Establishment of magnetofection [Elektronische Ressource] : a novel method using superparamagnetic nanoparticles and magnetic force to enhance and to target nucleic acid delivery / vorgelegt von Franz Scherer

Franz Scherer

Sujets

• Gesundheit

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 13
• Langue: English

Extrait

Dissertation
zur Erlangung des Doktorgrades der Fakultät für Chemie und
Pharmazie der Ludwig-Maximilians-Universität München




Establishment of magnetofection –
a novel method using superparamagnetic nanoparticles and
magnetic force to enhance and to target nucleic acid delivery


vorgelegt von
Franz Scherer
aus Bad Tölz
2006
Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Herrn Professor Dr. Ernst Wagner betreut.



Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.



München, am 21.02.06, Franz Scherer












Dissertation eingereicht am 21.02.06
1. Gutachter Prof. Dr. Ernst Wagner
2. Gutachter PD Dr. Christian Plank (TU München)
Mündliche Prüfung am 23.05.06








To my parents














ACKNOWLEDGMENTS

Lots of thanks to Prof. Dr. Ernst Wagner for the supervision of this thesis and for his
understanding and patience.
Foremost, I want to thank group leader PD Dr. Christian Plank (group “nonviral gene
vectors”, Institute of Experimental Oncology and Therapy Research, Klinikum Rechts der
Isar) for his intensive supervision in the experiments and in writing the dissertation. Somehow
he understood to fill me with enthusiasm for scientific work.
Many thanks also to Prof. Dr. Bernd Gänsbacher for the possibility to work in his “Institute of
Experimental Oncology and Therapy Research” (Klinikum Rechts der Isar) and for his
interest and encouragement.
For help and support in my project thanks to all colleagues in laboratory 1.39 (group
“nonviral gene vectors”), especially to Ursula Putz and Dr. Ulrike Schillinger.
Very much I enjoyed the many after-work discussions, usually with Christian Plank, Christian
Koch and Ulrike Schillinger in “Unions Bräu”, “Pinguin” or a “Biergarten”.
In particular, I feel the wish to thank the following people for their extraordinary engagement
during my illness: Dr. Ulrike Schillinger, Ursula Putz, Dr. Hubert Schönberger, Sabine
Brandt, PD Dr. Christian Plank, Prof. Dr. Axel Stemberger, Matthias Strobl, Prof. Dr. Bernd
Gänsbacher (all from the Institute of Experimental Oncology and Therapy Research) and PD
Dr. Joseph Rosenecker (Department of Pediatrics, LMU Munich). You all helped me very
much!
Finally, my very special thanks to my parents who always encouraged me in my plans and
without whom all my education would not have been possible. TABLE OF CONTENTS
1 INTRODUCTION..............................................................................................................9
1.1 Nucleic acids as drugs................................................................................................ 9
1.2 Delivery of nucleic acids............................................................................................ 9
1.3 Localized drug and nucleic acid delivery................................................................. 11
1.3.1 The importance of localized delivery............................................................... 11
1.3.2 Hierarchies of localization (targeting).............................................................. 13
1.3.3 Passive and active targeting ............................................................................. 13
1.4 Biological methods of targeting applied in research up to now............................... 14
1.4.1 Receptor-ligand interactions............................................................................14
1.4.2 Localization sequences.....................................................................................16
1.4.3 Site-specific genomic integration..................................................................... 17
1.5 Biological methods of local control applied in research up to now ......................... 18
1.5.1 Tissue-specific and inducible promoters (“transcriptional targeting”) ............ 18
1.5.2 Activation of prodrugs 19
1.5.3 Triggering localized drug delivery................................................................... 19
1.6 Physical methods of targeting applied in research up to now.................................. 20
1.6.1 Gravitational force............................................................................................20
1.6.2 Local injection..................................................................................................21
1.6.3 Intravascular delivery combined with occlusion of the blood outflow from the
target organ ....................................................................................................... 21
1.6.4 Hydrodynamic force.........................................................................................22
1.6.5 Aerosolization
1.6.6 Ballistic methods..............................................................................................23
1.6.7 Systems for controlled drug release .................................................................
1.6.8 Electric fields....................................................................................................24
1.6.9 Magnetic drug targeting ................................................................................... 24
1.7 Physical methods of local control applied in research up to now ............................ 25
1.7.1 Stress-inducible promoters (“transcriptional targeting”) ................................. 25
1.7.2 Triggering localized drug delivery................................................................... 25
1.8 The development of magnetic drug targeting and its current state .......................... 26
1.9 Topic of this thesis ................................................................................................... 29
2 MATERIALS AND METHODS ..................................................................................... 31
2.1 Abbreviations, reagents and materials ..................................................................... 31
2.2 General methods.......................................................................................................37
322.2.1 Radioactive ( P) labeling of plasmid DNA by nick translation......................
2.2.2 Cell culture, transfection and reporter gene assays.......................................... 37
2.2.2.1 Cells..............................................................................................................37
2.2.2.2 Transfection..................................................................................................38
2.2.2.3 Luciferase assay...........................................................................................39
2.2.2.4 β-Galactosidase assay................................................................................... 40
2.2.3 Preparation of DOTAP-Cholesterol cationic liposomes .................................. 40 2.2.4 Preparation of polyethylenimine (PEI) ............................................................ 41
2.2.5 Biotinylation of PEI (bPEI).............................................................................. 41
2.2.6 Coupling of streptavidin to trMAG-PEI (trMAG-PEI-Sta) ............................. 41
2.3 Characteristics of magnetic nanoparticles (trMAGs) used in this study.................. 43
2.3.1 Measurement of particle size by dynamic light scattering............................... 43
2.3.2 Transmission electron microscopy of trMAGs ................................................ 43
2.4 Binding of DNA to magnetic particles..................................................................... 44
2.4.1 Examination of trMAG-PEI as representative for positively charged magnetic
beads with a monolayer of PEI......................................................................... 44
2.4.1.1 Ninhydrin assay to determine the amount of PEI in trMAG-PEI particle
suspensions................................................................................................... 44
2.4.1.2 DNA-binding curves....................................................................................45
2.4.1.3 Measurement of zeta potential by laser Doppler velocimetry (LDV).......... 46
2.4.1.4 Particle sizes in 150 mM NaCl 47
2.4.1.5 Transmission electron microscopy............................................................... 47
2.4.2 Examination of trMAG-16/1 as representative for positively charged magnetic
beads with a multilayer of PEI.......................................................................... 48
2.4.2.1 Ninhydrin assay to determine the amount of PEI in trMAG-16/1 particle
suspensions................................................................................................... 48
2.4.2.2 DNA-binding curve......................................................................................48
2.4.2.3 Measurement of zeta potential by laser Doppler velocimetry (LDV).......... 48
2.4.2.4 Transmission electron microscopy.......................................................