Mistry, Alpesh (2009) The development and application of biological models for evaluation of direct

264 Pages·2017·10.68 MB·English

by  Christine Middleton

The Development and Application of Biological Models for
Evaluation of Direct Nose-to-Brain Drug Delivery Systems

Alpesh Mistry
MPharm(Hns), MRPharmS

Thesis submitted to the University of Nottingham
for the degree of Doctor of Philosophy

August 2008
ABSTRACT
The olfactory neuroepithelium is the only part of the central nervous system that is
exposed directly to the external environment. Therefore, it is the only non-invasive drug
delivery route to the brain. Surface modification of PS nanoparticles with chitosan
(C-PS), polysorbate 80 (P80-PS) and polysorbate 80+FCS (P80-FCS-PS) changed the
toxicity and distribution of these nanoparticles in olfactory mucosae. In addition, a
reduction in nanoparticle diameter from 200nm to 20nm increased nanoparticle mucosal
penetration and possibly also their cellular toxicity.

In vitro vertical Franz diffusion chamber and in vivo mouse models were adapted to
investigate the transport of nanoparticles via the olfactory system. For the in vitro
model, preliminary studies found that olfactory epithelium lined the caudal portion of
the dorsal nasal turbinate in the porcine nasal cavity. To ensure the scientific validity of
the diffusion chamber studies, it was necessary to prove that the experimental
procedures themselves (without the addition of nanoparticles) had no effect on the
mounted tissue. Therefore, viability and cellular morphology of the dissected olfactory
epithelia were assessed prior to application of nanoparticles to tissues. Alamar Blue™
viability and histological findings showed that the diffusion chamber experiment did not
affect the olfactory tissue when compared to samples that were not mounted on the
apparatus. Citrate buffer (pH6.0) had significantly reduced the viability (PD, I and
sc
Alamar Blue™) of the porcine olfactory epithelium compared to SNS buffer (pH7.4)
but it did not kill it. Citrate buffer may have depleted the mucosal pH gradient in the
epithelium. Overall both SNS buffered and citrate buffered porcine olfactory epithelia
were suitable for nanoparticle transport studies in the vertical Franz diffusion cell.
The in vitro and in vivo biological models showed surface modification had changed the
distribution of nanoparticles within the epithelia. There was good agreement between
particle losses from donor chamber, fluorescence microscopy images and stereology
results that C-PS particles adhered to extracellular mucus to a greater extent compared
to PS and P80-FCS-PS. P80-PS nanoparticles were taken into the nasal epithelial cells
to a greater extent than C-PS. Nanoparticles were not transported to the receiver
chamber in vitro or the olfactory bulbs in vivo. The size of the nanoparticles was also
important. Fluorescence microscopy and stereology showed that greater numbers of
100nm PS and 100nm P80-FCS-PS were taken up into mouse olfactory epithelial cells
compared to 200nm diameter equivalents. Larger particles may not have penetrated
mucus as effectively as smaller ones.

Bright field microscopy images of olfactory epithelia dismounted from the diffusion
chamber apparatus after transport study with C-PS nanoparticles showed that these
particles caused the greatest amount of cellular damage compared to PS, P80-PS and
P80-FCS-PS systems. Greater damage was observed for progressively smaller particles.
For example, 20nm C-PS may have accessed subcellular organelles such as
mitochondria to cause cell death by oxidative stress. However, similar findings in the
mouse model were not observed. It was hypothesised that, unlike the in vitro model, the
mouse model may have been able to maintain a pH gradient across the mucous layer by
neutralising the acidity from the citrate buffer using blood borne HCO - ions. This
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would protect the epithelial cells by causing C-PS to aggregate in the mucus thereby
preventing them from accessing the epithelial cells.
ACKNOWLEDGMENTS

I would like to thank my PhD supervisors Prof. Lisbeth Illum and
Dr. Snjezana Stolnik for their guidance and knowledge.

The Division of Drug Delivery & Tissue Engineering (School of Pharmacy, University
of Nottingham) and technical support staff (in particular Mrs. Christine Grainger-
Boultby and Mrs. Teresa Marshall) are also credited for their contribution. Sys Glud
Zoffman (University of Aarhus, Denmark) is thanked for the histological and
stereological work that was undertaken and for her constructive comments during the in
vivo study. Fredrick Dagnaes-Hansen (University of Aarhus, Denmark) is
acknowledged for supervision of animals during the in vivo study. John Corbett (Sutton
Bonnington Campus, University of Nottingham, UK) is thanked for procurement of
animals used in the in vitro study. I am also grateful to the Engineering and Physical
Sciences Research Council (EPSRC) and The School of Pharmacy, Nottingham, UK for
funding the project.

