 − 54.50 37.12 ± 4.42 31.39 38.14 ± 3.47 31.62 17 549.0 ± 6.98 − 54.79 28.18 ± 3.12 28.99 26.52 ± 5.13 28.47 18 561.4 ± 7.45 − 54.98 21.37 ± 5.03 26.59 18.77 ± 4.83 25.46

Results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate.

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Figure 5B.7: Main effects plot for S/N ratios of (a) particle size (B) % entrapment efficiency of RSV and (c) % entrapment efficiency of PBN.

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Table 5B.5: Final formulation compositions of the investigated NLCs

Ingredients Quantity [(%) w/w] Placebo RSV PBN RSV-PBN NLCs NLCs NLCs NLCs Resveratrol - 0.1 - 0.1 PBN - - 0.1 0.1 Gelucire 50/13 1.8 1.8 1.8 1.8 Labrafil M 1944 CS 0.2 0.2 0.2 0.2 LS 11 0.5 0.5 0.5 0.5 Double distilled water ≈ 97.5 ≈ 97.5 ≈ 97.5 ≈ 97.4

In order to validate the results obtained, three confirmation experiments (combination-NLCs) were conducted for each of the response characteristics (particle size and entrapment efficiency) at optimal levels of the formulation as well as process parameters. The average values of the characteristics were obtained and compared with the predicted values. Minitab software was used to predict the values of S/N ratios and mean. The results are given in Table 5B.8. The predicted results had very close values with the experimental results. The optimal combination-NLCs batch had PDI and zeta potential values 0.248 and −19.02 ± 1.52, respectively. RSV-NLCs and PBN-NLCs were prepared using same optimal levels of combination-NLCs and had particle size, PDI, zeta potential and EE values 98.23 ± 1.95, 91.74 ± 3.27; 0.224, 0.216; −16.11 ± 1.52, −18.84 ± 3.09 and 84.61 ± 3.15, 86.91 ± 1.08, respectively. From the above result, we can conclude that the combination of both antioxidants presented similar characteristics, when compared to the formulations containing each antioxidant individually.

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Table 5B.6: ANOVA for S/N ratio of particle size and % EE of RSV and PBN

Source DF SS MS F Contribution P Value % value Particle size Surfactant 5 306.987 61.397 20.95 87.44 0.001 concentration Lipid concentration 2 3.899 1.949 0.67 1.11 0.548 Sonication amplitude 2 10.028 5.014 1.71 2.86 0.258 Sonication time 2 12.598 6.299 2.15 3.59 0.198 Error 6 17.587 2.931 5.01 Total 17 351.099 100 % EE of RSV Surfactant 5 140.529 28.105 20.44 86.19 0.001 concentration Lipid concentration 2 1.823 0.911 0.66 1.12 0.549 Sonication amplitude 2 8.994 4.496 3.27 5.52 0.110 Sonication time 2 3.449 1.724 1.25 2.12 0.351 Error 6 8.250 1.375 5.06 Total 17 163.045 100 % EE of PBN Surfactant 5 144.268 28.854 20.19 84.06 0.001 concentration Lipid concentration 2 2.569 1.285 0.90 1.50 0.455 Sonication amplitude 2 11.856 5.928 4.15 6.91 0.074 Sonication time 2 4.368 2.184 1.53 2.54 0.291 Error 6 8.573 1.429 4.99 Total 17 171.634 100

DF - degrees of freedom, SS - sum of squares, MS - mean squares (Variance), F- ratio of variance of a source to variance of error, p < 0.10 - determines significance of a factor at 95 % confidence level

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Table 5B.7: Response table for particle size and % EE of RSV and PBN

Level Surfactant Lipid Sonication Sonication time concentration concentration amplitude Particle size 1 − 57.94 − 52.16 − 52.96 − 53.35 2 − 52.42 − 51.61 − 52.38 − 51.69 3 − 45.04 − 52.75 − 51.17 − 51.48 4 − 49.16 5 − 53.71 - 6 − 54.76 Delta 12.90 1.14 1.79 1.87 Rank 1 4 3 2 % EE RSV PBN RSV PBN RSV PBN RSV PBN 1 31.31 32.37 33.78 34.08 33.07 33.31 33.08 33.13 2 34.43 35.42 34.02 34.11 33.31 33.20 33.91 34.17 3 37.09 37.28 33.26 33.30 34.68 34.98 34.08 34.19 4 36.32 35.66 5 33.98 33.72 - 6 29.00 28.52 Delta 8.10 8.76 0.76 0.82 1.60 1.78 1.00 1.06 Rank 1 4 2 3

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Table 5B.8: Results of the prediction and confirmation experiment for particle size and % EE

Levels Particle size % EE RSV PBN Mean S/N Mean S/N Mean S/N (nm ± ratio (nm ± ratio (nm ± ratio S.D) (dB) S.D) (dB) S.D) (dB) Prediction 115.48 − 42.77 81.34 38.80 83.74 39.06 ± 3.17 ± 1.04 ± 4.17 A3B2C3D3 Experiment 131.61 − 47.29 77.15 35.41 79.07 37.06 ± 1.08 ± 3.74 ± 1.84

5B.3.4 TEM investigations Particle size is one of the most important aspects to be controlled in the development of new nanoparticulate system formulations. Thus, a variety of techniques, including PCS and TEM were used to measure particle size and size distribution, respectively. As demonstrated in earlier section (confirmation experiment, Table 5B.8), particle size distribution of combination-NLCs formulation was in the nanoscale range (131.61 ± 1.08 nm). Concerning the morphological characteristics of combination-NLCs, the TEM analysis confirmed ideal spherical lipid nanoparticles with particle size ≈ 128 nm (Fig.5B.8). Note also that the width of the particles is in line with the z-ave measurement by DLS (131.61 ± 1.08 nm). Moreover, no free drugs (crystals) were observed at or near the surface of the nanoparticles during the analysis. The absence of free drug mainly attributed to the higher entrapment efficiency feature of the optimized NLCs system.

5B.3.5 In vitro drug release studies The drug release study was another key parameter to be investigated to determine the potentiality of NLCs as a carrier to control the release of both the lipophilic drugs. As shown in Fig.5B.9, the release profile of the two drugs from NLCs was compared with pure-drug. Both pure-drugs (i.e. RSV-PD and PBN-PD) under investigation exhibited burst release profile ( ≈ 30 % release at 1 h) when compared to RSV and PBN from combination-NLCs ( ≈ 10 % release). The slow-

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Chapter 5B: Photoprotection Aspects of Combination-NLCs sustained release profile of combination-NLCs is due to the observed high solubility (Table 5B.1) and encapsulation efficiency (Table 5B.4) of both the drugs in selected lipid components and in NLC system, respectively. Note also that co-encapsulation of the two drugs in the NLCs demonstrated indistinguishable release profile, that is, ≈ 90% drug release at the end of 24 h. The obtained data demonstrate that NLCs were capable of accommodating two drugs efficiently and showed sustained release profile.

Figure 5B.8: Transmission electron microscopy (TEM) image of combination- NLCs ( A and B samples prepared in duplicate) original magnification 55,000 ×

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Figure 5B.9: Release profiles of resveratrol pure-drug (RSV-PD), PBN pure-drug (PBN-PD) and, resveratrol (RSV) and PBN (PBN) from combination-NLCs. Experiments were carried out at 37 °C ± 1 °C. Values were the mean of three independent experiments in triplicate ± SD.

5B.3.6 In vitro occlusion and ex-vivo drug penetration studies For cosmetic applications it is important that the cosmetic actives are not systemically absorbed, but it is crucial a certain penetration into the skin for the desired effect to take place. In order to confirm the same, we have investigated the occlusion and penetration profile of combination-NLCs. As reported in Fig.5B.10 (at end of 12 h), the NLCs were able to significantly increase the amount of both resveratrol ( ≈ 85 %) and PBN ( ≈ 89 %) in skin when compared to pure-drug (that is, the RSV ≈ 40 % and PBN ≈ 45 %). Moreover, no drug was detected in receptor compartment from combination-NLCs, due to the fact that NLCs are known to make closer contact with corneocytes clusters and furrows present between corneocytes islands and hence favours accumulation of drug for several hours [41, 42]. Regarding, the higher deposition of two drugs from NLCs in skin is mainly attributed to their nearly 3-fold higher occlusion factor (36.09 ± 4.17 for 158

Chapter 5B: Photoprotection Aspects of Combination-NLCs combination-NLCs) when compared to pure-drug (that is, the RSV 11.24 ± 2.75 and PBN 13.71 ± 1.08). Skin occlusion is directly related to lipid film formation of NLCs on the skin which resulted in higher occlusion factor and skin hydration [43]. Skin hydration after applying NLCs leads to a reduction of corneocytes packing and an increase in the size of the corneocytes gaps. This will facilitate the percutanious absorption and drug penetration to the deeper skin layers, hence higher deposition of NLCs. Highest occlusion factor will be reached by using low temperature melting lipids (around 40 °C) and very small particles (sub-100 nm) [44].

Figure 5B.10: A Comparison of the resveratrol pure-drug (RSV-PD), PBN pure- drug (PBN-PD) and, resveratrol (RSV) and PBN (PBN) from combination-NLCs. Bars represent mean ± S.D. from three independent experiments, each performed in triplicate.

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5B.3.7 Stability investigations To investigate the stability performance of prepared combination-NLCs, the formulations were subjected to different stability conditions as per ICH guidelines (25 °C ± 2 °C/ 60 % ± 5 % RH and at 40 °C ± 2 °C/ 75 % ± 5 % RH) for 90 days. Effects of temperature and relative humidity on particle size and % EE were studied at initial period as well as at the end of 7, 15, 30 and 90 days. Table 5B.9 indicates that no significant differences in the particle size and % EE were observed throughout the stability period. Moreover, instabilities such as gelation, phase separation (oil and water seperation) and aggregation are not observed, indicating a good physical stability of the optimized NLCs system. Thus from these results we can conclude that the formulation had satisfactory stability over the entire stability period.

Table 5B.9: Stability study (90 days, at varying temperature) data of combination- NLCs.

Time Particle size PDI ζζζ (mV) ± EE (%) ± S.D. (days) (z-ave) S.D.

(nm) ± S.D. RSV PBN 25 °C ± 2 °C/ 60 % ± 5 % RH Initial 121.81 ± 1.15 0.248 −19.02 ± 1.52 82.32 ± 2.38 86.19 ± 4.65 7 days 134.03 ± 3.41 0.251 −18.36 ± 1.36 79.67 ± 4.96 83.53 ± 2.59 15 days 133.92 ± 1.03 0.249 −20.67 ± 2.27 81.54 ± 4.16 86.37 ± 0.84 30 days 134.40 ± 1.72 0.253 −16.12 ± 1.08 75.03 ± 1.50 84.69 ± 1.73 90 days 136.13 ± 2.23 0.258 −17.05 ± 0.63 75.21 ± 2.76 82.13 ± 3.52 40 °C ± 2 °C/ 75 % ± 5 % RH 7 days 136.10 ± 2.06 0.261 −17.64 ± 1.31 77.28 ± 1.07 83.63 ± 4.95 15 days 135.06 ± 4.19 0.257 −16.20 ± 0.91 79.59 ± 5.42 82.03 ± 2.68 30 days 134.47 ± 4.52 0.249 −17.75 ± 2.74 79.30 ± 4.26 85.17 ± 1.05 90 days 138.01 ± 1.37 0.267 −16.96 ± 1.58 78.43 ± 2.75 81.55 ± 1.92

Results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate.

