Synthesis, characterization and application of calcium phosphate nanoparticles for the transfection of cells [Elektronische Ressource] / vorgelegt von Viktoriya Sokolova

Hu201

Sujets

• Biologie

Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2006
• Nombre de lectures: 48
• Langue: English
• Poids de l'ouvrage: 19 Mo

Extrait

Synthesis, Characterization and Application of
Calcium Phosphate Nanoparticles for the
Transfection of Cells

Dissertation


zur Erlangung des Grades
eines Doktors der Naturwissenschaften
vorgelegt von


Viktoriya Sokolova
aus Kharkiv / Ukraine


Fachbereich Chemie
der
Universität Duisburg-Essen

Essen 2006







Dedicated to my husband and my parents
Viktoriya Sokolova, Dissertation 2006










Die vorliegende Arbeit wurde in der Zeit von September 2003 bis Oktober 2003
am Lehrstuhl für Anorganische Chemie I, Arbeitskreis Prof. Dr. Matthias Epple
(Festkörperchemie), an der Ruhr-Universität Bochum und in der Zeit von
Oktober 2003 bis Mai 2006 am Institut für Anorganische Chemie, Arbeitskreis
Prof. Dr. Matthias Epple, an der Universität Duisburg-Essen angefertigt.


1. Gutachter: Prof. Dr. M. Epple
2. Gutachter: . W. Streit

Tag der mündlichen Prüfung: 17.10.2006
2Viktoriya Sokolova, Dissertation 2006
List of contents
1 INTRODUCTION...................................................................................6
2 THEORETICAL BACKGROUND..........................................................8
2.1 Nanoparticles and Nanotechnology............................................................. 8
2.1.1 Colloids..................................................................................................... 9
2.1.2 The electrical double layer...................................................................... 10
2.1.3 Colloid stability and DLVO theory ........................................................ 12
2.1.4 Aging and coagulation of colloids.......................................................... 13
2.1.5 Stabilization of colloidal systems ........................................................... 15
2.1.6 Calcium phosphate.................................................................................. 15
2.2 Biopolymers applied for the stabilization of calcium phosphate colloids
..................................................................................................................... 18
2.2.1 Desoxyribonucleic acid (DNA) .............................................................. 18
2.2.2 Ribonucleic acid (RNA) ......................................................................... 20
2.2.3 Oligonucleotides ..................................................................................... 20
2.2.4 Proteins ................................................................................................... 21
2.3 Transfection methods.................................................................................. 24
2.3.1 Viral gene delivery systems.................................................................... 24
2.3.2 Physical methods .................................................................................... 25
2.3.3 Chemical methods................................................................................... 26
2.4 RNA interference......................................................................................... 31
3 RESULTS AND DISCUSSION ...........................................................33
3.1 Synthesis and characterization of DNA-functionalized calcium
phosphate nanoparticles and their role as carriers for transfection .... 33
3.1.1 Plasmid DNA, pcDNA3-EGFP, applied for transfection....................... 37
3.1.2. Optimization of calcium and phosphate concentration and the amount of
DNA ....................................................................................................... 38
3.1.3. Optimization and characterization of calcium phosphate/DNA
nanoparticles with additions of magnesium and aluminum................... 41
3.1.4 Transfection experiments with calcium phosphate/DNA nanoparticles 49
3.1.5 Conclusion .............................................................................................. 54
3.2 Multi-shell calcium phosphate/DNA nanoparticles as carriers for the
transfection................................................................................................. 55
3.2.1 Characterization of multi-shell calcium phosphate/DNA nanoparticles 55
3.2.2. Transfection efficiency of multi-shell nanoparticles............................. 62
3.2.3 Conclusion 68
3Viktoriya Sokolova, Dissertation 2006
3.3 Calcium phosphate colloids containing protamine: Colloidal and
biochemical properties .............................................................................. 69
3.3.1 Mechanism of the transfer of calcium phosphate/DNA/protamine
nanoparticles........................................................................................... 70
3.3.2 Characterization of colloids with protamine .......................................... 72
3.3.3 Transfection efficiency of calcium phosphate/DNA/protamine
nanoparticles 75
3.3.4 Conclusion .............................................................................................. 83
3.4 Tracking the pathway of calcium phosphate/DNA.................................. 84
3.4.1 Particle characterization.......................................................................... 85
3.4.2 Experiments with cells............................................................................ 87
3.4.3 Conclusions............................................................................................. 93
3.5 Functionalization of calcium phosphate nanoparticles by
oligonucleotides and their application for gene silencing...................... 94
3.5.1 Characterization of calcium phosphate/oligonucleotide colloids........... 95
3.5.2. Gene silencing experiments on HeLa-EGFP cells .............................. 102
3.5.3 Conclusion ............................................................................................ 107
3.6 Synthesis of carbonated apatite with the model protein ubiquitin by the
co-precipitation method .......................................................................... 108
3.6.1 Characterization of the ubiquitin-calcium phosphate samples............. 109
3.6.2 Measurement of the incorporation of radioactive ubiquitin into the
particles................................................................................................. 114
3.6.3 Conclusion 118
4 MATERIALS AND METHODS..........................................................119
4.1 Applied materials for cell culture experiments ...................................... 119
4.1.1 Cell culture solutions/antibiotics 119
4.1.2 Chemicals.............................................................................................. 119
4.1.3 Devices.................................................................................................. 119
4.1.4 Applied consumables und kits.............................................................. 120
4.2 Molecular-biological methods .................................................................. 120
4.2.1 Preparation of plasmid-DNA from bacterial culture ............................ 120
4.2.2 Synthesis of oligonucleotides ............................................................... 121
4.2.3 Fluorescence microscopy...................................................................... 122
4.3. Cell culture methods ................................................................................ 122
4.3.1 Cultivation of secondary cell lines ....................................................... 122
4.3.2 Cryoconservation and defrosting of cells ............................................. 123
4.3.3 Cell transfection.................................................................................... 123
4.3.4 Transport of nanoparticles into the cells............................................... 125
4Viktoriya Sokolova, Dissertation 2006
4.3.5 Gene silencing experiments.................................................................. 126
4.4 Physicochemical methods ......................................................................... 128
4.4.1 Analytical ultracentrifugation............................................................... 128
4.4.2 Dynamic light scattering (DLS)............................................................ 128
4.4.3 Zeta potential measurements (ZP)........................................................ 129
4.4.4 Infrared spectroscopy (IR).................................................................... 130
4.4.5 X-ray powder diffractometry (XRD).................................................... 131
4.4.6 Scanning electron microscopy (SEM).................................................. 132
4.4.7 Transmission electron microscopy (TEM) ........................................... 133
4.5 Experimental procedures ......................................................................... 134
4.5.1 The preparation of calcium phosphate/DNA colloids .......................... 134
4.5.2 Preparation of multi-shell nanoparticles............................................... 134
4.5.3 Preparation of calcium phosphate/DNA/protamine nanoparticles....... 135
4.5.4 Prepar