Investigation on factors affecting drug delivery using polymers and phospholipids 1

ACKNOWLEDGMENTS At the end of four years of research, I would like to thank all the people that have contributed to the realization of my thesis. I wish to acknowledge with gratitude the contribution of my supervisor A/P Chan Sui Yung for providing an excellent environment for research. Her availability, and numerous interesting discussions I have got with her, has been a powerful stimulant during these years. Financial assistance from the Agency for Science Technology and Research (A Star) and the National University of Singapore is gratefully acknowledged. I am grateful to my numerous friends and my colleagues at NUS who helped to make this endeavor an enriching and enjoyable experience. I wish to acknowledge my parents and my brother. Their love and support through the years have been invaluable to me and I dedicate this thesis to them. Finally, I wish to thank God for his blessings. i TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS SUMMARY LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS CHAPTER 1.1 1.2 1.3 1.4 1.5 CHAPTER ii vi x xi xiv General Introduction Skin Structure Topical and Transdermal Drug Delivery Skin Permeation Models Recent Formulation Developments Hypothesis and Objective Effect of Non-Ionic Surfactants in Proniosomes on the Permeation of Drugs Across Human Skin 2.1 2.2 Introduction Materials and Methods 2.2.1 Materials 2.2.2 HPLC Analysis 2.2.3 Phase Solubility and Surface Tension Studies 2.2.4 Proniosome Formulations 2.2.5 Encapsulation Efficiency and Stability of Proniosomes 2.2.6 Scanning Electron Microscopy (SEM) 2.2.7 Preparation of Human Epidermis 2.2.8 In vitro Skin Permeation Studies 2.2.9 Confocal Laser Scanning Microscopy (CLSM) 2.2.10 Statistics 2.3 Results and Discussion 2.3.1 Solubility and Surface Tension Studies 2.3.2 Encapsulation Efficiency and Vesicle Stability 2.3.3 SEM Imaging 2.3.4 In vitro Skin Permeation Studies 2.4 Conclusion CHAPTER i Effect of Drug Complexation and Drug Ionization on the Permeation of Haloperidol Across Human Skin 3.1 Introduction 3.2 Materials and Methods 3.2.1 Materials 3.2.2 HPLC Analysis 3.2.3 Molecular Modeling 28 28 29 29 30 30 31 33 33 34 34 36 36 36 36 37 38 40 46 47 47 49 49 50 50 ii 3.2.4 Phase Solubility Studies 3.2.5 Surface Tension and Contact Angle Measurements 3.2.6 In vitro Skin Permeation Studies 3.3 Results and Discussion 3.3.1 Molecular Modeling 3.3.2 Solubility Studies 3.3.3 Surface Tension and Contact Angle Measurements 3.3.4 In vitro Skin Permeation Studies 3.4 Conclusion CHAPTER Development of a Multilayered/Multicomponent Fiber Mat for Improved Topical Delivery of L-Ascorbic Acid and Retinoic Acid 4.1 Introduction 4.2 Materials and Methods 4.2.1 Materials 4.2.2 Electrospinning 4.2.3 Field Emission Scanning Electron Microscopy (FESEM) 4.2.4 Fourier Transform Infrared Measurements (FTIR) 4.2.5 HPLC Analysis 4.2.6 Drug Release Profile 4.2.7 In vitro Skin Permeation Studies 4.3 Results and Discussion 4.3.1 Characterization of Nanofiber 4.3.2 FTIR Studies 4.3.3 In vitro Drug Release Studies 4.3.4 In vitro Skin Permeation Studies 4.4 Conclusion CHAPTER Development of a Nutrient-Rich Facial Mask for the Topical Delivery of Ascorbic Acid and Retinoic Acid 5.1 Introduction 5.2 Materials and Methods 5.2.1 Materials 5.2.2 Electrospinning 5.2.3 FESEM and Energy Dispersive X-Ray Spectroscopy (EDS) Analysis of the Fiber Mat 5.2.4 UV Spectroscopy 5.2.5 In vitro Skin Permeation Studies 5.2.6 Skin Histology 5.2.7 Statistical Analysis 5.3 Results 5.3.1 Fiber Morphology and EDS Analysis 