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5B.3.8 Cell uptake studies In order to confirm the uptake of NLCs by the HaCaT cells in the study, cells were incubated with combination-NLCs and pure-drug (RSV-PD and PBN-PD) for various lengths of time. As shown in Table 5B.10, a quantitative estimation of the RSV and PBN ( g/mL), intracellular and extracellular (medium) from combination- NLCs and pure-drug (RSV-PD and PBN-PD) were time dependent.

Table 5B.10: In vitro Cell uptake studies of RSV-PD, PBN-PD, and RSV and PBN from combination-NLCs in HaCaT cells. Values represent the mean ± SD (n = 3)

Formulation Cellular uptake at time points (h) 2 6 24 Cells Med Cells Med Cells Med RSV-PD 1.418 ± 12.104 ± 5.032 ± 9.104 ± 1.928 ± 6.144 ± 0.310 2.988 1.084 1.988 2.031 0.729 PBN-PD 2.922 ± 10.051 ± 7.162 ± 6.051 ± 3.774 ± 7.205 ± 1.183 3.121 1.274 2.121 1.008 1.096 Comb RSV 13.439 ± 1.705 ± 12.507 ± 2.009 ± 8.271 ± 4.038 ± -NLCs 0.952 a 0.175 a 1.973 a 0.851 a 1.021 a 2.738 a PBN 15.063 ± 0.835 ± 13.724 ± 1.724 ± 7.092 ± 7.014 ± 1.025 b 0.094 b 1.381 b 0.603 b 0.903 b 1.793 b a b p < 0.05 vs. RSV-PD; p < 0.05 vs. PBN-PD; Med = Medium

The obtained data demonstrated that the concentration of both RSV and PBN in cells was highest at 2 h and 6 h from combination-NLCs and pure-drug, respectively. The rapid internalization of NLCs was due to its spherical appearance, negative zeta potential charge and mean particle size well below 200 nm. However, the size of NLCs alone may not be the prepotent factor to influence the uptake. It was reported that the affinity between fatty acid and cell membrane was related with the melting point of fatty acid, and the length and saturation degree of the carbon chain [45, 46]. The cellular uptakes of the lower melting point materials were higher [47]. The results outlined above are consistent with our previous reports that indicated that an incubation time of 2 h was sufficient to achieve maximal cellular uptake of the NLCs.

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5B.3.9 Photoprotective effects of NLCs To investigate the protective effects of combination-NLCs, placebo-NLCs, RSV-NLCs and PBN-NLCs against oxidative stress in UVB-irradiated cells, first we performed the cell viability assay; next we evaluated and recorded the level of intracellular ROS production. Cell viability results showed that (Fig.5B.11) all the NLCs were non-toxic, which means NLCs increases the survival of HaCaT cells (that is, the combination- NLCs 98.01 ± 1.28 %, placebo-NLCs 81.03 ± 4.02 %, RSV-NLCs 98.26 ± 1.16 %, PBN-NLCs 97.03 ± 2.29 %) significantly ( p < 0.01) by suppressing UVB induced apoptosis when compared with positive control cells (52.16 ± 6.29 %). Note also that, inclusion of RSV or PBN into NLCs boosted the proliferation of HaCaT cells, due to their ability to absorb UVB wavelengths and higher antioxidant activity (as evidenced in screening and selection of antioxidant combination section). However, the combination-NLCs did not show any significant protection to HaCaT cells when compared to individual NLCs (i.e. RSV-NLCs and PBN-NLCs), the observed effect could be due to the fact that microplate reader only record the total fluorescence not single cell fluorescence and also its low sensitivity towards the determination of cell viability when compared with flow cytometry. Therefore, to cross-check this effect we evaluated the level of ROS using a flow cytometer. As presented in Fig.5B.12, a significantly increased level of ROS was observed in positive control cells (untreated cells but irradiated by UVB) when compared to all the NLCs formulation tested. The results show that the increase of intracellular ROS formation (increase in MFI value is an indication of ROS formation/generation), induced by UVB treatment, was thwart by all the NLCs. Note that, combination-NLCs resulted in 12.6, 9.2, 2.2, 1.71 and 1.2-fold lower MFI (24.36) when compared to positive control group (307.12), placebo-NLCs + UVB (226.20), negative control group (54.52), RSV-NLCs + UVB (41.76) and PBN- NLCs + UVB (31.32), respectively. The observed effect is due to the synergistic antioxidant activity of two drugs and literature evidence of different chemical and biological activity (mainly photoprotection) exhibited by these two drugs on many cell and animal types [15, 34, 48-51].

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Figure 5B.11: Protective effect of combination-NLCs, placebo-NLCs RSV-NLCs 2 2 and PBN-NLCs against 55 W/cm UVB (200mJ/cm for 1 h)-mediated phototoxicity in HaCaT cells. Results are represented as percentage of cell viability compared to negative control group set on 100 %. Bars represent mean ± S.D. from three independent experiments, each performed in triplicate. *** p < 0.001 vs. – control, ooo p < 0.001 vs. + control (One-Way ANOVA test, Dunnett’s Multiple comparison test); oo p < 0.01 vs. RSV-NLCs, PBN-NLCs and Combination-NLCs (One-Way ANOVA test, Tukey’s Multiple comparison test).

Taken together in order to achieve the higher photoprotection effect, following points needs to considered: (1) intelligent selection of antioxidant combination (i.e. two or more antioxidant with quite different mechanism of action, ability to absorb UVB wavelength and non-toxic concentration), (2) NLCs with sub- 100 nm particle size and negative zeta potential as a drug carrier offer enhance cellular uptake, photostability, occlusion and subsequent penetration of drug into skin and (3) selection of novel biodegradable, biocompatible and multifunctional surfactant to stabilize combination-NLCs, preferably non-PEG for e.g. amino acid-

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Chapter 5B: Photoprotection Aspects of Combination-NLCs based surfactant, are known to be biocompatible, non-toxic to HaCaT cells, and are precursors for the synthesis of GSH (the main protective mechanism against UVB radiation) due to their L-glutamic acid as a base amino acid.

2 Figure 5B.12: Photoprotective effect of different NLCs against 55 W/cm UVB 2 (200 mJ/cm for 1 h)-mediated oxidative stress in HaCaT cells. Each histogram represents the median fluorescence intensity (MFI) values and intracellular ROS levels in HaCaT cells.

5B.4 Conclusion In the current work, our results demonstrate that stable combination-NLCs with sub-100 nm particle size, negative zeta potential and high entrapment efficiency can be prepared when appropriate concentration of Amisoft LS 11 (optimized using Taguchi’s mixed level-design) as a surfactant was used. The co- encapsulation of RSV and PBN into NLCs did not alter the properties of NLCs containing individual drug. Moreover, combination-NLCs showed slow-sustained release profile leading to increased photostability of both RSV and PBN and hence

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Chapter 5B: Photoprotection Aspects of Combination-NLCs exhibited higher photoprotection activity against UVB-mediated phototoxicity and oxidative stress in HaCaT cells. The above findings show that NLCs are attractive drug delivery systems for cosmetics and dermatological applications and that two or more drugs could be encapsulated efficiently via proper selection of lipid phase component, also higher occlusion can be achieved by tuning the physico-chemical properties of the nanoparticles. Since, RSV and PBN are promising antioxidants and can be used to prevent and treat several diseases associated oxidative stress, for instance neurodegenerative diseases like Alzheimer’s disease, further in that direction we initiated the work which include co-encapsulation of RSV and PBN into brain targeted NLCs stabilized by natural protein, albumin.

5B.5 References

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16) Kyadarkunte AY, Patole MS, Pokharkar VB. Cellular interactions and photoprotective effects of idebenone-loaded nanostructured lipid carriers stabilized using PEG-free surfactant. Int J Pharm. 2015; 479:77-87. 17) Kyadarkunte A, Patole M, Pokharkar V. In Vitro cytotoxicity and phototoxicity assessment of acylglutamate surfactants using a human keratinocyte cell line. Cosmetics 2014; 1:159-170. 18) Boots the Chemist Ltd., 1991. The Guide to Practical Measurement of UVA/UVB Ratios. The Boots Co. PLC, Nottingham, England. 19) Diffey, B.L., 1994. A method for broad spectrum classification of sunscreens. Int. J. Cosmet. Sci. 16, 47–52. 20) Stevanato R, Bertelle M, Fabris S. Photoprotective characteristics of natural antioxidant polyphenols. Regul Toxicol Pharmacol. 2014 ;69:71-7. 21) Baxter RA. Anti-aging properties of resveratrol: review and report of a potent new antioxidant skin care formulation. J Cosmet Dermatol. 2008; 7:2-7. 22) http://www.beautymagonline.com/beauty-articles-4/1112-spin-traps-2 (accessed 06.11.2014) 23) http://geronova.com/pbn-overview/ (accessed 06.11.2014) 24) McDaniel, D.H., Neudecker, B.A., DiNardo, J.C., Lewis, J.A., Maibach, H.I., 2005a. Idebenone a new antioxidant - Part I. Relative assessment of oxidative stress protection capacity compared to commonly known antioxidants. J. Cosmet. Dermatol. 4, 10–17. 25) Urbán-Morlán Z, Ganem-Rondero A, Melgoza-Contreras LM, Escobar-Chávez JJ, Nava-Arzaluz MG, Quintanar-Guerrero D. Preparation and characterization of solid lipid nanoparticles containing cyclosporine by the emulsification-diffusion method. Int J Nanomedicine. 2010; 7:611-20. 26) Date AA, Vador N, Jagtap A, Nagarsenker MS. Lipid nanocarriers (GeluPearl) containing amphiphilic lipid Gelucire 50/13 as a novel stabilizer: fabrication, characterization and evaluation for oral drug delivery. Nanotechnology. 2011; 22:275102. 27) http://www.gattefosse.com/en/applications/gelucire-5013.html (accessed 07.11.2014) 28) http://www.gattefosse.com/node.php?articleid=10 (accessed 08.12.2014) 29) R. H. MUller, M. Radtke and S. A. Wissing: Nanostructured lipid matrices for improved microencapsulation of drugs. International Journal of Pharmaceutics 2002; 242(1-2):121-128. 30) M. Radtke, E. B. Souto and R. H. MUller: Nanostructured lipid carriers: A novel generation of solid lipid drug carriers. Pharmaceutical Technology Europe 2005; 17(4):45-50. 31) M. Radtke and R. H. Muller: Nanostructured lipid carriers. New Drugs 2001; 2:48-52. 32) Teeranachaideekul, V., Souto, E.B., Junyaprasert, V.B., Müller, R.H., 2007. Cetyl palmitate- based NLC for topical delivery of Coenzyme Q(10) – development, physicochemical characterization and in vitro release studies. Eur. J. Pharm. Biopharm. 67, 141–148. 33) Vittorio B, Natascia L, Donatella N, Elena P, Chiara S. Host–Guest Interaction Study of Resveratrol With Natural and Modified Cyclodextrins. J. Incl. Phenom. Macro. 2006; 55:270- 287. 34) Caddeo C, Teskac K, Sinico C, Kristl J. Effect of resveratrol incorporated in liposomes on proliferation and UV-B protection of cells. Int J Pharm. 2008; 363:183-91. 35) Gokce EH, Korkmaz E, Dellera E, Sandri G, Bonferoni MC, Ozer O. Resveratrol-loaded solid lipid nanoparticles versus nanostructured lipid carriers: evaluation of antioxidant potential for dermal applications. Int J Nanomedicine. 2012; 7:1841-50. 36) Jung KH, Lee JH, Park JW, Quach CH, Moon SH, Cho YS, Lee KH. Resveratrol loaded polymeric nanoparticles suppress glucose metabolism and tumor growth in vitro and in vivo. Int J Pharm. 2014; 478:251-257. 37) Pinarbasli O, Aktas Y, Dalkara T, Andrieux K, Alonso MJ, Fernandez-Megia E, Novoa-Carballal R, Riguera R, Couvreur P, Capan Y. Preparation and evaluation of alpha-phenyl-n-tert-butyl nitrone (PBN)-encapsulated chitosan and PEGylated chitosan nanoparticles. Pharmazie. 2009; 64:436-9. 38) Kuram E, Tasci E, Altan AI, Medar MM, Yilmaz F, Ozcelik B. Investigating the effects of recycling number and injection parameters on the mechanical properties of glass-fibre reinforced nylon 6 using Taguchi method. Mater Design 2013; 49:139-150. 39) Chaulia PK, Das R. Process parameter optimization for fly ash brick by Taguchi method. Mater Research 2008; 11:159-164. 40) Kamyabi-Gol A, Zebarjad SM, Sajjadi SA. Fabrication of NiO/SiO 2 nanocomposites using sol– gel method and optimization of gelation time using Taguchi robust design method. Colloids Surf A Physicochem Eng Asp. 2009; 336: 69-74. 41) Alvarez-Román R, Naik A, Kalia YN, Guy RH, Fessi H. Skin penetration and distribution of polymeric nanoparticles. J. Controlled Release. 2004; 99:53-62. 42) Cevc G. Lipid vesicles and other colloids as drug carriers on the skin. Adv. Drug Delivery. Rev. 2004; 56:675-711.

