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Delivery of Messenger RNA to platelets using lipid nanoparticles Novakowski, Stefanie Kim 2019

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DELIVERY OF MESSENGER RNA TO PLATELETS USING LIPID NANOPARTICLES by  Stefanie Kim Novakowski  B.Sc., Queen’s University, 2012  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Biochemistry and Molecular Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  January 2019  © Stefanie Novakowski, 2019  ii  The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Delivery of messenger RNA to platelets using lipid nanoparticles  submitted by Stefanie Kim Novakowski in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology  Examining Committee: Dr. Christian J. Kastrup, Biochemistry and Molecular Biology Supervisor  Dr. Dana Devine, Pathology & Laboratory Medicine Supervisory Committee Member  Dr. Hugh Kim, Biochemistry and Molecular Biology University Examiner Dr. Marcel Bally, Pathology & Laboratory Medicine University Examiner  Additional Supervisory Committee Members: Dr. Eric Jan, Biochemistry and Molecular Biology Supervisory Committee Member  iii  Abstract Platelets are small, anucleate cells that circulate in the blood stream and mediate hemostasis, inflammation, and angiogenesis. Platelet transfusions are used to treat active bleeding as well as to prevent bleeding during thrombocytopenia or prior to surgery. Yet there are situations where transfusions do not adequately stop bleeding, such as during trauma, which is associated with platelet dysfunction. A method for genetically modifying platelets might enhance their efficacy and lead to new therapeutic uses for platelets. As platelets are anucleate, directly modifying platelets requires messenger RNA (mRNA). Attempts to transfect platelets with mRNA have not been successful, and it is unknown whether lipid-based materials could be used as mRNA transfection agents for platelets. Lipid nanoparticles (LNPs) have been used for nucleic acid delivery in vitro and in vivo. In this thesis, the ability of four different classes of LNPs to deliver mRNA to platelets was compared. These classes consisted of LNPs containing cationic lipids (cLNPs) that are highly effective in vitro, LNPs containing ionizable cationic lipids (icLNPs) developed for in vivo use, LNPs without a cationic lipid commonly used to encapsulate proteins or small molecules, and a commercially available agent previously used for short interfering RNA delivery to platelets. To identify ideal conditions for transfection with mRNA, uptake under various storage conditions and the ability of the LNPs to alter platelet activation was quantified. Finally, the ability of platelets to translate and release the mRNA was assessed. Two approaches were taken for mRNA delivery. In one approach, mRNA was synthesized inside of liposomes, indicating proteins, DNA, and small molecules can be delivered to platelets using LNPs. In the second approach, in vitro transcribed mRNA was directly delivered to platelets using icLNPs and cLNPs, and mRNA delivered to platelets using cLNPs was released in microparticles. These iv  were the first examples of direct delivery of mRNA to platelets, and the first step towards creating genetically modified platelets. While protein synthesis in LNP-treated platelets was not detected, optimizing the LNP formulations used here may lead to a transfection agent for platelets that allows for de novo synthesis of exogenous proteins in the future.   v  Lay Summary Platelets are small cells that are required to stop the flow of blood during injury. They are used in hospitals to prevent and stop bleeding in patients with reduced or impair platelet function, such as during trauma or certain cancers, yet they are not effective in all situations. The ability to alter a cell’s content in order to modify its function is an important tool in biological and medical research, however commonly used tools for delivering materials such as proteins and nucleic acids to cells have no or limited effect in platelets. This thesis compares the ability of different materials to deliver nucleic acids to platelets, identifying the ideal conditions for delivery and characterizing the effect of each material on platelets. This may ultimately lead to platelets with an improved ability to stop bleeding, and potentially extend the range of diseases that can be treated using platelets.  vi  Preface Approval for the study was given by the University of British Columbia’s research ethics board (certificate number: H12-01516) and the Canadian Blood Services’ research ethics board (certificate number: REB 2016.006). A modified version of chapter 2 has been published in Angewandte Chemie: V. Chan, S.K. Novakowski, S. Law, C. Klein-Bosgoed, C.J. Kastrup (2015). Controlled transcription of exogenous mRNA in platelets using protocells. V.C. and S.K.N. were co-first authors on the paper, designed and performed all experiments, interpreted all data, and wrote the paper. S.K.N. performed experiments testing uptake of liposomes by platelets, and functionality of RNA transcribed in light-sensitive liposomes. V.C. performed experiments demonstrating light-sensitive RNA synthesis in platelets and collected data for Figures 2.7b, 2.8 and Appendix Figures A.1 and A.2. S.L. helped developed methods for testing uptake of liposomes by platelets. C.K-B. helped develop methods for platelet isolation. C.J.K. helped design experiments, interpret data and write the paper.  A modified version of chapters 2, 3, and 4 is in press at Scientific Report: S.K. Novakowski, K. Jiang, G. Prakash, C. Kastrup (2018). Delivery of mRNA to platelets using lipid nanoparticles. S.K.N. designed and performed all experiments, analyzed and interpreted data and wrote the paper. K.J. helped collect data in Figure 3.6. G.P. helped develop methods for imaging platelets and testing translation of mRNA within platelets, and edit the paper. C.J.K. helped design experiments, interpret data and write the paper. The reuse and reprint of all published work is with permission from the joural referenced.  vii  Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Abbreviations ...................................................................................................................xv Acknowledgements ......................................................................................................................xx Dedication ................................................................................................................................... xxi Chapter 1: Introduction ................................................................................................................1 1.1 Thesis Overview ............................................................................................................. 1 1.1.1 Rationale ..................................................................................................................... 1 1.1.2 Objectives ................................................................................................................... 3 1.2 Background and Literature Review ................................................................................ 5 1.2.1 Platelet physiology ...................................................................................................... 5 1.2.1.1 Platelet formation and clearance ......................................................................... 5 1.2.1.2 Platelets mediate hemostasis ............................................................................... 6 1.2.1.3 Platelets outside of hemostasis............................................................................ 9 1.2.2 The role of platelets in the clinic............................................................................... 12 1.2.2.1 Indications for platelet transfusions .................................................................. 12 1.2.2.2 Generating transfusable platelets ...................................................................... 13 1.2.2.3 Adverse reactions to transfusion ....................................................................... 14 viii  1.2.2.4 Platelet storage  ................................................................................................. 16 1.2.3 Creating modified or synthetic platelets for the clinic .............................................. 19 1.2.3.1 The platelet transcriptome ................................................................................. 19 1.2.3.2 Protein synthesis in platelets ............................................................................. 22 1.2.3.3 Creating genetically modified platelets ............................................................ 26 1.2.3.4 Culturing platelets ex vivo ................................................................................. 29 1.2.3.5 Synthetic platelets ............................................................................................. 31 1.2.4 Platelets take up and release materials ...................................................................... 33 1.2.4.1 Mechanisms of uptake of biological materials by platelets  ............................. 33 1.2.4.2 Endocytosis of synthetic materials.................................................................... 35 1.2.4.3 Regulation of platelet secretion ........................................................................ 36 1.2.4.4 Platelet microparticles ....................................................................................... 38 1.2.5 LNPs and their application in gene therapy .............................................................. 39 1.2.5.1 Challenges in gene therapy ............................................................................... 39 1.2.5.2 Development of lipid-based delivery agents..................................................... 40 1.2.5.3 Interaction of LNPs with the cellular environment ........................................... 41 1.2.5.4 Applications of LNPs in mRNA delivery ......................................................... 42 1.2.5.5 Examples of direct platelet transfusion ............................................................. 44 Chapter 2: Identifying strategies for the delivery and synthesis of mRNA in platelets ........46 2.1 Rationale ....................................................................................................................... 46 2.2 Methods......................................................................................................................... 48 2.2.1 Preparing washed platelets ........................................................................................ 48 2.2.2 Preparing mRNA-LNPs ............................................................................................ 48 ix  2.2.3 Preparing RNA-synthesizing nLNPs ........................................................................ 50 2.2.4 Generating PCR templates for in vitro transcription ................................................ 50 2.2.5 Generating biotinylated mRNA ................................................................................ 50 2.2.6 Treating platelets with LNPs .................................................................................... 51 2.2.7 Quantifying uptake of LNPs by flow cytometry....................................................... 51 2.2.8 Quantifying uptake of LNPs by confocal microscopy .............................................. 52 2.2.9 Measuring transcription in RNA-synthesizing nLNPs by qPCR .............................. 53 2.2.10 Measuring protein expressionn from synthesized RNA ........................................... 53 2.2.11 Statistical analysis ..................................................................................................... 54 2.3 Results ........................................................................................................................... 54 2.3.1 Specific LNP formulations can be internalized by platelets ..................................... 54 2.3.3 Developing RNA-synthesizing nLNPs ..................................................................... 58 2.3.3 Engineering platelets to synthesize RNA ................................................................. 61 2.4 Discussion ..................................................................................................................... 64 Chapter 3: Characterizing the interaction of mRNA-LNPs with platelets ............................70 3.1 Rationale ....................................................................................................................... 70 3.2 Methods......................................................................................................................... 71 3.2.1 Quantifying uptake of LNPs by flow cytometry and confocal micrscopy ............... 71 3.2.2 Quantifying alpha granule release by flow cytometry .............................................. 72 3.2.3 Measuring thrombin generation ................................................................................ 72 3.2.4 Measuring platelet spreading .................................................................................... 73 3.2.5 Measuring platelet aggregation ................................................................................. 73 3.3 Results ........................................................................................................................... 73 x  3.3.1 Platelets internalize mRNA-LNPs only under specific storage conditons ............... 73 3.3.3 LNPs do not impair platelet function ........................................................................ 78 3.4 Discussion ..................................................................................................................... 81 Chapter 4: Stability, translation, and release of mRNA following delivery by LNPs ...........88 4.1 Rationale ....................................................................................................................... 88 4.2 Methods......................................................................................................................... 89 4.2.1 Quantifying uptake of LNPs by flow cytometry and confocal microscopy ............. 89 4.2.2 Quantifying protein expression ................................................................................. 90 4.2.3 Isolating platelet microparticles ................................................................................ 90 4.2.4 Quantifying mRNA by qPCR ................................................................................... 91 4.2.5 Preparing modified cLNPs ........................................................................................ 91 4.3 Results ........................................................................................................................... 92 4.3.1 Stability of the mRNA depends on LNP class and storage conditions ..................... 92 4.3.3 Transfection of platelets with icLNPs or cLNPs does not lead to protein expression     ................................................................................................................................... 95 4.3.3 Delivered mRNA is released in platelet microparticles ............................................ 98 4.3.4 Optimizing delivery of LNPs .................................................................................. 100 4.4 Discussion ................................................................................................................... 103 Chapter 5: Conclusion and Future Directions ........................................................................113 5.1 Summary ..................................................................................................................... 113 5.2 Future directions ......................................................................................................... 116 5.2.1 Optimizing LNP and mRNA delivery .................................................................... 116 5.2.2 Assessing the in vivo safety and efficacy of mRNA-LNPs .................................... 118 xi  5.2.3 Potential uses of modified platelets in the clinic  ................................................... 119 5.2.4 Potential uses of modified platelets for studying platelet function  ........................ 121 Bibliography ...............................................................................................................................124 Appendices ..................................................................................................................................152 Appendix A: CHAPTER 2 SUPPLEMENTARY DATA  ................................................. 152  xii  List of Tables Table 1.1. Proteins synthesized in platelets following activation with platelet agonists ............. 24  xiii  List of Figures Figure 1.1. Schematic of modified platelets and their potential applications  ............................... 2 Figure 1.2. Overview of protein synthesis in platelets ................................................................. 24 Figure 1.3. Overview of platelet secretion  .................................................................................. 37 Figure 2.1. LNPs have similar size characteristics except for Lf complexes .............................. 55 Figure 2.2. mRNA delivered by icLNPs and cLNPs is internalized ........................................... 56 Figure 2.3. Mechanism of uptake depends on LNP formulation ................................................ 57 Figure 2.4. Optimizing RNA synthesis in nLNPs  ....................................................................... 58 Figure 2.5. Freeze-thawing and purification on an anionic exchange column allows for transcription inside of nLNPs ...................................................................................................... 59 Figure 2.6. Transcription of FLuc mRNA in nLNPs is controlled by light ................................ 60 Figure 2.7. nLNPs were internalized by platelets ........................................................................ 62 Figure 2.8. nLNPs internalized by intact platelets synthesized RNA ......................................... 63 Figure 3.1. Only binding of icLNPs increases with longer incubation times ..............................74 Figure 3.2. Binding of LNPs depends on pH, presence of plasma, and platelet activation, as well as the class of LNP ........................................................................................................................ 75 Figure 3.3. Internalization of icLNPs but not cLNPs depends on initial pH and presence of thrombin ........................................................................................................................................77 Figure 3.4. cLNPs induce alpha granule release...........................................................................78 Figure 3.5. LNPs do not induce or impair aggregation.................................................................79 Figure 3.6. cLNPs activate the coagulation cascade .................................................................... 80 Figure 3.7. LNPs do not impair platelet spreading ..................................................................... 81 xiv  Figure 4.1. icLNPs and cLNPs are stable in resting platelets but cLNPs are not stable in activated platelets or platelets in plasma .......................................................................................93 Figure 4.2. icLNPs and cLNPs remain internalized after uptake unless platelets are stored in plasma ...........................................................................................................................................94 Figure 4.3. Alpha granule release following removal of LNPs depends on storage conditions and presence of cationic lipids .............................................................................................................95 Figure 4.4. Platelets do not translate mRNA delivered with icLNPs or cLNPs .......................... 97 Figure 4.5. Platelet microparticles range from 100 nm to 400 nm ..............................................98 Figure 4.6. mRNA delivered by LNPs is released in platelet microparticles ..............................99 Figure 4.7. Use of DOPE in cLNPs is not sufficient for protein expression in platelets .......... 101 Figure 4.8. RNA binding may be enhanced through increased delivery of nLNPs, Lf or icLNPs, but not cLNP .............................................................................................................................. 103 Figure A.1. Unencapsulated transcriptional components are removed from nLNPs using an anionic exchange column ........................................................................................................... 152 Figure A.2. Transcription of GFP mRNA in nLNPs is controlled by light .............................. 153  xv  List of Abbreviations ADP  Adenosine diphosphate ATP Adenosine triphosphate AGO2 Argonaute2 APC Allophycocyanin ATP Adenosine triphosphate Bcl-3   B-cell lymphoma encoded protein B.D. Below detection CD41 Glycoprotein IIb CD42b Glycoprotein Ib CD61 Glycoprotein IIIa CD62 P-selectin CGS Citrate glucose saline buffer Chol Cholesterol CircRNA Circular RNA cLNP  Cationic lipid nanoparticle COX-1 Cyclooxygenase-1 CTP Cytosine triphosphate Cy5 Cyanine 5 DC-SIGN Dendritic cell-specific intracellular adhesion molecule-3-grabbing-non integrin DHPE 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine DLin-KC2-DMA 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane xvi  DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid DOPSA  2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOTAP 1,2-dioleoyl-3-trimethylammonium-propane DSPE-PEG2000 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000 EDTA Ethylenediaminetetraacetic acid  eIF Eukaryotic initiation factor 4E-BP eIF4E-binding protein ePC Egg phosphatidylcholine ECM Extracellular matrix FITC Fluorescein isothiocyanate FIXa Activated coagulation factor IX FLuc  Firefly luciferase FV(a) (Activated) coagulation factor V FVIII Coagulation factor VIII FX(a) (Activated) coagulation factor X FXI Coagulation factor XI FXIII(a) (Activated) coagulation factor XIII GAPDH Glyceraldehyde 3-phosphate dehydrogenase  xvii  GFP  Green fluorescent protein GP Glycoprotein GTP Guanosine triphosphate HBSS  Hank’s buffered salt solution HEK293  Human embryonic kidney cells 293 HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIV Human immunodeficiency virus HLA  Human leukocyte antigen HSC  Hemopoietic stem cell icLNP   Ionizable cationic lipid nanoparticle IL-1β Interleukin-1 Beta IRES Internal ribosome entry site IVT In vitro transcribed MCS Multiple cloning site m7G cap 5’-7-methylguanosine cap miRNA   Micro ribonucleic acid mRNA  Messenger ribonucleic acid mTOR Mammalian target of rapamycin MVB Multivesicular bodies NET Neutrophil extracellular trap nLNP  Neutral lipid nanoparticle Lf Lipofectamine LPS Lipopolysaccharide xviii  LNP  Lipid nanoparticle OCS  Open canicular system OG Oregon Green PAR Protease-activated receptor PAS Platelet additive solution PBS Phosphate buffered saline PCR Polymerase chain reaction PDGF Platelet-derived growth factor PEG Polyethylene glycol PEG-c-DMA N-[(methoxy poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine PGE1  Prostaglandin E1 PLGA Poly(lactic-co-glycolic acid Pre-miRNA Premature microRNA PRP Platelet-rich plasma PSL Platelet storage lesion P2Y Purogenic receptor qPCR  Quantitative polymerase chain reaction RANTES Regulated on activation normal T cell expressed and secreted RISC RNA-induced silencing complex RNA  Ribonucleic acid RRL Rabbit reticulocyte lysate sCD40L Soluble CD40L xix  SEM Standard error of mean siRNA Small interfering ribonucleic acid SNARE Soluble N-ethylmaleimide-sensitive fusion attachment protein receptor TF  Tissue factor TGF-β1 Transforming growth factor beta 1 TRAIL  Tumor necrosis factor-related apoptosis-inducing ligand TRALI Transfusion-related acute lung injury TP α  Thromboxane receptor TxA2  Thromboxane A2 TBS Tris-buffered saline TLR Toll-like receptor UTP Uridine triphosphate UTR Untranslated region VAMP Vesicle associated membrane protein VGDF Vascular derived growth factor vWF von Willebrand factor v/v Volume to volume ratio w/w Weight to weight ratio xx  Acknowledgements  Thank you to all current and past members of the Kastrup Lab. Dr. V. Chan, G. Prakash, K. Jiang, N. Ashok and S. Law all contributed to data collection. Dr. V. Chan and W.S. Hur were also indispensable resources while navigating my way through my graduate studies. I thank my supervisor, Dr. Christian Kastrup, and my supervisory committee Dr. D. Devine and Dr. E. Jan, for their support and research guidance. I also thank our collaborators, Dr. D. Devine and Dr. P. Cullis, and the members of their labs for their contributions, particularly Drs. C. Klein-Bosgoed., S. Chen and J. Kulkarni.   