5.3.3 In vitro Skin Permeation Studies 5.4 Conclusion 51 51 52 53 53 54 55 58 65 66 66 68 68 68 70 70 70 70 71 71 71 72 74 76 77 79 79 81 81 81 82 83 83 83 84 84 84 87 89 iii CHAPTER Development of a Thermosensitive Mat for Sustained Topical Delivery of Levothyroxine 6.1 Introduction 6.2 Materials and Methods 6.2.1 Materials 6.2.2 HPLC Analysis 6.2.3 Electrospinning of PVA/PNIPAM Nanofibers 6.2.4 FTIR Studies of the Nanofibers 6.2.5 FESEM and Fluorescence Microscopy of the Nanofibers 6.2.6 In vitro Drug Release Studies 6.2.7 In vitro Skin Permeation Studies 6.2.9 Confocal Laser Scanning Microscopy (CLSM) 6.3 Results and Discussion 6.3.1 FTIR Measurements of the Drug-loaded Nanofibers 6.3.2 FESEM and Florescence Image of Nanofibers 6.3.3 In vitro Drug Release Studies 6.3.4 In vitro Skin Permeation Studies 6.4 Conclusion CHAPTER Effect of Polymer Transition on the Topical Delivery of Levothyroxine 7.1 Introduction 7.2 Materials and Methods 7.2.1 Materials 7.2.2 Preparation of Microparticles 7.2.3 Encapsulation Efficacy and Stability Studies 7.2.4 Microparticle Characterization 7.2.5 HPLC Analysis 7.2.6 In vitro Drug Release Studies 7.2.7 In vitro Skin Permeation Studies 7.2.8 FTIR of Skin Sample 7.2.9 Confocal Studies of the Treated Skin 7.3 Results and Discussion 7.3.1 FESEM Characterization of Microparticles 7.3.2 Determination of Encapsulation Efficiency 7.3.3 In vitro Release of Levothyroxine 7.3.4 In vitro Skin Permeation Studies 7.3.5 FTIR of Human Skin Samples 7.4 Conclusion CHAPTER Effect of Skin Lipid Fluidization and Drug Encapsulation on the Transdermal Delivery of Diclofenac 8.1 Introduction 8.2 Materials and Methods 8.2.1 Materials 8.2.2 Preparation of Diclofenac Sodium-Loaded Vesicles 90 90 91 91 91 92 92 93 93 93 94 94 94 95 98 100 102 104 104 106 106 106 106 107 107 107 107 108 108 108 108 110 111 112 117 118 120 120 122 122 122 iv 8.2.3 Determination of Encapsulation Efficiency 8.2.4 Storage Stability of Vesicles 8.2.5 In vitro Skin Permeation Studies 8.2.6 FTIR Studies of the Human Skin 8.2.7 HPLC Assay of Diclofenac Sodium 8.3 Results and Discussion 8.3.1 Vesicle Size Measurement 8.3.2 Determination of Encapsulation Efficiency 8.3.3 In Vitro Drug Permeation Studies 8.3.4 FTIR Studies of the Human Skin 8.4 Conclusion CHAPTER Conclusion and direction for future work REFERENCES LIST OF PUBLICATIONS 125 125 125 126 126 126 126 128 128 131 134 136 142 167 v SUMMARY Topical drug delivery helps to achieve therapeutic concentrations at the site of application to achieve a localized effect, while transdermal delivery is defined as delivery of the drug through intact skin so that it reaches the systemic circulation at sufficient concentrations to attain therapeutic levels. The objective of this thesis was to study the effect of some physicochemical factors of the drug molecule and the carrier, including their solubility, hydrophilicity, contact angle and surface area on the human skin permeation or accumulation of drug molecules. In vitro flow-through diffusion cells were employed to explore the skin permeation of hydrophilic (ascorbic acid, diclofenac) and lipophilic molecules (levothyroxine, haloperidol, retinoic acid) from various lipid vesicles namely, liposome, niosome, proniosome, transferosome, ethosome, cerosome, or polymeric, poly N-isopropylacrylamide (PNIPAM), poly vinylalcohol (PVA), randomly methylated β- cyclodextrin (RM β-CD), poly lactide (PLA), poly lactide co glycolide (PLGA), ethyl cellulose (EC), microparticles and nanofibers. Proniosomal formulations with non-ionic surfactant, Spans and Tweens, were studied. The effect