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43) Wissing S, Müller R. The influence of the crystallinity of lipid nanoparticles on their occlusive properties. Int J Pharm. 2002; 242:377-9. 44) Wissing S, Lippacher A, Müller R. Investigations on the occlusive properties of solid lipid nanoparticles (SLN). J Cosmet Sci. 2001; 52:313-24. 45) Tranchant T, Besson P, Hoinard C, Delarue J, Antoine JM, Couet C, Gore J. Mechanisms and kinetics of alpha-linolenic acid uptake in Caco-2 clone TC7. Biochim Biophys Acta. 1997; 1345:151–161 46) Hu H, Liu D, Zhao X, Qiao M, Chen D. Preparation, characterization, cellular uptake and evaluation in vivo of solid lipid nanoparticles loaded with cucurbitacin B. Drug Dev Ind Pharm. 2013; 39:770-9. 47) Yuan H, Miao J, Du YZ, You J, Hu FQ, Zeng S. Cellular uptake of solid lipid nanoparticles and cytotoxicity of encapsulated paclitaxel in A549 cancer cells. Int J Pharm. 2008; 348:137-45. 48) Park K, Lee JH. Protective effects of resveratrol on UVB-irradiated HaCaT cells through attenuation of the caspase pathway. Oncol Rep. 2008; 19:413-7. 49) Coradini K, Lima FO, Oliveira CM, Chaves PS, Athayde ML, Carvalho LM, Beck RC. Co- encapsulation of resveratrol and curcumin in lipid-core nanocapsules improves their in vitro antioxidant effects. Eur J Pharm Biopharm. 2014; 88:178-85. 50) Lee JH, Park JW. Protective role of alpha-phenyl-N-t-butylnitrone against ionizing radiation in U937 cells and mice. Cancer Res. 2003; 63:6885-93. 51) Tomita H, Kotake Y, Anderson RE. Mechanism of protection from light-induced retinal degeneration by the synthetic antioxidant phenyl-N-tert-butylnitrone. Invest Ophthalmol Vis Sci. 2005; 46:427-34.

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Chapter 6

Neuroprotection aspects of intranasally administered combination-NLCs

This chapter deals with albumin stabilized NLCs as a drug delivery system to facilitate RSV and PBN into the brain after intranasal administration. The combination-NLCs were prepared by melt-emulsification ultrasound homogenization technique. Combination-NLCs were rapidly taken up by and detected maximum in brain after i.n. administration, with no detection in rat plasma. This study successfully demonstrate the benefits of co-encapsulation of two antioxidants into NLCs which eventually provided improved neuroprotective activity when compared to the single antioxidant loaded NLCs (synergistic action). Hence, current study proves that developed combination-NLCs are of great potential for treating neurologic diseases, AD in particular.

Chapter 6: Neuroprotection Aspects of Combination-NLCs

6.1 Genesis and outline of the work Brain, the most vital organ of the central nervous system (CNS) is extremely susceptible to oxidative stress or damage mediated by reactive oxygen species (ROS) as compared to other tissues due to its limited antioxidant capacity, higher energy requirement, and higher amounts of lipid content [1,2]. Generation of ROS leads to cellular dysfunction as damage to cellular components takes place [3,4]. Antioxidants are natural or synthetic compounds that inhibit the oxidation of other compounds. They act in different ways including removal of oxygen (O 2), scavenging reactive oxygen species or their precursors, inhibiting ROS formation and binding metal ions needed for catalysis of ROS generation. Human body is evolved with endogenous defense mechanisms to help protect against free radical induced cell damage. Glutathione peroxidase, catalase, and superoxide dismutases are such endogenous antioxidant enzymes, which metabolize toxic oxidative Blood–brain barrier (BBB), which is considered to be the most formidable obstacle of the treatment of brain disorder, prevents drugs from penetrating into the central nervous system (CNS). Excitingly, intranasal (i.n.) administration offers an alternative non-invasive delivery of many drugs [5,6]. Lipid nanoparticles are promising strategy for drug delivery to the brain, due to its rapid uptake by the brain, bioacceptability, biodegradability and less toxicity compared to the polymeric nanoparticles. Indeed, feasibility in scale-up and absence of burst effect make them promising carriers for drug delivery [7,8]. NLCs is an improved generation of lipid nanoparticles and is preferred over solid lipid nanoparticles due to its potential limitations like low loading capacity and drug expulsion during storage [9]. Albumin, the predominant transport protein in the blood, is emerging as a versatile transport molecule for targeted drug delivery [10], and albumin has recently gained attention in the field of pharmaceutical development for its ability to prolong the half-life and stability of bioactive compounds [11]. Whereas albumin binding proteins, such as gp60 (albondin), facilitate transcytosis of albumin in peripheral capillaries, there is low expression in brain endothelial cells [12], and albumin has been shown not to cross the BBB in vivo or in vitro [13], however, a study has suggested that albumin may be taken up by brain after i.n. administration [14]. Here, we assessed albumin stabilized NLCs as a drug delivery system to facilitate RSV and PBN into the brain after i.n. administration. Optimized NLCs was 168

Chapter 6: Neuroprotection Aspects of Combination-NLCs administered through IN route to deliver drug in the brain to improve its clinical utility and therapeutic efficacy. The neuroprotective effects of the formulations on memory impairments were evaluated in AD model rats induced with A β25–35 using Y-maze test and brain acetyl cholinesterase (AChE) activity in vivo.

6.2 Experimental 6.2.1 Pre-formulation studies

6.2.1.1 Lipid phase screening The solubility of RSV and PBN in various solid lipid and liquid lipids was determined as previously described (section 4.2.1). The amount of RSV and PBN dissolved in liquid lipid was quantified by reverse-phase high performance liquid chromatography (RP-HPLC) method.

6.2.1.2 Surfactant selection Selection of ideal surfactant for the preparation of different NLCs was evaluated by method reported previously [15]. Briefly, 100 mg of binary mixture (i.e. Gelucire 44/14 and Labrafil M 1944 CS) was dissolved in 3 mL of dichloromethane (DCM) and added to 10 mL of 1 % surfactant solutions under magnetic stirring. DCM was removed by rotary evaporation at 40 °C and the resultant suspensions were diluted with 10-fold double distilled water (DDW). Percentage transmittance of the resultant samples was observed using UV spectrophotometer at 510 nm.

6.2.1.3 DSC studies The DSC measurements of RSV, PBN, Gelucire 44/14 (G 44/14), binary mixture of G 44/14 and Labrafil M 1944 CS (LMC) at 9:1 ratio, ternary mass mixture containing drug and binary mixture (i.e. RSV, G 44/14 and LMC at 0.5:9:1; PBN, G 44/14 and LMC at 0.5:9:1; RSV+PBN, G 44/14 and LMC at 1:9:1) were carried out as previously described (section 5B.2.2.2).

6.2.1.4 HPLC analysis of RSV and PBN 6.2.1.4.1 Instrumentation

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The amount of RSV and/or PBN in various samples was quantified by RP- HPLC method reported previously, with minor modifications [16]. The HPLC system equipped with an Intelligent LC Pump (model Jasco PU-2080, Japan) connected to UV-visible detector (Jasco UV-2075, Japan) with an auto sampler programmed at 20 L capacity per injection was used for the analysis. The mobile phase consisted of a mixture of methanol/DDW/glacial acetic acid (50/50/0.05, v/v/v, ≈ pH 4.5) and was delivered at a flow rate of 1mL/min. The separation was carried out at 25°C, using a guard column (RP-18, 33mm Kromasil ®) and a reversed phase C18 column (a Thermo Scientific ODS Hypersil, 250 × 4.6 mm, 5 m particle size). Detections were carried out at respective wavelength (i.e. RSV at 306 nm, PBN at 293 nm and simultaneous estimation of RSV PBN at 296 nm) and data was integrated using JascoBorwin version 1.5, LC II/ADC system.

6.2.1.4.2 Standard solutions and spiked plasma samples The stock solutions of RSV, PBN and carbamazepine (CBZ, internal standard) were prepared separately by dissolving 10 mg of drug in 10 mL methanol to yield the final concentration 1mg/mL (1000 g/mL). This stock solution was stable for at least three months when stored in refrigerator at 4 ºC. Working standard solutions were prepared by diluting the stock solution to desired concentration with mobile phase. Plasma standards were prepared by spiking blank rat plasma (100 L) with RSV, PBN, RSV+PBN and CBZ standard solutions, respectively. For calibration in plasma, standards from 0.2 – 10 g/mL were prepared by spiking blank rat plasma with 50 L of working stock solutions of RSV, PBN and RSV+PBN, respectively.

6.2.1.4.3 Preparation of biological matrices samples Liquid-liquid extraction method was used for the preparation of plasma and brain samples. To 100 L of blank plasma, 50 L of internal standard solution (10 g/mL) and 300 L of methanol (as an extraction solvent) was added. The resulting sample was vortexed for 10 min followed by centrifugation at 7000 rpm for 20 min at 4 ºC. The supernatant was collected and a volume of 20 L was injected into the HPLC system. The brain tissue samples (200 mg was mixed with 50 L internal standard solution, 2000 L methanol) were prepared by homogenizing tissue

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Chapter 6: Neuroprotection Aspects of Combination-NLCs samples followed by centrifugation 7000 rpm for 20 min at 4 ºC. The supernatant was collected, and a volume of 20 L was injected into the HPLC system.

