Exploring extracellular vesicles for siRNA delivery and Raman-based diagnostics Stephan Stremersch Pharmacist Master of Science in Drug Development 2016 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 Promotor Prof.dr.apr. Jan Van Bocxlaer Prof.dr.apr. Stefaan De Smedt Promotor Promotor Dr.apr. Koen Raemdonck Prof.dr. Kevin Braeckmans Members of the Exam Committee: Prof.dr.apr. Serge Van Calenbergh (chairman) Ghent University Prof.dr.apr. Filip Van Nieuwerburgh (secretary) Ghent University Prof.dr.apr. Olivier De Wever University Hospital Ghent Prof.dr. Raymond Schiffelers University Medical Center Utrecht Prof.dr. Roos Vandenbroucke VIB-Ghent University The author and the promotors give the authorization to consult and to copy parts of this thesis for personal use only. Any other use is limited by the Laws of Copyright, especially the obligation to refer to the source whenever results from this thesis are cited. De auteur en de promotoren geven de toelating dit proefschrift voor consultering beschikbaar te stellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting uitdrukkelijk de bron te vermelden bij het aanhalen van resultaten uit dit proefschrift. Ghent, June 24th 2016 The promotors: The author: Prof.dr.apr. Stefaan De Smedt Apr. Stephan Stremersch Dr.apr. Koen Raemdonck Prof.dr. Kevin Braeckmans Table of Contents List of abbreviations 9 Aim and outline of this thesis 13 Chapter 1 Therapeutic and diagnostic applications of extracellular 15 vesicles (EVs) 1. A general introduction to EVs 18 2. Therapeutic applications of EVs 25 3. Diagnostic applications of EVs 41 4. General conclusions 44 Chapter 2 Electroporation-induced siRNA precipitation obscures the 63 efficiency of siRNA loading into extracellular vesicles Chapter 3 Inadequate purification of extracellular vesicles can lead to 97 misinterpretation of downstream experimental data Chapter 4 Comparing exosome-like vesicles with liposomes for the 125 functional cellular delivery of small RNAs Chapter 5 Identification of individual exosome-like vesicles by surface 165 enhanced Raman spectroscopy Chapter 6 Broader international context, relevance and future 191 perspectives Summary and conclusions 213 Samenvatting en conclusies 217 Curriculum Vitae 221 Acknowledgements / Dankwoord 229 List of abbreviations AAV adeno-associated virus ADSC adipose-derived stem cells AgNP silver nanoparticles AGO2 argonaute 2 APC antigen presenting cell API active pharmaceutical ingredient AUC area under the curve AuNP gold nanoparticles BALF bronchoalveolar lavage fluid BBB blood brain barrier BSA bovine serum albumin CARS coherent anti-stokes Raman spectroscopy (c)CCM (concentrated) conditioned cell medium CD cluster of differentiation CEA carcinoembryonic antigen CHEMS 3β-hydroxy-5-cholestene 3-hemisuccinate chol-siRNA cholesterol-conjugated siRNA DC dendritic cell DLS dynamic light scattering DMAP 4-dimethylaminopyridine DMEM dulbecco's modified eagle medium DNA deoxyribonucleic acid DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine dsRNA double stranded RNA EBV epstein-bar virus EDTA ethylenediaminetetraacetic acid eGFP enhanced green fluorescent protein EGFR epidermal growth factor receptor EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid ELV(s) exosome-like vesicle(s) EP electroporation EPCAM epithelial cell adhesion molecule EPR enhanced permeability and retention EV(s) extracellular vesicle(s) FBS fetal bovine serum FDA food and drug administration List of abbreviations│ 9 FFS fluorescence fluctuation spectroscopy FITC fluorescein isothiocyanate gag group specific antigen GM-CSF granulocyte macrophage colony stimulating factor GP100 glycoprotein 100 HDL high density lipoprotein HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HER2 human epidermal growth factor receptor 2 HRP horseradish peroxidase Hsp heat shock protein ILV intraluminal vesicle iNKT invariant natural killer T cell IP intraperitoneal ISEV international society of extracellular vesicles IV intravenous L1CAM L1 cellular adhesion molecule LBPA lysobisphosphatidic acid LDL low density lipoprotein MCR-ALS multivariate curve resolution alternating least squares MFI mean fluorescence intensity MHC major histocompatibility complex miRNA microRNA MPS mononuclear phagocyte system mRNA messenger RNA MSC mesenchymal stem cell MVB multivesicular body NK-cells natural killer cells NSCLC non-small-cell lung carcinoma nt nucleotides OMV outer membrane vesicle ORF open reading frame PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PCR polymerase chain reaction pDNA plasmid DNA PEG polyethylene glycol PK pharmacokinetics PLS-DA partial least square discriminative