of hydrophilic-lipophilic balance (HLB value) of one or two surfactants on drug solubility, proniosome surface structure and stability and skin permeation of haloperidol from different formulations were investigated. It was found that a balance of hydrophilicity is needed for an efficient drug release from proniosomes and a high diffusion rate to the skin. Formulations with single surfactants were found to increase the permeation of HP more than mixtures of surfactants. Mixtures of surfactants may form a new type of mixed micelle that vi could behave differently than the two single surfactants and thus decrease the skin permeation rate. Surfactant characteristics such as HLB value, surface tension, number of carbons in the alkyl chain influence the skin permeation of the drug molecule. The effect of RM β-CD on the solubility and complexation energy of haloperidol was studied. Highest increase in drug solubility was observed when the drug was in its degree of ionized form in RM β-CD, resulting in a 128-fold increase in the intrinsic solubility of the drug. Interfacial tension of various concentrations of this CD derivative was explored and it was found that controversial results regarding the role of CD as a penetration enhancer reported by various scientists may be related to the surface active behavior and the CMC value of the CD. It was found that contact angle of the vehicle influences the extent of drug permeation across the skin layers. Mixture of hydrophilic, (poly vinyl alcohol, PVA, and randomly methylated βcyclodextrin, RM β-CD), and hydrophobic (poly d,l-lactide, PLA, and poly d,llactide-co-glycolide, PLGA) polymers were electrospun to make a multilayered/multicomponent nanofiber mat. The release characteristic of the drug was modified using the layer by layer approach to help compensate the limitation of the individual materials. Incorporation of RM β-CD to the PVA solution significantly decreased the degradation rate of the resulting fiber mat from a few weeks to a few seconds. Polyesters, PLA and PLGA, releases drug via hydrolysis of the polymer and could provide sustained and controlled release rate of the drug. Blends of these hydrophilic and hydrophobic polymers could effectively prolong drug release and decrease physiological toxicity resulting from fast release of drugs. A novel anti-wrinkle polymeric nanofiber of PVA and RM β-CD face mask containing ascorbic acid, retinoic acid, collagen and gold nanoparticles was vii developed. The formulation is dry in nature and would become wetted only when applied on the skin. This would maintain the chemical stability of the ascorbic acid and thus the shelf life of the product compared to the pre-moistened commercially available facial masks. RM β-CD could help to increase the solubility of the low water soluble compounds such as retinoic acid. The high surface area-to-volume ratio and porosity of the fibers will ensure maximum contact with the skin surface and enhance the permeation rate of the active compounds when compared to cotton facial masks available in the market. Polymeric nanofibers of poly (N-isopropylacrylamide) (PNIPAM) and PVA and blends of the two polymers were developed to modify the drug release patterns. PNIPAM is a thermosensitive polymer with low critical solution temperature (LCST) of around 32oC in aqueous solution. The release of T4 from mixed polymer mat was found to be a function of PNIPAM concentration used. PNIPAM nanofibers sustained the permeation of levothyroxine to the skin and therefore maintained the effective drug concentration in the skin layers for a longer