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The Repurposing of Low Molecular Weight Drugs to Induce Lysosomal Release of Sirna and in Vitro Approaches to Probe the Toxicity of Inorganic Nanoparticles

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The Repurposing of Low Molecular Weight Drugs to Induce Lysosomal Release of Sirna and in Vitro Approaches to Probe the Toxicity of Inorganic Nanoparticles The repurposing of low molecular weight drugs to induce lysosomal release of siRNA and in vitro approaches to probe the toxicity of inorganic nanoparticles. Freya Joris Pharmacist Master of Science in Drug Development 2017 Thesis submitted to obtain the degree of Doctor in Pharmaceutical Sciences Proefschrift voorgedragen tot het bekomen van de graad van Doctor in de Farmaceutische Wetenschappen Dean Promotors Prof.dr.apr. Jan Van Bocxlaer Prof.dr.apr. Stefaan De Smedt Prof.dr.apr. Koen Raemdonck Members of the Exam Committee: Prof.dr.apr. Serge Van Calenbergh (chairman) Ghent University Prof.dr.apr. Christophe Stove (secretary) Ghent University Prof.dr.apr. Tom Coenye Ghent University Prof.dr. Arwyn Jones Cardiff University Prof.dr.apr. Véronique Préat Université catholique de Louvain Prof.dr. Roos Vandenbroucke VIB-Ghent University This document may contain confidential information proprietary to the Universiteit Gent. It is stricktly forbidden to publish, cite or make public in any way this document or any part thereof without the express written permission by the Universiteit Gent. Under no circumstances this document may be communicated to or put at the disposal of third parties; photocopying or duplicating it in any other way is strictly prohibited. Disregarding the confidential nature of this document may cause irremediable damage te the Universiteit Gent. Dit document bevat geheime informatie toebehorend aan de Universiteit Gent. Het is ten strengste verboden om dit document of enig onderdeel ervan te publiceren, te citeren of op enige wijze publiek bekend te maken zonder uitdrukkelijke schriftelijke toestemming vanwege de Universiteit Gent. Onder geen enkele voorwaarde mag dit document gecommuniceerd of ter beschikking gesteld worden van derden; het is niet toegestaan het te fotokopiëren of op enige andere manier te dupliceren. Het niet respecteren van de vertrouwelijkheid van dit document kan de Universiteit Gent onherstelbare schade toebrengen. Ghent, March 9th 2017 Promotors Author Prof.dr.apr. Stefaan De Smedt Apr. Freya Joris Prof.dr.apr. Koen Raemdonck – The journey is the reward – Steve Jobs, 1983 TABLE OF CONTENTS List of abbreviations 11 Aim & outline 17 PART I: THE REPURPOSING OF LOW MOLECULAR WEIGHT DRUGS TO INDUCE LYSOSOMAL RELEASE OF SIRNA Chapter 1 General introduction to RNA interference, nanogels as 23 nanocarriers and functional inhibitors of acid sphingomyelinase Chapter 2 Boosting non-viral nucleic acid delivery and transfection 41 efficiency: small molecules aid to convey big messages Chapter 3 The repurposing of cationic amphiphilic drugs to enhance 79 cytosolic siRNA delivery Chapter 4 Exploring the broader applicability of a low molecular weight 115 adjuvant approach to improve nucleic acid delivery to the cytosol PART II: IN VITRO APPROACHES TO PROBE THE TOXICITY OF INORGANIC NANOPARTICLES Chapter 5 Assessing nanoparticle toxicity in cell-based assays: 149 influence of cell culture parameters and optimized models for bridging the in vitro-in vivo gap Chapter 6 The impact of species and cell type on the nanosafety profile of 201 iron oxide nanoparticles in neural cells Chapter 7 Choose your cell model wisely: the in vitro nanoneurotoxicity of 229 differentially coated iron oxide nanoparticles for neural cell labelling Chapter 8 Broader international context, relevance and future perspectives 251 Appendix A: Supporting Information to Chapter 6 & 7 281 Summary & conclusion 299 Samenvatting & conclusie 303 Curriculum Vitae 307 Acknowledgements 313 LIST OF ABBREVIATIONS PART I: THE REPURPOSING OF LOW MOLECULAR WEIGHT DRUGS TO INDUCE LYSOSOMAL RELEASE OF SIRNA 2OHOA 2-hydroxy oleic acid 5A2dC 5-aza-2’-deoxycytidine Ago2 Argonaute 2 ASM Acid sphingomyelinase ASO Antisense oligonucleotide BMP Bis(monoacylglycero)phosphate CAD Cationic amphiphilic drug CaP Calcium phosphate CD Carvedilol CHAP31 Cyclic hydroxamic acid containing peptide 31 Chol-siRNA Cholesterol-siRNA conjugate CLQ Chloroquine CPP Cell penetrating peptide CRISPR Clustered regularly interspaced short palindromic repeats Dex-HEMA Dextran hydroxyethyl methacrylate Dex-NGs Dextran nanogels DL Desloratadine DLS Dynamic light scattering DMSO Dimethylsulfoxide DNMT DNA methyl transferase DPC Dynamic polyconjugate DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOTAP (2,3-dioleoyloxy-propyl)-trimethylammonium 11 LIST OF ABBREVIATIONS DSF Disulfiram dsRNA Double stranded RNA DX Dextrometorphan eGFP Enhanced green fluorescent protein FBS Fetal bovine serum FD FITC-labeled dextran FIASMA Functional inhibitor of the acid sphingomyelinase GalNAc