Thank you to the donors and staff at the Canadian Blood Services’ netCAD facility, as well as all my fellow graduate students and co-workers who donated their time to our lab, even if it meant letting me perform phlebotomy on them.   Thank you to the Canadian Institute of Health Research, Natural Sciences and Engineering Research Council, and University of British Columbia for financial support.   Special thanks to my friends and family for their support xxi  Dedication  To my parents for their continual support. 1  Chapter 1: Introduction 1.1 Thesis Overview 1.1.1 Rationale Platelets are key players in many physiological and pathological processes, including hemostasis, thrombosis, inflammation and cancer [1]. Platelet transfusions are used extensively in the clinic for thrombocytopenia, myelodysplastic syndromes, chemotherapy-induced thrombocytopenia, aplastic anemia, trauma, and major cardiac surgery [1]. Blood products are carefully managed, and while there have not been major shortages of platelet products in Canada in the past two decades, shortages have occurred in the United States [2-6]. However, the limited shelf-life of platelets makes managing the supply of transfused platelets challenging. Artificial platelet substitutes are being developed, but only act as hemostatics, agents which stop the flow of blood, and lack the functional complexity of platelets [7]. Platelet can be produced from stem cells ex vivo, but this technology is still being scaled-up and will require clinical testing before it is ready for use in patients [8]. Modified platelets may help address this issue (Fig. 1.1). Platelets are already natural delivery vehicles within the blood [9]. They carry and protect their contents from degradation, and once activated at sites of vasculature damage release cargo for targeted drug delivery. De novo synthesis of proteins inside of platelets could allow for increased production and release of pro-coagulant or anti-fibrinolytic molecules, or alternatively, release of exogenous RNA that could be delivered to nearby cells to alter their activity [10, 11]. Such technology could also be used to extend the activity of platelets into new directions, through synthesis of anti-angiogenic factors to tumors or pro-angiogenic factors to enhance wound healing. 2   Figure 1.1 Schematic of modified platelets and their potential long-term applications. Following uptake of mRNA, platelets may a) translate the mRNA during storage or b) translate the mRNA at sites of active bleeding following transfusion into a patient. c) The newly synthesized protein may also be secreted upon activation. d) Alternatively, the mRNA may be released in platelet microparticles and delivered to nearby cells. 3   There are challenges in modifying platelets. Currently, hematopoietic stem cells need to be transfected to increase protein expression in platelets. While these have been used to treat inherited platelet disorders, stem cell transplantations require chemotherapy prior to the transplantation, which can lead to increased risk of infection and does not eliminate the possibility of graft-versus-host disease [12, 13]. While modified platelets can be obtained from genetically-modified stem cells, methods for generating platelets ex vivo are not yet advanced enough to produce clinically-relevant numbers of platelets [8]. An alternative approach would be to directly alter donor-derived platelets ex vivo prior to transfusion. However, methods for transfecting platelets are limited. Common commercial-based lipid agents have low efficiency [14]. A polyamine-based mixture has shown increased efficiency for short interfering RNA (siRNA) and plasmid delivery, however the effects of this agent on platelet function are uncharacterized [15]. There is currently no method for the delivery of messenger RNA (mRNA) to platelets. Since platelets are anucleate, modifying protein expression within the mature platelet requires delivery of RNA-based agents. Even after delivery of the mRNA to the cell is achieved, there are many challenges in obtaining efficient protein expression. Unmodified in vitro transcribed mRNA can trigger an innate immune response through interaction with toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns such as double-stranded RNA, leading to impaired to degradation of the RNA and impaired protein synthesis [16-19]. Furthermore, escape of mRNA from endosomal compartments is a limiting factor for nucleic acid delivery to all cell types [20, 21]. Overcoming the limited translational activity of platelets is an additional challenge [22]. As such, the focus of this thesis was on developing a 4  method for efficiently delivering mRNA to platelets without activating them, which is the first step for direct transfection of transfusable platelets.  1.1.2 Objectives In chapter 2, I identified lipid nanoparticle (LNP) formulations that efficiently deliver mRNA to platelets. LNPs are used to deliver nucleic acids to cells in vitro and in vivo, however their transfection efficiency and biocompatibility is highly dependent on the lipid composition. The ability of four classes of LNPs to deliver in vitro transcribed mRNA were compared, and uptake was determined by use of small molecule inhibitors and flow cytometry, as well as confocal microscopy. As one of these four classes lacked the cationic lipid that typically enhances mRNA encapsulation and cellular uptake, platelets were also engineered to synthesize RNA using this class of LNPs as an alternative delivery approach. In chapter 3, I determined the ideal conditions for LNP uptake by platelets. Platelet function is dependent on storage conditions, including time, pH, and storage media, as well as on activation state. Flow cytometry and confocal microscopy were used to compare uptake under conditions for the LNP formulations tested in chapter 2. LNPs or foreign RNA can induce platelet activation, which may increase the risk of thrombosis following transfusion. In vitro assays quantified the ability of LNPs to induce or impair four different measures of platelet activation: granule release, aggregation, pro-coagulant activity, and spreading. From these assays, I identified storage conditions which maximize mRNA delivery to the platelet while minimizing activation. In chapter 4, I characterized the stability and release of the LNP formulations that showed the best uptake, based on the work in chapters 2 and 3. The stability of the internalized RNA in different storage conditions was compared by confocal microscopy, flow cytometry and 5  quantitative PCR (qPCR). The ability of the platelets to translate the delivered RNA was tested, although no protein expression was detected. Conditions under which the RNA was released in microparticles were identified using flow cytometry and qPCR. 1.2 Background and Literature Review 1.2.1 Platelet Physiology 1.2.1.1 Platelet formation and clearance Platelets are small, 1 to 3 µm cells that circulate within the blood for 7 to 10 days and mediate many physiological and pathological processes [1]. About 100 x 109 platelets per liter of blood are produced and cleared each day, allowing the body to maintain a normal platelet count of 150 x 109 to 400 x 109 L-1 [23]. Anucleate, platelets are the end product of megakaryocytes, large polyploid cells residing in the bone marrow. Following maturation, megakaryocytes extend pseudopodial structures termed proplatelets through bone marrow sinusoids and into the bloodstream [24]. This process can also occur in the lung circulation, in megakaryocytes that have migrated from the bone marrow [25]. Organelles and platelet cellular content are transported to the tips of proplatelets with the help of an active microtubule system, which forms two platelet-sized microtubule loops at the end of the proplatelets [26]. These barbells break into two individual mature platelets. Disc-shaped cells larger than a mature platelet and capable of converting back into barbell-shaped pro-platelets have also been observed in culture, although its unknown if these preplatelets are present in vivo [27]. Platelets are cleared by the reticuloendothelial system, primarily the liver or spleen. As with nucleated cells, platelets undergo apoptosis [23]. This process in platelets is regulated by many of the same pro- and anti-apoptotic proteins found in other mammalian cells, including the pro-apoptotic Bak and Bax [28] and the anti-apoptotic Bcl-xL [29]. Knockout of Bcl-xL.induces 6  thrombocytopenia in mice, which can be rescued by a double knockout of Bak and Bax [28, 30]. During storage of human platelets, Bcl-xL gradually declines, leading to apoptosis [30]. The signalling pathways regulating apoptosis within platelets, and how they trigger platelet clearance, is unclear. In typical cells, the phosphatidylserine exposure upon apoptosis serves as a marker for phagocytotic clearance. While this does occur in apoptotic platelets [31], glycoproteins and modification of glycoprotein receptors platelets seems to play a larger role in mediating platelet clearance. Desialylation of the glycoprotein Ib (GPIb/CD42b) receptor leads to recognition by the Ashwell-Morell receptor on the surface of hepatocytes and liver macrophages, termed Kupffer cells, causing clearance of the platelets [32, 33]. While GPIb contains 70% to 80% of the total sialic acid in platelets, injection of neuraminidase into mice lacking GPIb still leads to platelet clearance, suggesting other glycoproteins can act as clearance signals [34]. For example, N-acetylglucosamine present on integrin αMβ2 has also been linked to platelet clearance of refrigerated platelets [35]. Additionally, Fc-receptor mediated clearance by macrophages occurs through alloantibodies to GPIIIb/IIa (CD41/CD61) and GPIb/IX [36]. 1.2.1.2 Platelets in hemostasis Platelets’ primary role is to mediate hemostasis [37]. They are the first responders following injury to the vessel well, leading to the formation of a platelet plug and eventually a stable blood clot. They are recruited to sites of injury by exposed extracellular matrix (ECM) proteins, particularly collagen and fibronectin [38]. Initial binding to the subendothelium is mediated by interactions between von Willebrand factor (vWF) and collagen. vWF circulates in the blood in an inactive form and is also secreted by endothelial cells activated by vessel damage as well as by activated platelets. During storage and in plasma, vWF binds to coagulation factor VIII (FVIII), protecting it from degradation and rapid clearance. FVIII is needed for activation of 7  the coagulation cascade, a series of enzymatic reactions required for a stable clot to form (discussed below) [39]. High shear forces also lead to unfolding of vWF in plasma, exposing binding sites for the platelet GPIb-V-IX receptor complex [40]. The collagen-vWF-GPIb complex recruits circulating platelets to the site of injury, where adhesion is strengthened by additional binding of collagen to platelet GPVI [41] and GPIa-IIa [42]. Several other platelet integrin-ECM protein pairs further enhance adhesion, including fibronectin and α5β1 [43] and laminin and α6β1 [44]. Following initial adhesion, platelets are activated to enhance recruitment and induce platelet aggregation. GPVI receptors promote activation through outside-in signalling [41], which in turn leads to release of platelet granular contents [45], including adenosine diphosphate (ADP), thromboxane A2 (TxA2), and serotonin. Binding of ADP to the purogenic receptors P2Y12 [46] and P2Y1 [47] and TxA2 to the thromboxane receptor TP α  [48] amplifies platelet activation [45], while binding of serotonin to the 5-hydroxytryptamin 2A receptor enhances this activity [49]. The purogenic and thromboxane receptors are G-protein coupled receptors [50, 51], and along with GPIb-V-IX and GPVI, cause activation of GPIIb/IIIa [52, 53].  Once activated, GPIIb/IIIa undergoes a conformational change, facilitating binding of the plasma protein fibrinogen and aggregation of platelets [54, 55]. Collagen, ADP and TxA2 also cause release of calcium from the dense tubular system of platelets, increasing the cytosolic concentration of calcium [56]. This activates downstream signalling pathways, promoting further granule release, aggregation and platelet shape change [45]. The most potent platelet agonist is thrombin, a serine protease that circulates in the blood as inactive prothrombin until it is activated by the prothrombinase complex, composed of activated FX (FXa), FVa, and calcium on a phospholipid surface [57]. Thrombin activates platelets through the protease-activated receptors 1 (PAR1) and 8  4 (PAR4) [58] and GPIb-IX-V [59], inducing aggregation, granule release, and calcium release [51]. Thrombin also cleaves multiple zymogens in the coagulation cascade, including FVIII, which then complexes with FIXa to generate FXa, amplifying thrombin generation [57]. Thrombin cleaves fibrinogen into fibrin, which is crosslinked by FXIIIa to form an insoluble meshwork [57]. Platelets play an important role is this process through the release of coagulation factors and calcium, stored in granules, and by providing a negatively-charged phospholipid surface to which the coagulation factors bind [38, 60]. Activation also involves major cytoskeletal rearrangement of the platelet. Initially, platelets change from a discoid to spherical shape, mediated by rearrangement of the platelet’s microtubular ring, and form lamellipodia and filopodia, caused by reorganization of the actin cytoskeleton [61]. The initial shape change is required for aggregation, while platelet spreading occurs in response to collagen or fibrinogen and outside-in signalling through GPVI, GPIa-IIa and GPIIb/IIIa [62]. The increase in surface area is possible due to the open canicular system (OCS), a network of channels connected to the surface and extracellular environment [63]. Upon activation, the OCS provides excess membrane for the platelet, and provides a path by which platelet granules can release their contents. Levels of receptors are also regulated via interactions with the OCS. GPIb and inactive GPIIIb/IIa are stored within the OCS prior to activation, and GPVI is sequestered back into the OCS following activation [64]. Activated platelets also mediate clot retraction through interactions between the actin cytoskeleton and GPIIb/IIIa [61]. Contractile forces along the cytoskeleton increase the internal density of a clot, improving its stability [65].  A stable clot has a complex architecture. Rather than an homogenous network of fibrin and activated platelets, platelets within different regions are morphologically and molecularly 9  distinct [66], with a dense core of highly activated and degranulated platelets covered by a shell of less activated, loosely associated platelets. Platelet activation encompasses a series of events and a gradient of activation stages. Shape change, GPIIb/IIIa activation, and granule release are early events [62, 67, 68], while later, irreversible events include phosphatidylserine exposure, which supports coagulation factor complex formation, as well as microparticle formation, mediating cell-to-cell communication and coagulation [60, 69-71]. Clinically, several points in this process are targeted to inhibit platelet activity and prevent thrombosis. Aspirin blocks TxA2 synthesis and thienopyridines such as clopidogrel block the P2Y12 receptor, preventing amplification of the activation response and reducing the risk of thrombosis [72, 73]. Antagonists of GPIIb/IIIa, eptifibatide and abciximab, are also used to inhibit platelet function [74]. Bleeding complications are observed in patients who have inherited disorders that cause defects in platelet activation. This includes Glanzmann’s thrombasthenia, caused by defective or absent GPIIb/IIIb [75], Hermansky-Pudlak Syndrome, caused by dense granules defects [76], and gray platelet syndrome, leading to the absence of alpha granule [77]. Dense granules primarily store small molecules such as ADP, serotonin, and calcium, required for activation of platelets and the coagulation cascade [78]. The alpha granules contain proteins essential for platelet activation, including vWF, fibrinogen, GPIIb/IIIa and GPVI, as well as FV, FXI, FXIII and prothrombin, which, along with calcium, promotes the pro-coagulant activity of platelets [79, 80]. However alpha granules also contain proteins that inhibit coagulation factors or promote fibrinolysis [80], indicating platelets’ play a regulatory role in hemostasis, rather than acting only to promote clot formation.  10  1.2.1.3 Platelets outside of hemostasis Initially platelets were thought only to mediate hemostasis and thrombosis through their role in clot formation; however, it is now clear platelets play important roles in inflammation and the innate and acquired immune systems, as well as in angiogenesis and tumor development. Upon activation, P-selectin (CD62) is transported from alpha granules to the platelet surface, facilitating the recruitment of leukocytes and activation of endothelial cells by binding to P-selectin glycoprotein ligand-1 on their surfaces [81, 82]. The release of cytokines and chemokines such as regulated on activation normal T cell expressed and secreted (RANTES) and platelet factor 4 (PF4) from alpha granules and serotonin from dense granules also helps recruit immune cells [83-85]. PF4 is one of the most abundant proteins in alpha granules, and promotes neutrophil granule release and adhesion to neutrophils, recruitment of monocytes to the endothelial and their differentiation into macrophages, and phagocytosis and generation of reactive oxygen species by macrophages [86]. Platelets also express CD40L, as well as release soluble CD40L (sCD40L), promoting secretion of chemokines and expression of adhesion molecules on endothelial cells [87, 88]. Platelet-derived CD40L induces differentiation and alters immunoglobulin production in B-cells [89], enhances protective T-cell responses [90], and decreases the production of pro-inflammatory cytokines by dendritic cells [91], indicating a role for platelets in adaptive as well as innate immunity. Due to the pro-inflammatory activities of platelets, they have been implicated in the development of acute organ injury [92], sepsis [93-95], and chronic inflammatory diseases, including atherosclerosis [81, 96] and rheumatoid arthritis [97].  Platelets also play a role in host defense. Platelets induce formation of neutrophil extracellular traps (NETs) via their interaction with neutrophils [93]. NETs, which are released 11  by neutrophils, are composed of extracellular DNA, DNA-associated nuclear proteins such as histones, and serine proteases, and mediate pathogen capture and phagocytosis by neutrophils. NET formation occurs following activation of platelets by bacteria-bound lipopolysaccharide (LPS), which interacts with toll-like receptor 4 (TLR4) on platelets. Platelets can also bind bacteria within bundles of fibrin, leading to leukocyte activation and NET formation [98]. Platelets can directly mediate microbial death as well. They release thrombocidins from their alpha granules, which are antibacterial proteins that can kill fungi or bacteria [99], and can directly mediate the destruction of red blood cells infected with Plasmodium spp. parasites, which cause malaria [100].  Platelets also aid in wound healing, the final step of the hemostatic response to injury, through the release of growth factors, angiogenic factors, and apoptotic regulators. These growth factors promote proliferation and migration of smooth muscle cells (SMCs) and endothelial progenitor cells, helping to re-establish vessel wall integrity [101]. Platelet derived growth-factor (PDGF) is a key regulator of SMC proliferation and migration [102], while stromal cell-derived factor-1 promotes migration of endothelial cell progenitors [103]. Pro- and anti-angiogenic factors, such as vascular derived growth factor (VEGF) and endostatin, regulate revascularization [101], while apoptotic regulators to help eliminate damaged cells [104]. Due to the ability of platelet-derived proteins to promote wound healing, platelet-rich plasma is used in the treatment of ulcers, burns, muscle repair, bone disease, and tissue recovery after surgery [105]. Pathologically, growth factors secreted from alpha granules also play a role in the development of cancer, where they are utilized by cancer cells to promote tumour growth and metastasis [106-109]. VEGF, PDGF, and platelet-derived transforming growth factor β1 (TGF-12  β1) have all been implicated in promoting cancer growth. Conversely, anti-angiogenic factors can limit tumour growth.  1.2.2 The role of platelets in the clinic 1.2.2.1 Indications for platelet transfusion Platelet transfusions are routinely used to prevent bleeding in patients that have dysfunctional or decreased platelets [110]. Platelets may be transfused to actively stop bleeding in patients with non-immune thrombocytopenia, immune-mediated thrombocytopenia, platelet dysfunction due to inherited or acquired causes, or during life-threatening hemorrhage and trauma [110, 111]. They may also be given prophylactically to patients undergoing surgery or with thrombocytopenia induced by bone marrow disorders or chemotherapy [110, 111]. In adults, there is generally an increased risk of severe spontaneous bleeding when the platelet count drops below 10 x 109 L-1, while the risk of bleeding complications during surgery increases when the platelet count drops below 30 to 50 x 109 L-l [110]. There are guidelines that include recommended platelet thresholds for transfusion depending on the specific indication and the associated risk of bleeding, however the decision to transfuse is typically made on a case-to-case basis, depending on a variety of factors [110-113]. This includes the patient`s platelet count, their age, the underlying cause of the thrombocytopenia, whether additional haemostatics such as antifibrinolytics can be used, and whether the patient may have increased risk of bleeding due to underlying factors such as fever, antibiotics, or anti-coagulants [110]. For instance, platelet transfusions may be given following head trauma or life-threatening hemorrhage, but not in patients on dual anti-platelet therapy experiencing spontaneous intracranial hemorrhage, where it may increase the risk of adverse reactions [110, 114]. In addition, if thrombocytopenia is due to destruction of platelets, such as during idiopathic thrombocytopenic purpura, thrombotic 13  thrombocytopenic purpura, or heparin-induced thrombocytopenia, transfusion is generally not recommended except in specific cases of severe bleeding [111, 115, 116]. Recommended platelet thresholds for transfusions also differ for children, based on whether the infant is stable or pre-term or whether there is active bleeding, and a single dose is generally less that used in an adult [117]. As with adults, transfusion decisions are made on a case-to-case basis. 1.2.2.2 Generating transfusable platelets In Canada, blood products are collected and produced by Canadian Blood Services, except in Quebec, where this is under the control of Héma-Québec. Platelets for transfusion are obtained either from pooled buffy coat preparations produced from whole blood or from plateletpheresis [116, 118]. Pooled buffy coat-derived platelets are produced by centrifuging whole blood, which separates into red blood cells at the bottom, the buffy coat containing the platelets and white blood cells in the middle, and the plasma on top. The buffy coat is then removed and centrifuged again to separate the platelets from leukocytes and residual red blood cells. Four or five ABO-matched platelet concentrations are pooled into the plasma from one of the donors and the unit undergoes leukoreduction by filtration  [116, 118]. Alternatively, single platelet concentrates can be derived directly from whole blood, stored as single units, and four to six units pooled prior to transfusion [111, 115]. While not used in Canada, platelets are produced this way in the United States, while the buffy coat method is predominantly used in Europe [111, 119]. Platelet concentrate from a single donor can also be obtained using automated apheresis system which separates the red blood cells and leukocytes from the platelets and plasma [116]. Both types of platelet products are collected into an anti-coagulant, generally citrate, and donor plasma is tested for ABO group and Rh type [111, 115, 116]. Following production, platelets are stored gently agitated at 20 to 24°C [111, 115, 116]. The length of storage varies between 14  countries. Storage is up to 7 days in Canada and in some parts of Europe, although in Europe this depends on the product and use of bacterial screening, while in the United States storage is limited to 5 days [111, 115, 116]. For prophylactic transfusions, one dose of platelets for an adult transfusion is typically considered to be one unit of pooled or apheresis platelets, although the number of units administered will vary depending on the patient [111, 115, 116]. Apheresis platelets are indicated for use during alloimmune refractoriness in Canada, but in general there are no universal guidelines for the use of pooled donor platelets over apheresis platelets [111, 115, 116]. 