period. Polymeric microparticles of PLA, PLGA, PNIPAM and EC were used as carriers for the skin delivery of levothyroxine. These polymeric microparticles varied in their surface morphology, drug encapsulation efficacy and stability profiles. The low transition temperature, Tg, of PLA and PLGA (~ 37oC), and low LCST of PNIPAM (~ 32oC) in aqueous solutions caused precipitation of the rubber-like polymer on the skin surface which created an impermeable barrier to prevent drug penetration across the epidermis. Skin permeation observed from EC microparticles was due to the high Tg of this polymer. Diclofenac-loaded conventional liposomes, ethosomes, transferosomes, niosomes, and PEG-PPG-PEG niosomes were studied for their effects on the skin viii lipid fluidization and skin permeation. The lipid structure of the skin was modified, however the skin permeation of diclofenac was not significantly enhanced. This suggests that there may be no correlation between drug encapsulation and skin lipid fluidization with skin permeation of hydrophilic drug. It can be concluded that modification of the characteristics of the drug and the carrier can help to increase the skin permeation/accumulation of drugs. ix LIST OF TABLES Table 1.1 Overview of lipid vesicle research in transdermal drug delivery. 14 Table 1.2 Overview of cyclodextrin-drug inclusion research in transdermal drug delivery. 19 Table 1.3 25 Table 1.4 Overview of polyester and thermosensitive polymer research in transdermal drug delivery. Scope of investigation of this thesis. Table 2.1 Composition and appearance of proniosomal formulations. 32 Table 2.2 Properties of the surfactants incorporated in proniosomes. 45 Table 2.3 Permeation profiles of different proniosomal formulations (n=3). 45 Table 3.1 Surface tension and contact angle values of the solutions (n=3). 58 Table 3.2 Flux value of HP across human epidermis (n=3). 60 Table 4.1 Details of the nanofiber formulations. 69 Table 5.1 Details and the composition of the face masks. 82 Table 6.1 Details of the nanofiber formulations. 92 Table 8.1 Composition of lipid suspensions. 124 Table 9.1 Scope of investigation and findings of this thesis. 141 27 x LIST OF FIGURES Fig. 1.1 Image of the human epidermis, (a) Binary image of the human epidermis and localization of green fluorescence, staining of cell nuclei with DAPI is shown as blue signal. Slice view of stratum granulosum is shown in red fluorescence. (b) Cross-section of human epidermis. Details on the method of sample preparation are mentioned in section 2.2.9 and 5.2.6. Fig. 2.1 Solubility and surface tension measurements of HP solutions (n=3). 37 Fig. 2.2 Encapsulation efficiencies (%) of proniosomal formulations (n=3). 38 Fig. 2.3 Scanning electron microscopy images of proniosome formulations, (a) HLB 1.8, (b) HLB 2, (c) HLB 6, (d) HLB 6.7, (e) HLB 10, (f) HLB 16 and (g) HLB 16.7. 39 Fig. 2.4 Permeation profile of HP across human epidermis (n=3). 41 Fig. 2.5 (a) Image of the epidermis and localization of red fluorescence incorporated in to the proniosomes as a function of depth into the skin. (b) For better visualization, skin samples where stained with fluorescein prior skin permeation studies. The image depths (from left to right) are 0, 4, 8, 12, 16, 20 and 24 µm. 44 Fig. 3.1 (a) Hypothetical structure of the haloperidol-DM β-CD complex, and (b) haloperidol-HP β-CD complex. (1) Side view; (2) Side view with electron surface; (3) Top view; and (4) Top view with electron surface. 54 Fig. 3.2 Phase solubility of haloperidol in CD solutions (n=3). 55 Fig. 3.3 Surface tension of RM β-CD and HP β-CD (n=3). 