N-acetylgalactosamine GR Glucocorticoid receptor HDAC Histon deacetylase HEPES N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid i.t. Intratumoral LDR Lysotracker® Deep Red LMP Lysosomal membrane permeabilization LNP Lipid nanoparticle LPS Liposome LPX Lipoplex LSCM Laser scanning confocal microscope LSD Lysosomal storage disorder MFI Mean fluorescence intensity miRNA microRNA mRNA messenger RNA MTOC Microtubule-organizing center NA Nucleic acid NG Nanogel NM Nanomedicine NP Nanoparticle NPC1 Niemann-Pick C1 12 LIST OF ABBREVIATIONS NPD Niemann-Pick disease NT Nortriptyline ON Oligonucleotide PAMAM Poly(amidoamine) PBS Phosphate buffered saline pDNA Plasmid DNA PEG Polyethylene glycol PEI Polyethylenimine PFA Paraformaldehyde PLA2 Phospholiase A2 PLD Phospholipidosis PLK1 Polo-like kinase 1 Pre-miRNA Precursor microRNA pri-miRNA Primary microRNA PTX Paclitaxel RISC RNA-induced silencing complex RNAi RNA interference SEM Standard error of the mean shRNA Short hairpin RNA siCTRL Scrambled control siRNA duplex sieGFP siRNA duplex targeting eGFP siNG siRNA-loaded nanogel siPLK1 siRNA duplex targeting PLK1 siRNA Small interfering RNA SSC Side scatter siSTABLE Nuclease-stabilized siRNA duplex SM Sphingomyelin SMoC Small molecule carrier 13 LIST OF ABBREVIATIONS SNALP Stable nucleic acid lipid nanoparticle SSO Splice switching oligonucleotide ST Salmeterol TM Tobramycin TMAEMA 2-(methacryloyloxy)ethyl)trimethylammonium chloride TSA Trichostatin A PART II: IN VITRO APPROACHES TO PROBE THE TOXICITY OF INORGANIC NANOPARTICLES ΔΨm mitochondrial membrane potential 2+ [Ca ]c Cytosolic free calcium concentration AgNP Silver nanoparticle ATP Adenosine triphosphate AuNP Gold nanoparticle Ca2+ Calcium Cd Cadmium CeO2NP Cerium oxide nanoparticle CNT Carbon nanotube dc Core diameter DLS Dynamic light scattering DMSA 2,3-meso-dimercaptosuccinic acid ECM Extracellular matrix ER Endoplasmatic reticulum GI Gastro-intestinal HCS High content screening hNSC Human neural stem cell ISDD In vitro sedimentation, diffusion and dosimetry model 14 LIST OF ABBREVIATIONS IONP Iron oxide nanoparticle MDDC Monocyte derived dendritic cell MDM Monocyte derived macrophage mNSC Murine neural stem cell MRI Magnetic resonance imaging NM Nanomaterial NP Nanoparticle NOAEL No observed adverse effect level NSC Neural stem cell NTC Not treated control OECD Organization for Economic Cooperation and Development PMA poly(isobutylene-alt-maleic anhydride) QD Quantum dot QSAR Quantitative structure-activity relationship REACH Registration, Evaluation, Approval and Restriction of Chemicals ROS Reactive oxygen species SiO2NP Silica nanoparticle SEM Standard error of the mean SS Shear stress TEM Transmission electron microscopy TiO2NP Titanium dioxide nanoparticle TNFα Tumor necrosis factor α ZnONP Zinc oxide nanoparticle 15 16 AIM & OUTLINE AIM & OUTLINE Nanotechnology is one of the main drivers of the current scientific advancements. Indeed, by engineering materials in the nanoscale range, materials may acquire unique properties that allow the development of novel and innovative applications for sometimes well-known materials. Distinct nanoparticles enjoy widespread popularity as they are being exploited by various industries, such as the automotive, aerospace, chemical, textile and cosmetic industry. In this spirit, the use of organic and inorganic nanoparticles (NPs) is also being avidly explored for biomedical use. NPs can be applied to improve biomarker detection, enhance imaging-mediated diagnosis or improve treatment strategies. Overall organic nanoparticles have evolved further in their clinical development, whereas the elusive safety profiles of inorganic nanoparticles hinder their clinical implementation. In this thesis we will look into the origin of the questionable safety of inorganic NPs and how the optimization of nanotoxicity testing may bring solace. First, we look into the use of organic particles as nanocarriers for nucleic acids (NA), and how their delivery potential could be enhanced using low molecular weight adjuvants. The therapeutic potential of nucleic acids has been well established for a myriad of disorders. In contrast, few NA-based therapeutics managed to transfer to the clinic. This can mainly be attributed to delivery challenges, as NA require delivery to their cytosolic or nuclear site of action to evoke a therapeutic effect. Nanocarriers were initially believed to be the magic bullet towards efficient delivery of NA. However, the intracellular endo-lysosomal entrapment upon endocytosis severely hampers their delivery efficiency. Indeed, only a minor fraction of the internalized NA dose manages to reach its intracellular site of action, whereas the bulk is destined for lysosomal degradation. Hence, escape to the cytosol remains a major intracellular hurdle, which we aim to tackle by improving cytosolic NA release through the repurposing of cationic amphiphilic drugs (CADs). This method was initially investigated in
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