6.2.1.4.4 Linearity and range To evaluate linearity, calibration curves were prepared and analysed in triplicate. Linearity was determined by calculation of a regression linear line from the peak area ratios of analyte/internal standard vs concentrations plot for ten standard solutions (0.2, 0.4, 0.6 0.8, 1.0, 2.0, 4.0, 6.0, 8.0 and 10.0 g /mL) in mobile phase, plasma and brain samples. The linearity of the calibration curve was tested and evaluated using linear regression model of internal standard calibration curve.

6.2.2 Preparation of NLCs RSV-NLCs, PBN-NLCs and RSV-PBN loaded NLCs (combination-NLCs) and placebo-NLCs were prepared by a melt-emulsification-ultrasonication method as previously described (section 4.2.5). Briefly, the solid lipid (G 44/14, melting point 43 °C) was melted 10 °C above their melting point; to the above solution liquid lipid (Labrafac M 1944 CS) was added and mixed together using cyclo-mixer (2 min). To the obtained mixture, a hot (50 - 55°C) aqueous albumin solution (as surfactant) was quickly added and finally subjected to ultrasonication procedure (amplitude 90 %, cycle 1, 1 min). The resulting NLCs were allowed to cool down to room temperature and initial particle size, PDI, zeta potential, entrapment efficiency and drug content were recorded.

6.2.3 Characterization of NLCs The prepared different NLCs were characterized for their z-ave, PDI, zeta potential, entrapment efficiency and drug content as previously described (section 4.2.6.1 and 4.2.6.2). RSV, PBN and RSV+PBN were quantified by RP-HPLC at 306 nm, 293 nm and 296 nm, respectively. in addition to the above-mentioned characterization, 90 days accelerated stability study of combination-NLCs was carried out at different temperature conditions (25 °C ± 2 °C / 60 % ± 5 % RH, 4 °C, and 40 °C ± 2 °C/ 75 % ± 5 % RH) (section 4.2.6.7).

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6.2.3.1 Environmental scanning electron microscopy The shape and surface characteristics of combination-NLCs were analyzed by environmental scanning electron microscopy (ESEM) method. Samples were analyzed in the form of aqueous dispersion using Quanta 200 ESEM (FEI, USA) (magnification: 20,000 ×; accelerating voltage: 20.0 kV). Analysis was performed at 25 ± 2 °C [17].

6.2.3.2 pH The pH was checked in order to assess the feasibility of NLCs dispersions for intranasal administration. The pH measurements were performed using a pH meter at room temperature. The electrode was introduced in the NLCs samples and the pH was recorded directly. pH was measured on the day of NLCs production and after 90 days of their storage at 4 °C.

6.2.3.3 In vitro release studies In vitro release studies of different samples (i.e. combination-NLCs and a dispersion containing combination of pure drugs of RSV and PBN) were carried out by the bulk-equilibrium reverse dialysis technique (using USP Type II dissolution test apparatus) as described elsewhere, with minor modification [18]. Phosphate buffer solution pH 6.0 (PBS, 500 mL) stirred at 50 rpm at a temperature of 37 ± 0.5 ºC was used as the dissolution medium. Prior to analysis, dialysis bags (molecular weight cut off 12–14 kDa) were equilibrated in release medium for 12 h. Aliquots containing 1 mg equivalent dose of different samples were filled in dialysis bags, tied at both the ends and were directly placed into the release medium. The sampling port was fitted with 0.45 m filter. Samples (3 mL) were collected at the predetermined time points (1, 2, 4, 6, 8 and 12 h) with the help of syringe and replaced with a fresh release medium. The amount of RSV and/or PBN present in the aliquots was quantified by HPLC.

6.2.4 In vivo pharmacokinetic studies 6.2.4.1 Animals In vivo experiments were performed in male Wistar rats (200 - 250 g) purchased from National Toxicology Centre, Pune, India. Animal handling was performed according to Good Laboratory Practices. Animals were housed together 172

Chapter 6: Neuroprotection Aspects of Combination-NLCs under standard conditions of temperature (24 ± 1 °C), relative humidity (55 ± 10 %) and 12 h light/dark cycles throughout the experiment. Animals had free access to commercially available standard pellet diet (Pranav Agro Industries, Sangli, Maharashtra, India) and filtered water, ad libitum. Animals were acclimatized for one week prior to the initiation of treatment. During this acclimatization period, the health status of the rats was monitored daily. All animals were without food overnight prior to the experiments.

6.2.4.2 Pharmacokinetics - single dose intranasal (i.n.) administration One hundred and fourty-seven male wistar rats (200 ± 10 g) were divided into seven groups (n = 21), administered i.n. with following formulations: 1) RSV in 1 % albumin solution; 2) RSV-NLCs; 3) PBN in 1 % albumin solution; 4) PBN- NLCs; 5) combination of RSV and PBN in 1 % albumin solution; 6) combination- NLCs and 7) 1 % albumin solution (control). I.n. administration was carried out after light anaesthesia with diethyl ether. Briefly, the formulations (60 L one shot in each nostril; i.e. ≈ 100 g/rat) were administered via a polyethylene (PE) 10 tube attached to a micropipette inserted 0.5 cm into the nostril of the rats. The procedure was performed gently, allowing the rats to inhale all the formulation. The rats (n = 3) were sacrificed humanely at different time intervals, viz , 0.083, 0.25, 0.5, 1, 2, 4 and 8 h and blood was collected by cardiac puncture. Blood samples were mixed thoroughly with di-sodium EDTA in order to prevent blood clotting. Samples were centrifuged at 7000 rpm for 20 min at 4 °C. Separated plasma samples were transferred into pre labelled tubes and internal standard solution was added followed by storage in refrigerator at − 80 °C until further analysis. Brains were harvested and after weighing, the brain samples were immediately frozen in liquid nitrogen and then stored at − 80 °C until further analysis. Both plasma and brains samples (biological matrices), were prepared as described previously (section 6.2.1.4.3). The protocol of this study was reviewed and approved by Institutional Animal Ethical Committee (IAEC) constituted as per guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India (CPCSEA/50/12).

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6.2.5 Neuroprotection effects of the NLC formulations on Alzheimer’s disease model induced by A β 25–35

6.2.5.1 In vivo model of Alzheimer’s disease Wayne A. Dornan et al. [19] have demonstrated that bilateral injections of

Aβ 25–35 into the hippocampus could produce a dramatic disruption in the acquisition of a spatial learning in the rat, and this validated animal model could be useful to develop and evaluate potential drugs for AD. In brief, A β 25–35 was dissolved in saline at the concentration of 5 mg/mL and then incubated at 37 °C for 7 days to form neurotoxic fibrils. Male SD rats were anaesthetized by i.p. of chloral hydrate and fixed in a stereotaxic apparatus. Two microliters of A β 25–35 was bilaterally injected slowly into hippocampus (± 2.0 mm lateral to be midland, 3.5 mm posterior to the bregma and 2.7 mm ventral to the skull surface) over 5 min. The needle was kept in the place for another 5 min before it was slowly withdrawn. Negative control group animals were i.n. administered with the same volume of saline. The animals were allowed to recover for one week in cages.

6.2.5.2 Drug treatment and experimental design Rats were divided into six groups (Table 6.1): negative control, positive control, standard and three test groups. After one-week recovery, the preparations were given to the rats according to the table, and were tested for behavioral parameter (Y-maze test).

Table 6.1: Groups and treatments Groups Treatments - Control i.n. 1 % albumin solution, 60 L/nostril/d for 14 days + Control (AD control) i.n. 1 % albumin solution, 60 L/nostril/d for 14 days Standard p.o. Donepezil*, 400 g/rat/d for 14 days RSV-NLCs i.n. RSV-NLCs, 60 L/nostril/d for 14 days PBN-NLCs i.n. PBN-NLCs, 60 L/nostril/d for 14 days Combination-NLCs i.n. Combination-NLCs, 60 L/nostril/d for 14 days

* Indicates marketed Donepezil (Aricep 5mg, Eisai Pharmaceuticals, India, Pvt. Ltd). 174

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6.2.5.3 Behavioral parameter. 6.2.5.3.1 Y maze-test The Y-maze analysis has been shown to be a reliable, non-invasive test to determine cognitive changes in wistar rat through the measurement of the spontaneous alteration behaviour in the Y-maze task [20, 21]. The Y-maze used in the present study consisted of three arms (40 cm long, 25 cm high and 10 cm wide, labeled A, B and C) constructed of plywood and painted black with an equilateral triangular central area. This maze was used in a testing room with constant illumination. Each rat was placed at the end of one arm and allowed to move freely through the apparatus for 8 min. The time limit in Y-maze test was 8 min, and every session was stopped after 8 min. An arm entry was counted when the hind paws of the rat were completely within the arm. Spontaneous alteration behaviour was defined as three consecutive entries in three different arms (i.e. A, B, C or B, C, A). The percentage alteration score was calculated using the following formula: Total alteration number / (Total number of entries − 2) × 100 Furthermore, total number of arm entries was used as a measure of general activity in the animals. The maze was wiped clean with 70 % ethanol between each animal to minimize odour cues.

6.2.5.4 Biochemical parameters 6.2.5.4.1 Reagents for tissue processing 1. Phosphate buffered saline (PBS) (pH 7.4): 1.38 g of disodium ethylenediamine tetraacetic acid, 0.19 g of potassium dihydrogen phosphate and 8 g of sodium chloride was dissolved in 900 mL of distilled water and adjusted pH using dilute hydrochloric acid. The volume was adjusted to 1000 mL using distilled water.

2. Tris hydrochloric buffer (10 mM, pH 7.4): Tris HCl (1.21 g) was dissolved in 900 mL of distilled water and the pH was adjusted to 7.4 with 1 M hydrochloric acid. The resulting solution was diluted to 1000 mL with distilled water.

6.2.5.4.2 Procedure for preparation of brain tissue homogenate Biochemical estimation such as brain AChE was performed at the end of behavioral test. Animals were sacrificed by cervical dislocation and the whole brain was quickly excised and transferred into ice-cold PBS pH 7.4. It was blotted free of 175

Chapter 6: Neuroprotection Aspects of Combination-NLCs blood and tissue fluids, weighed on a single pan electronic balance. The brain was cross-chopped with surgical scalpel into fine slices, suspended in chilled 0.25 M sucrose solution and quickly blotted on a filter paper. The tissues were then minced and homogenized in chilled tris hydrochloride buffer (10 mM, pH 7.4). After centrifugation at 7000 rpm for 25 min at 4 °C, the clear supernatant was obtained and was used for subsequent biochemical estimation.

6.2.5.4.3 Estimation of brain acetyl cholinesterase activity in vivo AChE activity is a common marker of extended loss of the cholinergic system in the brain and the quantitative measurement of AChE activity in the brain was performed according to the method of Ellman’s et al. with slight modification [22]. Samples derived from brain homogenate were added to a cuvette containing 2.6 mL phosphate buffer (0.1M, pH 8.0) and 100 L of dithiobisnitrobenzoic acid (DTNB). The contents of the cuvette were mixed thoroughly and absorbance was measured at 412 nm using UV-spectrophotometer. It was recorded as the basal reading when absorbance reached a stable value and then 20 L acetylthiocholineiodide (ATCI) was added as substrate. Change in absorbance was recorded for a period of 10 min at 30 sec intervals. One unit of AChE activity was defined as the number of micromoles of ATCI hydrolyzed per min per mg of protein. The specific activity of AChE is expressed in mol/min/mg protein.