1.2.2.3 Adverse reactions to transfusion Adverse reactions due to infectious diseases is an ongoing concern. Blood donors are screened for a number of pathogens, which commonly includes human immunodeficiency virus (HIV), hepatitis B virus, hepatitis C virus (HCV), and syphilis [111, 115, 116]. In Canada and the United States the risk of transfusion-transmitted viral infections is low for these pathogens [120, 121]. However, there is still the risk of transmitting pathogens which are not readily detected or there is no donor screening test for, including tick-borne diseases caused by parasites, such as babesiosis, which appeared in Canada in 2015, and travel-related infections, which have included Zika virus and Ebola virus in recent years [111, 121-123]. Bacterial contamination is also a particular concern for transfusion of platelets because their storage conditions are  favourable for bacterial growth [124]. In Canada, all platelets units are screened for bacterial contamination, although this does not eliminate all cases of bacterial sepsis [124]. From 2010 to 2016, an estimated 1 in 10,000 units were contaminated, leading to an estimated 1 in 100,000 cases of bacterial sepsis, which can be fatal [124]. Pathogen inactivation techniques, where platelet products are treated with ultraviolet light and alkylating agents to damage the nucleic 15  acids of pathogens prior to transfusion, further reduce the risk of infectious adverse affects [125]. These are already used in the US and European Union [111, 126], and as of 2018 one such technology has been licenced by Health Canada for use with platelets [122]. In addition to infectious adverse reactions, there are also non-infectious adverse effects. These include mild to severe allergic reactions, febrile non-hemolytic transfusion reactions (FNHTR), anaphylaxis, transfusion association circulatory overload (TACO) and transfusion-related acute lung injury (TRALI), as well as several other rare reactions [111, 115, 116, 126]. The use of leukoreduction techniques has greatly reduced the number of transfusion complications in the past 20 years, including FNHTR, which is caused by cytokines or recipient antibody-donor leukocyte antigen interactions [6, 127]. Human error remains a major cause of transfusion-related adverse reactions, which can result from as mis-labelling of components, handling and storage errors, and mis-transfusion of blood components [127-129]. In Canada and the United Kingdom, TACO is a leading cause of transfusion-related death, arising from impaired cardiac function due to an excessively rapid rate of transfusion [127-129]. Human error can also lead to hemolytic transfusion reactions if ABO-incompatible products are transfused, as well as fatal delays in obtaining the appropriate products for a patient [120, 127, 129]. TRALI is also a major cause of transfusion-related mortality, and while it occurs less frequently than TACO, the mechanisms behind TRALI are not well understood [116, 122, 127, 129]. Biologically active lipids and cytokines such as sCD40L as well as platelet-induced NET formation are associated with TRALI [130, 131].  A concern unique to platelet transfusion, compared to transfusion of other blood products, is platelet alloimmune refractoriness, which occurs if the recipient`s immune system immediately destroys the transfused platelets, preventing an increase in platelet count [110]. This 16  can be caused by anti-human leukocyte antigen (HLA) antibodies or anti-human platelet antigen (HPA) antibodies and in Canada is generally managed with the use of HLA and HPA-matched apheresis platelets [110, 116]. However, leukoreduction has greatly reduced the occurrence of alloimmune refractoriness [110, 132]. Non-immune refractoriness is more common, but this is typically due to an underlying condition, such as fever, infection, drugs, splenomegaly, or disseminated intravascular coagulation, rather than a reaction to the platelet product itself [132]. Differences in the platelet product, including the dose, donor characteristics, and duration of storage, may also affect post‐transfusion platelet increments, which can complicate management of platelet refractoriness [132]. Management of platelet refractoriness is dependent on the underlying cause and patient factors [110, 132]. As platelets are important mediators of hemostasis, thrombosis also remains a risk during transfusion. Patients with thrombotic thrombocytopenic purpura and heparin-induced thrombocytopenia have an increased risk of arterial thrombosis and mortality and consequently, platelet transfusions are contraindicated for both disorders in Canada [116, 133].  1.2.2.4 Platelet storage Maintaining adequate supplies of platelet products is challenged by the need to minimize bacterial contamination, which limits the shelf-life of room-temperature stored platelet products [134].  While improved protocols for bacterial screening allowed Canadian Blood Services to extend storage periods from 5 to 7 days in in 2017 [121], platelets also undergo deleterious changes to platelet structure and function during in vitro storage, terming platelet storage lesion (PSL), leading to concerns over the effectiveness of older platelets in vivo [134]. After 10 or more days of storage at room temperature, platelets have increased exposure of P-selectin and phosphatidylserine, microparticle formation, loss of GPIbα and GPVI surface expression, and 17  activation of GPIIb/IIIa conformational change, which are indicators of platelet activation [135-137]. PSL is also linked to an increase in apoptosis, based on increased expression of pro-apoptotic proteins, including Bax and Bak and the cleavage of caspase-3 [137]. There are multiple factors that induce activation and apoptosis, including oxidative stress induced by handling of blood components during isolation, as well as ongoing glycolysis by platelets during storage, leading to lactate production and, without buffering, a decline in pH [138, 139].  Compared to platelets that are 0 to 2 days old, platelets that are 3 or more days old have decreased in vivo recovery and survival, are associated with shorter times until the next transfusion, a higher risk of bleeding, and an increased need for platelet transfusions in hematological patients [140, 141]. The relationship between platelet age and adverse reactions is still not clear, which may in part be due to differences in how adverse transfusion reactions are defined and reported, study sizes, and platelet preparation. While a recent meta-analysis of the literature did not identify any correlation between platelet age and adverse reactions for leuko-reduced platelets, a clinical trial in the United States with over 50,000 participants demonstrated inflammatory, but not allergic reactions, were specifically correlated with apheresis, irradiated platelets that were 2 to 5 days old [6, 141].  Predicting the efficacy of platelets following transfusion is still challenging. In vivo recovery and survival can be directly measured by labelling transfused platelets with radioisotopes (15Cr or 111In) or chromophores, but these assays are time-consuming and complex to perform [30, 142-144]. In vitro indicators of platelet quality include markers of platelet metabolism and activation, including pH, glucose and lactate concentration, P-selectin exposure, the extent shape change, and the hypotonic shock response, which assesses the ability of platelets to perform active transport and measures of membrane integrity and metabolism [142, 145, 146]. 18  However, these markers only weakly correlate with in vivo platelet recovery and survival [142, 144].  To improve platelet quality, platelets are stored in platelet additive solutions (PAS) in many countries, including the United States and Europe [115, 120]. These solutions replace the plasma such that the final solution is between 20 to 50% plasma, reduce the frequency of adverse reactions to platelets, and enable the use of pathogen inactivation technologies [138, 139]. They are several PAS available with varying compositions, but generally include citrate, acetate, phosphate, potassium and magnesium, and in some, gluconate [138]. Acetate is a key component that prevents the decline in pH, acting both as a buffer and as alternative energy source to glucose, as it can be consumed by platelets through oxidative phosphorylation and reduce lactate production [138]. Newer generations of PAS include glucose and bicarbonate and can maintain acceptable in vitro platelet quality with as little as 5% to 10% of plasma [145, 146].  Storage of platelets at lower temperatures, around 4°C, are of interest because lower temperatures reduce bacterial growth and decrease platelet metabolism [134]. In vivo studies indicate that shortly after transfusion, cold-stored platelets demonstrate increased hemostatic efficacy, however, the half-life of cold-stored platelets is only about 1 day, compared to 4 days for room-temperature platelets [33, 134]. This has led to the hypothesis cold-stored platelets might be ideal for use in trauma and surgery patients to control active bleeding, while room-temperature platelets would be suited for prophylactic transfusions [147]. Currently, 3-day cold-stored platelets are approved in the United States specifically for use during active bleeding [148].  19  1.2.3 Creating modified or synthetic platelets for the clinic 1.2.3.1 The platelet transcriptome Platelets receive an abundant array of RNA from their parent megakaryocyte. This includes mRNA [149, 150], ribosomal RNA, and transfer RNA for protein translation [151-153], as well as regulatory miRNA [154] and circular RNA (circRNA) [155]. Generally, the platelet transcriptome matches that of the parent megakaryocyte. However there are exceptions where only the protein and not mRNA template is found in platelets, and vice versa, indicating megakaryocytes preferentially sort mRNAs during platelet production [149]. Platelets can also endocytose mRNA from cancer cell-derived microparticles [156]. While anucleate, platelets have some transcriptional activity in their mitochondria, where they synthesize DNA and perform DNA-dependent RNA synthesis [157].  The structure of platelet mRNA is typical of a mammalian cell, with transcripts containing a 5’-7-methylguanosine (m7G) cap, a 3’ poly(A) tail, and 5’- and 3’-untranslated regions (UTRs) [158, 159]. Relative to leukocytes, platelets contain less mRNA per cell, which is expected since they are small and anucleate [160]. The transcriptome is varied, with thousands of different mRNAs identified using RNA-sequencing approaches [158, 161]. The majority contain exon-exon junctions, indicating they are mature, spliced mRNA [161]. The half-life of typical platelet mRNA is about 6 hours in newly formed platelets, with ribosomal protein mRNA having slightly longer half-lives closer to 7 hours [22, 162]. Consistently, translational activity is higher in younger, reticulated platelets and declines sharply within 24 hours of storage  [22]. However, there are subsets of platelet mRNA that have long half-lives, such as GPIIIa mRNA, which has a half-life of 2.9 days, as well as mRNA for proteins important in signalling pathways, including myeloid differentiation primary response gene 88 and Ras-related protein Rap-1A 20  [151, 162]. This suggests there are mechanisms for regulating platelet mRNA stability in platelets. In general, the 3`UTR in platelets is longer and more complex than in nucleated cells, and enriched for cytoplasmic polyadenylation elements that facilitate translation by extending the poly(A) tail as well as AU-rich mRNA decay elements that regulate transcript stability and translation [163]. Platelets contain proteins that bind these elements, including a cytoplasmic form of the poly(A) binding protein [164], T-cell internal antigen-1 (TIA-1), TIA-1-related protein, and Hu protein R [165]. Release of TIA-1 from SERPINE1 mRNA leads to translation of the mRNA into plasminogen activator inhibitor-1 [166], indicating a functional role for the protein. The majority of small RNAs in platelets are miRNA, which play an active role in regulating protein expression and platelet function [167, 168]. There are 500 to 2,000 detectable mature miRNAs present in platelets, although 90% of all platelet miRNAs are represented by just 15 different families of miRNA [169]. Platelets contain the necessary proteins for splicing premature (pre-miRNA) into mature miRNA, including the ribonuclease Dicer1, as well as components of the RNA-induced silencing complex (RISC), which directs miRNA and mRNA pairing and ultimately mRNA degradation or translational repression. This includes Argonaute-1, -2, and -3 (AGO1, 2 and 3), key components of RISC [154, 170]. Platelets derived from megakaryocyte lacking Dicer1 exhibit increased uptake of fibrinogen and activation of GPIIb/IIa in response to PAR4 stimulation, which enhances in vivo hemostasis and thrombotic potential [168].  In healthy individuals, differential expression of specific miRNAs are correlated with reactivity to epinephrine [167], as well as to race and PAR4 reactivity [171]. Specifically, the amount of miR-376c was inversely proportional to the amount of its target mRNA, which 21  encodes phosphatidylcholine transfer protein (PCTP). Higher levels of PCTP were in turn correlated with increased PAR4-mediated platelet aggregation. Additionally, miR-96 is negatively correlated with expression of its target mRNA, VAMP-8, and with platelet hyperreactivity to epinephrine, suggesting a role for miRNAs in regulating granule secretion [172]. Platelet miRNAs may also regulate pathways beyond hemostasis. Upon stimulation with thrombin, platelet miR-27b levels are reduced, increasing protein expression of its mRNA target, anti-angiogenic thrombospondin-1, and lowering the ability of platelet releasate to promote endothelial tube formation [173]. This suggests that miR-27b is a negative regulator of platelet’s angiogenic activities. Platelet miRNAs are also important in disease. Differential expression and release of miRNAs has also been detected in various patient populations, including patients with ST-elevated myocardial infarction [174], and patients with diabetes also exhibit reduced miRNA levels due to calpain-mediated cleavage of Dicer [168]. As in other mammalians cells, platelet miRNAs can be post-transcriptionally modified, generally by uridylation or adenylation at the 3’ end, however the seed regions from nucleotide 2 to 8, which is responsible for targeting to its mRNA partner, are well preserved [169]. There are also numerous miRNA isoforms present in platelets, arising from imperfect cleavage of the pre-miRNA species or post-translational modification, increasing the diversity of mRNAs that can be targeted by platelet miRNAs. Platelets are also enriched for circRNA due to the rapid decay of linear mRNA [155], and release circRNA in microparticles [175]. CircRNA is resistant to degradation by exonucleases and can regulate protein translation [176], but the physiological relevance of platelet circRNA is not known. 22  1.2.3.2 Protein synthesis in platelets An overview of protein synthesis in platelets is provided in Fig. 1.3. Platelets contain active ribosomes, located along the rough endoplasmic reticulum [177]. Initial studies in the late 1960’s demonstrated global protein synthesis in platelets by observing the incorporation of radiolabelled amino acids, demonstrating larger, younger platelets have greater synthetic activity that older platelets, and that protein synthesis in platelets could be altered by extracellular stimuli   Figure 1.2. Overview of protein synthesis in resting and activated platelets. Platelets contain multiple types of mRNA, including (a) miRNA and (b) mRNA. Pre-miRNA can be processed by the endonuclease Dicer to generate mature miRNA, which represses translation of certain mRNA, while other mRNAs are constitutively expressed. (c) Upon activation, pre-mRNA containing introns is spliced, leading to signal-dependent translation. (d) Global levels of translation increase due to activation of eukaryotic initiation factors, while changes in pre-miRNA processing alters miRNA levels and the protein expression profiles. e) Certain miRNAs will be released in platelets microparticles, leading to increased expression of their target mRNA. Intact mRNA may also be released. 23  [178-180]. GPIb, GPIIb, GPIIIa, fibrinogen, thrombospondin, albumin, vWF, HLA, and coagulation factor XIII a-chain (FXIIIA) are synthesized in freshly isolated platelets [165, 179, 181], and proteomic analysis on stored platelet concentrates has identified several additional proteins that are constitutively translated [182]. The pathophysiological relevance of platelet translation is highlighted by the differential protein expression of transcripts in patients with thrombosis [183, 184], sickle cell anemia [185], and systemic lupus erythematosus [186]. Platelets are also capable of activation-dependent translation in response to hemostatic agonists, such as thrombin, or markers of inflammation, including LPS and α-toxin (Table 1.1, Fig 1.3) [85, 103, 149, 152, 173, 187-192]. Despite lacking nuclei, platelets can splice pre mRNA into mature mRNA to regulate expression of interleukin 1β (IL-1β) [85], TF [187, 188] and FXI [192]. Recently, a combined transcriptomic and proteomic approach correlated splicing of pre-mRNA to protein expression of specific functional subgroups in platelets treated with collagen or thrombin receptor-activating peptides, indicating splicing may be a general mechanism of regulation for protein synthesis in platelets [193]. The molecular mechanisms controlling translation in platelets are not fully understood. Classic translational inhibitors, including cycloheximide, chloramphenicol, and puromycin, decrease translation in platelets [152, 194]. Platelets express eukaryotic initiation factors (eIFs), specifically eIF4E and eIF-2α, which are essential to translational initiation in nucleated cells and major regulators for translational initiation. In the canonical pathway for protein synthesis, initiation of protein synthesis involves eIF2 ternary complex formation, which is required for binding of the ribosome to the mRNA. The eIF2 ternary complex is composed of the three eIF2 subunits (α, β, ɣ), GTP, and the initiating methionyl-tRNA, which binds the 40S subunit of the ribosome to form the 43S preinitiation complex [195]. mRNA is bound by the eIF4F cap-binding  24  Table 1.1. Proteins synthesized in platelets following activation with platelet agonists. Protein Platelet agonist Function Bcl-3 Thrombin with or without fibrinogen [152] Immobilized fibrinogen or collagen S. aureus α-toxin [190] Clot retraction FXI ADP [192] Procoagulant COX-1  Thrombin and fibrinogen [196] TXA2 synthesis COX-2  LPS, CD14, and LPS-binding protein [197]  IL-1β Thrombin with or without fibrinogen [85] Platelet activating factor with fibrinogen [85] ADP [85] Collagen [85] Epinephrine [85] LPS, CD14, and LPS-binding protein [197] Platelet aggregation and granule release, Neutrophil binding MAP1LC3B2 All-trans-retinoic acid [198] Microtubule assembly and reorganization Plasminogen activator inhibitor-1 Thrombin [191] Inhibits clot lysis Ras-related protein 1 Rap1 ADP [199] GPIIb/IIIa activation and granule release Stromal cell-derived factor 1α Thrombin [200] Proangiogenic Sodium vitamin C transporter 2 Thrombin Phorbol 23-myristate 13-acetate Regulation of platelet redox state, thrombus rigidity Thrombospondin-1 Thrombin [173] Antiangiogenic TF Thrombin and fibrinogen [188] Procoagulant Tissue inhibitor of metalloproteinase-2 Thrombin [149] Inhibits platelet aggregation and ECM degradation  complex, which includes the cap-binding protein eIF4E, the helicase eIF4A and the scaffold eIF4G, which also binds the poly(A) binding protein to allow circularization of the mRNA [201]. Along with eIF4B, a co-factor for eIF4A, this facilitates unwinding of the mRNA at the 5’ end 25  and attachment of the 43S pre-initiation complex to the mRNA [201]. Once attached, the 43S complex scans along the mRNA until reaching the start codon, leading to 48S complex formation. Once the GTP in the eIF2 complex is hydrolyzed to GDP, the eIFs are displaced, and the 60S subunit of the ribosome associates [201]. To initiate further rounds of translation, the GDP in the eIF2 ternary complex must be exchanged for GTP, a process mediated by eIF2B [201]. Regulation of translation initiation is primarily through reversible phosphorylation, which regulates the availability of eIF2 and eIF4F. Phosphorylation of eIF2α, which occurs in response to cellular stress, prevents eIF2 from dissociating with eIF2B and recycling of the GDP to GTP, slowing global translation [195]. Translation can also be inhibited by eIF4E-binding protein 1 (4E-BP1), which binds to eIF4E when hypo-phosphorylated and prevents formation of the eIF4F cap-binding complex. Phosphorylation of 4E-BP1 is mediated by mammalian target of rapamycin (mTOR), which is activated by mitogenic signals [201]. In activated platelets, phosphorylation of 4E-BP1 by mTOR facilitates synthesis of B-cell lymphoma 3 (Bcl-3) [189] and cyclooxygenase-1 (COX-1) [196]. Platelet activation is also linked to redistribution of eIF4E to the outer actin ring of platelets [202], as well as phosphorylation of ribosomal protein S6 kinase beta-1 (S6K1) by mTOR [189]. Phosphorylation of S6K1 promotes translation by activating eIF4B and relieving inhibition of eukaryotic elongation factor 2 [203].  Platelets also contain nuclear receptors that play signalling roles within platelets. The retinoid A receptor-α binds specific mRNAs, including its own encoding mRNA and microtubule-associated protein 1 light chain 3 beta 2 (MAP1LC3B2) mRNA, preventing translation [198]. When treated with all-trans retinoic acid, a vitamin A metabolite and natural ligand for RARα, expression of MAP1LC3B2 increased.  26  Ribosome profiling demonstrates that treatment of platelets with thrombin leads to a global increase of ribosome occupancy on mRNA, consistent with increased in total protein synthesis observed upon activation with thrombin and/or fibrinogen [152, 162, 204]. Activation also leads to differential changes across transcripts, increasing ribosome occupancy on some transcripts and decreasing occupancy on others. Platelets lack the ribosome recycling factor ABCE1 and the ribosome rescue factor Pelota (PELO), both of which are required for dissociation of terminal or stalled ribosomes from mRNA [205]. This leads to an abundance of ribosomes at the 3’UTR and slows mRNA decay. It has also been estimated platelets had about 1000 less rRNA compared to HeLa cells [206], as well as lower levels of eIF4E and eIF-2α [207], which along with the stalled ribosomes, may contribute to the low translational activity in resting platelets. Platelet translational machinery may also be hijacked by invading viruses. In dengue-virus infected platelets, the positive-sense single-stranded RNA genome of the virus was replicated by platelets over 7 days of storage, presumably by the viral RNA-dependent RNA polymerase [208]. Dengue viral proteins were expressed in infected platelets, and treatment with cycloheximide, a translation elongation inhibitor, reduced viral production. 1.2.3.3 Creating genetically modified platelets Genetic approaches to altering platelets have focused on correcting inherited platelet defects using modified hemopoietic stem cells (HSCs), as megakaryocytes only have a lifespan of about 5 days and platelets are anucleate [209]. Megakaryocyte-specific gene promoters, which include members of GPIBA-GPIX-GPV complex [210], ITGA2B (GPIIb) [211], GPVI [212], the thrombopoietin receptor (c-mpl) [213], and PF4 [214], direct transcription of transgenes during the early and mid-stages of megakaryocyte formation. GPIBA, ITGA2B, and PF4 are the most 27  commonly used promoters, driving moderate to high-levels of protein expression preferentially within megakaryocytes [10, 215, 216].  Defects in genes that play a role in platelet function or formation occur in about 1 in 10,000 individuals, and can lead to uncontrolled bleeding [209]. Typically, this is treated by platelet transfusions, anti-fibrinolytic agents, or recombinant factor VIIa, however patients can become refractory to transfusions and infusions of antifibrinolytic agents or recombinant proteins can be short-lived and costly [209]. Bone marrow transplantation of modified HSCs has been used to treat patients with Glanzmann thrombasthenia, caused by absent or defective GPIIb/IIIa. Use of this technology is still limited by transplant-related complications, which include graft-versus-host disease or graft failure as well as thrombocytopenia arising from antibodies to GPIIb/IIIa [12, 13]. Recently, monocytes from Glanzmann thrombasthenia patients were reprogrammed into pluripotent stem cells, which in turn were used to regenerate GPIIb-expressing megakaryocytes and platelets [215]. This may address some of the complications that arose with the bone marrow transplantations. In a murine model, transplantation of lentiviral transduced HSCs has also been used to correct the genetic defect causing Bernard-Soulier syndrome, causing by mutations in GPIb or GPIX, although this has not been tested in humans [217]. This approach was also used to induce expression of urokinase-type plasminogen activator in mice, confirming the role of this protein in Quebec platelet disorder. This also prevented thrombosis in wild-type mice, indicating that shifting the hemostatic balance of platelets through expression of fibrinolytic proteins might be a therapeutic strategy for managing thrombosis [218]. Hemophilia A, caused by defects in FVIII, is currently treated with plasma-derived and recombinant factor concentrates, given prophylactically and in response to serious bleeds [209]. 