Finally, I express my sincerest gratitude to my family for giving me the strength to
follow my dreams.
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“What will tell in the end will be character and not a knowledge of letters.”
Mohandas Karamchand Gandhi
(b. Porbandar, 2 Oct. 1869; d. Delhi 30 Jan. 1948)
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ABSTRACT 2
ACKNOWLEDGMENTS 4
ABBREVIATIONS 11
1. INTRODUCTION AND OVERVIEW 12
1.1 INTRODUCTION 12
1.2 NASAL ANATOMY AND PHYSIOLOGY 13
1.2.1 The External Nose and the Nasal Cavity 14
1.2.2 The Structure of Nasal Epithelia 18
1.2.3 Respiratory Epithelium and Mucocillary Clearance 20
1.2.4 Olfactory Epithelium and Neuronal Supply 23
1.2.5 Junctional Complexes 27
1.2.6 Interspecies Variability of Nasal Anatomy 28
1.3 CELLULAR MECHANISMS FOR TRANSMUCOSAL DRUG DELIVERY 30
1.4 EARLY NOSE-TO-BRAIN THERAPEUTICS 35
1.5 VIRAL PARTICLES AND ENVIRONMENTAL POLLUTANTS IN THE OLFACTORY SYSTEM 35
1.6 THE TRANSPORT OF DRUG SOLUTIONS DIRECT FROM NOSE-TO-BRAIN 36
1.7 NOSE-TO-BRAIN TRANSPORT OF SYNTHETIC PARTICLES 45
1.8 SUMMARY: DIRECT NOSE-TO-BRAIN TRANSPORT OF DRUG DELIVERY FORMULATIONS 52
1.9 NANOPARTICLE FORMULATION STRATEGIES FOR ENHANCING NOSE-TO-BRAIN DRUG
DELIVERY 55
1.9.1 Chitosan 55
1.9.2 Polysorbate 80 60
1.10 PROJECT OUTLINE 64
1.11 STUDY PLAN 64
2. IN VITRO EXPERIMENTAL SETUP AND METHOD VALIDATION 66
2.1 INTRODUCTION 66
2.2 MATERIALS AND METHODS 67
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2.2.1 Materials 67
2.2.1.1 Diffusion Chamber Experiments 67
2.2.1.2 Viability Assays 67
2.2.1.3 Microscopy Sample Preparation and Differential Stains 68
2.2.2 Preparation of Physiological Buffering Solutions 68
2.2.3 Dissection of Nasal Epithelia from the Pig 68
2.2.4 Vertical Franz Diffusion Chamber Studies 72
2.2.5 Sample Viability Measurements 77
2.2.5.1 Electrophysiological Viability Measurements 78
2.2.5.2 Alamar Blue™ 81
2.2.5.3 Hoechst DNA Quantification 83
2.2.6 Comparison of Olfactory and Respiratory Tissue Metabolism 86
2.2.7 Preparation of Tissue Samples for Microscopy 86
2.2.8 Staining Samples for Light Microscopy 88
2.2.9 Staining Samples for TEM 89
2.2.10 Statistical Analyses 89
2.2.10.1 Comparison between grouped continuous data 89
2.2.10.2 Testing for correlation between continuous datasets 90
2.2.10.3 Relative Standard Deviation (RSD) 91
2.3 RESULTS 91
2.3.1 Morphological Features of Porcine Nasal Epithelia 92
2.3.2 Effect of Different Buffers and Sodium Azide on Olfactory Epithelium 99
2.3.3 Assessment of Equilibration Period 106
2.3.4 Comparison of Olfactory and Respiratory Tissue Metabolism 107
2.3.5 Correction Effect of Alamar Blue™ Values with DNA Content 108
2.4 DISCUSSION 111
2.4.1 The Histology of the Porcine Nasal Epithelium 111
2.4.2 Tissue Viability: The Effect of pH Buffers and Sodium Azide 114
2.4.3 The Bioelectrical Equilibration Period for Porcine Olfactory Epithelium 122
2.4.4 Comparison of Olfactory and Respiratory Tissue Metabolism 124
2.4.5 Hoechst Assay 128
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2.4.6 Assessment Methods for Epithelial Viability / Metabolic Rate 128
2.4.7 Viability Acceptance Criteria for Porcine Olfactory Epithelium 131
2.5 CONCLUSIONS 132
3. DEVELOPMENT OF SURFACE MODIFIED NANOPARTICLES 135
3.1 INTRODUCTION 135
3.2 MATERIALS AND METHODS 136
3.2.1 Materials 136
3.2.2 FluoSpheres™ 136
3.2.3 Nanoparticle Physicochemical Characterisation 137