analysis pre-miRNA precursor-miRNA List of abbreviations│ 10 pri-miRNA primary-miRNA PS phosphatidylserine PSA prostate specific antigen PSMA prostate-specific membrane antigen PVDF polyvinylidene difluoride PVP polyvinylpyrrolidone RBC red blood cell RISC RNA-induced silencing complex RNA ribonucleic acid RNAi RNA interference RNase ribonuclease RT-PCR reverse transcription polymerase chain reaction SC subcutaneous SD standard deviation SDS sodium dodecyl sulfate SEC size exclusion chromatography SEM standard error of the mean SERS surface enhanced Raman spectroscopy siCD45 siRNA targeted against CD45 siCTRL siRNA control sequence siGFP siRNA targeted against eGFP siRNA small interfering RNA SPR surface plasmon resonance SPT single particle tracking TAA tumor associated antigen TEM transmission electron microscopy TERS tip-enhanced Raman spectroscopy TLR toll-like receptor Tris tris(hydroxymethyl)aminomethane TRL technology readiness level TYRP2 tyrosinase-related protein 2 UC ultracentrifugation UF ultrafiltration UTR untranslated region VSV vesicular stomatitis virus List of abbreviations│ 11 List of abbreviations│ 12 AIM AND OUTLINE OF THIS THESIS The first observation of vesicular structures outside of the cell dates back to the 1960s. Initially, these vesicles were believed to be mainly involved in extracellular processes like blood clotting and in maintaining the cellular homeostasis by acting as waste removal entities. However, during the last decade these extracellular vesicles (EVs) are increasingly regarded as important mediators of intercellular communication. It was this novel insight that sparked the mounting interest in these nanosized vesicles from both the therapeutic and diagnostic field, which is accompanied by numerous new applications exploiting EVs in different pharmaceutical domains. Because of their natural function as transporters of macromolecular components, it is hypothesized that EVs are endowed with unique features that rationalize their exploitation as a drug carrier system. Macromolecular therapeutics with an intracellular target, including nucleic acids and pharmaceutical proteins, require formulation into nanoparticles (i.e. so-called nanomedicines) to guide them across the many extra- and intracellular barriers. Unfortunately, many synthetic nanomedicines fail to merge drug delivery efficiency with acceptable biocompatibility. In light of these shortcomings, the first aim of this thesis is to explore EVs as bio-inspired carriers for small interfering RNA (siRNA) delivery. Indeed, the field of RNA interference (RNAi) is still struggling to translate its wide potential into a clinical applicable technology due to a lack of targeted and functional delivery. Chapter 1 provides a general introduction to EVs with a focus on their therapeutic and diagnostic applications. An overview of the main historical findings that shaped the further development of the field is followed by a more in depth discussion of the different EV types and their respective biogenesis pathways. Next, different methods to purify EVs are listed and discussed for their potential in a clinical setting. Finally, a comprehensive overview of the different fields of application for which EVs have been explored is given. Here, the main emphasis is put on exploiting EVs as a drug delivery vehicle with an outline of the most important strategic choices to be considered. In parallel, we aimed to pinpoint some of the major hurdles that need to be overcome to accelerate the development of this application. One of the first critical steps in harnessing EVs as a therapeutic carrier is developing robust methods to load these nanosized membranous vesicles with the therapeutic cargo of interest. In chapter 2 we thoroughly investigated the value of electroporation to load isolated EVs with siRNA and provide details on the emergence of electroporation artifacts that substantially overestimate the EV loading efficiency. Aim and outline of this thesis│ 13 Furthermore, it is important to bear in mind that EVs are typically present in complex media (i.e. cell medium, serum, etc.). The strategies to isolate EVs are continuously evolving, though the field has not yet reached consensus on a gold standard protocol. In chapter 3 we provide a comparative analysis of different EV purification strategies, discuss the purity of the final isolate, both for endogenous as well as exogenous contaminants, and indicate some misinterpretations that impurities can entail in downstream experimental readouts. In light of the results obtained in the previous chapters, a new strategy to associate small RNAs to EVs is described