Dynamic endosomolytic polymer conjugates for pDNA and siRNA delivery [Elektronische Ressource] / vorgelegt von Martin Meyer

Martin Meyer

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 23
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

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



Dynamic endosomolytic polymer conjugates for
pDNA and siRNA delivery

vorgelegt von
Martin Meyer
aus Ludwigshafen am Rhein
2008Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 der Promotionsordnung vom
29. Januar 1998 von Professor Dr. Ernst Wagner betreut.



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


München, am …………………….







……………………………
(Unterschrift des Autors)



Dissertation eingereicht am 13.11.2008
1. Gutacher: Prof. Dr. Ernst Wagner
2. Gutacher: Prof. Dr. Wolfgang Frieß
Mündliche Prüfung am 15.01.2009


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Meinen Eltern













3



The outcome of any serious research can only be to make
two questions grow where only one grew before.



Thorstein Veblen (1857-1929)
amerikanischer Soziologe und Ökonom














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Table of Contents
1 INTRODUCTION ........................................................................................... 1
1.1 Nucleic acid-based therapy .................................................................................................. 1
1.2 pDNA and siRNA delivery ..................................................................................................... 2
1.3 Physiological barriers of nucleic acid delivery .................................................................. 4
1.4 Overcoming endosomal entrapment ................................................................................... 6
1.4.1 Natural models......................................................................................................................... 6
1.4.2 Artificial approaches................................................................................................................. 9
1.5 Design of programmed nucleic acid carriers.................................................................... 11
1.5.1 Redox- and enzyme-sensitive systems ................................................................................. 12
1.5.2 pH-sensitive systems............................................................................................................. 13
1.6 Aims of the thesis ................................................................................................................ 15
2 MATERIAL AND METHODS....................................................................... 17
2.1 Chemicals and reagents...................................................................................................... 17
2.2 Conjugate synthesis............................................................................................................ 18
2.2.1 Synthesis of 3-(2-pyridyldithio)propionate-modified PLL....................................................... 18
2.2.2 Synthesis of PEG-modified PEI and PLL............................................................................... 18
2.2.3 Synthesis of 3-(2-pyridyldithio)propionate-modified PEI- and PLL-PEG ............................... 19
2.2.4 Synthesis of PLL-Mel ............................................................................................................. 19
2.2.5 Synthesis of DMMAn-Mel-modified PEI and PLL .................................................................. 19
2.2.6 Synthesis DMMAn-Mel-modified PEI- and PLL-PEG ............................................................ 20
2.2.7 Synthesis of INF-modified PEI- and PLL-PEG ...................................................................... 20
2.2.8 Synthesis of Mel-modified PEI- and PLL-PEG ...................................................................... 21
2.2.9 Synthesis of succinic anhydride-melittin-modified PEI-PEG ................................................. 21
2.2.10 Deprotection of thiol-modified siRNA..................................................................................... 21
2.2.11 Synthesis of PLL-PEG-DMMAn-Melittin-siRNA..................................................................... 22
2.2.12 Quantitative analysis of PEI and PLL (TNBS assay)............................................................. 22
2.2.13 Determination of PEG to polycation ratio............................................................................... 23
2.2.14 Ellman’s assay....................................................................................................................... 24
2.3 Polyplex formation............................................................................................................... 24
2.4 Measurement of zeta-potential ........................................................................................... 24
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2.5 Measurement of particle size via dynamic light scattering (DLS) .................................. 25
2.6 Measurement of particle size via fluorescence correlation spectroscopy.................... 25
2.7 Ethidium bromide exclusion assay.................................................................................... 26
2.8 Agarose gel electrophoresis .............................................................................................. 27
2.9 Erythrocyte leakage assay.................................................................................................. 27
2.10 Cell culture ........................................................................................................................... 27
2.11 Luciferase gene silencing................................................................................................... 28
2.12 Luciferase reporter gene expression................................................................................. 28
2.13 Cell viability assay (MTT assay) ......................................................................................... 29
2.14 Cytotoxicity assay (LDH release assay)............................................................................ 29
2.15 Transmission light microscopy.......................................................................................... 29
2.16 Cy5 labeling of pDNA .......................................................................................................... 29
2.17 Flow cytometric analysis of cellular polyplex internalization......................................... 30
3 RESULTS.................................................................................................... 31
3.1 A dimethylmaleic acid–melittin-polylysine conjugate for pDNA delivery...................... 31
3.1.1 Design and synthesis of the DMMAn-modified PLL conjugate.............................................. 31
3.1.2 pH-dependent lytic activity..................................................................................................... 33
3.1.3 Gene transfer activity............................................................................................................. 34
3.1.4 Biophysical characterization .................................................................................................. 37
3.1.5 Cellular toxicity....................................................................................................................... 39
3.2 PEGylated endosomolytic conjugates for pDNA and siRNA delivery ........................... 42
3.2.1 Design and synthesis of the conjugates ................................................................................ 42
3.2.2 pDNA-delivery........................................................................................................................ 45
3.2.2.1 PEI-PEG-conjugates................................................................................................................. 45
3.2.2.2 PLL-PEG-conjugates ................................................................................................................ 48
3.2.3 siRNA-delivery ....................................................................................................................... 51
3.2.3.1 PEI-PEG-conjugates................................................................................................................. 52
3.2.3.2 PLL-PEG-conjugates ................................................................................................................ 56
3.3 Conjugate containing covalently attached siRNA for improved delivery...................... 60
3.3.1 Design and synthesis of the programmed siRNA carrier....................................................... 60
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3.3.2 siRNA delivery efficiency and cytotoxicity.............................................................................. 63
3.3.3 Glutathione induced release of siRNA................................................................................... 65
3.3.4 Particle size................................................................................