6.2.6 Statistical analysis All data were expressed as mean ± S.E.M. Statistical differences were determined using ANOVA, followed by Tukey’s Multiple Comparison Test performed using GraphPad Prism 5.0 software (Graph pad software, San Diego, California, USA.). A value of p < 0.05 was considered statistically significant.

6.3 Results and discussion

6.3.1 Pre-formulation studies Lipid phase component (i.e. solid lipid and liquid lipid) of NLCs were selected based on their ability to dissolve the RSV and PBN either alone or in combination. A good affinity of the lipid phase component may warrant for high entrapment efficiency, a key qualification of a drug carrier system. Higher solubility

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(> 0.1 %) of RSV and PBN was observed in Gelucire 44/14 (mixture of mono-, di-, and triglycerides and mono- and di-fatty acid esters of PEG 1500 with lauric acid

C12 as a main fatty acid i.e. 30 to 50 %) and Labrafil M 1944 CS (mixture of mono-, di-, and triglycerides and mono- and di-fatty acid esters of PEG 300, with oleic acid

C18:1 as a main fatty acid i.e. 58 to 80 %) hence was used as a solid lipid and liquid lipid (Table 6.2), respectively. Generally, the presence of mono- and diglycerides in the lipid used as matrix material promotes drug solubilization [23]. Moreover, lipids that are mixture of mono-, di-, and triglycerides and lipids containing fatty acids of different chain lengths form less perfect crystals with many imperfections, offering space to accommodate the drugs [23].

Table 6.2: Solubility studies of RSV and PBN in Gelucire 44/14, Labrafil M 1944 CS, and at their 9:1 combination.

Lipids RSV PBN RSV + PBN 0.1 % 0.1 % 0.2 % w/w w/w w/w (total) Solid lipid Gelucire 44/14 + + +* Liquid lipid Labrafil M 1944 CS − + − Solid : liquid lipid (9:1) Gelucire 44/14 + Labrafil M 1944 CS + + +*

Data are expressed as mean ±SD, n = 3. “ −” Indicates insoluble (presence of drug crystals); + indicates soluble (absence of drug crystals) and NP indicates not performed. * Indicates drug soluble more than 0.1%.

Based on our experience, Gelucire 44/14 and Labrafil M 1944 CS at 9:1 was selected as lipid phase component. As Gelucire 44/14 was observed to have better

RSV and PBN solubilization capacity ( > 0.1 %), a higher ratio of solid lipid could be useful for the higher drug entrapment. The physical appearance of the said lipid ratio revealed very little drop of liquid lipid on Whatman filter paper (Fig.6.1j), indicating good miscibility between both the lipids. Moreover, microscopic 177

Chapter 6: Neuroprotection Aspects of Combination-NLCs examination and DSC studies of this mixture with RSV and PBN showed absence of drug crystals (Fig.6.1k) and melting temperature of lipids was well above (38.77 °C) the body temperature (Fig. 6.2g), respectively, indicating that both drugs were dissolved in lipids and after NLCs production nanoparticles will remain in the solid state at both room temperature as well as body temperature. The solid state is an important feature of nanoparticle, hence provide higher physical stability, restrict drug mobility in the lipid core and sustain the release of a drug [24]. Since, the stabilizers strongly influence the particle size and surface properties of the NLCs, the second focus point of pre-formulation study was selection of appropriate surfactant and hence emulsification capability of the some of the surfactant solution was considered and tested. A higher percentage transmittance corresponds to the smaller sized particles and hence better emulsification [25]. As per the observed results in Table 6.3, albumin, Tween 80 and Pluronic F68 produced an emulsion with the highest percentage transmittance for the system when compared with Pluronic F108, Pluronic F127 and Tween 60. In such condition albumin with aforementioned multifunctional properties and due to its ability to form cohesive films around droplets providing barriers that bestow long steric hinderance [26], without significantly decreasing surface tension, are better emulsifiers in terms of imparting stability to emulsions than smaller nonprotein amphiphile surfactants (e.g., Tweens) which function primarily by lowering surface tension. Moreover, albumin form cohesive film possessing mechanical strength which resists rupture when pressure is applied [27]. Therefore, albumin which is reported to be very suitable for forming and stabilizing o/w emulsions [28], was selected for the preparation and stabilization of NLCs.

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Figure 6.1: Steps involved in lipid phase screening: (a) solid lipid (Gelucire 44/14) at room temperature, (b) liquid lipid (Labrafil M 1944 CS) at room temperature, (c) binary lipid mixture i.e. a+b at 9:1 ratio (when melted at 50 °C), (d) RSV pure drug, (e) PBN pure drug, (f) when RSV and PBN were added in c, (g) f mixture without cyclomixing, (h) when g cyclomixed for 2 min., (i) when h solidified at room temperature, (j) when i placed on Whatman filter paper and placed at 37°C in humidity chamber, circle indicates very little drop of liquid lipid on filter paper, (k) Polarized light microscopic images depicting absence of drug crystals in binary lipid mixture.

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Table 6.3: Clarity of emulsions produced by different surfactants

Surfactant % Transmittance ± SD Albumin 86.18 ± 3.86 Tween 80 85.97 ± 7.02 Tween 60 81.17 ± 6.52 Pluronic F68 85.03 ± 4.27 Pluronic F108 79.37 ± 2.39 Pluronic F127 81.18 ± 3.06

Figure 6.2: DSC thermograms: (a) RSV, (b) PBN, (c) Gelucire 44/14 (G40/14), (d) G 44/14 +Labrafil M 1944 CS at 9:1 ratio, (e) RSV + G 44/14 + Labrafil M 1944 CS at 0.5:9:1 ratio, (f) PBN + G 44/14 + Labrafil M 1944 CS at 0.5:9:1 ratio and (g) RSV+PBN + G 44/14 + Labrafil M 1944 CS 1:9:1 ratio.

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6.3.2 RP-HPLC chromatographic method The reproducibility of reported RP-HPLC method was carried out with a view to simultaneously analyze RSV and PBN present in different NLCs formulation. The mobile phase methanol/DDW/glacial acetic acid (50/50/0.05, v/v/v, ≈ pH 4.5) at a flow rate rate of 1 mL/min provided acceptable retention times (5.33 min and 6.37 min for RSV and PBN, respectively) with good resolution for both drugs in pure form (Fig. 6.3). The RP-HPLC method was then explored to achieve simultaneous determination of RSV and PBN from a rat brain tissue samples in a single chromatographic run within a very short period of time. Liquid- liquid extraction method proved to be helpful in achieving better extraction of RSV and PBN (Fig.6.7). To evaluate linearity, calibration curves were constructed by plotting the peak area ratios of RSV/CBZ and PBN/CBZ against the concentration of RSV and PBN (Table 6.4). In a binary gradient mobile phase with 0.05 % glacial acetic acid in water and methanol, the assay demonstrated good linearity in the tested range for both RSV and PBN, in all the matrices (R 2 > 0.997). Regression analysis of calibration curves for individual RSV and PBN, and simultaneous estimation of RSV and PBN in plasma and brain tissue over the specified concentration rate are depicted in Table 6.4. Calibration curves for RSV and PBN in biological matrices (plasma and brain) were also linear for the range tested (0.2 – 10 g/mL) with a regression coefficient (R 2) higher than 0.996 (Table 6.4). Hence, this method was used for the quantification of RSV and PBN in rat brain and plasma samples.

Figure 6.3: HPLC chromatogram of simultaneous determination of RSV and PBN in mobile phase

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Figure 6.4: HPLC chromatogram of extracted blank brain

Figure 6.5: HPLC chromatogram of RSV in brain tissue samples

Figure 6.6: HPLC chromatogram of PBN in brain tissue samples

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Figure 6.7: HPLC chromatogram of brain spiked with RSV, PBN and carbamazepine (CBZ).

Table 6.4: Regression analysis of calibration curves for individual RSV and PBN, and simultaneous estimation of RSV and PBN in plasma and brain tissue over the specified concentration rate.

Matrix Range Slope Intercept Correlation coefficient (r) RSV at 306 nm Mobile phase 0.2 – 10 29746 8273 0.9993 Plasma (g/mL) 23469 2986.7 0.9966 Brain 0.0552 0.0219 0.9960 PBN at 293 nm Mobile phase 0.2 – 10 22923 6812 0.9993 Plasma (g/mL) 19771 981.14 0.9983 Brain 0.0459 0.0046 0.9999 RSV at 296 nm Mobile phase 0.2 – 10 29173 1068 0.9979 Plasma (g/mL) 27501 − 728.32 0.9975 Brain 0.0546 0.0164 0.9969 PBN at 296 nm Mobile phase 0.2 – 10 21519 573.26 0.9982 Plasma 19447 − 3016.1 0.9967 Brain 0.0470 − 0.0033 0.9988

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6.3.3 Preparation and characterization of NLCs In the current study, first co-encapsulated RSV and PBN solid lipid nanoparticles (combination-SLNs) were prepared (Table 6.5, formulation 1 to 4) by varying albumin concentrations between 0.5 to 5 % w/w. After SLNs production, the macroscopic appearance of all dispersions was milky-whitish of low viscosity with few drug crystals. Upon physicochemical characterization (Table 6.6) combination- SLNs revealed particle size in the range of 386 to 672 nm with % EE of 51 to 62 and 53 to 64 for RSV and PBN, respectively. According to the literature, the small size nanoparticles ( < 200 nm) may partition better through a biological membrane (in present case its nasal mucosa), which is ideal for direct nose to brain targeting [29]. Moreover, low % EE of RSV and PBN in SLNs could affect the therapeutic outcome of the said antioxidants. Hence, SLNs were not further evaluated and were eliminated from the current study.

Table 6.5: Composition of lipid nanoparticles (Batch size 10 mL)

Formulation no. Drug % (w/w) Lipid % (w/w) Surfactant % (w/w) RSV PBN G 44/14 LMC Albumin 1 (Combo-SLNs 1) 0.1 0.1 2.0 - 0.5 2 (Combo-SLNs 2) 0.1 0.1 2.0 - 1.0 3 (Combo-SLNs 3) 0.1 0.1 2.0 - 2.5 4 (Combo-SLNs 4) 0.1 0.1 2.0 - 5.0 5 (Combo-NLCs 1) 0.1 0.1 1.8 0.2 0.5 6 (Combo-NLCs 2) 0.1 0.1 1.8 0.2 1.0 7 (Combo-NLCs 3) 0.1 0.1 1.8 0.2 2.5 8 (Combo-NLCs 4) 0.1 0.1 1.8 0.2 5.0 9 (RSV-NLCs) 0.1 - 1.8 0.2 1.0 10 (PBN-NLCs) - 0.1 1.8 0.2 1.0 11 (Placebo-NLCs) - - 1.8 0.2 1.0

Formulations no. 1 – 11, pH in between 4.7– 4.9

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Figure 6.8: Images depicting different NLCs on the day of production

Table 6.6: Physicochemical characteristics of prepared NLCs. particle si ze (z-ave, nm), polydispersity index (PDI), zeta potential (ZP, ζ) and entrapment efficiency (% EE) assessed on the day of NLCs production.

