Polymeric nanoparticles as original theranostic approach for alzheimer￿s disease Davide Brambilla To cite this version: Davide Brambilla. Polymeric nanoparticles as original theranostic approach for alzheimer￿s disease. Human health and pathology. Université Paris Sud - Paris XI, 2012. English. ￿NNT: 2012PA114801￿. ￿tel-00692581￿ HAL Id: tel-00692581 https://theses.hal.science/tel-00692581 Submitted on 1 May 2012 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. UNIVERSITÉ PARIS-SUD 11 FACULTÉ DE PHARMACIE DE CHÂTENAY-MALABRY ECOLE DOCTORALE : INNOVATION THÉRAPEUTIQUE : DU FONDAMENTAL A L‟APPLIQUÉ PÔLE : PHARMACOTECHNIE ET PHYSICO-CHIMIE ANNÉE 2011-2012 SÉRIE DOCTORAT N° 1149 THÈSE Présentée À L‟UNITÉ DE FORMATION ET DE RECHERCHE FACULTE DE PHARMACIE DE CHÂTENAY-MALABRY UNIVERSITÉ PARIS-SUD 11 pour l‟obtention du grade de DOCTEUR DE L‟UNIVERSITÉ PARIS-SUD 11 par M. Davide BRAMBILLA POLYMERIC NANOPARTICLES AS ORIGINAL THERANOSTIC APPROACH FOR ALZHEIMER‟S DISEASE soutenue le : 11 Janvier 2012 JURY : Prof. Seyed Moein Moghimi Rapporteur Prof. Claus Michael Lehr Rapporteur Prof. Charles Duyckaerts Examinateur Prof. Patrick Couvreur Co-directeur de Thèse Dr. Julien Nicolas Co-directeur de Thèse Dr. Karine Andrieux Co-directeur de Thèse Table of contents Sommaire / Table of contents Introduction générale / General introduction ..................................................................................... 7 Partie 1: Etude bibliographique / Part 1: Bibliographic study ................................................... 11 Nanotechnologies for Alzheimer’s disease: diagnosis, therapy and safety issues ............................ 13 Résumé / Abstract ........................................................................................................................................ 14 1. Introduction .............................................................................................................................................. 15 2. Imaging-based nanotechnologies for AD diagnosis ................................................................................. 16 3. Nanotechnologies for detection of AD biomarkers in biological fluids ................................................... 20 4. Nanotechnologies for AD treatment ......................................................................................................... 25 5. Nanotechnologies for in vitro evaluation of drugs activity ...................................................................... 39 6. Safety issues ............................................................................................................................................. 39 7. Conclusions .............................................................................................................................................. 42 8. Acknowledgements .................................................................................................................................. 42 9. References ................................................................................................................................................ 43 Partie 2: Travail expérimental / Part 2: Experimental work ...................................................... 53 Chapitre 1: Nanoparticules polymériques et barrière hématoencéphalique .................................... 55 Chapter 1: Polymeric nanoparticles and blood-brain barrier .......................................................... 55 Section 1 ....................................................................................................................................................... 59 Implications dans le projet NAD / Implications in the NAD project ........................................................... 59 Design of fluorescently tagged poly(alkyl cyanoacrylate) nanoparticles for human brain endothelial cells imaging ......................................................................................................................................................... 61 Résumé / Abstract .................................................................................................................................... 62 1. Communication .................................................................................................................................... 62 2. Acknowledgments ................................................................................................................................ 68 3. References ............................................................................................................................................ 68 Supporting Information ............................................................................................................................ 69 Section 2 ....................................................................................................................................................... 83 Implications dans le projet NAD / Implications in the NAD project ........................................................... 83 Quantum dots-loaded PEGylated poly(alkyl cyanoacrylate) nanoparticles for in vitro and in vivo imaging 85 Résumé / Abstract .................................................................................................................................... 86 1. Introduction .......................................................................................................................................... 87 2. Experimental part ................................................................................................................................. 88 3. Analytical techniques ........................................................................................................................... 89 Table of contents 4. Results and discussion ......................................................................................................................... 91 5. Conclusions .......................................................................................................................................... 97 6. Acknowledgements .............................................................................................................................. 97 7. References ............................................................................................................................................ 98 Supporting Information .......................................................................................................................... 101 Section 3 ..................................................................................................................................................... 103 Implications dans le projet NAD / Implications in the NAD project ......................................................... 103 Fine evaluation of polymeric nanoparticles endocytosis and transcytosis across a human Blood-Brain Barrier in vitro model ................................................................................................................................. 105 Résumé / Abstract .................................................................................................................................. 106 1. Introduction ........................................................................................................................................ 107 2. Experimental section .......................................................................................................................... 109 3. Results and discussion ....................................................................................................................... 114 4. Conclusions ........................................................................................................................................ 123 5. References .......................................................................................................................................... 124 Supporting Information .......................................................................................................................... 126 Conclusions et perspectives futures / Conclusions and future perspectives ................................... 127 Chapitre 2 : Interaction de nanoparticules polymériques avec le peptide Aβ ............................ 129 1-42 Chapter 2: Polymeric nanoparticle interaction with Aβ peptide ................................................ 129 1-42 Section 1 ..................................................................................................................................................... 133 Implications dans le projet NAD / Implications in the NAD project ......................................................... 133 A new method based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) to monitor interaction between nanoparticles and the amyloid-β peptide ...................................................... 135 Résumé / Abstract .................................................................................................................................. 136 1. Introduction ........................................................................................................................................ 