56 Fig. 3.4 Schematic aggregation of CD. 57 Fig. 3.5 Permeation profile of haloperidol across human epidermis. Influence of (a) different RM β-CD concentrations, (b) limonene and RM β-CD, (c) ionization and RM β-CD, (d) RM β-CD and sink condition in receptor compartment, R and D denote receptor and donor compartment of the flow through diffusion cells, respectively (n=3). 63 Fig. 4.1 Schematic presentation of single layered and multilayered 69 nanofibers. Fig. 4.2 FESEM images of single-layered haloperidol-loaded nanofibers. 72 xi Fig. 4.3 FTIR spectra of (a) PLA, (b)PLGA 48:52 , (c)PLGA 73:27, (d) PVA-RM β-CD, (e) PVA-RM β-CD and PLGA 48:52 (f) PVA-RM β-CD and PLGA 73:27 (g) PVA-RM β-CD and PLA and (h) PVA nanofibers. 73 Fig. 4.4 In vitro release profile of haloperidol from electrospun fiber mat in phosphate buffer saline (pH 7.4) at body temperature (37oC), n=3. 75 Fig. 4.5 Cumulative HP permeation across human skin (n=3). 77 Fig. 5.1 FESEM morphology of the electrospun fiber mats and digital image of the fiber mat. 85 Fig. 5.2 X-ray energy spectrum of nanofiber face mask, demonstrating the presence of the gold element signals using area analysis and spot analysis. 86 Fig. 5.3 Cumulative AA and RA across human epidermis (n=3). 88 Fig. 5.4 Morphology of human epidermis, before and after skin permeation of gold nanoparticle-loaded nanofibers. The nucleated cells of the epidermis have been stained blue, unsaturated lipids, including fatty acids and esters have been stained red. 89 Fig. 6.1 FTIR spectra of nanofiber mats of (a) 10% w/v PVA - No drug, (b) 10% w/v PVA, (c) 10% w/v PVA - 5% w/v PNIPAM, (d) 10% w/v PVA - 10% w/v PNIPAM, (e) 10% w/v PNIPAM and (f) 10% w/v PNIPAM - No drug. All formulations contain drug unless otherwise mentioned. 95 Fig. 6.2 FESEM images of T4-loaded nanofibers of (a) 10% w/v PVA, (b) 10% w/v PNIPAM in ethanol, (c) 10% w/v PNIPAM in water, (d) 10% w/v PVA - 5% w/v PNIPAM, (e) 10% w/v PVA - 10% w/v PNIPAM, (f) fluorescein -loaded 10% w/v PNIPAM. 97 Fig. 6.3 In vitro release profile of T4 from electrospun mat in phosphate buffer (pH 7.4) at body temperature (37oC), n=3. 98 Fig. 6.4 In vitro release profile of T4 from electrospun mat in phosphate buffer (pH 7.4) at room temperature (20oC), n=3. 99 Fig. 6. Cumulative T4 permeation across human epidermis (n=3). 100 Fig. 6.6 (a) Image of the epidermis and localization of green fluorescence incorporated in to the PNIPAM nanofibers as a function of depth into the skin. The image depths (from left to right) are 0, 8, 16 and 24 µm. (b) Binary image of the skin. 102 Fig. 7.1 FESEM images of T4 loaded (a) PLA, (b) PLGA, (c) EC and (d) PNIPAM microparticles. 110 xii Fig. 7.2 Stability of lipid suspensions: Encapsulation efficacy of the vesicles over time at room temperature (20oC) and fridge temperature (4oC), n=3. 111 Fig. 7.3 In vitro release profile of T4 from microparticles in phosphate buffer (pH 7.4) at body temperature (37oC), n = 3. 112 Fig. 7.4 Permeation profile of T4 across human epidermis (n=6-8). 113 Fig. 7.5 Binary image of the epidermis and localization of green fluorescence on the skin after treatment with the polymeric particles. For better visualization cell nuclei were counter stained with DAPI. 116 Fig. 7.6 FTIR spectrum of (a) untreated human epidermis, and skin treated with (b) 10% w/v PVA mat, (c) 10% w/v PNIPAM mat and (d) PBS. 118 Fig. 8.1 Stability of lipid formulations: formulations (nm) with time, (n=3). Mean diameter of vesicle 127 Fig. 8.2 Stability of lipid suspensions: vesicles with time, (n=3). Encapsulation efficacy of the 129 Fig. 8.3 Cumulative concentrations of diclofenac sodium across human epidermis (n=3). 