3.2.3.1 Particle size distribution 137
3.2.3.2 Measurement of ζ potential 138
3.2.4 Preparation of Chitosan-coated Nanoparticles 138
3.2.4.1 Varying pH conditions for adsorption (100nm C-PS) 138
3.2.4.2 Evaluating the Effect of Adjusting Aqueous Phase Ionic Strength 139
3.2.5 Preparation of 100nm and 200nm P80-PS 141
3.2.6 Preparation of 20nm P80-PS 141
3.2.7 Preparation of P80-FCS-PS 142
3.2.8 Preventing FCS Adsorption to Vessels during Formulation 143
3.3 RESULTS 144
3.3.1 Formulation Development of C-PS Nanoparticles 145
3.3.1.1 Varying pH conditions for Adsorption 145
3.3.1.2 Evaluating the Effect of Adjusting Aqueous Phase Ionic Strength 146
3.3.2 100nm and 200nm P80-PS 149
3.3.3 20nm P80-PS 154
3.3.4 P80-FCS-PS 155
3.3.4.1 Zeta potential and Particle Size 155
3.3.4.2 Bradford’s Protein Assay 157
3.3.4.3 FCS Adsorption to Glass- and Plastic- ware 161
3.4 DISCUSSION 163
3.4.1 Interactions between FluoSphere™ Surfaces and Chitosan 163
3.4.1.1 Effect of pH on Chitosan Adsorption 164
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3.4.1.2 Effect of Excess Chitosan in Suspension 168
3.4.1.3 Chitosan Adsorption and pH Control 169
3.4.1.4 Chitosan Adsorption and Surface Curvature 170
3.4.1.5 Chitosan Adsorption and Aqueous Phase Ionic Strength 172
3.4.1.6 Other factors Influencing Chitosan Adsorption to Surfaces 173
3.4.2 Interactions between FluoSphere™ Surfaces and Polysorbate 80 174
3.4.2.1 Thickness of Adsorption Layer 175
3.4.2.2 The Influence of Adsorption on ζ Potential 177
3.4.2.3 Estimated Adsorption of Polysorbate 80 to FluoSpheres™ 178
3.4.3 Adsorption of FCS to 100nm P80-PS 178
3.5 CONCLUSIONS 182
4. DIRECT NOSE-TO-BRAIN NANOPARTICLE TRANSPORT STUDIES 185
4.1 INTRODUCTION 185
4.2 TRANSPORT STUDY USING THE FRANZ DIFFUSION CELL 186
4.2.1 Materials and Methods 186
4.2.1.1 Materials 186
4.2.1.2 Transport Study Protocol 187
4.2.1.3 Test Formulations 189
4.2.1.4 Spectrofluorometry 189
4.2.1.5 Histological Sample Preparation 191
4.2.1.6 Statistical Analysis 193
4.2.2 Results 193
4.2.2.1 The Influence of Particle Surface Modifications on Particle Transport 194
4.2.2.2 The Influence of Particle Diameter on Particle Transport 202
4.3 TRANSPORT STUDY USING THE IN VIVO MOUSE MODEL 205
4.3.1 Materials and Methods 205
4.3.1.1 Materials 205
4.3.1.2 Animals 206
4.3.1.3 Intranasal Administration 206
4.3.1.4 Test formulations 207
4.3.1.5 Histological and Imaging Procedures 208
4.3.1.6 Estimating Tissue Particle Numbers 209
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4.3.2 Results 210
4.3.2.1 Condition of Epithelia after Exposure to Citrate Buffer 211
4.3.2.2 The Influence of Particle Surface Modifications on Particle Transport 212
4.3.2.3 The Influence of Particle Diameter on Particle Transport 214
4.3.2.4 Affect of Olfactory and Non-Olfactory Nasal Epithelia on Particle Transport 215
4.4 DISCUSSION 215
4.4.1 The Influence of Particle Surface Modifications on Particle Transport 216
4.4.2 The Influence of Particle Diameter on Particle Transport 222
4.4.3 Toxicological Aspects of Nanoparticle Exposure to Olfactory Epithelium 224
4.4.4 Comparison of In vitro and In vivo Biological Models 231
4.4.5 Particle Transport into Olfactory and Non-olfactory Epithelia 234
4.4.6 Implications of Study Outcomes 235
4.5 CONCLUSIONS 236
5. SUMMARY 240
5.1 FINAL CONCLUSIONS 240
5.2 SIGNIFICANCE AND FUTURE POSSIBILITIES 243
REFERENCES 246
APPENDIX 263
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