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Liposomal drug carrier systems for inhalation treatment of lung cancer [Elektronische Ressource] / von Samah Anabousi

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145 pages
Ajouté le : 01 janvier 2006
Lecture(s) : 24
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Liposomal Drug Carrier Systems for Inhalation Treatment of Lung Cancer
















Dissertation
zur Erlangung des Grades
des Doktors der Naturwissenschaften
der Naturwissenschaftlich-Technischen Fakultät III
Chemie, Pharmazie, Bio- und Werkstoffwissenschaften
der Universität des Saarlandes











Von
Samah Anabousi

Saarbrücken
2006






"Gedruckt mit Unterstützung des Deutschen Akademischen Austauschdienstes"






































Tag des Kolloquiums: 04.September 2006

Dekan: Prof. Dr. Kaspar Hegetschweiler

Berichterstatter: Prof. Dr. Claus-Michael Lehr

Dr. Carsten Ehrhardt

Prof. Dr. Alexandra K. Kiemer

Die vorliegende Dissertation entstand unter der Betreuung von

Prof. Dr. Claus-Michael Lehr

in der Fachrichtung Biopharmazie und Pharmazeutische Technologie
der Universität des Saarlandes


Bei Herrn Prof. Lehr möchte ich mich für die Überlassung des Themas und die
wertvollen Anregungen und Diskussionen bedanken








































Dedicated to:





Mum's spirit and dad
Brother and sister,
My beloved husband,
The heart who flourishes my life



















Happiness keeps you sweet…
Trials keep you strong……...
Sorrows keep you human….
Failures keep you humble…
Success keeps you glowing…
But only God keeps you going.






TABLE OF CONTENTS
Page

Chapter 1
1. General introduction 1
1.1 Lung anatomy 2
1.2 cancer 3
1.3 Doxorubicin 6
1.4 Liposomal drug delivery systems 8
1.5 TfR-based targeting for anti-cancer therapeutics 12
1.6 Inhalation therapy 15
1.7 Liposomes for inhalation 17
1.8 Fate of inhaled particles 23
1.9 Conclusions 27
1.10 Setting objectives 29
1.11 References 30

Chapter 2
2. Expression pattern of CD71 in respiratory epithelial cells 34
2.1 Abstract 35
2.2 Introduction
2.2.1 Transferrin/transferrin receptors in tumors 36
2.2.2 Role of transferrin 37
2.2.3 Transferrin in the lung 37
2.2.4 receptor and lung cancer 38
2.3 Materials and methods
2.3.1 Cell culture conditions 41
2.3.2 FACS analysis 42
2.3.3 Immunofluorescence confocal laser scanning microscopy 43
2.4 Results
2.4.1 FACS 44
2.4.2 Transferrin receptor localisation 47
2.5 Discussion 48
2.6 References 49
Chapter 3
3. Tf conjugation to liposomal surfaces 52
3.1 Abstract 53
3.2 Introduction
3.2.1 Advantages of ligand-targeted liposomes 54
3.2.2 Choice of target receptor 55
3.2.3 Internalisation 56
3.2.4 Chemical linkage 56
3.2.5 Techniques for coupling ligands to liposomes 58
3.2.6 Coupling strategies 59
3.2.7 Transmission electron microscopy 60
3.2.8 Atomic force microscopy 60
3.3 Materials and methods
3.3.1 Materials 63
3.3.2 Liposome preparation 63
3.3.3 Coupling of transferrin to the liposomes 63
3.3.4 Photon correlation spectroscopy 66
3.3.5 ζ-potential measurements 66
3.3.6 Phospholipid concentration 66
3.3.7 Protein assay and Tf-binding efficacy 67
3.4.8 Transmission electron microscopy 67
3.3.9 Atomic force microscopy 68
3.3.10 Statistical analysis 68
3.4 Results
3.4.1 Characterisation of liposomes 69
3.4.2 Tf-binding efficacy 70
3.4.3 Transmission electron microscopy 70
3.4.4 Atomic force microscopy72
3.5 Discussion 74
3.6 References 75