130 Fig. 8.4 Representative FTIR spectra of (a) untreated human epidermis, and skin in the presence of (b) PBS, (c) Ethosome, (d) transferosome, (e) proniosome, (f) niosome, (g) PEG-PPG-PEG niosome and (h) cerosome. 134 xiii LIST OF ABBREVIATIONS A AA ANOVA Au AZT C Co Cf Cion Cmax Ct Cunion CD CLSM CM β-CD CMC CP D/L2 DAPI DM β-CD DMSO DSC EC EDS EI EE FDA FESEM FTIR G GJP HBsAg HLB HP HP β-CD HPMC HPLC IPA IV Jss Jtot Kpion Kpunion KD/L Area L-ascorbic acid One- way analysis of variance Gold Azidothymidine Carbon Initial drug concentration in the donor cell Concentration of the free drug Concentration of the ionized drug Maximum plasma concentration Concentration of the total drug Concentration of the unionized drug Cyclodextrin Confocal laser scanning microscopy Carboxymethyl-β-cyclodextrin Critical micelle concentration Capsaicin Drug diffusion parameter 4’, 6-diamidino-2-phenylindole Dimethyl- β –cyclodextrin Dimethyl sulfoxide Differential thermal analysis Ethyl cellulose Energy Dispersive X-Ray Spectroscopy Enhancement index Encapsulation efficacy Food and drug administration Field emission scanning electron microscopy Fourier transform infrared spectroscopy Gauge Gap junction protein Hepatitis B surface antigen Hydrophilic-lipophilic balance Haloperidol Hydroxypropyl β-CD Hydroxypropyl methyl cellulose High-performance liquid chromatography Isopropyl alcohol Intravenous Steady state flux Total flux Drug permeability of the ionized drug Drug permeability of the unionized drug Drug permeability xiv KL kV LCST M MPZ Na O PBS PEG PEG-PPG-PEG PG PLA PLGA PM β-CD PNIPAM PVA PVP Q RA RM β-CD SB SC SD SEM SG SS T4 Tg tL TDD TEWL TRA UV Partition parameter Kilo volt Lower critical solution temperature Molar Metopimazine Sodium Oxygen Phosphate buffer saline Polyethylene glycol Polyethylene glycol-block-polypropylene glycol-block-polyethylene glycol Propylene glycol Poly D,L lactide Poly D,L lactide co glycolide Partialy methylated β-CD Poly (N-isopropylacrylamide) Poly vinyl alcohol Poly vinyl pyrrolidone Cumulative amount of released drug 13-cis retinoic acid Randomly methylated β-CD Stratum basale Stratum corneum Standard deviation Scanning electron microscopy Stratum granulosum Stratum spinosum Levothyroxine Glass transition temperature Lag time Transdermal drug delivery Transepidermal water loss Tretinoin Ultra violet xv [...]... Azidothymidine Carbon Initial drug concentration in the donor cell Concentration of the free drug Concentration of the ionized drug Maximum plasma concentration Concentration of the total drug Concentration of the unionized drug Cyclodextrin Confocal laser scanning microscopy Carboxymethyl-β-cyclodextrin Critical micelle concentration Capsaicin Drug diffusion parameter 4’, 6-diamidino-2-phenylindole Dimethyl-... w/v PVA - No drug, (b) 10 % w/v PVA, (c) 10 % w/v PVA - 5% w/v PNIPAM, (d) 10 % w/v PVA - 10 % w/v PNIPAM, (e) 10 % w/v PNIPAM and (f) 10 % w/v PNIPAM - No drug All formulations contain drug unless otherwise mentioned 95 Fig 6.2 FESEM images of T4-loaded nanofibers of (a) 10 % w/v PVA, (b) 10 % w/v PNIPAM in ethanol, (c) 10 % w/v PNIPAM in water, (d) 10 % w/v PVA - 5% w/v PNIPAM, (e) 10 % w/v PVA - 10 % w/v PNIPAM,... measurements of HP solutions (n=3) 37 Fig 2.2 Encapsulation efficiencies (%) of proniosomal formulations (n=3) 38 Fig 2.3 Scanning electron microscopy images of proniosome formulations, (a) HLB 1. 