Specific enzymatic cleavage and payload release from peptide-based hybrid nanocapsules [Elektronische Ressource] / Julien Andrieu

Julien Andrieu

Sujets

• Gesundheit

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2011
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

Specific enzymatic cleavage
and payload release
from peptide-based hybrid nanocapsules



Dissertation


zur Erlangung des Grades
"Doktor der Naturwissenschaften"
im Promotionsfach Chemie


am Fachbereich Chemie, Pharmazie und Geowissenschaften
der Johannes Gutenberg-Universität in Mainz


vorgelegt von


Julien Andrieu
geboren in Nantes (Frankreich)






Mainz, 2011
















Diese Dissertation wurde in der Zeit von Oktober 2008 bis November 2011 im Arbeitskreis
Physikalische Chemie der Polymere am Max-Planck-Institut für Polymerforschung in Mainz
erstellt.
Ich versichere hiermit, dass ich diese Arbeit selbstständig angefertigt habe und keine
anderen als die angegeben Quellen und Hilfsmittel benutzt sowie die wörtlich oder inhaltlich
übernommenen Stellen als solche kenntlich gemacht habe.







































Tag der mündliche Prüfung: 7. Dezember 2011




















A mes quatre grands-parents,





Table of contents


I Introduction & Motivation……………………… 1


II Theoretical background……………………..…. 5

II.1 Responsive materials……………………………………………………………. 6
II.1.1 Temperature-responsive materials………………………………………. 6
II.1.2 pH-responsive materials………………………………………………… 7
II.1.3 Light-responsive materials………………………………………………. 8
II.1.4 Redox-responsive materials……………………………………………… 9
II.1.5 Enzyme-responsive materials 10

II.2 Bioconjugates……………………………………………………………………. 11
II.2.1 Presentation of bioconjugates…………………………………………... 11
II 2.2 Defined peptide-polymer conjugates…………………………………… 11

II.3 Triggered release via enzymatic cleavage……………………………………… 14

II.4 Solid-phase peptide synthesis…………………………………………………… 16
II.4.1 Principle of the method 16
II.4.2 Mechanistic approach… 19

II.5 Miniemulsion…………………………………………………………………….. 24
II.5.1 Polymerization in heterophase………………………………………….. 24
II.5.2 Miniemulsion principle………………………………………………….. 25
II.5.3 Inverse miniemulsion…………………………………………………… 29
II.5.4 Preparation of capsules via miniemulsion………………………………. 30

II.6 Polyaddition……………………………………………………………………… 36
II.6.1 Characteristics of polyaddition…………………………………………. 36
II.6.2 Reactivity of the isocyanate group……………………………………… 38


III Relevant methods for characterization…….….. 43

III.1 MALDI-TOF mass spectrometry……………………………………………. 44

III.2 Size-exclusion chromatography……………………………………………… 45

III.3 DOSY-NMR spectroscopy…………………………………………………… 46

III.4 Electron Microscopy …………………………………………………………. 48



III.5 Light scattering……………………………………………………………….. 50

III.6 Absorption and fluorescence………………………………………………… 51
III.6.1 Absorption and UV-Vis Spectroscopy ………………………………. 51
III.6.2 Fluorescence ………………………………………………………… 52


IV. Results & discussion…………………………. 59

Strategy……………………..……………………………………… 60

IV.1 Synthesis, characterization and cleavage of linear peptide-polymer conjugates 62
IV.1.1 Peptide-polymer conjugates via coupling in solution ……………………. 62
IV.1.2 Triblock peptide-polymer conjugates via coupling on solid phase ……… 63
IV.1.3 Enzymatic cleavage of triblock peptide-polymer conjugates…………. 73

IV.2 Development of a method to prepare peptide based hybrids nanocapsules … 75
IV 2.1 Choice of the surfactant ………………………………………………… 76
IV 2.2 Optimization of synthetic parameters…………………………………… 76
IV 2.3 Characterization of the peptide-based structures by electron microscopy.. 81

IV.3 Synthesis, characterization, and cleavage of hybrid nanocapsules with
FRET pair Mant/Y(NO )…………………………………………………………… 84 2
IV.3.1 Peptide used for capsules preparation …………………………………. 84
IV.3.2 Preparation and characterization of cleavable capsules with FRET pair… 88
IV.3.3 Redispersion in water and enzymatic cleavage experiments……………. 94
IV.3.4 Change to the FRET pair Mca/Dnp ………………………………….. 97
IV.3.5 Redispersion in water and enzymatic cleavage experiments…………… 103
IV.3.6 On the solubility of the polymer and phase separation………………… 105

IV.4 Synthesis, characterization, and cleavage of peptide based hybrid
nanocapsules loaded with fluorescing polymer……………………………… 107
IV.4.1 Synthesis of the capsules & possible influence of encapsulated polymer
on the capsules…...............…………………………………………………….. 107
IV.4.2 Investigation with electron microscopy………………………………… 109
IV.4.3 Redispersion into water and preconditioning…………………………… 110
IV.4.4 Cleavage of nanocapsules with encapsulated fluorescent polymer ……… 112

IV.5 Synthesis, characterization, and cleavage of peptide based hybrid
nanocapsules with release of fluorophore………………………………….... 113
IV.5.1 Synthesis of the capsules – Size and morphology……………………… 113
IV.5.2 Improved redispersion into water with mild ultrasonication…………… 115
IV.5.3 Influence of TDI equivalent and functionality of the peptide after the
encapsulation of sulforhodamine…………….……………….………………… 118
IV.5.4 Release of payload via enzymatic cleavage of peptide-based nanocapsules 122
IV.5.5 Quantitative approach of cleavage and release…………………………. 126

IV.6 Hepsin-cleavable nanocapsules……………………………………………… 129
IV.6.1 Identification of hepsin-cleavable sequence ……….. 129
IV.6.2 Preparation of the capsules ……………………………………….….. 131
IV.6.3 Cleavage of the capsules in cell culture ………………………………. 132




V Conclusion and perspectives…………………… 135

VI Experimental part………… 139

VI.1 Materials………………………………………………………………………… 140
VI.1.1 Chemicals………………………..……………………………………... 140
VI.1.2 Synthesis of carboxylic acid end-functionalized polystyrene by anionic
polymerization ……………………..………………………………..…………………. 141
VI.1.3 Radical synthesis of poly(vinylpyrrolidone-co-bodipy)…………………... 142

VI.2 Synthesis and preparation of peptides………………………………………… 144
VI.2.1 Solid-Phase Peptide Synthesis (SPPS)…………………………………... 144
VI.2.2 Peptides…………………………………………………………………. 145

VI.3 Peptide-polymer coupling reactions…………………………………………… 150
VI.3.1 Peptide-polymer coupling in solution…………………………………... 151
VI.3.2 P-polying on solid phase……………………………… 151

VI.4 Preparation of peptide-based nanocapsules in miniemulsion……………….. 152

VI.5 Appendix………………………………………………………………………… 154
VI.5.1 Methods………………………………………………………………… 154
VI.5.2 Summary of FRET-peptide based hybrid nanocapsules………………… 158


Abbreviation & Symbols…………………………………………………………… 159

Bibliographic references …………………………………………………………… 161

Acknowledgments …………………………………………….…………….……… 168

Curriculum Vitae …………………………………………….…………….………. 170

Publications and presentations……………………………………………………. 171