28  A strategy for continuously producing FVIII at the site of vascular injury from platelets might reduce the cost and improve the quality of life for hemophilia patients. Adeno-associated viral vectors targeting the liver have been utilized for gene therapy in hemophilia B patients with defective FIX [219], and may be utilized to treat hemophilia A, but patients with pre-existing liver damage or antibodies against FVIII or the viral capsid would not be able to use this therapy. To overcome these limitations, production and storage of FVIII within platelets using lentiviral-modified HSCs was used and corrected bleeding defects in murine and canine hemophilia A models [10, 220]. Fusing an alpha granule targeting peptide to FVIII prevented leakage of FVIII from the cytoplasm into the plasma, which helped protect FVIII from degradation and reduce complications due to pre-existing FVIII antibodies.  In addition to treating bleeding, platelets are being developed to treat cancer. Platelets have been modified to produce an antioncogenic agent, tumstatin, using HSCs transduced with a lentiviral vector and a non-specific gene promoter [221]. These platelets stored tumstatin within alpha granules and the releasate from thrombin-activated platelets caused an antiangiogenic effect in lung tumor cells in vitro. Lentivirals were also used to transduce HSCs with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which induces apoptosis in tumor cells. TRAIL-expressing platelets killed cancer cells in vitro and reduced metastasis in a mouse model of prostate cancer after bone marrow transplantation of the HSCs [222].  There are several issues with the use of modified HSCs that are common to all viral-based gene therapies, including side effects from pre-transplant preconditioning with immunosuppressive agents, risk of insertional mutagenesis, and development of an acquired immune response to the transgene product or the vector [209]. A method for intraosseous delivery of lentiviral vectors was developed as an approach that would not require the uses of 29  preconditioning agents. While this technique was used to express FVIII in platelets in a murine model of hemophilia A, it has not yet been tested in large animal models [223]. With regards to off-target effects caused by modifying the megakaryocyte lineage, there is one report of platelet-derived FVIII causing decreased platelet production and altered clot formation, however in animal models no defects have been noted [224].  1.2.3.4 Culturing platelets ex vivo An alternative approach to transplanting transduced HSCs is the direct delivery of modified platelets cultured ex vivo from the transduced HSCs. These would be useful in scenarios where permanent changes to the genome are not desired, such as during episodes of acute bleeding that are not due to an underlying genetic defect. There are still challenges to overcome before ex vivo generated platelets can be used in the clinic. The formation of megakaryocytes to HSCs involves two microenvironments, the osteoblastic and vascular niche [225, 226]. Both erythrocytes and megakaryocytes arise from the same progenitor after the initial differentiation of HSCs, however the signals controlling this final stage of separation are not well understood [227]. Several transcription factors and cytokines that are involved in megakaryopoeisis and platelet production have been identified, including thrombopoietin, which is a major regulator of platelet production [228-230]. However this process is complex, and still not fully understood. Before platelet formation, megakaryocytes undergo endomitosis, replicating its DNA but not its nucleus [231], in order to generate the large number of organelles and proteins to be packaged into platelets [27, 232]. Following maturation, megakaryocytes form proplatelets, with a single megakaryocyte breaking into 2,000 to 10,000 platelets [233]. High shear forces can significantly increase the rate of platelet release ex vivo, yet beyond this the 30  mechanisms controlling megakaryocyte maturation and platelet release are still being characterized [8, 27]. There are two major challenges to developing donor-independent platelets: generating sufficient numbers of megakaryocytes to produce a single platelet transfusion unit and generating physiological numbers of functional platelets per megakaryocytes [8]. Cultured platelets are generated from CD34+ progenitor cells, derived from umbilical cord blood, fetal liver, peripheral blood, human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs) or bone marrow [8, 234-239]. Only hESCs and iPSCs have the potential to be a renewable and unlimited source of progenitor cells, while the remaining sources require a continuous supply of donors. Since ESCs and iPSCs are tumorgenic and form teratomas in vivo, complete removal of residual pluripotent cells is required [240]. While methods for large-scale production of megakaryocytes from hESCs have been improved, yields of platelets are still at about 7 platelets per megakaryocyte [234]. However, the rate and yield of platelet formation was increased using a three-dimensional microfluidic bioreactor which mimics the bone marrow environment and hemodynamics of the blood [8]. With this device, the rate of platelet release increased from 18 hours to 2 hours, and the percentage of megakaryocytes forming proplatelet extensions rose from 10% to 90%, leading to the formation of about 42 platelets per megakaryocyte were formed. Universal platelets, negative for any HLA major histocompatibility antigens, have also been generated from hiPSC by deleting the β2-microglobulin gene, but only about 6 platelets per megakaryocyte were generated [235]. These technologies are currently being scaled-up and developed for clinical use by Platelet BioGenesis, a company in the United States. In an alternative approach, mature megakaryocytes have also been infused into mice and platelets formed in vivo, generating 100 to 200 platelets per infused megakaryocyte [241]. While 31  functional, the half-life of these platelets was slightly shorter, and generating sufficient mature megakaryocytes for this process remains a challenge. 1.2.3.5 Synthetic platelets Synthetic platelets that mimic the hemostatic properties of platelets have been designed as alternatives to transfused platelets. These primarily mimic the aggregatory or adhesive abilities of platelets, and are composed of albumin, polymeric, or lipid particles coated with fibrinogen or fibrinogen-related peptides [7]. Advances have been made in materials that mimic multiple functions of the platelets. SynthoPlate (Haima Therapeutics) is composed of liposomes decorated with a vWF-binding and collagen-binding peptides for adhesion, and an GPIIb/IIIa binding fibrinogen-mimetic peptide for adhesion [242-244]. It is the first example of a platelet mimetic tested in a large animal model, improving survival in a porcine model of traumatic injury [244]. These same ligands were also coated on layered poly(allylamine hydrochloride) and bovine serum albumin to create a flexible outer shell, mimicking the discoid shape of platelets and enhancing adhesion of the particle compared to their spherical counterparts [245]. Additionally, liposomes encapsulating ADP and coated with a fibrinogen-binding peptide improve survival in a rabbit model of trauma [246]. Some researchers have focused on the mechanical properties of platelets. An alternative approach to lipid-based platelet mimetics has been used in the design of deformable, crosslinked poly(N-isopropylacrylamide-co-acrylic acid) microparticle gel, surface modified with fibrin fiber antibodies [247]. This material mimicked the clot retracting activity mediated by the platelet cytoskeleton, yet could still spread like platelets, and lowered the bleeding time in rat models. Alternatively, shear-responsive materials composed of poly(lactic-co-glycolic acid) (PLGA) and coated with tissue plasminogen activator were used to created clot-busting materials that reduced 32  vessel occlusion in mouse models [248]. The nanoparticles form microaggregates that break apart in response to shear stress, leading to targeted delivery of the tPA at the site of obstruction.   Platelet membranes have also been coated onto nanoparticles to create platelet mimetics for use outside of hemostasis. In a mouse model of rheumatoid arthritis, these were used to deliver an anti-arthritic drug [249]. Similarly, platelet membrane-coated PLGA particles localized to atherosclerotic plaques in mice, targeting regions susceptible to rupture [250], and can be used as an antibody decoy in mouse models of immune thrombocytopenia [251]. Silica particles were also used to target circulating tumor cells in mice, and after being functionalized with a tumor-specific apoptosis-inducing ligand, reduced lung metastasis in a mouse breast cancer model [252].   Pre-clinical and clinical testing is still needed before these materials can supplement or replace transfused platelets. With regards to platelet-coated materials, there are concerns about maintaining the hemostatic function of the platelet membrane during extraction, as well as a source of platelet concentrates to generate the membranes from [7]. Alternative approaches using synthetic or recombinant protein fragments are still limited by cost and steric hindrance on the nanoparticles themselves, since multiple proteins are needed to replicate a platelets hemostatic function [7]. Costs can be reduced with the use of peptides, such as those used in the manufacture of SynthoPlates, however additional studies addressing dosing and the administrative time window are still needed to establish protocols for how artificial platelets can be used in hospitals [244]. 33  1.2.4 Platelets take up and release materials  1.2.4.1 Mechanisms of uptake of biological materials by platelets Platelets endocytose proteins [253-255], viruses [208, 256, 257], and bacteria [256, 258]. Plasma proteins endocytosed by platelets include albumin, IgG, and fibrinogen through GPIIb/IIIa [253, 254, 259], while endocytosis of IgG can also occur through the FcɣRIIA receptor [260]. In addition, uptake of vWF and fibronectin has been observed [255], as well as uptake of the purinergic receptors P2Y1 and P2Y12 [261].  Endocytosis can occur through multiple mechanisms, which are classified as clathrin-dependent or clathrin-independent [262]. Both mechanisms requiring adaptor proteins and GTPases for vesicle formation and fission from the membrane, while clathrin is a structural protein that can coat the outside of the vesicles. Clathrin-dependent mechanisms use dynamin as the GTPase, while clathrin-independent mechanisms are more varied and can be classified based on whether they utilize dynamin or another family of GTPases. In platelets, fibrinogen, vWF and fibronectin co-localize with clathrin-coated vesicles on the cytoplasmic surface of alpha granules, OCS and the plasma membrane [255]. However, the ADP-ribosylation factor 6 (Arf6), a GTP utilized in a dynamin-independent pathway, can also regulate trafficking of fibrinogen through endocytosis of GPIIb/IIIa [263], as well as internalization of P2Y12 following activation of the platelet, which allows resensitization of the platelet to ADP [261].  Platelets are also capable of fluid-phase endocytosis, or pinocytosis [264]. Compared to receptor-mediated endocytosis, pinocytosis involves membrane ruffling and formation of a membrane protrusion, rather than invagination of the membrane [265]. The endocytotic vesicle is them formed by collapse and fusion of the membrane protrusion onto the plasma membrane [265]. Unlike receptor-mediated endocytosis, which involves specific interactions between cell-34  surface proteins and extracellular ligands, pinocytosis occurs through nonspecific binding of materials to the cell membrane, and the amount of uptake is not saturable with respect the concentration of the extracellular material [265].  Following uptake, internalized vesicles fuse with early endosomes and are either recycled back to the surface via recycling endosomes or sorted into late endosomes and then lysosomes for degradation [262]. Multivesicular bodies are formed as an intermediate between early and late endosomes. Platelets contain markers for these organelles [263, 266], and endocytosed fibrinogen co-localizes with markers of early and recycling endosomes [263]. Phagocytosed materials enter the phagosomes, acidic compartments where their content is degraded [267]. Movement between these compartments are mediated by soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) complexes. In platelets, the SNARE cellubrevin/vesicle-associated membrane protein-3 (VAMP-3) is essential for uptake of fibrinogen, which passes through early and recycling endosomes before ending in alpha granules [268].  Like endocytosed proteins, human immunodeficiency viruses (HIV) and adenoviruses 100 nm to 200 nm in diameter were found in the OCS and possibly endosomes following uptake [256, 257]. Dengue virus is also endocytosed by platelets and replicates follow uptake, indicating its escape from endocytotic compartments [208]. Uptake of viruses is energy-dependent, and activation enhances the association between the platelet and virus [208, 256, 257]. Viruses can bind numerous receptors on the platelet surface to facilitate uptake. Binding of dengue is mediated through dendritic cell-specific intracellular adhesion molecule-3-grabbing-non integrin (DC-SIGN) and heparan sulfate proteoglycans [208], while HIV binds platelets through DC-SIGN and C-type lectin receptor-2 [269]. Platelets can also endocytose bacteria around 800 nm 35  in diameter. As with uptake of viruses, this requires platelet activation and involves the OCS, but then leads to formation of 1 or 2 large engulfment vacuoles that fuse with alpha granules and lysosomes [256].  1.2.4.2 Endocytosis of synthetic materials by platelets Platelets can endocytose a variety of inert particles, including latex beads [258, 270], liposomes [271], and gold nanoparticles [258]. As with plasma proteins and platelet receptors, endocytosed nanoparticles 100 nm or less in size appear in the OCS and alpha granules, and gold nanoparticles can reach the cytoplasm [271]. The process is energy-dependent, and impaired by disruption of the cytoskeleton [270]. Aspirin or other anti-coagulants including EDTA, sodium citrate, or heparin have no effect on uptake over short time periods [271]. When coated with fibrinogen or IgG, this process was mediated through the GPIIIb/IIa or FcɣRIIA receptor, respectively [254, 260]. Uptake of large latex beads 3 to 6 µm in diameter involves formation of pseudopods and the complete spreading of the platelet over the surface of the bead [258].  The effect of the endocytosed material on platelet function varies with the composition of the nanoparticle and size. Latex beads 87 nm in diameter caused minor dilation of the OCS, which became more pronounced with beads 312 nm in diameter, while micron sized beads led to complete evagination of the OCS [258]. Latex beads around 200 nm do not induce platelet aggregation [257]. Similarly, neutral liposomes had no effect on the ability of platelets to aggregate, while negatively charged liposomes prevented aggregation in response to thrombin and positively charged liposomes prevented aggregation in response to ADP but not to thrombin [272, 273]. In vivo, negatively charged liposomes produced transient thrombocytopenia in rats and platelet aggregation in guinea pigs, while positively-charged or neutral liposomes produced a smaller or no response [274, 275].  36  Modifying the surface of the nanoparticle with targeting peptides can enhance uptake. Liposomes conjugated to fibrinogen-mimicking peptides have been tested for their hemostatic and drug delivery capabilities [242, 246, 273, 276]. These liposomes bind to platelets, and activation of platelets enhanced the interaction [273, 276]. A positively-charged octa-arginine sequence in a fibrinogen-binding peptide was required to be internalized by the platelet [273]. In general, these liposomes enhance clotting and reduced bleeding in vivo, but depending on the peptide and size of the liposome they can cause aggregation or impair platelet function [246, 277]. 1.2.4.3 Regulation of platelet secretion Secretion allows platelets to act as natural delivery vehicles, through the release of small molecules, proteins, and polymers that regulate coagulation, inflammation, innate immunity, and angiogenesis (Fig. 1.3). Release only occurs upon activation, allowing for targeted delivery of these molecules. Within mammalian cells, including platelets, secretion is mediate by SNARE complexes present on the granule or vesicle (v-SNARE) or on the target membrane (t-SNARE). The t-SNARE and v-SNAREs form a heteromeric complex between the two bilayers to mediate fusion of the membranes. In platelets, the key components of the t-SNARE are synaptosomal-associated protein 23 and syntaxin-8 or syntaxin-11 for dense granule release [278, 279], while syntaxin-2, -4 and -11 have all been implicated in alpha granule and lysosome release [279-281], The key v-SNARE is VAMP-8 [282], and VAMP-7 also plays a major regulatory role, linking granule exocytosis with the actin cytoskeletal rearrangement that occurs during secretion [283].  37   Figure 1.3. Overview of platelet secretion. Platelets store small molecules and proteins in (a) alpha and dense granules (gray). The proteins may be endocytosed from the environment and trafficked through endosomes (blue) before reaching the granules. (b) Multi-vehicular bodies (MVBs) may serve as a midpoint during trafficking. Upon treatment with a platelet agonist, MVBs and storage granules fuse with the plasma membrane, releasing (c) exosomes and (d) granular contents into the extracellular environment. (e) Microparticles can also bud from the plasma membrane, carrying platelet proteins and RNA which are transferred to nearby cells. Some RNA may also be bound by proteins and released directly into the plasma.  How the platelet regulates release of proteins with varied and occasionally opposing biological roles is still not full understood, although redundancy in the v-SNAREs and t-SNARES likely plays a role. Release from granules first involves centralization of the granules [284], which is not observed during exocytosis in most nucleated cells [285]. Release of dense granule contents occurs rapidly, followed by alpha granules, and then lysosomes [68]. The actin cytoskeleton plays an important role in regulating granule release, as small molecules that disrupt the cytoskeleton have different effects on alpha and dense granules [286]. Using confocal microscopy, specific alpha granule contents have been observed to localize into different zones 38  [287, 288]. However, no functional grouping could be consistently identified. Similarly, while there is variation in the kinetics of release for different cargoes depending on the agonist used, the functional relevance of this variation has not been demonstrated [288].  1.2.4.4 Platelet microparticles Upon activation, platelets release a heterogenous population of extracellular vesicles, which are enriched in the blood of both healthy individuals and in patients [69, 289]. Extracellular vesicles vary in size, morphology, content, and source, with smaller 50 nm to 150 nm exosomes deriving from multi-vesicular bodies or alpha granules and larger 100 nm to 1,000 nm microparticles produced by membrane budding and plasma shedding (Fig. 1.2) [290]. Proinflammatory and procoagulant proteins [289, 291], bioactive lipids [291, 292], and RNA [70, 293] have been found in extracellular vesicles, and intact, active mitochondria are also present in platelet microparticles [294]. The majority of extracellular vesicles in the blood are derived from platelets, however extracellular vesicles can also be generated by leukocytes, erythrocytes, endothelial cells, smooth muscle cells, and cancer cells [291]. The source of blood-derived microparticles can be identified by surface antigens unique to their parent cell, such as CD42b and CD41 for platelet-derived microparticles, which allows identification of the origin cell by flow cytometry [291, 295]. Platelet microparticles can alter clot structure and support thrombin generation through exposure of phosphatidylserine and tissue factor [71]; however only a subset of platelet microparticles are pro-coagulant [291, 295]. Elevated levels of microparticles are also detected in patients with thrombosis [296], cancer [297], inflammatory and autoimmune diseases [97, 298], and metabolic disorders [299], and as a result, platelets microparticles are of clinical interest as biomarkers for disease. 39  Platelet microparticles have potential as drug delivery systems, as they can be internalized by endothelial cells [70], macrophages [300], neutrophils [301], and tumor cells [302] to mediate cell-to-cell communication. In solid tumors, transfer of microRNA (miRNA) led to reduced tumor growth [302], and in endothelial cell cultures transfer of both endogenous platelet miRNA and transfected synthetic miRNA led to knockdown in the endothelial cells [70, 174]. Microparticles have also transferred reporter green fluorescent protein (GFP) mRNA to hepatocytes [293], monocytes [300], and endothelial cells [300]. This led to de novo protein translation in these cells, although is it still unclear whether this process occurs in vivo. 1.2.5 LNPs and their applications in gene therapy 1.2.5.1 Challenges in gene therapy A major challenge in nucleic acid-based therapies is delivery. Nucleic acids are large, hydrophilic, negatively charged molecules, and on their own cannot cross the cell membrane [303]. The use of synthetic polymers and lipids, as well as viral vectors, have tried to address this problem. The ideal in vivo delivery agent would protect the nucleic acid from degradation and immune detection, prevent non-specific interactions with proteins or non-target cells and renal clearance, and allow for extravasation to reach target sites if necessary [304]. Furthermore, once inside the cell, the nucleic acid needs to be released from the endocytotic compartment into the cytosol. Each type of nucleic acid comes with its own unique challenges [305]. DNA needs to reach the nucleus to exert its effect and carries the additional risk of insertional mutagenesis [305]. Unmodified exogenous mRNA is unstable, leading to high initial protein expression that rapidly declines within a day [303]. It also needs to be recognized by ribosomes, while RNAi-based therapeutics need to be incorporated into RNAi machinery [305]. 40  To address the challenges of mRNA delivery, modifications to the mRNA itself and identification of a suitable delivery vehicle are necessary. Unmodified mRNA can activate TLRs, triggering cytokine formation and toxicity [16-19]. Incorporation of 2-thiouridine and 5-methyl-cytidine decrease binding to TLR-3, TLR-7 and TLR-8 and retinoid-inducible gene-1 (RIG-1) in peripheral blood mononuclear cells, reducing the innate immune response and increasing the stability of the mRNA [16]. This leads to enhanced protein expression in vivo following intramuscular injection and after lung administration, detectable for up to a week. Similarly, mRNA modified with 5-methylcytidine and pseudouridine was successfully used following intramyocardial delivery, co-delivered with the commercial transfection agent RNAiMAX [306]. Even in the absence of modifications, mRNA complexed to the commercial transfection agent Stemfect has been delivered intravenously, leading to protein expression in the spleen [307]. 