F. No Particle size PDI ζ (mV) ± S.D. Entrapment efficiency (z-ave) (% ± S.D) (nm) ± SD RSV PBN 1 492.4 ± 3.7 0.582 − 15.72 ± 3.12 59.72 ± 4.1 58.27 ± 9.2 2 385.9 ± 5.1 0.486 − 17.14 ± 2.61 61.81 ± 2.4 63.48 ± 6.3 3 515.3 ± 6.5 0.610 − 16.83 ± 2.89 58.15 ± 1.8 57.03 ± 5.2 4 671.8 ± 8.3 0.628 − 15.94 ± 3.74 51.22 ± 6.4 53.10 ± 4.8 5 217.2 ± 3.7 0.347 − 15.08 ± 2.97 74.17 ± 6.9 75.24 ± 5.2 6 91.6 ± 5.2 0.228 − 17.20 ± 1.38 82.46 ± 4.7 87.07 ± 2.5 7 279.6 ± 6.1 0.375 − 14.60 ± 1.94 69.65 ± 3.1 73.83 ± 4.0 8 403.1 ± 6.3 0.515 − 14.55 ± 2.01 61.39 ± 3.9 63.40 ± 1.4 9 85.5 ± 7.7 0.219 − 18.01 ± 1.47 79.52 ± 2.7 - 10 82.0 ± 6.9 0.206 − 17.12 ± 1.06 - 83.81 ± 4.3 11 69.7 ± 5.4 0.209 − 17.58 ± 1.50 - -

ζ recorded when pH of the formulation was adjusted to pH 6.5

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To improve the % EE of RSV and PBN into lipid nanoparticles, small amount of liquid lipid (Labrafil M 1944 CS) was incorporated in the lipid phase resulting in NLCs. The albumin concentration was kept between 0.5 to 5 % w/w (Table 6.5, formulation 5 to 11). The macroscopic appearance of combination- NLCs, RSV-NLCs, and PBN-NLCs are depicted in Fig. 6.8. As can be seen in Fig. 6.8, the NLCs showed a characteristic clear transparent bluish tint and no drug crystals/ particles were observed, which is a good visual indicator of a monodispersed, < 200 nm size particles. The particle size measurement of NLCs by

DLS confirmed the < 100 nm size particles (Table 6.6). Note that combination- NLCs (formulation 6), RSV-NLCs (formulation 9) and PBN-NLCs (formulation 10) did not have significant impact on particle size when compared with placebo-NLCs (formulation 10). This indicates that the 1 % w/w albumin concentration was capable of stabilizing the particle of NLCs, regardless of number of antioxidant encapsulated into NLCs. Mixing of Labrafil M 1944 CS in Gelucire 44/14 resulted in two important changes in combination-NLCs, first, it reduced the particle size (Table 6.6) significantly from 385.9 ± 5.1 nm to 91.6 ± 5.2 nm. This could be due to the fact that addition of liquid lipid reduced the viscosity inside lipid nanoparticles, and subsequently, reduced the surface tension to form smaller particles [30]. Second, it increased the % EE (Table 6.6) of RSV and PBN from 61.81 ± 2.4 and 63.48 ± 6.3 to 82.46 ± 4.7 and 87.07 ± 2.5, respectively. This occurred when more complex liquid lipid is used in preparation of lipid nanoparticles, which form less perfect crystals with many imperfectations offering space to accommodate the drugs, hence higher EE in case of NLCs [31]. Regarding surface charge, all NLCs dispersions showed negative zeta potential (between − 14.60 ± 1.94 to − 18.01 ± 1.47) which is primarily attributed to albumin own negative charge and indicated successful coating of albumin over particles. Observed PDI values were well below 0.25 (Table 6.6). The ESEM image (Fig. 6.9) of combination-NLCs revealed uniform particle size in nanometer range with spherical shape. Generally, a lipid mixture of mono-, di-, and triglycerides favors the formation of spherical lipid nanoparticles [32]. Note also that, no drug crystals were observed during microscopy investigation, mainly attributed to higher entrapment efficiency of NLCs.

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The release of RSV and PBN from NLCs was investigated for 12 h (Fig. 6.10) and compared with their pure drug solution. The PBS pH 6.0 as a release medium was used due to its relevance to the nasal pH. Combination-NLCs under investigation exhibited slow, sustained and same release profile ( ≈ 58 % RSV and 53 % PBN at 4 h) when compared to pure drug solution ( ≈ 80 % RSV and ≈ 90 % PBN at 4 h), which demonstrate that NLCs are capable of accommodating two drugs efficiently. Slow and sustained release of drug from NLCs indicates homogenous entrapment of the drug throughout the system [33], this means that selection of lipid phase component with proper solid to liquid lipid ratio and surfactant concentration governs the drug release from the lipid nanoparticles.

Figure 6.9: ESEM image of combination-NLCs, scale bar represent 200 nm.

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Figure 6.10: Release profiles of PBN-PD ( ), RSV-PD ( ), PBN ( ) and RSV ( ) from combination-NLCs in PBS pH 6.

The stability performances of combination-NLCs were carried at different stability conditions as per ICH guidelines. Table 6.7 indicates that no significant differences in the particle size and % EE were observed when NLCs stored at 4°C for 90 days, whereas NLCs stored at 25 °C ± 2 °C/ 60 % ± 5 % RH and 40 °C ± 2 °C/ 75 % ± 5 % RH showed significant differences in particle size and % EE after 7 days. This could be due to the thermal decomposition / degradation of the albumin coatings resulting higher particle size and lower % EE of combination-NLCs.

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Table 6.7: Stability study (90 days, at varying temperature) data of combination- NLCs.

Time Particle size PDI ζζζ (mV) ± EE (%) ± S.D. (days) (z-ave) S.D. (nm) ± S.D. RSV PBN Initial 91.6 ± 5.2 0.228 − 17.20 ± 1.38 82.46 ± 4.7 87.07 ± 2.5 4°C 7 days 94.1 ± 4.7 0.231 − 16.59 ± 3.14 86.94 ± 8.1 91.18 ± 4.2 15 days 92.4 ± 3.1 0.229 − 17.01 ± 1.78 85.20 ± 5.4 89.07 ± 6.1 30 days 91.9 ± 3.8 0.226 − 18.16 ± 2.50 82.03 ± 6.2 84.24 ± 4.7 90 days 93.2 ± 1.5 0.230 − 16.93 ± 1.23 80.48 ± 4.5 82.93 ± 5.5 25 °C ± 2 °C / 60 % ± 5 % RH 7 days 93.7 ± 6.3 0.240 − 18.73 ± 4.52 77.82 ± 2.85 85.44 ± 5.07 15 days 102.3 ± 4.0 0.263 − 14.81 ± 3.90 76.72 ± 3.04 79.86 ± 1.75 30 days 109.0 ± 7.2 0.296 − 12.37 ± 2.73 73.19 ± 1.00 74.63 ± 4.69 90 days 136.3 ± 5.1 0.311 − 11.29 ± 4.95 64.59 ± 7.04 67.17 ± 5.71 40 °C ± 2 °C / 75 % ± 5 % RH 7 days 113.0 ± 6.6 0.278 −13.10 ± 5.04 71.62 ± 7.1 74.83 ± 1.30 15 days 157.7 ± 2.7 0.295 −11.65 ± 2.48 63.03 ± 6.0 68.25 ± 4.24 30 days 163.1 ± 1.9 0.316 −11.70 ± 5.73 55.26 ± 8.3 59.47 ± 7.11 90 days 247.9 ± 7.2 0.398 −9.31 ± 2.51 39.20 ± 5.9 45.14 ± 9.46

Results are expressed as mean ± S.D. from three independent experiments, each performed in triplicate. PDI indicates polydispersity index; ζ indicates zeta potential; EE indicates entrapment efficiency

6.3.4 Brain distribution of NLCs As mentioned in previous section, RSV and PBN distribution in brain was detected using the RP-HPLC method. Different time points for animal sacrifice were selected to measure RSV and PBN uptake, namely 0.083, 0.25, 0.5, 1, 2, 4 and 8 h after administration. Brain distribution profile of different formulation and pure drug samples (i.e. RSV-PD, PBN-PD, RSV-NLCs, PBN-NLCs, combination-NLCs, and co-administered RSV and PBN in pure solution form) after intranasal (i.n.) administration are depicted in Fig. 6.11 and 6.12.

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Results revealed remarkable differences between NLCs and the pure drug solutions ( p < 0.05). As previously reported, nanoparticles could significantly change the body distribution of the incorporated drugs [34]. At 0.083 h (i.e. 5 min) post-i.n. administration of combination-NLCs, several fold higher increasing levels in the brain concentration of RSV and PBN were observed in comparison with pure drug solution, and the same tendency was maintained till 8 h. At 2 h post-i.n. administration of RSV-NLCs, PBN-NLCs, combination-NLCs coated with albumin, the drug contents in the brain were 0.798 %, 0.614 %, 0.649 % and 0.621 % of the i.n. dose (i.e. 100 g/rat), respectively. Usually, low concentrations of drugs (0.2 %) in the brain are adequate to result in a therapeutic effect [35]. For the majority of the CNS drugs available in the market, less than 0.2 % of the peripheral dose is taken up into brain [36]. Additionally, both RSV and PBN could be detected till 8 h in brain after the i.n. administration of NLCs. On the other hand, pure drug solutions were detected in the brain only till 2 h after i.n. (Fig. 6.11 and 6.12).

A B

Figure 6.11: Concentration (in brain) vs. time curves of different samples after intranasal administration of A) Resveratrol pure drug (RSV-PD; ) and Resveratrol-NLCs (RSV-NLCs; ) and B) PBN pure drug (PBN-PD; ) and PBN-NLCs ( ).

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Overall pharmacokinetic results are depicted in Table 6.8 and 6.9. A brain distribution study of NLCs formulations during 8 h was performed giving detailed information about the potentiality of NLCs formulations for targeting incorporated RSV and PBN to the brain. A prolonged residence in brain was achieved with NLCs. The MRT of NLCs increased in brain compared with the same dose of free drug solutions (Table 6.8 and 6.9). In fact, the MRT of RSV and PBN in brain almost doubles when they were encapsulated into NLCs.

Figure 6.12: Concentration (in brain) vs. time curves of combination samples after intranasal administration of Resveratrol (RSV) from combo-PD ( ), RSV from combo-NLCs ( ), PBN form combo-PD ( ) and PBN from combo-NLCs ( ).

The higher maximum RSV and PBN concentration in brain was achieved with NLCs formulation (i.e. 2892 ng/g brain, 2347 ng/g brain, 3174 ng/g brain and 2751 ng/g brain for RSV-NLCs, PBN-NLCs and combination-NLCs, respectively).

The maximum concentration (C max ) of RSV and PBN in the brain was reached at 5 min (T max ) after the i.n. administration of NLCs formulations (Table 6.8 and 6.9).

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This phenomenon is probably related to the unique pathway for albumin via fluid- phase transcytosis [37]. The pattern of NLCs uptake could be due to rapid transport from nasal epithelium along with the olfactory nerves into the cerebrospinal fluid (CSF), diffusion into arterial perivascular spaces, and rapid distribution throughout the brain [37]. This explanation is supported by the findings that none of the formulations were detected in plasma and suggests that NLCs transported to the brain directly from the nasal cavity and not via absorption into systemic circulation. Moreover, it also suggests that albumin may act to keep NLCs within brain. The

AUC 0-8 of RSV and PBN in the brain following i.n. administration of RSV-NLCs, PBN-NLCs and combination-NLCs was ≈ 23, 18, 26 and 20 folds higher when compared with i.n. administered pure drug (PD) solutions, respectively. The observed higher values of C max and AUC 0-8 indicate that albumin coated NLCs could help enhance the delivery of RSV and PBN into the brain.