8, (b) HLB 2, (c) HLB 6, (d) HLB 6.7, (e) HLB 10 , (f) HLB 16 and (g) HLB 16 .7 39 Fig 2.4 Permeation profile of HP across human epidermis (n=3) 41 Fig 2.5 (a) Image of the epidermis and localization of red fluorescence... view with electron surface 54 Fig 3.2 Phase solubility of haloperidol in CD solutions (n=3) 55 Fig 3.3 Surface tension of RM β-CD and HP β-CD (n=3) 56 Fig 3.4 Schematic aggregation of CD 57 Fig 3.5 Permeation profile of haloperidol across human epidermis Influence of (a) different RM β-CD concentrations, (b) limonene and RM β-CD, (c) ionization and RM β-CD, (d) RM β-CD and sink condition in receptor... 11 2 Fig 7.4 Permeation profile of T4 across human epidermis (n=6-8) 11 3 Fig 7.5 Binary image of the epidermis and localization of green fluorescence on the skin after treatment with the polymeric particles For better visualization cell nuclei were counter stained with DAPI 11 6 Fig 7.6 FTIR spectrum of (a) untreated human epidermis, and skin treated with (b) 10 % w/v PVA mat, (c) 10 % w/v PNIPAM mat and. .. nanofibers as a function of depth into the skin The image depths (from left to right) are 0, 8, 16 and 24 µm (b) Binary image of the skin 10 2 Fig 7 .1 FESEM images of T4 loaded (a) PLA, (b) PLGA, (c) EC and (d) PNIPAM microparticles 11 0 xii Fig 7.2 Stability of lipid suspensions: Encapsulation efficacy of the vesicles over time at room temperature (20oC) and fridge temperature (4oC), n=3 11 1 Fig 7.3 In vitro... PNIPAM mat and (d) PBS 11 8 Fig 8 .1 Stability of lipid formulations: formulations (nm) with time, (n=3) Mean diameter of vesicle 12 7 Fig 8.2 Stability of lipid suspensions: vesicles with time, (n=3) Encapsulation efficacy of the 12 9 Fig 8.3 Cumulative concentrations of diclofenac sodium across human epidermis (n=3) 13 0 Fig 8.4 Representative FTIR spectra of (a) untreated human epidermis, and skin in the presence... (e) proniosome, (f) niosome, (g) PEG-PPG-PEG niosome and (h) cerosome 13 4 xiii LIST OF ABBREVIATIONS A AA ANOVA Au AZT C Co Cf Cion Cmax Ct Cunion CD CLSM CM β-CD CMC CP D/L2 DAPI DM β-CD DMSO DSC EC EDS EI EE FDA FESEM FTIR G GJP HBsAg HLB HP HP β-CD HPMC HPLC IPA IV Jss Jtot Kpion Kpunion KD/L Area L-ascorbic acid One- way analysis of variance Gold Azidothymidine Carbon Initial drug concentration in... Fig 1. 1 Image of the human epidermis, (a) Binary image of the human epidermis and localization of green fluorescence, staining of cell nuclei with DAPI is shown as blue signal Slice view of stratum granulosum is shown in red fluorescence (b) Cross-section of human epidermis Details on the method of sample preparation are mentioned in section 2.2.9 and 5.2.6 2 Fig 2 .1 Solubility and surface tension measurements... proniosomes as a function of depth into the skin (b) For better visualization, skin samples where stained with fluorescein prior skin permeation studies The image depths (from left to right) are 0, 4, 8, 12 , 16 , 20 and 24 µm 44 Fig 3 .1 (a) Hypothetical structure of the haloperidol-DM β-CD complex, and (b) haloperidol-HP β-CD complex (1) Side view; (2) Side view with electron surface; (3) Top view; and . Carbon C o Initial drug concentration in the donor cell C f Concentration of the free drug C ion Concentration of the ionized drug C max Maximum plasma concentration C t Concentration of the total drug C union Concentration. Determination of Encapsulation Efficiency 11 0 7.3.3 In vitro Release of Levothyroxine 11 1 7.3.4 In vitro Skin Permeation Studies 11 2 7.3.5 FTIR of Human Skin Samples 11 7 7.4 Conclusion 11 8 CHAPTER. Skin 13 1 8.4 Conclusion 13 4 CHAPTER 9 Conclusion and direction for future work 13 6 REFERENCES 14 2 LIST OF PUBLICATIONS 16 7 vi SUMMARY Topical drug delivery helps to achieve therapeutic concentrations