1.2.5.2 Development of lipid-based delivery agents Liposomes are spherical vesicles containing at least one bilayer, and have been used for the delivery of DNA, mRNA and siRNA, as well as small molecules [304]. Their main component are phospholipids, typically phosphatidylcholine and phosphatidylethanolamine, leading to an amphipathic structure with an aqueous core and an hydrophobic membrane which prevents the passage of hydrophilic agents. Fusogenic helper lipids, including 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), are believed to promote disruption of the endosome bilayer and therefore aid in the escape of the nucleic acid into the cytoplasm [308]. While the earliest transfection agents were comprised of just a cationic a fusogenic lipid, forming lipoplexes, more sophisticated agents containing cholesterol and a lipid modified with polyethylene glycol (PEG) have since been developed [308, 309]. Cholesterol is often added to promote stability [310], while PEGylated lipids regulate particle size, minimize particle 41  aggregation, and limit unwanted nonspecific immune responses by providing shielding from macrophages [311]. PEGylated lipids prevent association with plasma proteins, which can lead to opsonization and uptake by macrophages. However, too high a concentration of PEG decreases internalization, as positive charges on the liposomes facilitates movement of liposomes across the cell membrane [312, 313]. Therefore, an optimal proportion of PEGylated is required. Inclusion of a cationic lipid or polymer greatly enhances the interaction between the lipid and nucleic acid and facilitates cellular entry and endosomal escape [304, 312]. Initially used in the delivery of DNA, synthetic cationic lipids have three main components: a positive head group, a hydrophobic tail and a linking group between the domains [308]. Once endocytosed by the cell, the head groups of cationic lipids and anionic endosomal membrane lipids form ion pairs, leading to adoption of a cone shape. This promotes transition from a bilayer to the non-bilayer hexagonal HII phase, leading to membrane disruption and endosomal escape [308].  Cationic lipids are rapidly cleared in vivo and induce inflammatory responses, which have prevented their widespread clinical use [314]. To overcome these limitations, second and third generation cationic lipids were developed, leading to ionizable cationic lipids which contain a protonizable head group and multiple unsaturated alkyl chains [308]. These lipids have been further modified by introducing an ester group into the hydrophobic chain, creating lipids that are more readily degraded to reduce toxicity [315]. Unlike liposomes, which contain an aqueous hollow core, LNPs formed with cationic lipids and siRNA have a core containing a mix of lipid-siRNA complexes surrounded by a monolayer of lipids [316].  1.2.5.3 Interaction of LNPs with the cellular environment Interactions between nanoparticles and proteins is a major determinant of their biodistribution, as most materials become coated with a protein corona upon contact with 42  biological substances [317]. Of the 3,700 proteins in plasma, at least 50 can associate with nanoparticles to form a protein corona around its outer surface [317]. These proteins include opsonins, such as immunoglobulins or complements proteins, which are recognized by phagocytic cells in the liver, spleen and lymph nodes [314, 317]. Consisting of dendritic cells, monocytes, and macrophages, these cells will degrade foreign materials in the blood. Surface charge, hydrophobicity, size, and shape can all influence interactions with plasma proteins [318]. In general, neutral, hydrophilic particles are opsonized more slowly. Hydrophobicity can influence which proteins bind the nanoparticles, while size and shape influence how much protein binds, but not which proteins [318]. Binding of proteins to liposomes can also aid in delivery to the desired location. For siRNA-LNPs, the binding of apolipoprotein E to LNPs acts as an endogenous targeting ligand and it is the main mechanism by which these LNPs are targeted to hepatocytes [319]. In contrast, DNA-cationic lipid complexes primarily transfect lung endothelial cells shortly after intravenous administration, and while they are rapidly cleared within 24 h, those that remain accumulate in the liver but retain low transfection efficiency [320, 321].  The toxicity and transfection efficiency of cationic lipid-DNA complexes can be affected by lipid/DNA charge ratio and inclusion of helper lipids, as well as the structure of the cationic lipid itself. Lower lipid/DNA charge ratios as well as smaller sized particles reduce accumulation in the lung, but lower lipid/DNA charge ratio also decrease overall transfection efficiency [322].  Reducing the overall molar percentage of cationic lipid also decreases overall transfection efficiency. Complexes composed of just cationic lipid and a helper lipid are highly toxic to macrophages, leading to production of nitric oxide, tumor necrosis factor-α, and interferon-ɣ [323]. These factors prevent efficient transfection following repeated dosing of lipoplexes, 43  possibly due to apoptosis of lung endothelial cells. Toxicity was reduced by use of phosphatidylcholine instead of phosphatidylethanolamine, as well as inclusion of a PEGylated lipid [324]. Toxicity was also associated with a higher positive charge, as free liposomes had higher toxicity compared to when complexed with DNA, and removal of free liposomes from mixtures led to reduced toxicity in vitro. The head group of cationic lipids induces the formation of reactive oxygen species, leading to DNA damage in the lung and spleen [314]. 1.2.5.4 Applications of LNPs in mRNA delivery Cationic lipid-RNA complexes are being tested as vaccines against viral and tumor antigens, as the immunostimulatory properties of the cationic carriers act as an adjunct to boost the immune response [325]. Identifications of factors that reduce mRNA’s immunogenicity and toxicity and increase its stability and translation has increased the feasibility of mRNA vaccines. Two types of mRNA are under investigation: non-replicating mRNA and virally derived, self-amplifying mRNA.  A variety of vectors against specific viral and cancer antigens have been tested in murine models, including a RNA-peptide complex within a liposome [326], self-amplifying mRNA in PEGylated, ionizable liposomes [327], mRNA complexed with the commercial agent Stemfect [328], mRNA in cationic lipid-phosphatidylethanolamine complexes [329], liposomes composed of a mannose-conjugated lipid and a PEGylated poly-L-lysine-mRNA complex [330], and mRNA complexed in a cationic lipid alone [331]. A cationic nanoemulsion, composed of cationic lipid, squalene, Tween 80 and Span 85, has also been used as an adjunct along with self-amplifying mRNA, inducing an immune response in mice, rats, rabbits and primates [332]. The immunostimulatory effects of mRNA can be controlled by how in vitro transcribed (IVT) mRNA is purified, as well as the introduction of modified nucleosides [16, 333-336]. IVT 44  mRNA contains double-stranded RNA contaminants, which is a potent pathogen-associated molecular pattern recognized by receptors in the cytosol and endosomes, including TLRs. This leads to type I interferon production and inhibition of translation and degradation of cellular mRNA and ribosomal RNA [335]. Purification of IVT mRNA by reverse-phase fast protein liquid chromatography or high-performance liquid chromatography reduces these contaminants [334]. This has enabled delivery of mRNA and exogenous protein expression in vivo, as previously discussed [16, 333-336]. An optimized LNP formulation for in vivo mRNA delivery has also been developed using in silico screening [337]. These LNPs contained a lipid-like material called a lipidoid, similar to ionizable cationic lipids but requiring less helper lipid [338]. Starting with an siRNA-optimized formulation, the new formulation had an increased lipidoid:mRNA weight ratio, used phosphatidylethanolamine instead of phosphatidylcholine as a helper lipid, and had altered molar ratios of the lipid components [337].  1.2.5.5 Examples of direct platelet transfection Electroporation has been tested as a method for siRNA delivery to platelets, however 2 of the 3 solutions tested led to a 10-fold reduction in platelet count, while the third solution did not yield detectable fluorescent signal in the platelets [14]. Alternatively, transfection with the commercial lipid-based reagent Lipofectamine (Lf) led to a transfection efficiency of 8.4%, based on flow cytometry analysis [14]. After isolating transfected cells, siRNA targeting glyceraldehyde 3-phosphate dehydrogenase (GAPDH) led to a 33% reduction in GAPDH mRNA after 24 hr, although no replicates were performed. The same protocol has been used to transfect platelets with synthetic miRNA, which was then released in platelet microparticles [70, 174]. In one study, P-selectin exposure and GPIIb/IIIa conformational change were used to measure platelet activation and phosphatidylserine exposure was used to measure of apoptosis. 45  No statistical analysis was performed, but after 24 hours there was no change in GPIIb/IIIa activation or phosphatidylserine exposure but an upward trend in P-selectin surface expression, indicating granule release [174].  A commercial reagent, RiboJuice, as been used to transfect platelets with much higher efficiency [15, 173]. Delivery of siRNA led to a transfection efficacy greater than 95%, and 96% of microparticles produced from these transfected platelets contained the siRNA. Using this method, a miR-27b mimic and control have also been delivered to platelets with 55% efficiency, leading to reduced protein expression of its mRNA target, thrombospondin-1 [173]. RiboJuice is a mixture of ethyl alcohol and polyamines, with the optimal formula using polyethylenimmine solution and a mixture of platelet phospholipid extracts [15]. Transfection of the platelets with a plasmid containing the sequence for GFP led to detection of the corresponding protein [15]. No data on how this agent effects the hemostatic function of platelets is available.  46  Chapter 2: Identifying strategies for the delivery and synthesis of mRNA in platelets 2.1 Rationale Platelets regulate diverse processes in part due to their ability to target and selectively release small molecules, nucleic acids, and proteins at sites of vasculature damage [9]. The routine use and natural ability of platelets to target sites of vasculature damage suggests modified platelets may be beneficial. Since platelets are anucleate, modifying protein expression within the mature platelet requires delivery of RNA-based agents. Despite lacking a nucleus, platelets have a diverse transcriptome and all the necessary components for translation [153, 173]. Upon activation by pro-coagulant and pro-inflammatory proteins, platelets undergo signal-dependent protein synthesis from mature mRNA [85, 103, 149, 152, 173, 187-192], which may allow for controlled modification the platelets. However, efficient methods for transfecting platelets with mRNA do not exist. LNPs are used to deliver nucleic acids to cells in vitro and in vivo, however their transfection efficiency and biocompatibility is highly dependent on the lipid composition [308, 315, 339]. Cationic lipids are typically used in lipid carriers of RNA to improve their stability, cellular uptake and endosomal escape [340, 341]. While cationic lipids have high efficiency in vitro, a separate class of lipids termed ionizable cationic lipids have improved pharmacokinetics and are used for in vivo delivery [308, 315]. Lf, a commercial lipid-based reagent containing a cationic lipid, has been used to deliver siRNA and miRNA to platelets [14, 70, 174], however it is unknown whether this approach can be used to deliver mRNA.  47  Platelets can also endocytose neutral LNPs lacking a cationic lipid [272, 273]. While these LNPs are typically not used for nucleic acid delivery, they have been used to encapsulate cell- and nuclei-free systems for synthesizing RNA and proteins, composed of phage RNA polymerases and translational machinery extracted from cells. These “protocells”, liposomes capable of protein expression [342], have been used to model early cellular life [343-345], and as a result significant advances have been made in studying and maximizing protein expression within nano- [346] and micro- [347] sized liposomes. Several applications for protocells in synthetic biology and drug delivery have been explored. These include using protein-synthesizing vesicles as synthetic vaccines [348], reactors for directed evolution [349], and stimuli-responsive vehicles toward in vivo drug delivery and for modifying bacterial cell behavior [350, 351]. I hypothesized that a lipid-based transfection agent could be formulated to deliver mRNA to platelets. Identifying a suitable class of transfection agents and a method for encapsulating functional mRNA is a necessary preliminary step. Four main classes of LNPs were tested: Lf, LNPs lacking cationic lipids (nLNPs), LNPs containing a cationic lipid that remains positively charged at physiological pH (cLNPs), and LNPs containing an ionizable cationic lipid that is neutral at physiological pH but becomes positively charged in acidic conditions (icLNPs). IVT mRNA was encapsulated within these LNPs and delivery to platelets was quantified by flow cytometry, confocal microscopy, and qPCR. Since encapsulation of mRNA within nLNPs was expected to be low, I also tested nLNPs capable of light-induced RNA synthesis, allowing transcription to be initiated only after nLNPs have been internalized by platelets. Components of a transcription reaction, consisting of T7 RNA polymerase, a linear DNA template, and ribonucleotide triphosphates, including photo-caged adenosine triphosphate (ATP), were 48  encapsulated into nLNPs. Protocells typically contain coupled RNA and protein synthesis, but we focused on transcription to maximize the amount of mRNA that could be synthesized and then delivered by these LNPs.  2.2 Methods 2.2.1 Preparing washed platelets Approval for the study was obtained from the ethics boards of the University of British Columbia (UBC) and the Canadian Blood Services (CBS), in accordance with the UBC and CBS Research Ethics Boards’ guidelines. Informed, signed consent was obtained from healthy volunteers before their blood was collected. Platelet rich plasma (PRP) was obtained from pooled donor plasma generated by the CBS netCAD facility or isolated from citrated (0.105 M) whole blood by centrifuging at 100 × g for 20 min and collecting the top 75% (v/v) of the upper layer. The source of PRP was kept consistent for a given experiment. PRP was then centrifuged at 250 × g for 20 min and the platelet poor plasma was removed. The platelets were washed once in a citrate-glucose-saline buffer (CGS; 120 mM NaCl, 30 mM D-glucose, 11 mM trisodium citrate, pH 6.5) and once in Tyrode’s-HEPES buffer (1.8 mM CaCl2, 1.1 mM MgCl2, 2.7 mM KCl, 137 mM NaCl, 0.4 mM NaH2PO4, 10 mM HEPES, 5.6 mM D-glucose, pH 6.5) at 250 × g for 10 min. Prostaglandin E1 (PGE1; 10 µM) was added at all wash steps. Washed platelets were suspended in Tyrode’s-HEPES at a concentration of 200 × 109 platelets L-1. 2.2.2 Preparing mRNA-LNPs nLNPs were prepared using a protocol previously described, with modifications [352]. A thin film of 12 µmol of lipids, consisting of egg-phosphatidylcholine, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ePC:chol:DSPE-PEG2000; 58.9:39.6:1.5 molar ratio; Avanti Polar Lipids, chol from Sigma Millipore) was 49  rehydrated with deionized water, extruded ten times through a 200 nm filter using a LIPEX extruder (Northern Lipids Inc.), and lyophilized. The lyophilized lipid was rehydrated with deionized water to 16 mM and extruded through a 200 nm filter using a Mini-extruder (Avanti Polar Lipids) eleven times. To encapsulate IVT mRNA, the formed nLNPs were mixed with mRNA (25 µg mL-1) in tris-buffered saline (TBS; 50 mM Tris-HCl, 150 mM NaCl) to a final concentration of 12 mM lipids, and the mixture was freeze-thawed once. IcLNPs and cLNPs were prepared using a method previously described for formulating siRNA-encapsulated icLNPs [308]. Briefly, 3 µmol of lipids, consisting of cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; Avanti Polar Lipids), N-[(methoxy poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA; gift from the lab of P. Cullis), and either 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA; gift from the lab of P. Cullis) for icLNPs or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; Avanti Polar Lipids) for cLNPs were mixed in a 10:38.5:1.5:50 molar ratio in an ethanol-sodium citrate solution (30% v/v, 50 mM sodium citrate, pH 4) for icLNPs or an ethanol-tris ethylenediaminetetraacetic acid (EDTA) solution (30% v/v, 10 mM Tris, 1 mM EDTA, pH 8) for cLNPs. Lipids were at a final concentration of 12 mM. The empty LNPs were then extruded three times through a 200 nm filter using a LIPEX extruder and mixed with mRNA (25 µg mL-1 ) to a final concentration of 6 mM lipids before equilibrating for 30 min at 22°C. The LNPs were then dialyzed against phosphate-buffered saline (PBS) for 24 h. Lf-mRNA complexes were formed by diluting Lf MessengerMax (5% v/v; ThermoFisher) and mRNA (25 µg mL-1) in PBS and incubating for 10 min at 22°C. Nanoparticle size for all classes of LNPs was measured using dynamic light scattering with Zetasizer Nano ZPS (Malvern).  50  2.2.3 Preparing RNA-synthesizing LNPs Empty nLNPs were formed as described above. Following extrusion through a Mini-extruder, a transcription mixture containing the supplier’s transcription buffer, 10 ng µL–1 of DNA template, 0.75 mM each of guanosine triphosphate (GTP), cytosine triphosphate (CTP), uridine triphosphate (UTP), and photo-caged ATP (Invitrogen), and 0.07 unit µL–1 of T7 RNA polymerase (Toyobo), was added to formed nLNPs while on ice and the entire mixture was freeze-thawed once. The linear DNA templates were prepared through amplification by polymerase chain reaction (PCR) of plasmid templates using specific primers. Unencapsulated material was removed from the nLNPs by passing the solution through an anionic exchange column (Vivapure Q Mini H, Sartorius).  2.2.4 Generating PCR templates for in vitro transcription The green fluorescent protein (GFP) coding sequence from pRSET-EmGFP (Invitrogen) was cloned into the multiple cloning site (MCS) of pT7CFE1-CHis (ThermoScientific) using EcoRI and XhoI (Fermentas) to create the GFP plasmid template. The firefly luciferase (FLuc) coding sequence from pEJ3 (a gift from E. Jan, UBC) was cloned into the MCS of pT7CFE1-CHis using PstI and PvuII (Fermentas) to create the FLuc plasmid template. PCR using primers forward 5’-GGC CTC TTC GCT ATT ACG C-3’ and reverse 5’-CGA GGA AGC CCG GAT ATA GT-3’ was used to amplify the sequences for GFP and FLuc transcription. Resulting PCR amplicons contained a T7 promoter for initiating transcription, an EMCV internal ribosome entry site (IRES) for mRNA translation, the protein coding sequence, and a poly(A) tail for stability.  2.2.5 Generating biotinylated mRNA Biotinylated mRNA was generated by incubating 50 mM FLuc PCR template with ATP, CTP and GTP (1 mM each), UTP (6.5 mM), biotin-16-UTP (3.5 mM), and 10% (v/v) each of T7 51  RNA polymerase and transcription buffer (MEGAscript T7 Transcription Kit, ThermoFisher) for 3 h at 37°C, then 0.1 U µL-1 TURBO DNase I for 1 h. RNA was purified by precipitating in 2.5 M lithium chloride overnight at 4°C, centrifuging for 15 min at 21,000 x g, washing the pellet with 70% (v/v) ethanol for 5 min at 21,000 x g, and resuspending in water at 1 mg mL-1. 2.2.6 Treating platelets with LNPs LNPs (1:100 v/v) containing purified IVT mRNA or the free mRNA (250 ng mL-1) were added to washed platelets, and the mixture was incubated for 2 h at 22°C. Unless otherwise stated, LNPs contained biotin-UTP-labelled FLuc mRNA, and uptake was performed in Tyrode’s-HEPES buffer (pH 6.5). To remove excess LNPs, platelets were centrifuged for 15 min at 250 × g, washed once with CGS, once with Tyrode’s-HEPES, and resuspended in Tyrode’s-HEPES to 200 × 109 platelets L-1. For RNA-synthesizing nLNPs, washed platelets were incubated with nLNPs (1.2 mM) for 30 min at 37°C while uptake occurred. To remove excess nLNPs, platelets and nLNPs were centrifuged at 250 × g for 5 min and the supernatant was removed. Platelets were then washed twice in Tyrode’s-HEPES buffer (pH 6.5) at 250 × g for 5 min. Samples used for qPCR analysis were further purified by running platelets through an ultrafiltration size exclusion column with an approximate pore size of 0.2 μm (Vivaspin 500; Sartorius). Platelets were then irradiated with white light for 30 sec using a fluorescence microscope and incubated for 1 h while transcription occurred. Samples were stored in TRIzol (ThermoFIsher) for qPCR analysis. 2.2.7 Quantifying uptake of LNPs by flow cytometry To quantify uptake of purified mRNA, LNPs contained mRNA that was capped and labelled with cyanine 5-UTP and 5-methoxy-UTP (Cy5 mRNA) encoding FLuc. For RNA-synthesizing nLNPs, Oregon Green 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine 52  (OG-DHPE; 0.2 – 0.5% w/w of total lipids; Invitrogen) was added to the lyophilized lipids before extruding and forming empty nLNPs. Platelets were stained with anti-human CD42b-FITC antibody (1 µg mL-1; ThermoFisher) or anti-human CD42b-APC antibody (5 μg mL-1; eBiosciences) for 30 min and analyzed by flow cytometry using a FACSCalibur (BD Biosciences) and CellQuest Pro software.  Platelets were identified using forward and side scatter, as well as CD42b staining. Single color controls were used to determine the background fluorescence of each stain and to draw quadrants.   To inhibit uptake of LNPs, platelets were pre-incubated for 30 min with dynasore (25 - 50 µM; Sigma Millipore), sodium azide (50 µM; Sigma Millipore), cytochalasin D (4 - 10 µM; Calbiochem), amiloride (1 mM; Sigma Millipore), phenylarsine oxide (PAO; 10 µM; Sigma Millipore) or dimethylsulfoxide (DMSO, 0.5% v/v; Sigma Millipore) before adding LNPs. When inhibitors were used, they were present in the platelet samples at all times.  2.2.8 Quantifying uptake of LNPs by confocal microscopy Platelets were directly fixed with 4% paraformaldehyde (v/v) and adhered to poly-L-lysine-coated cover slips. Cover slips were washed once with PBS, treated with 0.5% Triton X-100 for 20 min, and then blocked with 10% (v/v) goat serum in PBS overnight at 4°C. Platelets were stained with mouse anti-human CD42b antibody (2 µg mL-1, ThermoFisher) for 1.5 h at room temperature, washed three times with PBS, and then stained with streptavidin-FITC (1 µg mL-1, ThermoFisher) and Brilliant Violet 421 goat anti-mouse secondary antibody (100 ng mL-1, BioLegend) for 1 h. After three PBS washes, slides were mounted and images acquired using an Olympus FV1000 confocal microscope and Olympus FluoView software. The relative amount of mRNA was quantified by measuring the pixel intensity within the ring of CD42b staining for approximately 20 platelets using ImageJ software, normalizing to untreated platelets.  53  For RNA-synthesizing nLNPs, Texas Red-DHPE (1 % w/w of total lipids, Invitrogen) was added to the nLNPs. After fixing and blocking as described above, platelets were stained with anti-human CD42b-FITC antibody (5 μg mL-1; ThermoFisher) overnight before washing and fixing. Images were acquired using a 60 x objective on a Nikon Eclipse TI microscope and NIS-Elements AR software. 2.2.9 Measuring transcription in RNA-synthesizing nLNPs by qPCR To activate transcription, nLNPs were irradiated with white light for 30 sec using a fluorescence microscope (Leica DMI6000B). After incubating the nLNPs for 1 h at 37ᵒC, RNA was extracted using TRIzol reagent (Invitrogen), digested with TURBO DNA-free (Invitrogen), reverse transcribed into cDNA using oligo-DT primers and MMLV-reverse transcriptase (Invitrogen), and quantified using qPCR with gene-specific primers and SYBR green reagents (Applied Biosystems). RNA was quantified using 2-ΔΔCt, with GAPDH as an internal control when applicable. Primers forward 5’-GAT GAC GGA AAA AGA GAT CG-3’ and reverse 5’-TTT TTC TTG CGT CGA GTT TT-3’ were used for quantifying FLuc. Primers forward 5’-CAA CAG CCA CAA GGT CTA T-3’ and reverse 5’-GGT GTT CTG CTG GTA GTG-3’ were used for quantifying EmGFP. Primers forward 5’-CAA GGC TGT GGG CAA GGT-3’ and reverse 5’-GGA AGG CCA TGC CAG TGA-3’ were used for quantifying GAPDH.  2.2.10 Measuring protein expression from synthesized RNA. nLNPs were prepared as described above using the FLuc template (10 ng uL-1). RNA was extracted from 50 uL of nLNPs using TRIzol reagent, with modifications from the manufacturer’s protocol. During isopropanol precipitation, 100 µg glycogen (Invitrogen) was added, and the precipitation was allowed to proceed for 30 min before being spun down at 21,000 × g for 10 min and washed with 70% (v/v) ethanol. The final pellet was re-suspended in 54  25 µL of nuclease-free water (Promega). A rabbit reticulocyte lysate (RRL) system (Promega) was used for cell-free protein expression. RNA (6.5 µL) isolated from nLNPs was used in a 25 µL reaction of the RRL system to express the protein. After incubating the reaction mixture at 30ᵒC for 90 min, 50 µL of D-luciferin reagent (6 M; ThermoFisher) was added to the reaction and luminescence was measured in a microplate reader (Tecan Infinite M200). 2.2.11 Statistical analysis All values are expressed as mean ± SEM. A Shapiro-Wilkes test was used to test for normality. When data were normally distributed, comparisons were made using a paired and two-tailed t-test if only 2 groups were compared or by a one-way ANOVA, with a Bonferroni post-hoc test, when more than 2 groups were compared. If data were not normally distributed, comparisons were made by a Friedman test with a Dunn’s multiple comparisons post-hoc test. A P-value < 0.05 was considered significant. 