Table 6.8: Pharmacokinetic parameters in rat brain for RSV and PBN following intranasal administration of pure drug solutions and single antioxidant loaded-NLCs (single dose)

Parameter RSV-PD RSV-NLCs PBN-PD PBN-NLCs -1 Cmax (ng g ) 91.54 ± 2892.01 ± 98.2 ± 32.04 2347.84 ± 46.19 149.28 160.82 Tmax (h) 1 0.083 1 0.083 AUC 0-8 171.95 ± 4078.58 ± 188.51 ± 3414.1 ± -1 (ng h g ) 19.1 227.82 18.11 202.91 AUC 0-∞ 228.34 ± 4226.04 ± 225.93 ± 3540.49 ± -1 (ng h g ) 15.23 516.20 21.47 615.19 AUMC 0-∞ 236.61 ± 8739.39 ± 305.772 ± 7183.89 ± 2 -1 (ng h g ) 23.19 485.5 52.03 592.04 MRT (h) 1.036 ± 0.08 2.067 ± 0.16 1.35 ± 0.12 2.02 ± 0.16 T1/2 (h) 1.071 ± 0.11 1.597 ± 0.18 1.20 ± 0.21 1.62 ± 0.25

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Table 6.9: Pharmacokinetic parameters in rat brain for RSV and PBN following intranasal administration of pure drug solutions and combination-NLCs (single dose)

Parameter RSV from RSV from PBN from PBN from combo-PD combo- combo-PD combo- NLCs NLCs -1 Cmax (ng g ) 84.45 ± 3174.73 ± 91.65 ± 27.5 2751.84 ± 30.77 173.92 194.39 Tmax (h) 1 0.083 1 0.083 AUC 0-8 159.036 ± 4186.45 ± 176.652 ± 3591.46 ± -1 (ng h g ) 31.73 392.84 39.69 164.39 AUC 0-∞ 177.686 ± 4284.56 ± 209.089 ± 3704.16 ± -1 (ng h g ) 49.09 613.61 31.08 431.34 AUMC 0-∞ 196.335 ± 7346.95 ± 273.456 ± 6797.81 ± 2 -1 (ng h g ) 21.35 385.26 46.51 312.93 MRT (h) 1.104 ± 0.14 1.714 ± 0.11 1.307 ± 0.09 1.835 ± 0.09 T1/2 (h) 0.95 ± 0.09 1.387 ± 0.08 1.15 ± 0.13 1.47 ± 0.08

6.3.5 Neuroprotection effects of the NLC formulations on Alzheimer’s disease model induced by A β 25–35 6.3.5.1 Y-maze test Since AD is characterized clinically by a progressive decline in learning and memory process, we evaluated the potential neuroprotective effects of i.n. administered different NLCs against neurotoxicity using behavioural test in A β 25-35 – treated rats. Spontaneous alteration was used to investigate behavioral impairment involved in A β-induced cognitive decline. In Y-maze test, we observed after 14 days administration a significant increase in spatial memory in animal treatment with standard, RSV-NLCs, PBN-NLCs and combination-NLCs, indicating an increase in spontaneous alteration percentage compared to positive control group (Fig. 6.13), suggesting effect on short term-memory. Furthermore, we compared the effect of treatment with standard to those of combination-NLCs. We observed no significant decrease in spontaneous alteration in the Y-maze (Fig. 6.13). However, it is important to note that the treatment with combination-NLCs, in a dose 2-fold lower, attenuated this impairment triggered by A β 25-35 . At the same time, the results of this study also indicate that single antioxidant loaded NLCs (i.e. RSV-NLCs and PBN- NLCs) had minimum impact on short term-memory when compared to standard and combination-NLCs group, suggesting importance of co-encapsulation of antioxidant

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Chapter 6: Neuroprotection Aspects of Combination-NLCs into NLCs would provide improved cognition effect. Therefore we confirm that improvement in spontaneous alteration in A β 25-35 treated animals reflects neuroprotective effect of combination-NLCs primarily due to rapid transportation and prolonged residence of drug within brain, when administered i.n.

Figure 6.13: Aβ25-35 (2 µL bilaterally injected slowly into hippocampus using stereotaxic coordinates)-induced memory impairment and neuroprotective effect of RSV-NLCs, PBN-NLCs, combination-NLCs and test or standard drug in in-vivo behavioral tests (Y-maze). Data expressed as mean ± S.E.M (n = 3). Statistical significance was determined using one way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison Test. *** p < 0.001 vs. negative control group, ooo p < 0.001 vs. positive control group, ### p < 0.001 vs. RSV-NLCs and PBN-NLCs group, and ns vs. std group.

6.3.5.2 AChE activity Acetylcholine (ACh) plays an essential role in learning and memory [38]. ACh is degraded by the enzyme AChE, terminating its physiological action of neurotransmitter. In addition to their role in cholinergic transmission, cholinesterases may also play a role during morphogenesis and neurodegenerative diseases [39]. AD was reported to affect the cholinergic system resulting in increased activity of

AChE [40]. In the present study, AChE activity was significantly increased (p <

0.001) in the brains of Aβ 25-35 treated rats as compared to negative control group 194

Chapter 6: Neuroprotection Aspects of Combination-NLCs leading to memory deficits, but combination-NLCs treated group significantly decreased ( p < 0.001) AChE activity thereby increasing spatial memory shown by Y-maze task (Fig. 6.13). Furthermore, the effect of combination-NLCs was comparable (non significant difference) to standard treatment group and negative control, whereas RSV-NLCs and PBN-NLCs treated group showed significant increase in AChE activity ( p < 0.01) (Table 6.10). Though both RSV and PBN had been showed to inhibit AChE activity in AD model induced by A β 25–35, but present study depicts the advantage of co-encapsulation of antioxidants into NLCs and their role in significant inhibiting AChE activity when compared to single antioxidant loaded NLCs. Results of both behavioral and biochemical testing confirmed the potential of albumin stabilized NLCs to deliver both RSV and PBN to the brain via i.n. route and facilitate its therapeutic effects to AD.

Table 6.10: The AChE activity in brains of AD rats treated with different formulations.

Groups AChE ( mol/min/mg protein) - Control 0.035 ± 0.009 + Control (AD control) 0.129 ± 0.015 * Standard 0.038 ± 0.010 RSV-NLCs 0.057 ± 0.009 # PBN-NLCs 0.059 ± 0.014 # Combination-NLCs 0.040 ± 0.013 $, ns

Data is expressed as mean ± S.E.M (n = 3). Statistical significance was determined using one way analysis of variance (ANOVA) followed by Tukey’s Multiple * # Comparison Test. p < 0.001 vs. – control group; p < 0.01 vs. combination-NLCs

$ ns group; p < 0.001 vs. + control group; Non-significant vs. standard and − control group.

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6.4 Conclusion In conclusion, the binary mixture lipid which was selected based on various screening studies showed relative higher encapsulation of RSV and PBN when compared to single lipid (i.e. solid lipid, SLNs). For the very first time albumin was used to stabilize the lipid nanoparticles and provided cohesive films around the nanoparticles which provided barrier that bestow long range steric hindrance, without significantly decreasing surface tension and hence are better emulsifiers in terms of imparting stability to nanoparticles than smaller surfactants which function primarily by lowering surface tension. We found that NLCs are rapidly taken up by and detected maximum in brain after i.n. administration, with no detection in rat plasma. The current study successfully demonstrate the benefits of co-encapsulation of two antioxidants into NLCs which eventually provided improved neuroprotective activity when compared to the single antioxidant loaded NLCs. These results definitely indicate that developed combination-NLCs are of great potential for treating neurologic diseases, AD in particular, and undoubtedly set a good example for other small/large molecules.

6.5 References

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13) Pardridge WM, Eisenberg J, and Cefalu WT (1985) Absence of albumin receptor on brain capillaries in vivo or in vitro. Am J Physiol 249:E264–E267. 14) Migliore MM, Vyas TK, Campbell RB, Amiji MM, and Waszczak BL (2010) Brain delivery of proteins by the intranasal route of administration: a comparison of cationic liposomes versus aqueous solution formulations. J Pharm Sci 99: 1745–1761. 15) Negi LM, Jaggi M, Talegaonkar S. Development of protocol for screening the formulation components and the assessment of common quality problems of nano structured lipid carriers. Int J Pharm. 2014; 461:403-10. 16) Lu X, Ji C, Xu H, Li X, Ding H, Ye M, Zhu Z, Ding D, Jiang X, Ding X, Guo X. Resveratrol- loaded polymeric micelles protect cells from Abeta-induced oxidative stress. Int J Pharm. 2009; 375:89-96. 17) Shah KA, Date AA, Joshi MD, Patravale VB. Solid lipid nanoparticles (SLN) of tretinoin: potential in topical delivery. Int J Pharm. 2007; 345:163-71. 18) Mandpe L, Kyadarkunte A, Pokharkar V. Assessment of novel iloperidone-and idebenone-loaded nanostructured lipid carriers: brain targeting efficiency and neuroprotective potential. Ther Deliv. 2013; 4:1365-83. 19) Dornan WA, Kang DE, McCampbell A, Kang EE. Bilateral injections of beta A (25-35) + IBO into the hippocampus disrupts acquisition of spatial learning in the rat. Neuroreport. 1993; 18:165-8. 20) Xu H, Yang HJ, McConomy B, Browning R, Li XM. Behavioral and neurobiological changes in C57BL/6 mouse exposed to cuprizone: effects of antipsychotics. Front Behav Neurosci. 2010; 18:4:8. doi: 10.3389/fnbeh.2010.00008. 21) Siedlak SL, Casadesus G, Webber KM, Pappolla MA, Atwood CS, Smith MA et al. Chronic antioxidant therapy reduces oxidative stress in a mouse model of Alzheimer’s disease. Free Radical Research 2009; 43:156-164. 22) Ellman GL, Courtney KD, Andres V, Featherstone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88–95. 23) Muller RH, Mader K, Gohla S. Solid lipid nanoparticles (SLN) for controlled drug delivery—a review of the state of the art. Eur J Pharm Biopharm. 2000;50:161Y177. 24) Kuntsche J, Mäder K. Solid lipid nanoparticles (SLN) for drug delivery. In: Torchillin V, Amiji MM (Eds.) Handbook of Materials for Nanomedicine. Pan Stanford Publ., Singapore, 2011, pp. 383- 443. 25) Negi LM, Jaggi M, Talegaonkar S. Development of protocol for screening the formulation components and the assessment of common quality problems of nano-structured lipid carriers. Int J Pharm. 2014; 461:403-10. 26) Friberg S. 1976. Emulsion stability. In “Food Emulsions,” Friberg, S. (Ed), p. l Marcel Dekker, Inc., New York and Basel. 27) Graham, D.E. and Phillips M.C. 1979a Proteins at liquid interfaces. 1. Kinetics of adsorption and surface denaturation. J. Colloid interface Sci. 70:403 28) Phillips, M.C. 1981. Protein conformation at liquid interfaces and its role in stabilizing emulsions and foams. Food Technol. 35: 50. 29) Alam S, Khan ZI, Mustafa G, Kumar M, Islam F, Bhatnagar A, Ahmad FJ. Development and evaluation of thymoquinone-encapsulated chitosan nanoparticles for nose-to-brain targeting: a pharmacoscintigraphic study. Int J Nanomedicine. 2012; 7:5705-18. 30) Hu FQ, Jiang SP, Du YZ, Yuan H, Ye YQ, Zeng S. Preparation and characterization of stearic acid nanostructured lipid carriers by solvent diffusion method in an aqueous system. Colloids Surf B Biointerfaces. 2005; 45:167–73. 31) W. Mehnert, A. zur MuÈhlen, A. Dingler, H. Weyhers, R.H. MuÈller, Solid Lipid Nanoparticles (SLN)-ein neuartiger Wirkstoff-Carrier fuÈ r Kosmetika und Pharmazeutika. II. Wirkstoff- Inkorporation, Freisetzung und Sterilizierbarkeit, Pharm. Ind. (1997) 511±514. 32) Mehnert W, Mäder K. Solid lipid nanoparticles: Production, characterization and applications. Adv Drug Deliv Rev. 2001; 47:165-196. 33) Paliwal R, Rai S, Vaidya B, Khatri K, Goyal AK, Mishra N, Mehta A, Vyas SP. Effect of lipid core material on characteristics of solid lipid nanoparticles designed for oral lymphatic delivery. Nanomedicine. 2009; 5:184-191. 34) M.L. Bondi et al., Brain-targeted solid lipid nanoparticles containing riluzole: preparation, characterization and biodistribution, Nanomedicine (London) 5 (2010) 25–32. 35) I. van Rooy et al., In vivo methods to study uptake of nanoparticles into the brain, Pharm. Res. 28 (2011) 456–471. 36) W. Banks, Developing drugs that can cross the blood–brain barrier: applications to Alzheimer’s disease, BMC Neurosci. Suppl. 9 (3) (2008) S2.