137 2. Experimental Section ......................................................................................................................... 139 3. Apparatus ........................................................................................................................................... 139 4. Methods ............................................................................................................................................. 140 5. Results and Discussion ...................................................................................................................... 143 6. Conclusions ........................................................................................................................................ 148 7. Acknowledgments .............................................................................................................................. 149 8. References .......................................................................................................................................... 149 Supporting Information .......................................................................................................................... 152 Section 2 ..................................................................................................................................................... 155 Implications dans le projet NAD / Implications in the NAD project ......................................................... 155 Table of contents Colloidal properties of biodegradable nanoparticles influence interaction with amyloid-β peptide .......... 157 Résumé / Abstract .................................................................................................................................. 158 1. Introduction ........................................................................................................................................ 159 2. Results and Discussion ...................................................................................................................... 160 3. Conclusions ........................................................................................................................................ 164 4. Acknowledgments .............................................................................................................................. 164 5. References .......................................................................................................................................... 164 Section 3 ..................................................................................................................................................... 167 Implications dans le projet NAD / Implications in the NAD project ......................................................... 167 Polyethylene Glycol at the surface of nanocarriers alters Aβ peptide conformation: toward a nanomedicine for Alzheimer‟s disease .............................................................................................................................. 169 Resumé / Abstract .................................................................................................................................. 170 1. Introduction ........................................................................................................................................ 171 2. Results and discussion ....................................................................................................................... 173 3. Conclusions ........................................................................................................................................ 185 4. Experimental section .......................................................................................................................... 186 5. Acknowledgements ............................................................................................................................ 193 6. References .......................................................................................................................................... 193 Supporting Information .......................................................................................................................... 197 Section 4 ..................................................................................................................................................... 199 Implications dans le projet NAD / Implications in the NAD project ......................................................... 199 Selegiline-Functionalized, PEGylated poly (alkyl cyanoacrylate) Nanoparticles: Investigation of Interaction with Amyloid-β Peptide ............................................................................................................................. 201 Résumé / Abstract .................................................................................................................................. 202 1. Introduction ........................................................................................................................................ 203 2. Materials and Methods ....................................................................................................................... 205 3. Results and discussion ....................................................................................................................... 210 4. Conclusion ......................................................................................................................................... 217 5. Acknowledgments .............................................................................................................................. 218 6. References .......................................................................................................................................... 218 Conclusions et perspectives futures / Conclusions and future perspectives ................................... 221 Discussion générale / General discussion ........................................................................... 223 Conclusion générale et perspectives futures ...................................................................... 239 General conclusion and future perspectives ...................................................................... 239 Table of contents Abbreviations / Abréviations............................................................................................... 243 Aknowledgments / Remerciements ..................................................................................... 247 Introduction générale General introduction 7 General introduction Recent statistics indicate that 35 million people worldwide are affected by dementia with 4.6 million new cases per year (one new case every 7 seconds). In Europe there are 5 million cases of dementia, 3 million of which are classified as Alzheimer's disease (AD). Given the continuing increase in life expectancy, these numbers are expected to rise dramatically. In 2040, cases are expected to double in Western Europe and to triple in Eastern Europe. Despite great progresses in research, Alzheimer‟s disease etiology remains a big question and its treatment an important challenge of modern medicine. A major limitation to the development of active drug against Central Nervous System (CNS)- related diseases is represented by the Blood-Brain Barrier (BBB), a formidable gatekeeper in the body towards exogenous substances. Generally, pharmaceuticals including most small molecules do not cross the BBB. Therefore, an ideal device for treatment of AD should provide two main properties: (i) be able to cross the BBB and (ii) to exert a beneficial effect by inhibiting the AD pathogenesis. In this view, an original solution could arise from nanotechnologies. Indeed, during the last decades, nanotechnology applied to medicine, refereed as nanomedicine, showed interesting results for the treatment of numerous severe diseases leading to the development and commercialization of more efficient drugs. This PhD thesis has been performed in the framework of the NAD (Nanoparticles for therapy and diagnosis of Alzheimer's disease) European research project aiming to develop nanoparticulate systems for the diagnosis and the therapy of Alzheimer's disease. The research is founded by the European Union's 7th Framework Program and includes 19 European research centers (Grant agreement no: CP-IP 212043-2 NAD). Prof. M. Masserini from University of Milano-Bicocca is the scientific coordinator of the project. The goal of this study was to create nanoparticlate systems able to cross the BBB in order to reach the brain, the main site of AD, and to capture the Amyloid peptide, especially A species, the 1-42 aggregates of which are known to play a pivotal role in the etiology of the disease. In particular, the PhD was oriented toward the design of polymeric nanoparticles and the study of their crossing of a novel in vitro BBB model (provided by P.O. Couraud, Institut Cochin) and to study the interaction between the polymeric nanoparticles and the A peptide. Different kinds of 1-42 nanoparticles have been designed during the project, mainly based on poly(alkyl cyanoacrylate) (PACA) and presenting various surface properties, in order to increase their affinity towards the peptide. All the obtained particles have been screened in vitro for their ability to capture the peptide. 9