2.3 Results The work in sections 2.3.2 and 2.3.3 was done in collaboration with Dr. V. Chan, a former PhD candidate in the lab. We contributed equally to experiments to develop and optimize RNA-synthesizing nLNPs suitable for cell delivery and demonstrate uptake of RNA-synthesizing nLNPs by platelets. Dr. Chan developed the method for controlling synthesis by including photo-caged ATP in the ribonucleotide mix encapsulated within nLNPs. Data for Fig. 2.7b, 2.8 and Appendix A Figures A.1, and A.2, were collected by Dr. Chan, and I collected data for all other figures.  2.3.1 Specific LNP formulations can bind and be internalized by platelets The ability of four different classes of LNPs to deliver RNA to platelets were compared using confocal microscopy and flow cytometry. The four classes were: Lipofectamine (Lf), 55  LNPs lacking cationic lipids (nLNPs), LNPs containing a cationic lipid that remains positively charged at physiological pH (cLNPs), and LNPs containing an ionizable cationic lipid that is neutral at physiological pH but becomes positively charged in acidic conditions (icLNPs). All LNPs had an average diameter of 160 to 200 nm, except Lf, which had an average diameter of 350 nm (Fig. 2.1). Different classes of LNPs were not formed by the same method, leading to differences in the final lipid concentration. An equivalent concentration of total mRNA for each class of LNP was added to platelet samples during experiments; however the encapsulation efficacy could not be quantified and therefore the total lipid concentration and ratio of free to encapsulated mRNA differs for each class of LNP. Platelets were stained with anti-CD42b, a cell-surface marker expressed only on platelets and megakaryocytes. For confocal microscopy, biotin-labelled mRNA was encapsulated within LNPs and the fluorescence intensity of biotin within the membrane ring was quantified to determine the amount of material internalized by the platelets. Preparing platelets with either cLNPs or icLNPs led to an increase in the maximum    Figure 2.1. LNPs have similar size characteristics except for Lf complexes. Dynamic light scattering was used to measure the average diameter of the different formulations. (n=3) 56   Figure 2.2. The contents of icLNPs and cLNPs are bound to and internalized by platelets. a) Confocal immunofluorescence microscopy of platelets (red) transfected with LNPs containing biotin-labelled RNA (green). Representative images from 4 separate donors are shown. Scale bars indicate 2 µm. b) Quantifying biotin-labelled RNA inside the platelets from images in panel A (n=4). c) The amount of Cy5-positive platelets containing bound or internalized Cy5-mRNA was quantified by flow cytometry (n=5). **P < 0.01, ***P < 0.001 fluorescence intensity within the cells, but preparing the platelets with free RNA, nLNPs or Lf did not (Fig. 2.2a, 2.2b).   In mammalian cells, utpake of LNPs is believed to occur primarily through endocytosis, involving trafficking of LNPs along a cell’s cytoskeleton [341, 353]. To determine whether 57  LNPs were endocytosed by a similar mechanism in platelets, Cy5-labelled mRNA was encapsulated in LNPs and the number of Cy5-positive platelets was quantified using flow cytometry in the presence or absence of small molecule inhibitors. Preparing platelets with either cLNPs or icLNPs in the absence of inhibitors led to an increase in the number of Cy5-positive platelets compared to untreated platelets, while platelets prepared with nLNPs and Lf did not (Fig. 2.2c). Pre-treating platelets with the metabolic inhibitor sodium azide [354] or the endocytosis inhibitor dynasore [355] led to a decrease in the percentage of Cy5-positive platelets when prepared with icLNPs or cLNPs, but not with free RNA, Lf or nLNPs (Fig. 2.3). This indicates that dynamin contributes to the interaction of icLNPs and cLNPs with platelets. Inhibiting actin polymerization with cytochalasin D [355] reduced the number of Cy5-positive platelets prepared with icLNPs but not cLNPs, suggesting there are differences in the mechanism of uptake between icLNPs and cLNPs. Inhibiting phagocytosis with amiloride [355] had no   Figure 2.3. Mechanism of binding and uptake depends on LNP formulation. LNPs encapsulating Cy5-mRNA were delivered to platelets prepared with various endocytosis inhibitors and the percentage of Cy5-positive platelets was quantified by flow cytometry (n=4-5). *P < 0.05, **P  <  0.01 58  effect on the percentage of Cy5-positive platelets prepared with icLNPs or cLNPs, suggesting phagocytosis is not involved in LNP uptake. 2.3.2 Developing RNA-synthesizing nLNPs To synthesize RNA in platelets, it was first necessary to develop a method for controlling transcription in nLNPs. Photo-induced transcription has previously been achieved in LNPs as small as 170 nm, but the composition and purification of these LNPs was not optimized for use in cells [351]. To optimize the LNPs for transcriptional activity and delivery to cells, modifications to the composition and preparation of the RNA-synthesizing LNPs were made. The concentrations of the reaction components were adjusted, increasing the DNA template concentration 10-fold and the ribonucleotide concentration two-fold (Fig. 2.4a). This led to a 20-fold increase in RNA synthesis, relative to the original formulation. Incorporating a lipid functionalized with PEG (Fig. 2.4b), which is important for in vivo stability and enhances protein   Figure 2.4. Optimizing RNA synthesis in nLNPs. a) The concentration of reagents required for transcription encapsulated within the nLNPs was increased, and b) a PEGylated lipid was incorporated to enhance transcription (n=3). Error bars represent SEM. No statistical analysis is shown as the values are technical replicates only. 59  expression within LNPs [347], also led to a 10-fold increase in RNA synthesis. This nLNP formulation, which include the PEGylated lipid, was used to encapsulate the IVT mRNA described above. Transcription reaction components were encapsulated by freeze-thawing the pre-formed LNPs (Fig. 2.5). Freeze-thawing disrupts the LNP bilayer and leads to movement of the reaction components across the existing concentration gradient and into the interior of the LNP [356]. LNPs were then purified using an anionic exchange column to remove unencapsulated T7 RNA polymerase and DNA (Fig. 2.5; see Appendix A – Fig. A.1). This quick, single-step method for purification is an alternative to published methods of adding EDTA or nucleases to inhibit transcription outside of LNPs, which are likely unsuitable for subsequent cellular delivery [345, 357-359].  Figure 2.5. Freeze-thawing and purification on an anionic exchange column allows for transcription inside of nLNPs. In nLNPs, transcriptional components were encapsulated and protected from the anionic exchange column, which allowed transcription of GFP mRNA, as measured by qPCR (n=3). Error bars represent S.E.M. No statistical analysis is shown as the values are technical replicates only. 60  To control transcription within platelets, we first tested whether transcription in purified nLNPs 220 nm in diameter could be initiated using light. nLNPs were irradiated for 30 seconds with white light (λ > 300 nm) to release ATP from photo-caged ATP (Amax = 360 nm) and incubated for one hour at 37°C for transcription to occur. Photo-caged ATP contains a  nitrophenylethyl esterified to its terminal phosphate, preventing its use by T7 RNA polymerase. A 2,300-fold increase in GFP mRNA was detected in purified nLNPs following irradiation and incubation (see Appendix A - Fig. A.2). Only a six-fold increase was observed in samples that were not irradiated. Another DNA template, for FLuc mRNA, was also transcribed in nLNPs. Incubating nLNPs containing a FLuc DNA template for one hour led to a 12-fold increase in mRNA in irradiated samples and no significant increase in samples that were not irradiated (Fig. 2.6a). To confirm that mRNA transcribed within nLNPs was functional, a cell-free expression system was used to translate FLuc mRNA into protein. A 6-fold increase in luminescence occurred when a substrate for the FLuc enzyme was added to samples containing RNA isolated from irradiated nLNPs, indicating that functional FLuc mRNA was synthesized inside of the    61  Figure 2.6. Transcription of FLuc mRNA in nLNPs is controlled by light. a) The amount of FLuc mRNA (n = 3) increased only when irradiated with light, measured using qPCR. b) FLuc mRNA extracted from nLNPs produces functional enzyme that activates a luminescent substrate in a cell-free translation system (n=3). *P < 0.05, ** P < 0.01, n.s. indicates not significant compared to 0 h. nLNPs (Fig. 2.6b). The lower transcriptional activity observed with the FLuc template (2 kb) compared to the GFP template (1 kb) suggests some optimization is needed to maximize RNA synthesis of larger templates. Taken together, these data demonstrate that RNA transcription can be suppressed and specifically initiated using light in nLNPs. 2.3.3 Engineering platelets to synthesize RNA To determine whether the RNA-synthesizing nLNPs could be delivered to cells, the ability of platelets to take up nLNPs was first assessed using flow cytometry and confocal microscopy. Empty, fluorescently-tagged nLNPs were incubated with isolated platelets in a Tyrode’s-HEPES buffer at pH 6.5. Excess nLNPs were removed, and platelets were stained for CD42b. LNPs co-localized with 35% to 65% of platelets, with variation between platelet donors (Fig. 2.7a). Confocal microscopy of nLNP-treated platelets confirmed that the majority of co-localization corresponded to internalized lipids (Fig. 2.7b). As described above, small molecule inhibitors where used to determine whether uptake of nLNPs by platelets occurred by endocytosis, as seen in other cells. Uptake was reduced using several inhibitors of endocytosis, further confirming that nLNPs were internalized. Flow cytometry showed that the co-localization of platelets and nLNPs decreased by over 60% by cytochalasin D, an inhibitor of actin polymerization [355], as well as by sodium azide, a general metabolic inhibitor [354] (Fig. 2.7c). 62  Using inhibitors specific to different endocytotic pathways, uptake was reduced by over 60% by dynasore, while PAO and amiloride did not significantly reduce uptake (Fig. 2.7c). Dynasore inhibits dynamin-dependent endocytosis pathways, including caveolae- and clathrin-mediated   Figure 2.7. nLNPs were internalized by platelets. a) Representative flow cytometry histograms of platelets depict an increase in fluorescence from nLNPs, corresponding to nLNP internalization (black curve). b) Confocal images of platelets (green) and internalized nLNPs (red). Scale bar is 10 µm. Data collected by Dr. V. Chan. c) Uptake of nLNPs was reduced when platelets were pre-treated with a metabolic inhibitor and inhibitors of endocytosis, quantified by flow cytometry (n=3). *P < 0.05, ** P < 0.001 63  endocytosis [355]; PAO is an inhibitor of clathrin-mediated endocytosis and pinocytosis depending on the cell type [360, 361], and amiloride is an inhibitor of phagocytosis and micropinocytosis [355]. These results suggest platelets take up nLNPs through dynamin-dependent endocytotic pathways.  To test if protocells could function in platelets, light-inducible RNA-synthesizing nLNPs were incubated with platelets. Following incubation at 37°C, a 59-fold increase in mRNA was detected in irradiated platelets, compared to only a 2-fold increase when platelets were first treated with dynasore (Fig 2.8). To confirm RNA-synthesis primarily occurred within internalized nLNPs, two control samples, consisting of nLNPs without platelets or nLNPs   Figure 2.8. nLNPs internalized by intact platelets (PLT) synthesized RNA. A significant increase in RNA occurred one hour after irradiation only in uninhibited, intact platelets. When platelets were lysed or absent, no RNA was detected. In dynasore-inhibited platelets, no significant increase in RNA was detected (n=3). * P < 0.01, n.s. indicates not significant compared to the respective 0 h sample, B.D. indicates below detection by qPCR.   Data collected by Dr. V. Chan 64  platelets that were first lysed by freeze-thawing, were purified in the same manner as samples containing intact platelets. In both control samples, no significant increase in mRNA occurred (Fig. 2.8). This data demonstrates that RNA was synthesized in nLNPs internalized by intact platelets. 2.4 Discussion Here, two methods were used to deliver RNA to platelets. Purified, IVT mRNA was encapsulated in four difference classes of LNPs, The contents of icLNPs or cLNPs were detected within platelets, while the contents of nLNPs and Lf were not. Using the same nLNP formulation, RNA was also synthesized in anucleate platelets. In all cases, binding and uptake occurred through an energy and dynamin-dependent mechanism. Both Lf and cLNPs contain cationic lipids that readily form electrostatic interactions with negatively charged nucleic acids, leading to high encapsulation [308, 340]. This suggests the poor RNA delivery by Lf is due to poor uptake of Lf-RNA complexes by platelets. IcLNPs and cLNPs were smaller relative to the Lf-mRNA complexes, which may also have facilitated increased uptake by platelets. However, platelets can endocytose latex beads up to 312 nm in diameter [258] and can be transfected with Lf-siRNA complexes [14]. The experiments here were performed over two hours, so it is possible larger lipid-based materials require longer than 2 hours for endocytosis, as transfection with Lf-siRNA was performed over 24 hours. In other mammalian cells, uptake of polymeric nanoparticles 100 to 150 nm is more efficient and occurs faster than uptake of 500 nm particles [362, 363]. In contrast, phagocytic uptake of 500 nm polymeric nanoparticles by macrophages was greater than that of 150 nm nanoparticles and is generally slower [363]. However, the inability of amiloride to prevent uptake of LNPs, including Lf, suggests uptake is not occurring by phagocytosis. To determine the role of LNP size on  65  uptake by platelets, direct comparisons between LNPs from the same class but with varying diameters would be required. The lipid composition of Lf is undisclosed and may differ significantly from cLNPs, There are several versions of Lf, optimized for delivery to different cell types and for different types of nucleic acids. These experiments were performed with Lf MessengerMAX, which is optimized for mRNA delivery, while platelet transfection with siRNA was previously performed with Lf 2000 [14]. It is unknown how these compare to early Lf formulations, which used a 3:1 ratio of 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA) and DOPE [309]. In contrast, cLNPs are composed of DOTAP, DOPC, cholesterol and a PEGylated lipid. DOSPA is a cationic lipid similar in structure to DOTAP, but DOTAP is more readily degraded within target cells and lacks an extra spermine on its hydrophobic chains, which alters packing of the nucleic acid [364]. DOPE is more fusogenic than DOPC, which enhances endosomal release [308]. In other mammalian cells, Lf-DNA and DOTAP/DOPC-DNA complexes followed different trafficking pathways after uptake, with DOTAP/DOPC complexes co-localizing with lysosomes with increased frequency [341]. Assuming Lf MessengerMax contains the same lipids as the original Lf, it is possible that in platelets mRNA can not be detected following delivery by Lf because it is more rapidly degraded following endosomal release. This question could be addressed by comparing cLNP formulations with different cationic or helper lipids. The use of DOPE to enhance uptake was addressed in chapter 4, however more high-throughput screening techniques are required to test the full range of variations possible. While nLNPs were similar in size compared to icLNPs and cLNPs, there was poor uptake of IVT mRNA encapsulated in nLNPs. Platelets did endocytose RNA-synthesizing nLNPs, 66  which were composed of the same materials and were similar in size to nLNPs containing IVT mRNA, suggesting they also endocytosed IVT mRNA-containing nLNPs. While uptake of IVT mRNA by LNPs was compared using biotin or fluorescently-labelled mRNA, uptake of RNA-synthesizing nLNPs was tested using fluorescently-labelled lipids and qPCR. Labelling the lipids allowed uptake of nLNPs rather than mRNA to be assessed by flow cytometry and demonstrated nLNPs can be endocytosed by platelets. However, a cationic lipid is necessary for efficient encapsulation of mRNA [340, 365], so it is expected that nLNPs would have poor encapsulation of IVT mRNA. Even if platelets were able to endocytose nLNPs to the same extent as icLNPs and cLNPs, only low amounts of IVT mRNA would be delivered by nLNPs. In the experiments comparing different classes of mRNA, the results were normalized to the total amount of mRNA added to platelets, rather than the final lipid concentration, and unencapsulated mRNA was not removed from the LNPs prior to treatment of the platelets. This can affect the amount of uptake by LNPs, as receptor-mediated endocytosis is dose-dependent and saturable, as well as the impact of the LNPs on platelet activation [319]. Despite this, these initial experiments demonstrated the importance of a cationic charge and small size for uptake by platelets. Once an LNP formulation that allows for protein synthesis is identified, additional dosing experiments would be important in determining the effect of the lipid concentration of uptake by platelets. RNA-synthesizing nLNPs were tested an alternative approach to delivering mRNA to platelets. This work showed for the first time that controlled transcription of exogenous RNA can occur in an anucleate cell and was proof-of-concept that protocells could be adapted for use in mammalian cells to express exogenous RNA. Delivery of mRNA was demonstrated by qPCR, compared to the flouresence-based approaches used to demonstrate IVT mRNA delivery by icLNPs and cLNPs. As a result, the amount of RNA delivered by these two approaches cannot be 67  directly compared. However based on qPCR analysis, RNA-synthesizing nLNPs only delivered approximately 1 copy of mRNA per platelet, and synthesized RNA lacked a m7G cap and full-length poly(A) tail, two elements that are important for RNA stability and translation in mammalian cells [201]. Therefore we chose to continue optimizing delivery of IVT mRNA, rather than RNA-syntheszing nLNPs. However synthesizing mRNA in platelets demonstrates that a range of biologically active macromolecules can be delivered to platelets by LNPs, including enzymes and DNA. These method were subsequently modified by our research group to deliver the pro-coagulant enzyme thrombin to platelets and enhance their coagulability [366]. Platelets have been reported to internalize nanoparticles through the OCS and trafficking to storage vacuoles, as well as by cell engulfment [258]. Proteins endocytosed by platelets are trafficked through clathrin-dependent pathways, passing through the endolysosomal system, which is also how LNPs are endocytosed by mammalian cells [262, 353]. Here, small molecule inhibitors were used to compare the interaction of different LNP classes with platelets. Uptake of nLNPs and Cy5-mRNA complexed with icLNPs and cLNPs was inhibited by the metabolic inhibitor sodium azide [354]. This was expected, as both receptor-mediated endocytosis and pinocytosis within platelets is energy-dependent [260, 270]. However, dynasore, which targets dynamin [355], also inhibited uptake of all three classes, while cytochalasin D, which disrupts actin polymerization [355], only inhibited uptake of nLNPs and icLNPs, but not cLNPs. The ability of dynamin to reduce uptake of all three LNP classes suggests there is overlap in the mechanism of uptake for the different LNPs. However, none of the small molecular inhibitors caused a complete reduction in the percentage of Cy5-positive platelets. In these experiments, uptake was assessed using flow cytometry. However, this technique cannot distinguish between bound or internalized fluorescence. To determine whether the percentage of the Cy5-positive 68  platelets corresponded to bound versus internalized LNPs, the platelets could be treated with the different classes of LNPs in cold temperatures, at 4°C, which is known to inhibit both receptor-mediated endocytosis and pinocytosis [208, 264]. As receptor-mediated uptake of plasma proteins and receptors by platelets is also dynamin-dependent [255], it is possible uptake of LNPs is caused by interactions between the LNPs and platelet membrane proteins. These proteins may be platelet receptors that are naturally endocytosed, such as GPIbα or GPIIb/IIIa, but could also include proteins that are part of the endocytotic machinery. LNPs coated with lipophilic, anionic polyelectrolytes are endocytosed by other mammalian cells through interactions with caveolin, a key protein in dynamin-dependent, caveolae-mediated endocytosis [367]. The positive charge on cLNPs, but not nLNPs and icLNPs, may preferentially direct it towards or away from specific pathways, leading towards increased interactions with negatively charged groups of the cell surface, such as a sialic acid. This can alter binding and the mechanism of uptake by cells [368]. As the mechanism of uptake for nanoparticles can also be cellular dependent, depending on expression of endocytotic proteins and their regulators within cell [367], studies using antibodies to target specific platelet receptors would help to determine the exact mechanism of LNP uptake by platelets.  Here, uptake of LNPs was performed in Tyrode’s buffer at pH 6.5 for two hours. An ideal transfection agent for platelets could be used in platelet concentrates, which are stored either in plasma with anti-coagulant or with plasma and around 65% PAS [137]. The effect of different storage conditions will be addressed in the next chapter, along with the effect of the different LNP classes on platelet function. The methods used to demonstrate internalization of IVT mRNA relyed on the use of biotin- or flourescently-labelled nucleotides, and therefore cannot be used to determine whether the RNA delivered is intact and functional. This will be 69  more fully addressed in chapter 4. However, the work in this chapter identifies an approach for delivering mRNA to platelets using LNPs, which is the first step in developing a transfection agent for platelets. In summary, two approaches were used to successfully deliver mRNA to platelets. IVT mRNA was encapsulated within 4 different classes of LNPs and could be detected by flow cytometry and confocal microscopy when icLNPs and cLNPs were used. Additionally, RNA could be synthesized within nLNPs following endocytosis by platelets, using light-controlled transcription. While nLNPs, icLNPs and cLNPs can be endocytosed by platelets, only icLNPs and cLNPs delivered detectable amounts of fluorescently-labelled mRNA, demonstrating the importance of high RNA encapsulation in mRNA delivery to platelets. The ability of cLNPs but not Lf to deliver detectable amounts of mRNA to platelets demonstrates that while a cationic lipid enhances delivery to platelets, other factors such as LNP size and the presence of helper lipids play important roles. RNA-synthesizing nLNPs provide a platform technology for the delivery of biological macromolecules to platelets but are better suited for the delivery of proteins and small molecules, rather than mRNA.   70  Chapter 3: Characterizing the interaction of mRNA-LNPs with platelets 3.1 Rationale Platelet function, including uptake of materials, depends on storage conditions and activation state. Previous studies testing endocytosis of nanosized liposomes and latex particles in platelets have been performed over short incubation periods, typically an hour [258, 271]. In contrast, transfection of platelets with siRNA-Lf complexes was performed over a day [14]. Whether longer incubations would enhance uptake of mRNA-LNPs is unknown. Low temperatures inhibit uptake of latex beads and small molecules, indicating uptake of materials is energy-dependent, and platelet metabolism and activation is dependent on pH [270, 369]. Our initial experiments were performed at in buffer at pH 6.5 to reduce platelet activation, but it may be possible to enhance uptake by raising the pH to physiological levels. Furthermore, LNPs spontaneously associate with plasma proteins [317], and liposomes coating with fibrinogen-binding peptides had improved internalization compared to unmodified liposomes [273], suggesting the presence of plasma may alter uptake of LNPs by platelets. Activation of platelets enhances uptake of viruses, similar in size to the LNPs used here, as well as larger bacteria [256]. Platelet activation also leads to exposure of negatively-charged phosphatidylserine, which may enhance the binding and uptake of postively charged cLNPs [60]. To identify conditions that maximize uptake for all four classes of LNPs tested in chapter 2, the effect of time, storage pH, presence of plasma, and presence of a platelet activator on LNP uptake was assessed by flow cytometry and confocal microscopy. Foreign materials, including nanoparticles, can induce activation of platelets [272, 370]. As this may increase the risk of thrombosis and alter platelets’ hemostatic abilities following transfusion, an ideal delivery agent would have minimal effect on platelet function [272]. Platelet 71  activation is a multi-faceted process, including granule release, aggregation, pro-coagulant activity and morphological changes. Initial pro-coagulant activity is mediated by release of coagulation factors from platelet granules as well as exposure of phosphatidylserine on the platelet membrane, which form binding sites at which coagulation factors are activated [38]. Following activation of the coagulation factors, fibrin is cross-linked to form the stable meshwork of a blood clot. While granule secretion and initial shape change occur within seconds [68], aggregation and phosphatidylserine exposure take one or two minutes to complete [60, 67], and platelet spreading requires ten to thirty minutes before completion [371].  