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37) Falcone JA, Salameh TS, Yi X, Cordy BJ, Mortell WG, Kabanov AV, Banks WA. Intranasal administration as a route for drug delivery to the brain: evidence for a unique pathway for albumin. J Pharmacol Exp Ther. 2014; 351:54-60. 38) Prado VF, Martins-Silva C, de Castro BM, Lima RF, Barros DM, Amaral E et al. Mice deficient for the vesicular acetylcholine transporter are myasthenic and have deficits in object and social recognition. Neuron. 2006;51:601-12. 39) Reyes AE, Perez DR, Alvarez A, Garrido J, Gentry MK, Doctor BP et al. A monoclonal antibody against acetylcholinesterase inhibits the formation of amyloid fibrils induced by the enzyme. Biochem Biophys Res Commun. 1997;232:652-5. 40) Dai J, Buijs RM, Kamphorst W, Swaab DF. Impaired axonal transport of cortical neurons in Alzheimer's disease is associated with neuropathological changes. Brain Res. 2002;948:138-44.

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Chapter 7

Conclusions

This chapter includes conclusions from the present studies.

Chapter 7: Conclusions

Conclusions The main goal of initiating this project was to improve the delivery of antioxidants using novel topical and intranasal lipid nanoparticles as means to protect the human skin and neurons, respectively. It was hypothesised that by using NLCs formulations the amount of drug that reach skin and brain will be increased without reaching systemic circulation and avoiding the blood-brain barrier (BBB), respectively. Firstly, the IDB-NLCs were prepared and we show that meticulously executed two-step preformulation study followed by Taguchi robust orthogonal design as “simple” and “economical” optimization process was capable of improving IDB-NLCs key physicochemical aspects. Regarding cellular interactions, NLCs markedly improved biocompatibility of IDB and efficiently transported it across the cell membrane and that intracellular uptake mechanism was clathrin- mediated endocytosis. The antioxidant potency of the IDB is well known and in the present investigation IDB and placebo-NLCs prompted HaCaT cell photoprotection in a dose-dependent manner, but the loading of IDB into NLCs system further boosted to thwart the oxidative stress induced by UVB irradiation. Overall, suggesting that NLC has ability to interact with skin components (owing to their nano-size and high occlusive effect) and subsequently deliver IDB in a sustained release manner that in turn improved the cellular uptake and exerted photoprotective effects. Since, NLCs proved to be a suitable topical delivery system for IDB, we emphasize on loading of two or more antioxidants in NLC system (combination strategy) to treat premature skin aging and skin cancer caused by overexposure to UV radiation. Hence, in second study we utilized the green surfactant for the stabilization of comination-NLCs. In this first we conclude that the shorter the fatty acid chain length of acylglutamate surfactants, the milder it functions on HaCaT cell line and vice versa. In addition, both amino acid and fatty acid are valuable for the barrier effect of the skin cells. Considering their biocompatible properties and low cytotoxicity profile, the sodium lauroyl glutamate would be the most suitable surfactant for the synthesis and stabilization of lipid nanoparticles for improved delivery of various cosmeceutical actives. Hence we utilize this surfactants for the stabilization of combination-NLCs. The study showed sub-100 nm particle size, 199

Chapter 7: Conclusions negative zeta potential and high entrapment efficiency can be achieved when appropriate concentration of Amisoft LS 11 (optimized using Taguchi’s mixed level-design) as a surfactant was used. The co-encapsulation of RSV and PBN into NLCs did not alter the properties of NLCs containing individual drug. Moreover, combination-NLCs showed slow-sustained release profile leading to increased photostability of both RSV and PBN and hence exhibited higher photoprotection activity against UVB-mediated phototoxicity and oxidative stress in HaCaT cells. The findings of this study show that NLCs are attractive drug delivery systems for cosmetics and dermatological applications and that two or more drugs could be encapsulated efficiently via proper selection of lipid phase component, also higher occlusion features can be achieved by tuning the physico-chemical properties of the nanoparticles. Since, RSV and PBN are promising antioxidants and can be used to prevent and treat several diseases associated oxidative stress, for instance neurodegenerative diseases like Alzheimer’s disease. Hence, we initiated the work which include co-encapsulation of RSV and PBN into brain targeted NLCs stabilized by natural protein, albumin. In third and final study, we utilized the albumin to stabilize the RSV and PBN loaded NLCs (combination strategy). The binary mixture lipid which was selected based on various screening studies showed relative higher encapsulation of RSV and PBN when compared to single lipid (i.e. solid lipid, SLNs). For the very first time albumin was used to stabilize the NLCs and provided cohesive films around the nanoparticles which provided barrier that bestow long range steric hindrance, without significantly decreasing surface tension and hence are better emulsifiers in terms of imparting stability to nanoparticles than smaller surfactants which function primarily by lowering surface tension. The current study successfully demonstrate the benefits of co-encapsulation of two antioxidants into NLCs which eventually provided improved neuroprotective activity when compared to the single antioxidant loaded NLCs.

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Appendix

This part includes standard operating procedures for in vivo studies

Appendix

Standard operating procedure

Poona College of STANDARD OPERATING Pharmacy PROCEDURE SOP No. 1 Dept. of Pharmaceutics Procedure for intranasal drug

installation

Objective: To provide standard operating procedure for drug administration through intranasal installation

Procedure: 1. Remove the rats from the cage and anesthetize the rat using anesthetic ether. 2. Place the animal on the right lateral side so that drug can be instilled into the left nostril. 3. Fill the required amount of drug solution in micropipette, remove air bubbles if any 4. Connect the polyethylene (PE) 10 tube with the 100μl micropipette and insert the PE tube approximately 10–15 mm into the rat nasal cavity. 5. Gently push the piston of syringe to administer the accurate volume of drug solution into the nasal cavity.

Prepared by Checked and approved by (Abhay Kyadarkunte) (Prof. Varsha Pokharkar)

201 201 Appendix

Poona College of STANDARD OPERATING Pharmacy PROCEDURE SOP No. 2 Dept. of Pharmaceutics Removal of blood by cardiac

puncture (terminal procedure)

Objective: To provide standard operating procedure for removal of blood by cardiac puncture (terminal procedure).

Procedure: 1. Procedure is to take place in a quiet, clean environment, separate from other animals. 2. Draw required dosages of anesthetic solutions into the syringe. 3. Place the animal on its back and wipe the chest and abdomen wiped with ethanol. 4. Attach an appropriately sized needle to a syringe and insert it at a 30 º angle just below the xyphoid process, angling the needle slightly toward the left shoulder. For rats, a 23G 1 inch or longer needle may be required. 5. Retract the plunger slightly to create a vacuum inside the syringe, and then advance the needle until blood appears in the hub of the needle. Slowly retract the plunger to collect the desired amount of blood. 6. Once the blood collection is complete, withdraw the needle and euthanize the animal by cervical dislocation. 7. Place the carcass in a biohazard bag and store in a waste freezer in ARF.

Prepared by Checked and approved by (Abhay Kyadarkunte) (Prof. Varsha Pokharkar)

202 202 Publications

This part comprises list of total 9 articles (7 published and 2 communicated) that include 5 articles that was published during PhD tenure but are out of scope of this thesis.

Publications

Publications

1. Kyadarkunte AY, Patole MS, Pokharkar VB. Cellular interactions and photoprotective effects of idebenone-loaded nanostructured lipid carriers stabilized using PEG-free surfactant. Int J Pharm. 2015;479:77-87. DoI: 10.1016/j.ijpharm.2014.12.044.

2. Kyadarkunte AY, Patole MS, Pokharkar VB. In vitro cytotoxicity and phototoxicity assessment of acylglutamate surfactants using a human keratinocyte cell line. Cosmetics 2014;1:159-170. DoI:10.3390/cosmetics 1030159

3. Kyadarkunte AY, Patole MS, Pokharkar VB. Co-encapsulation of resveratrol and alpha-phenyl-N-tert-butylnitrone in L-glutamic acid stabilized nanostructured lipid matrices: An innovative strategy to improve skin photoprotection (communicated)

4. Kyadarkunte AY, Patil-Gadhe A, Aswar U, Khawade A, Pokharkar VB. Improved delivery and neuroprotection via co-encapsulation of resveratrol and alpha-phenyl-N-tert-butylnitrone in albumin stabilized nanostructured lipid carriers (communicated)

5. Patil-Gadhe, Kyadarkunte AY, Pereira M, Jejurikar G, Patole MS, Risbud A, Pokharkar VB. Rifapentine-proliposomes for inhalation: in vitro and in vivo toxicity. Toxicol Int. 2014;21:275-82. DoI: 10.4103/0971- 6580.155361.

6. Patil-Gadhe, Kyadarkunte AY, Patole MS, Pokharkar VB. Montelukast- loaded nanostructured lipid carriers: part II pulmonary drug delivery and in vitro-in vivo aerosol performance. Eur J Pharm Biopharm. 2014;88:169- 77. DoI: 10.1016/j.ejpb.2014.07.007.

203

Publications

7. Pokharkar VB, Mendiratta C, Kyadarkunte AY, Bhosale SH, Barhate GA. Skin delivery aspects of benzoyl peroxide-loaded solid lipid nanoparticles for acne treatment. Ther Deliv. 2014;5:635-52. DoI: 10.4155/tde.14.31.

8. Mandpe L, Kyadarkunte AY, Pokharkar VB. Assessment of novel iloperidone- and idebenone-loaded nanostructured lipid carriers: brain targeting efficiency and neuroprotective potential. Ther Deliv. 2013;4:1365- 83. DoI: 10.4155/tde.13.101.

9. Mandape L, Kyadarkunte AY, Pokharkar VB. High performance liquid chromatographic method for simultaneous determination of iloperidone and idebenone in spiked plasma. International Journal of Chemical and Analytical Science. 2013;4:19-23.

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