Calcium phosphate nanoparticles as carriers of nucleic acids [Elektronische Ressource] / vorgelegt von Anna Kovtun

Anna Kovtun

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2009
• Nombre de lectures: 25
• Poids de l'ouvrage: 4 Mo

Extrait

Calcium phosphate nanoparticles as carriers of
nucleic acids

Dissertation

Zur Erlangung des akademischen Grades
eines
Doktors der Naturwissenschaften
– Dr. rer. nat. –

vorgelegt von

Anna Kovtun
aus Kharkiv / Ukraine


Institut für Anorganische Chemie
der
Universität Duisburg-Essen
Essen 2009 Anna Kovtun, Dissertation 2009









Dedicated to my parents and my wonderful friend Olga
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Anna Kovtun, Dissertation 2009

Die vorliegende Arbeit wurde im Zeitraum von April 2006 bis September 2009
am Institut für Anorganische Chemie, Arbeitskreis Prof. Dr. Matthias Epple, der
Universität Duisburg-Essen durchgeführt.


1. Gutachter: Prof. Dr. M. Epple
2. Gutachter: Prof. Dr. C. Schmuck
3. Gutachter: Prof. Dr. W. E. G. Müller
Vorsitzender: Prof. Dr. B. Siebers

Tag der mündlichen Prüfung: 26.05.2010

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Anna Kovtun, Dissertation 2009

List of contents

1 Introduction ........................................................................................................ 8
2 Theoretical background .................... 10
2.1 Nucleic acids .............................. 10
2.1.1 Chemical structure of desoxyribonucleic acid (DNA) ........................ 10
2.1.2 Chemical structure of ribonucleic acid (RNA) .................................... 11
2.1.3 Biological role of nucleic acids ............................................................ 13
2.2 Mechanism of RNA interference ............................... 13
2.3 Oligonucleotides for the maturation of dendritic cells .............................. 16
2.4 Transfection ................................................................................................ 17
2.5 Calcium phosphate nanoparticles as carriers of nucleic acids ................... 19
2.6 Colloid stability and DLVO theory ............................ 22
3 Materials and Methods ..................................................................................... 24
3.1 Applied materials for cell culture experiments .......... 24
3.1.1 Cell culture solutions/antibiotics.......................... 24
3.1.2 Chemicals ............................................................................................. 24
3.1.3 Instruments ........................... 24
3.1.4 Applied consumables und kits 25
3.2 Molecular-biological methods ................................................................... 25
3.2.1 Preparation of plasmid DNA from bacterial culture ............................ 25
3.3 Cell culture methods and experimental procedures ... 26
3.3.1 Cultivation of secondary cell lines ....................................................... 27
3.3.2 Cryoconservation of cells .................................... 27
3.3.3 Defrosting of cells ................................................ 28
3.3.4 Fluorescence microscopy ..... 28
3+3.3.5 Preparation of calcium phosphate nanoparticles substituted with Al
2+and Mg ........................................................................................................ 28
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Anna Kovtun, Dissertation 2009

3.3.6 Cell transfection ................................................................................... 29
3.3.7 Calculation of transfection efficiency of magnesium- or aluminum-
substituted calcium phosphate nanoparticles ................ 31
3.3.8 Ca-imaging using Fura-2AM ............................................................... 32
3.3.9 Staining of cells with propidium iodide ............................................... 32
3.3.10 Toxicity tests ...................................................... 33
453.3.11 Experiments with radioactive Ca .................... 33
3.3.12 Preparation of cell lysates .................................................................. 36
3.3.13 Determination of the protein concentration ....... 36
3.3.14 Preparation of metal surfaces coated with calcium
phosphate/PEI/DNA-nanoparticles ............................................................... 37
3.3.15 Transfection from metal surfaces....................... 37
3.3.16 Cell fixation ........................................................ 39
3.3.17 Flow cytometry of NIH3T3 cells ....................................................... 39
3.3.18 Preparation of multi-shell calcium phosphate nanoparticles
functionalized with siRNA............................................................................ 40
3.3.19 Preparation of calcium phosphate nanoparticles functionalized with
CpG and Poly(I:C) ........................................................................................ 40
3.3.20 Preparation of calcium phosphate nanoparticles functionalized with
shRNA ........................................................................................................... 41
3.4 Physicochemical methods .......... 42
3.4.1 Dynamic light scattering ...... 42
3.4.2 Zeta potential measurements ................................................................ 42
3.4.3 Scanning electron microscopy (SEM) ................................................. 43
3.4.4 Transmission electron microscopy (TEM) .......... 44
4 Results and Discussion ..................................................... 45
3+ 2+4.1 Calcium phosphate nanoparticles substituted with Al and Mg ............ 45
4.1.1 Synthesis and characterization of calcium phosphate substituted with
3+ 2+Al and Mg ................................................................................................ 45
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Anna Kovtun, Dissertation 2009

4.1.2 Transfection with calcium phosphate nanoparticles substituted with
3+ 2+Al and Mg ................................................................................................ 55
4.1.3 Conclusion ........................... 57
4.2 Tracking of the intracellular calcium level during the transfection of cells
.......................................................................................................................... 59
45 2+4.2.1 Marking of nanoparticles with Ca .................. 59
4.2.2 Monitoring of the internal calcium level using the calcium-sensitive
dye Fura-2 ..................................................................................................... 70
4.2.3 Conclusion ........................... 80
4.3 Calcium phosphate nanoparticles functionalized with siRNA against EGFP
.......................................................................................................................... 81
4.3.1 Synthesis and characterization of siRNA-functionalized calcium
phosphate nanoparticles ................ 81
4.3.2 Gene silencing on HeLa-EGFP cells by siRNA-functionalized calcium
phosphate nanoparticles ................................................................................ 83
4.3.3 Conclusion ........................... 89
4.4 Calcium phosphate nanoparticles functionalized with shRNAs ................ 90
4.4.1 Synthesis and characterization of the nanoparticles functionalized with
shRNAs ......................................................................................................... 90
4.4.2 Gene silencing using calcium phosphate nanoparticles functionalized
with shRNAs . 93
4.4.3 Conclusion ........................................................................................... 99
4.5 Functionalization of nanoparticles with oligonucleotides for the activation
of dendritic cells ............................. 100
4.5.1 Synthesis and characterization of the nanoparticles functionalized with
Poly(I:C) and CpG ...................................................................................... 100
4.5.2 Activation of dendritic cells using nanoparticles functionalized with
Poly(I:C) and CpG ...................... 102
4.5.3 Conclusion ......................................................................................... 105
4.6 Surface-mediated transfection with DNA-functionalized calcium
phosphate nanoparticles ................. 106
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Anna Kovtun, Dissertation 2009

4.6.1 Preparation of electrophoretically coated titanium substrates ........... 106
4.6.2 Surface-mediated transfection of cells ............................................... 108
4.6.3 Conclusion ......................................................... 119
5 Summary......................................................................... 120
6 Literature ........................................ 122
7 Appendix ........ 133
7.1 List of abbreviations ................................................................................. 133
7.2 Publications .............................. 135
7.2.1 Regular papers in refereed journals ................... 135
7.2.2 Other publications .............................................................................. 136
7.3 Presentations and posters ......... 138
7.4 Patents ...................................... 144
7.5 Awards ............................................................