Chapter 4
4. Binding and uptake of liposomal formulations 78
4.1 Abstract 79
4.2 Introduction
4.2.1 Drug resistance in cancerous tissues 80
4.2.2 Overcoming drug resistance by liposomes 80
4.2.3 Liposomal doxorubicin as anti-neoplastic 82
4.2.4 Loading of liposomes with drugs 82
4.3 Materials and methods
4.3.1 Cell culture conditions 85
4.3.2 Liposome preparation 86
4.3.3 Uptake studies 87
4.3.4 Cytotoxicity assay 87
4.3.5 Statistical analysis 87
4.4 Results
4.4.1 Characterisation of liposomes 88
4.4.2 Uptake studies 88
4.4.3 Cytotoxicity assay89
4.5 Discussion 91
4.6 References 94

Chapter 5
5. Nebulisation of liposomal formulations 96
5.1 Abstract 97
5.2 Introduction
5.2.1 Inhalation therapy 98
5.2.2 Advantages of liposomes for inhalation 99
5.2.3 Inhalational chemotherapeutic agents for treatment of
lung cancer 102
5.2.4 Nebulisers 103
5.2.4.1 Air-jet nebuliser 104
5.2.4.2 Ultrasonic nebuliser 106
5.2.5 Lung surfactant 106 5.2.6 Challenges 106

5.3 Materials and methods
5.3.1 Liposome preparation 108
5.3.2 Photon correlation spectroscopy 108
5.3.3 ζ-potential measurements 109
5.3.4 Atomic force microscopy 109
5.3.5 Liposome membrane integrity after nebulisation 109
5.3.6 Liposome membrane integrity in lung surfactant 110
5.3.7 Statistical analysis 111
5.4 Results
5.4.1 Characterisation of liposomes 112
5.4.2 Stability after nebulisation 112
5.4.3 Stability in lung surfactant116
5.5 Discussion 117
5.6 References 120

Summary and outlook 122
1. Summary 123
2. Zusammenfassung 126
3. Outlook 129

Appendices 130
List of abbreviations 131
Curriculum vitae 133
List of publications 134
Acknowledgements 136



1. General introduction















Chapter 1


General introduction
















1 1. General introduction

1.1 Lung anatomy
The lung is the body’s organ of respiration. The trachea carries air into the body from the
nose/mouth. It then splits into two bronchi which branch off into the left and right lungs.
These primary bronchi branch into secondary bronchi, which in turn branch into tertiary
bronchi which then become bronchioles. Each level of branching sees a decrease in diameter
and an increase in the number of bronchioles. The bronchi can have diameters > 5 mm while
terminal bronchiole diameter can be as small as 5-10 µm. Their diameter is controlled by
bronchial smooth muscle which is under autonomic control. At the end of the terminal
bronchioles are the alveoli. The alveoli are the site of gaseous exchange in the lung.
Pulmonary capillaries surrounding the alveoli carry CO and O to and from the alveoli, 2 2
respectively. The alveolar and micro capillary walls together make up what is known as the
respiratory membrane. This consists of the alveolar epithelial lining (a layer of type I and type
II alveolar cells and associated alveolar macrophages), an epithelial basement membrane, a
capillary basement membrane and endothelial cells of the capillary (figure 1). Despite having
several layers, the respiratory membrane is very thin (~ 0.5 µm). This allows for the rapid
diffusion of O and CO across the membrane. It has been estimated that the lungs contain 2 2
8 2over 3 ×10 alveoli giving it a surface area of about 70-100 m for the exchange of gases [1].
In air-breathing animals, respiratory anatomy has evolved in such a way as to actively thwart
inhalation of putative airborne particulates.
A B
Figure 1: A) The anatomy of the human lung (taken from www.aduk.org.uk/gfx/lungs) B) Alveoli of the
terminal bronchioles. They are covered in capillaries and it is here that gaseous exchange takes place (taken from
http://en.wikipedia.org/wiki/Alveoli).
2

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