Previous studies assessing the effect of LNPs on platelets are limited. Electron microscopy studies performed over the course of a one hour incubation with small latex beads indicate no morphological changes occur when beads are less than 300 nm, but no other measures of activation were assessed [258]. Small neutral liposomes do not impair aggregation, while liposomes incorporating a cationic lipid do when ADP, but not thrombin, is used [272, 273]. siRNA-Lf complexes do not induce platelet activation, based on P-selectin exposure, GPIIb/IIIa activation, and phosphatidylserine exposure, but these studies were relative to untreated platelets also stored for 24 hours in media, and therefore these platelets were already partially activated [174]. To provide a more complete understanding on the effect of different classes of LNPs on platelets ex vivo, the ability of platelets to induce or impair granule release, aggregation, thrombin generation, and platelet spreading was quantified.  3.2 Methods 3.2.1 Quantifying uptake of LNPs by flow cytometry and confocal microscopy Platelets were treated and samples prepared as previously described, with minor modifications. Uptake was performed in Tyrode’s-HEPES (pH 6.5) in the presence or absence of 72  human thrombin (2 U mL-1; Hematological Technologies Inc.), in modified Tyrode’s-HEPES (134 mM NaCl, 3 mM KCl, 0.3 mM NaH2PO4, 2 mM MgCl2, 5 mM HEPES, 5 mM D-glucose, 12 mM NaHCO3, 3.5 mg mL-1 BSA, pH 7.4), or citrated human plasma (Affinity Biologicals). For confocal microscopy, platelets were stained with rabbit anti-human actin antibody (4 µg/mL, ThermoFisher) for 1.5 h at room temperature, washed, and then stained with streptavidin-FITC (1 µg mL-1, ThermoFisher) and AlexaFluor 594 goat anti-rabbit secondary (2 µg mL-1, ThermoFisher) for 1 h. The relative amount of mRNA was quantified by measuring the pixel intensity within the ring of actin staining for approximately 20 platelets using ImageJ software, normalizing to untreated platelets.  3.2.2 Quantifying alpha granule release by flow cytometry To quantify alpha granule release by flow cytometry, platelets were incubated with LNPs in Tyrode’s buffer (pH 6.5) for 1.5 h and stained with anti-human CD42b-FITC antibody (1 µg mL-1; ThermoFisher) and anti-human CD62-APC (1 µg mL-1, ThermoFisher) for 30 min. Gating for P-selectin staining was determined by activating untreated platelets with thrombin (2 U mL-1) prior to staining. Platelets were analyzed by flow cytometry using a FACSCalibur (BD Biosciences) and CellQuest Pro software.  Platelets were identified using forward and side scatter, as well as anti-CD42b-FITC staining. 3.2.3 Measuring thrombin generation Following treatment and removal of excess LNPs, platelets were resuspended in citrated human plasma containing a fluorescent thrombin substrate (1 µM, Boc-Asp(OBzl)-Pro-Arg-MCA, Peptide International). Following addition of calcium buffer (40 mM CaCl2, 90 mM NaCl) at a 3:1 volumetric ratio of plasma to buffer, platelets were incubated at 37˚C and 73  fluorescence was monitored using a plate reader (Tecan Genios). The lag time was determined as the time at which thrombin generation began. 3.2.4 Measuring platelet spreading Following treatment and removal of excess LNPs, platelets were resuspended in modified Tyrode’s (pH 7.4) supplemented with 5 mM calcium and placed on collagen-coated coverslips (Neuvitro). Platelets were fixed with 4% paraformaldehyde either immediately or incubated with thrombin (2 U mL-1) at 37°C for 1 h before fixing. After fixing, platelets were permeabilized and blocked overnight as previously described. Platelets were stained for 1.5 h with an F-actin probe (1:50,000 v/v, ActinGreen 488 ReadyProbes, ThermoFisher) before washing and mounting. To quantify platelet area, ImageJ was used to determine the surface area of individual platelets, with one to two dozen platelets quantified per condition. 3.2.5 Measuring platelet aggregation PRP was isolated from whole blood as previously described. Gel-filtered platelets were obtained by running PRP over a Sepharose 2B column equilibrated in modified Tyrode’s buffer (pH 7.4). Platelet aggregation was measured using a lumiaggregonometer (Chrono-log) after the addition of ADP (10 µM) and human fibrinogen (250 µg mL-1), either immediately after collection of platelets or 2 h after treatment with LNPs or free RNA. 3.3 Results 3.3.1 Platelets internalize mRNA-LNPs only under specific storage conditons Platelet function depends on their storage conditions and whether they have been activated, so the effects of storage time, pH, presence of plasma, and presence of a platelet activator during uptake was tested. Cy5 labelled-mRNA was encapsualted within LNPs and the co-localization between platelets and LNPs was quantified by flow cytometry. Increasing the 74  storage time from two hours to six hours increased the percentage of Cy5-positive platelets treated with icLNPs, but not free RNA, nLNPs, Lf, or cLNPs (Fig. 3.1). Therefore for the remaining experiments here and in chapter 4, uptake continued to be performed for two hours. When platelets were treated with icLNPs or cLNPs, the percentage of Cy5-positive platelets was greater in Tyrode’s buffer at pH 6.5 compared to when platelets were stored in plasma (Fig. 3.2a). Storing platelets at pH 7.4 did not change the precentage of Cy5-positive platelets when treated with cLNPs, but decreased the percentages of Cy5-positive platelets when treated with icLNPs (Fig. 3.2a). When platelets were treated with free RNA, Lf,  or nLNPs, storing platelets in plasma or at pH 7.4 did not change the percentage of Cy5-positive platelets (Fig. 3.2a). Activating platelets with thrombin, a potent platelet agonist, prior to adding LNPs, decreased the percentage of Cy5-positive platelets with cLNPs and increased the percentage of   Figure 3.1. Only binding of icLNPs increases with longer incubation times. LNPs encapsulating Cy5-mRNA were delivered to platelets and Cy5-positive platelets were quantified by flow cytometry after incubating in Tyrode’s buffer at pH 6.5 (n=4). *P < 0.05, **P  < 0.01. n.s. means not significant. 75   Figure 3.2. Binding and internalization of LNPs depends on pH, presence of plasma, and platelet activation, as well as the class of LNP. a) Binding of LNPs to platelets in Tyrode’s buffer at pH 6.5 or pH 7.4 or in plasma was compared by quantifying Cy5-positive platelets by flow cytometry (n=6). b) Binding in the presence and absence of thrombin, quantified by flow cytometry (n=10). c) Comparing internalization of mRNA in different storage conditions by quantifying biotin-labelled RNA in confocal immunofluorescence images of platelets prepared with LNPs (n=5). *P < 0.05, **P  < 0.01, ***P  < 0.001. n.s. means not significant.   76  Cy5-positive platelets with free RNA or Lf, while the percentage of Cy5-positive platelets with nLNPs or icLNPs was unchanged (Fig. 3.2b). Confocal microscopy was then used to determine whether internalization of the LNPs occurred under these different storage conditions, as flow cytometry cannot distinguish between bound or internalized RNA. Biotin-labelled mRNA was encapsulated in LNPs, and actin staining was used to visualize the platelet. Only icLNPs and cLNPs were examined, as altering the storage conditions of resting platelets did not improve delivery with free RNA, nLNPs, or Lf. Platelets prepared with icLNPs had an increased flourescence intensity inside of the platelets compared to untreated platelets only when unactivated at pH 6.5, and not when activated with thrombin at pH 6.5 or prepared at pH 7.4 or in plasma (Fig. 3.2c, Fig. 3.3a). Platelets prepared with cLNPs had an increased flourescence intensity when prepared at pH 6.5 in the absence or presence of thrombin and when unactivated at pH 7.4, but not when prepared in plasma (Fig. 3.2c, Fig. 3.3b). Based on these experiments, platelets were stored in Tyrode’s buffer at pH 6.5 for two hours during further experiments testing delivery by LNPs.  77   Figure 3.3. Internalization of icLNPs but not cLNPs depends on initial pH and the presence of thrombin. Confocal immunofluorescence microscopy of platelets (red) treated with a) icLNPs and b) cLNPs containing biotin-labelled RNA (green). Representative images for 5 different donors are shown, with untreated platelets from 2 representative donors for comparison. Scale bars indicate 2 µm. 78  3.3.2 LNPs do not impair platelet function An ideal transfection agent would cause minimal platelet activation and not impair platelet function. To determine whether LNPs activate platelets or inhibit activation, multiple markers of platelet activation were assessed. Granule release occurs during the early stages of activation and involves the movement of protein receptors from the platelet granules to the cell surface [63]. P-selectin, which is stored in alpha granules, is one such receptor [57]. To measure granule release, flow cytometry was used to quantify exposure of P-selectin following treatment with LNPs. Compared to untreated platelets, increased exposure of P-selectin occurred only with cLNPs or thrombin-activated platelets (Fig. 3.4). Following granule release, platelets form stable aggregates [67]. As even untreated platelets aggregate to a lesser extent following a short storage period in buffer, platelets were stimulated with ADP immediately after adding LNPs and two hours after adding LNPs in order to detect any minor differences that would only be evident in fresh platelets. None of the  Figure 3.4. cLNPs induce alpha granule release. P-selectin exposure on platelets, quantified using flow cytometry after incubating with LNPs for 2 h in Tyrode’s buffer (n=6). **P  < 0.01, ***P  < 0.001. 79  LNPs induced aggregation of gel-filtered platelets in buffer under either condition, nor did the LNPs inhibit platelets from aggregating in response to ADP at either timepoint (Fig. 3.5a). LNPs were also incubated with platelets in plasma, as isolation of the platelets led to slightly impaired aggregation. In plasma, LNPs also did not induce aggregation or impair the response to ADP immediately after adding LNPs (Fig 3.5b). As the formulations tested here could not be endocytosed in plasma, the ability of platelets to aggregate two hours after treatment with LNPs in plasma was not assessed.  Figure 3.5. LNPs do not induce or impair aggregation. a) Platelet aggregation was quantified in buffer before and after stimulation with ADP (10 µM), either with no incubation or a 2 h pre-incubation with LNPs or free RNA (n=5). b) Platelet aggregation was quantified in plasma in the presence of LNPs or free RNA, before and after stimulation with ADP (n=4). ***P < 0.001 80  Platelet activation leads to pro-coagulant activity, through exposure of phosphatidylserine on the platelet membrane and release of coagulation factors stored within platelet granules [38]. To determine if LNPs enhanced the coagulability of platelets, the time to initiate thrombin generation was measured using a fluorescent thrombin substrate. When platelets were treated with LNPs and reconstituted in plasma following removal of excess LNPs, thrombin generation was faster for platelets treated with cLNPs compared to untreated platelets (Fig. 3.6a). This is consistent with increased P-selectin expression in cLNP-treated platelets, which indicated faster granule release. In platelet-poor plasma, thrombin generation was also faster when cLNPs when added directly to plasma, but not when icLNPs, Lf, nLNPs, or free RNA was added to plasma (Fig. 3.6b). This suggests cLNPs can interact directly with components of the coagulation cascade.  Figure 3.6. cLNPs activate the coagulation cascade. a) The time for platelets to generate thrombin following treatment with LNPs was quantified using a fluorescent thrombin substrate (n=10). b) The time to generate thrombin in platelet-poor plasma treated with LNPs was quantified using a fluorescent thrombin substrate (n=4). **P < 0.01, ***P < 0.001 81  Platelet activation also involves early shape change, followed by spreading of adhered platelets [371]. When platelets were treated with LNPs and then adhered to a collagen-coated surface, spreading in response to thrombin was not inhibited for any formulation (Fig. 3.7). This, along with the ability of platelets to aggregate fully, suggests platelet function was not impaired by treatment with LNPs.  Figure 3.7. LNPs do not impair platelet spreading. a) Quantifying confocal microscopy images of platelets spreading on collagen-coated coverslips following activation with thrombin (n=5). b) Representative images of spread platelets (n=5). Scale bars indicate 5 µm. ***P  <  0.001 3.4 Discussion Here, uptake was tested in different storage conditions to determine which class of LNPs were the best delivery agents for platelets under longer storage times and more physiological conditions. This information is also necessary for designing experiments which test the stability, translation, and release of the materials delivered by the LNPs, as described in chapter 4, and will aid in translating this technology to the clinic, as storage conditions affect the hemostatic 82  abilities and survival of platelets following transfusion. In addition, the ability of platelets to induce or impair platelet activation, measured using granule release, aggregation, thrombin generation, and platelet spreading, was compared for the four classes of LNPs. Under most conditions tested, the contents of icLNPs or cLNPs could be detected within platelets, but not the contents of Lf or nLNPs. None of the LNPs classes impaired platelet activation, while cLNPs induced granule release and faster thrombin generation, indicating cLNPs induce platelet activation.  Increasing the storage time for two hours to six hours increased the percentage of Cy5-positive platelets treated with icLNPs, but not cLNPs. The binding of cLNPs to platelets occurs rapidly, as about 50% of the platelets are already Cy5-positive immediately after adding cLNPs, and after two hours about 80% of the platelets were Cy5-positive. While the percentage of Cy5-positive platelets did increase slightly after six hours, from 82% to 90%, this difference was not significant. It may be possible that after six hours increased amounts of RNA can be delivered to platelets, as the flow cytometry results only indicate the number of platelets that are associated with RNA, not the amount of RNA internalized by the platelets. However, after two hours of treatment with cLNPs, P-selectin exposure and the pro-coagulability of platelets was increased when treated with cLNPs. As further activation may enhance the risk of a thrombotic event or platelet clearance following transfusion, longer incubation times with cLNPs were not used for further experiments. However, longer incubation times may be feasible by modifying the cLNPs or mRNA to reduce the amount of platelet activation, and may be used to enhance delivery and protein translation if an optimized LNP formula that allows for protein translation is identified. Raising the pH during uptake from 6.5 to 7.4 reduced the percentage of Cy5-positive platelets treated with icLNPs, but not cLNPs. This is likely due to the ionizable lipid in icLNPs, 83  which has a pKa of 6.7, while cLNPs have a pH-insensitive cationic lipid. At pH 6.5 icLNPs are slightly cationic, but not at pH 7.4. Raising the pH to 7.4 also decreased the fluorescence intensity within icLNP-treated platelets, and not cLNP-treated platelets. This suggests cationic lipids are important for both the binding and internalization of LNPs to platelets. In other mammalian cells, increasing the positive charge on nanoparticles leads to increased uptake [340], and the results here indicate this is also the case for platelets. Consistent with this, platelets treated with cLNPs show increased binding as well as internalization compared to icLNPs. When platelets were incubated with icLNPs or cLNPs in plasma, the percentage of Cy5-positive platelets and the fluorescence intensity of internalized biotin was reduced for both formulations. Plasma proteins can bind to LNPs, forming a protein corona around the surface of the particle [317]. In vivo, this leads to rapid clearance of nanoparticles by the liver and spleen and alters uptake of the LNPs by cells [313, 321]. Increasing the amount of PEGylated lipid can increase PEG density and reduce protein adsorption, which reduces uptake by macrophages [317]. However, plasma proteins are naturally endocytosed by platelets. Incorporation of fibrinogen-mimicking peptides or integrin-binding peptides on liposomes enhances their uptake by platelets [273, 276]. Apolipoproteins in plasma bind both icLNPs and LNPs containing a cationic cholesterol-derivative, allowing uptake by hepatocytes [319], and apolipoproteins can bind platelets through low density lipoprotein protein receptors [372].  It was expected that the interaction between plasma proteins and the LNPs might enhance their uptake in a system composed only of platelets. The poor uptake suggests LNPs are not interacting with proteins that enhance their internalization. An alternative strategy for enhancing uptake by platelets would be to modify the LNP exterior to contain fibrinogen-mimicking peptides. If this does not work, uptake can be tested using varying percentages of plasma, as platelet concentrates used for 84  transfusion in the United States and Europe are typically composed of up to 65% PAS and only 35% plasma [111, 138]. Even solutions up to 95% PAS have been tested ex vivo, suggesting uptake in plasma may not be essential for a platelet transfection agent in the future [145, 146]. While platelet activation enhances binding and uptake of viruses and bacteria by platelets [208, 256, 258], its effect on LNP uptake depended on the class of LNPs. This is consistent with the results from chapter 2, which demonstrated there are differences in the mechanism of uptake for icLNPs and cLNPs. Activation of platelets leads to exposure of negatively charged phosphatidylserine as well as expression of platelet proteins on the cell surface [38, 60]. However, platelet receptors are also recycled back into the platelet or cleaved by proteases following activation [63, 373]. This means it is difficult to predict the effect of platelet activation on LNP internalization, as we have not determined which platelet proteins interact with LNPs. When platelets were activated by thrombin and treated with icLNPs, the percentage of Cy5-positive platelets was unchanged, indicating binding of icLNPs was not impaired. In contrast, the relative intensity of internalized RNA was no longer significantly higher compared to untransfected cells, suggesting impaired internalization of icLNPs. When platelets were treated with cLNPs the percentage of Cy5-positive platelets decreased, while the relative fluorescent intensity within platelets was unchanged. It is unclear whether this is due to changes in the binding and degradation of internalized RNA, or release of the RNA following delivery by cLNPs. To address this question, stability studies with icLNPs and cLNPs were performed in chapter 4. This is important when determining the ideal method for transfecting platelets, as the potential applications of the modified platelets will depend on intracellular location of the delivered mRNA following activation within the vasculature. If the mRNA does not remain 85  internalized by the platelet, that the LNP-treated platelets may be used for delivery of the mRNA to nearby cells instead of de novo protein synthesis inside the modified platelet. Activation of platelets with thrombin did increase the association of platelets with free RNA and Lf, while the association with nLNPs was unchanged. As with icLNPs and cLNPs, the reason for these changes are unclear, although they may be due to enhanced electrostatic interactions between the negatively charged free RNA and positively-charged platelet proteins and between the positively charged Lf complexes and negatively-charged platelet membrane. Activation of platelets may also enhance the phagocytic-like activity of platelets, which would lead to increased uptake for the larger free RNA and Lf-RNA complexes. While it may useful to deliver LNPs to activated platelets in vivo, allowing for targeted delivery of mRNA to platelets at sites of vasculature damage, free RNA or Lf complexes have poor pharmacokinetics and are not suited for in vivo use. Furthermore, increasing storage time or pH and performing uptake in plasma did not enhance binding of free RNA, nLNPs or Lf to platelets, so these formulations were not selected for further characterization following uptake. Here, free RNA, nLNPs, Lf, and icLNPs did not activate the platelets or affect their ability to aggregate or spread. Only cLNPs induced granule release and caused faster thrombin generation. This could be due to the presence of a cationic lipid, as cationic liposomes have previously been observed to induce aggregation in platelets [313], and cationic solid LNPs lead to platelet activation and aggregation in vivo [374]. In addition, cLNPs induced faster thrombin generation in the absence of platelets. Positively-charged surfaces enhance coagulation through multiple mechanisms, including direct interaction with coagulation factors and activators of coagulation factors [375]. While only minor platelet activation was observed, these effects may increase with higher concentrations of cLNPs. Increasing the PEG density and reducing the 86  overall surface charge may reduce cLNP-induced activation. However cLNPs only increased P-selectin exposure to 20%, which is still within the range typically observed for transfused platelets [144], suggesting cLNP-treated platelets would retain sufficient hemostatic activity for their use in the clinic. The RNA encapsulated by cLNPs may bind TLRs within the endo-lysosomal compartments after uptake, leading to platelet activation. Compared to the other formulations tested, the contents of cLNPs had the highest amount of internalization. Activation of platelet TLRs leads to platelet aggregation, adhesion, and granule release [376, 377]. This includes both TLR3, which recognizes double-stranded RNA [376], and TLR7, which recognizes single-stranded RNA, although stimulating TLR7 in vivo led to a pro-inflammatory but not thrombotic response [377]. Use of modified nucleotides can reduce the immune response to foreign RNA, but these were not used in the activation assays here. Including these nucleotides may in the future may reduce platelet activation in response to cLNPs. If the pro-inflammatory response cannot be managed, the LNP-treated platelets could potentially be used to deliver mRNA for apoptotic proteins to leukocytes, such as during arthritis, as a novel approach for reducing inflammation. In addition, cLNPs did not impair the ability of platelets to aggregate or spread. While the effect of incubating platelets in buffer before reconstituting in plasma was not fully characterized, platelets prepared with protein-loaded LNPs can still clot blood in vitro after short periods of storage in buffer [366]. Future in vivo studies will be required to determine whether platelets prepared with mRNA-LNPs maintain their ability to contribute to hemostasis. The in vitro tests used here are routinely used to assess platelet function ex vivo [136, 139]. However, the quality of transfused platelets is typically determined by additional morphological and 87  metabolic markers, such as pH, the extent of shape change, and the hypotonic shock response, which was not assessed in these experiments [137, 142]. However, there are generally not strong correlations between ex vivo markers of platelet quality and in vivo survival and recovery [142, 144]. The in vivo survival of the transfused platelets may determine which applications the modified platelets can be used for. If the transfused platelets are rapidly cleared, as are cold-stored platelets, they may be useful for treating acute bleeding, such as in trauma, but unsuited for prophylactic use.  Together, these results indicate that icLNPs or cLNPs would be the best classes for mRNA delivery to platelets, compared to free RNA, nLNPs or Lf, under a variety of storage conditions. While cLNPs show enhanced delivery compared to other formulations, they induce platelet activation. These effects may be reduced by optimizing the LNP formulation, as has been done to optimize nucleic acid formulations for other mammalian cells [337, 378]  Free RNA, nLNPs, Lf and icLNPs did not induce platelet activation, but only icLNPs and cLNPs led to internalization of RNA in resting platelets. However, delivery of mRNA to the cell is only the first step in identifying a successful transfection agent. The mRNA must still escape the endosomal compartments and remain intact long enough to be translated. To address this, the stability, release, and translation of the RNA delivered by icLNPs and cLNPs was tested in chapter 4.  88  Chapter 4: Stability, translation, and release of mRNA following delivery by LNPs 4.1 Rationale Genetically modified platelets have been used to treat inherited bleeding disorders in animal models and humans [10, 12, 13]. This has been accomplished by transduction of HSCs with viral vectors encoding for missing or defective proteins important to platelet function. This approach has been particularly effective in treating hemophilia A in large animal models [10]. However, therapeutic application of this method involves bone marrow transplantation, which requires intensive pre-conditioning procedures to reduce the likelihood of host rejection [12, 13]. An alternative approach would be to directly alter donor-derived platelets ex vivo prior to transfusion. This would be advantageous when the underlying disorder causing reduced or defective platelets is not due to an inherited genetic defect, as the treatment would only last as long as the lifespan of a platelet. For instance, trauma is often associated with platelet dysfunction, which could be improved through delivery of pro-coagulant or anti-fibrinolytic proteins to the platelets [366]. Alternatively, platelets may also be engineered to prevent cancer growth and used during transfusion in cancer patients only until remission. However, major advances in platelet culturing are needed before sufficient numbers of platelets derived from modified HSCs can be produced for the clinic [8]. An alterative approach is directly modifying platelets with mRNA. Platelets are capable of translation, receiving the necessary machinery from megakaryocytes [178-180]. They perform de novo protein synthesis during storage as well as after activation with pro-coagulant or pro-inflammatory stimuli [85, 149, 152, 187-190]. Platelet mRNA contains the typical elements of 89  eukaryotic mRNA, including a 5’-methylguanosine cap, 5’ and 3’ UTR, and poly(A) tail [158, 159]. Platelets also contain abundant miRNAs, which regulate platelet translation [158, 159], and are released in platelet microparticles and transferred to nearby cells, including leukocytes [70], endothelial cells [70], and tumors [302]. This provides an means by which platelets control protein translation in these cells. Platelet mRNAs and proteins can also be transferred and used by recipient cells [293, 300]. As microparticle release is activation-dependent, this suggests modified platelets may also be used as vehicles for targeted delivery to cells at sites of vasculature damage, where platelet activation occurs.  Building on the LNP formulations tested in chapter 2, and the uptake conditions identified in chapter 3, the stability of the mRNA following uptake by platelets was assessed by flow cytometry and confocal microscopy. Only the two LNP classes that delivered the highest amount of mRNA, icLNPs and cLNPs, were compared. Translation was tested using a reporter mRNA encoding GFP that contained modified nucleotides to improve stability and enhance translation. The release of mRNA in platelet microparticles was quantified by flow cytometry and qPCR to test the potential of using LNP-transfected platelets as mRNA-delivery vehicles. Preliminary experiments using modified cLNPs and higher concentrations of LNPs were also performed using flow cytometry to determine possible methods of increasing mRNA delivery to platelets in the future.  4.2 Methods 4.2.1 Quantifying uptake of LNPs by flow cytometry and confocal microscopy Platelets were treated and samples prepared as previously described. Uptake was performed in Tyrode’s-HEPES (pH 6.5) for 2 h, using equivalent concentrations of mRNA for each LNP formulation. In experiments testing the stability of delivered mRNA, excess LNPs 90  were removed and platelets re-suspended at a concentration of 200 × 1010 platelets L-1 and stored at 22°C for 2 to 4 h. For confocal microscopy experiments, platelets were stained with rabbit anti-human actin antibody (4 µg/mL, ThermoFisher) for 1.5 h at room temperature, washed, and then stained with streptavidin-FITC (1 µg mL-1, ThermoFisher) and AlexaFluor 594 goat anti-rabbit secondary (2 µ/mL, ThermoFisher) for 1 h. The relative amount of mRNA was quantified by measuring the pixel intensity within the ring of actin staining for approximately 20 platelets using ImageJ software, normalizing to untreated platelets.  4.2.2 Quantifying protein expression Following removal of excess LNPs, platelets were suspended in M199 media at a concentration of 200 × 1010 platelets L-1 and activated with human thrombin (52 U mL-1) and collagen (10 µg mL-1, Sigma Millipore) or left untreated. After 16 h at 37°C, platelets were centrifuged for 20 min at 250 x g, washed once in PBS, and resuspended in PBS. Human embryonic kidney 293 (HEK293) cells (ATCC) were grown in FluoroBrite Dulbecco’s Modified Eagle Medium (ThermoFisher), supplemented with Penicillin/Streptavidin (5% v/v, ThermoFisher) and heat-inactivated fetal bovine serum (5% v/v, ThermoFisher), and transfected with icLNPs or cLNPs (0.5 µg mRNA mL-1). After 24 h, cells were washed once with PBS, treated with trypsin-EDTA (12.5 µg mL-1, ThermoFisher) for 5 min and diluted with an equivalent volume of PBS. To measure protein expression in platelets and HEK293 cells, the fluorescent intensity (λEx/ λEm = 485/535 nm) was measured using a Tecan Genios plate reader. 4.2.3 Isolating platelet microparticles  After removing excess LNPs, platelets were suspended in modified Tyrode’s buffer (pH 7.4) or platelet-poor plasma. Platelet-poor plasma was obtained by centrifuging PRP isolated by the CBS’ netCAD facility for 10 min at 1000 × g, removing the supernatant, and centrifuging the 91  supernatant for 10 min at 1000 × g. The resulting supernatant was considered platelet-poor plasma. Following resuspension, only platelets in buffer were intentionally activated by incubating with calcium chloride (5 mM), thrombin (2 U mL-1) and collagen (10 µg mL-1) for 2 h at 37°C. Platelets were then centrifuged for 20 min at 250 × g and the resulting supernatant centrifuged for 10 min at 3,200 x g to clear platelet debris. The supernatant was removed and centrifuged for 90 min at 21,000 x g to isolate microparticles. The microparticle pellet was resuspended in PBS before flash-freezing in liquid nitrogen. After thawing, microparticles were stained with anti-human CD42b-FITC for 30 min at 4°C before analysis by flow cytometry using a CytoFLEX Flow Cytometer (Beckman Coulter) and CytExpert software. Gating was performed using CD42b staining. Microparticle size was measured using dynamic light scattering with Zetasizer Nano ZPS (Malvern).  4.2.4 Quantifying mRNA by qPCR Platelets or microparticles were resuspended in TRIzol reagent. RNA was extracted using the manufacturer’s protocol, digested with a TURBO DNA-free Kit (ThermoFisher), reverse transcribed into cDNA using oligo-D(T) primers and M-MLV reverse transcriptase, and detected using qPCR with gene-specific primers and SYBR green reagents. RNA was quantified using 2-ΔΔCt, with GAPDH as an internal control for platelets and exogenous GFP mRNA added as an internal control for microparticles.  4.2.5 Preparing modified cLNPs Modified cLNPs were prepared using a previously published method for mRNA transfection [379]. A thin lipid film of DOTAP and DOPE (1:1 molar ratio, Avanti Polar Lipids) was rehydrated with deionized water to a concentration of 2.9 mM lipids and extruded ten times through a 200 nm filter using a LIPEX extruder. The lipids were mixed with mRNA (125 µg mL-92  1) and HEPES (5 mM) to a final concentration of 0.36 mM total lipids and incubated for 10 min at 22°C before use.   4.3 Results 4.3.1 Stability of the mRNA depends on LNP class For mRNA to be translated, it must remain internalized by the platelets after delivery. As previously discussed, platelet function is sensitive to storage conditions, including pH and the presence of plasma. To assess the stability of the RNA in platelets treated with LNPs under a range of storage conditions, the percentage of Cy5-positive platelets was quantified by flow cytometry four hours after removal of LNPs. Only icLNPs and cLNPs were compared, based on the results of chapter 3. When platelets were prepared with icLNPs, the percentage of Cy5-positive platelets was unaltered during storage in buffer at pH 6.5 or pH 7.4, either in the presence or absence of thrombin, and during storage in plasma (Fig. 4.1).  When platelets were prepared with cLNPs, the percentage of Cy5-positive platelets decreased during storage in plasma and when activated with thrombin at pH 7.4, but did not change during storage in buffer at pH 6.5 or pH 7.4 in the absence of any platelet agonist (Fig 4.1). Confocal microscopy was then used to determine whether the fluorescence remained internalized within the platelets under these conditions, using actin to visualize the platelets. When platelets were prepared with icLNPs, there was no significant difference in the fluorescence intensity inside of the platelets under any storage conditions two hours after removal of icLNPs, relative to the fluorescence intensity immediately after uptake (Fig. 4.1b, Fig 4.2a). When platelets were prepared with cLNPs, the fluorescence intensity inside of the platelets decreased with storage in plasma, but not in buffer at pH 6.5 or 7.4 (Fig. 4.1b, Fig 4.2b).  93   Figure 4.1. The contents of icLNPs and cLNPs remain internalized in resting platelets but the contents of cLNPs do not in activated platelets or platelets in plasma. a) The number of Cy5-positive platelets 4 h after excess LNPs were removed and platelets were resuspended in Tyrode’s buffer at pH 6.5 or pH 7.4 or in plasma were quantified using flow cytometry (n=5). b) The amount of biotin that remains inside platelets during storage for 2 hr under different conditions was assessed by quantifying biotin-labelled RNA in confocal immunofluorescence images of platelets (n=5). P < 0.05, **P < 0.01, ***P < 0.001. n.s. means not significant.   94   95  Figure 4.2. The contents of icLNPs and cLNPs remain internalized after uptake unless platelets are stored in plasma. Confocal immunofluorescence microscopy of platelets (red) transfected with a) icLNPs and b) cLNPs containing biotin-labelled RNA (green) 2 h after removal of LNPs. Representative images for 5 different donors are shown. Scale bars indicate 2 µm. To determine whether these changes were caused by activation of platelets during storage, P-selectin exposure was compared by flow cytometry. In platelets prepared with either icLNPs or cLNPs, P-selectin exposure did not significantly increase in platelets stored in buffer at pH 6.5 or pH 7.4 two hours after removal of LNPs (Fig. 4.3). P-selectin exposure increased when platelets were stored in plasma and had been prepared with icLNPs, but not when they have been prepared with cLNPs (Fig. 4.3). This suggests that the decrease in bound and   96  Figure 4.3. Alpha granule release following removal of LNPs differ depending on storage conditions and presence of cationic lipids. P-selectin exposure on platelets, quantified using flow cytometry 2 h after excess LNPs were removed (n=4). *P  < 0.05,  **P  < 0.01, ***P  < 0.001.  internalized biotin for cLNP-prepared platelets stored in plasma is not due to increased activation of the platelets while in storage, compared to when stored in buffer at pH 6.5 or pH 7.4. The exception to this is thrombin-treated platelets, which still exhibit complete P-selectin exposure even when prepared with icLNPs or cLNPs (Fig. 4.3). 4.3.2 Transfection of platelets with icLNPs or cLNPs does not lead to protein expression As flow cytometry and confocal microscopy cannot distinguish between single nucleotides and intact mRNA, the amount of mRNA after treatment with icLNPs and cLNPs was quantified by qPCR. A short fragment of mRNA, 127 base pairs long, could be detected in platelets with icLNPs and cLNPs (Fig. 4.4a), indicating the mRNA is partially intact in platelets. While further experiments would be required to determine whether the mRNA is fully intact and functional, the ability of platelets to translate the delivered mRNA was still assessed. To ensure the LNPs were functional, HEK293 cells were first transfected with icLNPs and cLNPs containing GFP mRNA. While HEK293 cells expressed GFP (Fig. 4.4b), platelets with icLNPs or cLNPs did not (Fig. 4.4c). As platelets are capable of signal-dependent translation upon activation with thrombin or markers of inflammation, GFP expression was assessed in both resting and thrombin-activated platelets; however protein synthesis was not observed under either condition (Fig. 4.4c). This may be due to degradation of the mRNA following delivery to  97   Figure 4.4. Platelets do not translate mRNA delivered with icLNPs or cLNPs. a) Quantifying the amount of mRNA using qPCR after excess LNPs were removed (n=12). b) HEK293 cells (n=8) and c) platelets (n=5) were transfected with LNPs containing GFP mRNA and after 24 h protein expression was quantified by measuring green fluorescence. *P < 0.05, ***P < 0.001. n.s. means not significant.  platelets, as only a short fragment of the mRNA was detected by qPCR. Alternatively, mRNA may be unable to escape from the endosome following uptake of the LNPs. 98  4.3.3 Delivered mRNA is released in platelet microparticles  To test if platelets released the delivered mRNA in microparticles, flow cytometry was used to detect whether Cy5 localized to microparticles ranging from 100 nm to 400 nm in diameter (Fig. 4.5) and expressing CD42b, a marker of platelet microparticles. Microparticles were formed by platelets activated by the oxidative and mechanical stress induced by ex vivo isolation and storage of the platelets, or by treatment with thrombin and collagen [144, 289]. Under all conditions, a significant increase in Cy5-mRNA was detected in microparticles from platelets prepared with cLNPs and stored in either buffer or plasma (Fig 4.6a). In addition, a significant increase in mRNA could also be detected under these conditions using qPCR (Fig. 4.6b). More than 50% of platelet microparticles were Cy5-positive when platelets were stored in buffer, but not when stored in plasma (Fig. 4.6a). When platelets were prepared with icLNPs and not intentionally activated, there a significant increase in Cy5-positive microparticles from platelets stored in buffer (Fig. 4.6a), and significant increase could be detected by qPCR under this condition (Fig. 4.6b). When platelets were prepared with icLNPs and either activated with thrombin and collagen or stored in plasma, a significant increase in mRNA was detected by  Figure 4.5. Platelet microparticles range from 100 nm to 400 nm. Dynamic light scattering was used to measure the size of microparticles released from platelets. Representative plots of 3 different donors are shown. 99    100  Figure 4.6. RNA delivered by LNPs is released in platelet microparticles. Microparticles were isolated from platelets prepared with LNPs and resuspended in either Tyrode’s buffer, with or without activation, or in plasma without activation. a) The number of Cy5-positive and Cy5-negative microparticles from 1 x 108 platelets, quantified using flow cytometry (n=6). b) The amount of FLuc mRNA in all microparticles from 1 x 108 platelets, quantified by qPCR (n=11).  *P < 0.05, **P < 0.01,  ***P < 0.001. qPCR (Fig. 4.6b), but under these conditions there was no conclusive increase in Cy5-positive microparticles (Fig. 4.6a). 4.3.4 Optimizing delivery of LNPs While cLNPs delivered the highest amount of mRNA to platelets, no protein translation could be detected in cLNP-treated platelets. Therefore, modified cLNPs were formulated and their interaction with platelets was quantified by flow cytometry. The modified cLNPs had an average diameter of 250 nm (Fig. 4.7a), lacked a PEGylated lipid, and contained DOPE instead of DOPC. DOPE is a fusogenic lipid that enhances endosomal escape and transfection efficiency. Uptake experiments in chapters 2 and 3 were performed using 250 ng of mRNA per mL of platelets as this was the minimal amount of mRNA required to detect protein synthesis in HEK293 cells. In testing the interaction of modified cLNPs with platelets, a range of mRNA concentrations were tested. Preparing platelets with modified cLNPs encapsulating 250 ng of mRNA did not led to a significant increase in the number of Cy5-positive platelets compared to untreated cells, while preparing platelets with modified cLNPs encapsulating 1250 ng or 6 µg of mRNA did lead to a significant increase in the number of Cy5-positive platelets (Fig. 4.7b). Next, the ability of the modified cLNPs to induce platelet activation was assessed by quantifying  101   Figure 4.7. Use of DOPE in cLNPs is not sufficient for protein expression in platelets. a) Dynamic light scattering was used to measure the average diameter of modified cLNPs (n=3). b) The number of Cy5-positive platelets and c) P-selectin exposure 2 h after treatment with increasing amounts of mRNA-modified cLNPs was quantified using flow cytometry (n=6). GFP expression in d) HEK293 cells and e) platelets after treatment with modified cLNPs. **P < 0.01, ***P < 0.001. 102  granule release, as this is one of the earliest events in platelet activation [63]. Preparing platelets with modified cLNPs encapsulating 250 ng of mRNA did not led to a significant increase in P-selectin exposure compared to untreated cells, while preparing platelets modified cLNPs encapsulating with 1250 ng or 6 µg of mRNA did lead to a significant increase in P-selectin exposure (Fig 4.7c). To minimize activation while maximizing uptake, 1250 ng mRNA was used to test for protein synthesis. GFP expression was detected in HEK293 cells transfected with modified cLNPs (Fig. 4.7d), but not in platelets treated with modified cLNPs (Fig 4.7e). As modified cLNPs did not lead to protein expression in platelets, preliminary experiments testing the interaction of platelets with higher concentrations of free RNA, Lf, nLNPs, icLNPs, and cLNPs were preformed. Preparing platelets with 1250 ng of mRNA increased the percentage of Cy5-positive platelets for icLNPs and Lf, while preparing platelets with 6 µg of mRNA increased the percentage of Cy5-positive platelets for all formulations (Fig. 4.8a). P-selectin exposure was then used as a marker of platelet activation. Compared to untreated platelets, P-selectin exposure was increased when platelets with prepared with 250 ng, 1250 ng or 6 µg of mRNA-cLNPs, in a dose-dependent manner. (Fig. 4.8b), No increase in P-selectin expression was detected for free RNA, Lf, nLNPs, or icLNPs at any mRNA concentration (Fig. 4.8b).    103   Figure 4.8. Binding of LNPs is enhanced through increased delivery of nLNPs, Lf or icLNPs. a) The number of Cy5-positive platelets and b) P-selectin exposure 2 h after treatment with increasing amounts of Cy5-labelled mRNA-LNPs was quantified using flow cytometry (n=6).  *P < 0.05, **P < 0.01. 4.4 Discussion Here, we show that the stability and release of the contents delivered to platelets by cLNPs depends on storage conditions. RNA from cLNP-treated platelets could be detected in microparticles generated when platelets were stored in plasma or activated in buffer, with a 104  corresponding decrease in the Cy5-RNA or biotin-RNA content of the platelets. In contrast, while RNA delivered by icLNPs could be detected in microparticles under some conditions, no corresponding decrease in the Cy5-RNA or biotin-RNA content of the platelets could be detected, indicating the majority remained internalized in platelets. Several storage conditions were identified under which the contents of both icLNPs and cLNPs did not change; however, protein translation could not be detected in icLNP or cLNP-treated platelets, even when platelets were transfected with modified cLNPs with improved transfection efficiency in HEK293 cells.  The stability of the icLNPs and cLNPs depended on the storage conditions and the class of LNPs. While there was no change in the percentage of Cy5-positive platelets or internalized btioin when platelets treated with icLNPs or cLNPs were stored in buffer at either pH 6.5 or pH 7.4, the amount of biotin in platelets decreased when platelets were stored in plasma only when platelets were prepared with cLNPs and not when platelets were prepared with icLNPs. These differences did not correlate with differences in activation of icLNP and cLNP-treated platelets during storage, suggesting the differences in stability of icLNP and cLNP-treated platelets may be due to differences in trafficking of the LNPs. The decrease in biotin observed in platelets treated with cLNPs and stored in plasma could either be caused by degradation or release of the RNA. While degradation of the RNA cannot be ruled out, flow cytometry and qPCR indicate that RNA delivered by cLNP-treated platelets stored in plasma is released in microparticles. Cy5 could be detected in microparticles from platelets prepared with cLNPs and stored under all conditions tested, and significant increase in RNA was detected by qPCR for microparticles generated from platelets stored under all conditions. Furthermore, confocal microscopy indicates that biotin from RNA in these platelets is no longer localized to the platelet interior, but has moved to the periphery of the platelet, which would be expected if it was being packaged into 105  microparticles. This suggests the contents of cLNPs internalized by platelets are released during storage in plasma. When platelets were activated with thrombin following treatment with LNPs, the percentage of Cy5-positive platelets prepared with cLNPs decreased. While there was a downward trend in the internalized fluorescent intensity of platelets prepared with cLNPs, there was no consistent movement of internalized biotin to the peripheral of the platelets. However, flow cytometry and qPCR analysis indicated microparticles generated from platelets prepared with cLNPs and activated with thrombin and collagen contained fragmented or intact RNA. This suggests that the contents of cLNPs can also be released in microparticles generated during activation of platelets with thrombin and collagen. In contrast, no change in the percentage of Cy5-positive platelets or internalized biotin could be detected in cLNP-treated platelets stored in buffer and not treated with thrombin and collagen, although RNA was detected in microparticles generated under these conditions. This suggests only minimal amounts of the delivered RNA are released if platelets are stored in buffer in the absence of any agonist. Increased amounts of RNA may be released upon activation of platelets with thrombin and collagen, or activation with thrombin and collagen also enhanced decay of the mRNA delivered to platelets with cLNPs. Microparticles are commonly generated ex vivo using platelets resuspended in buffer and treated with thrombin, or a combination of thrombin and collagen. It was expected agonist-treated platelets would generate the highest amount of microparticles. We detected a large number of microparticles from platelets stored in plasma, and the majority were Cy5-negative, even in cLNP-treated platelets. It is possible this was a result of using platelet concentrates as the source of platelet-poor plasma in these experiments. Microparticles can be produced by platelets during storage, and would have been present in the platelet poor plasma, increasing the overall 106  number of microparticles in these samples, although we did not quantify the number of microparticles present in the platelet poor plasma to confirm this [137]. In buffer, the majority of microparticles from cLNP-treated platelets were Cy5-positive, suggesting that the contents of cLNPs are non-selectively packaged into platelet microparticles following uptake of cLNPs by platelets. However, these results could be due to release of intact RNA, or release of Cy5 generated if the mRNA is degraded following delivery to the platelets. Here, we used flow cytometry and qPCR to test whether mRNA delivered by platelets was released in microparticles. While flow cytometry only detected the fluorescent label from delivered mRNA, which could be released if the mRNA was degraded following delivery into the platelet, qPCR detected a fragment about 100 base pairs long. Without further experiments it is not possibly to conclude whether the RNA released in microparticles is intact and functional mRNA, potentially allowing delivery of mRNA for therapeutic proteins to target cells. However, only low quantities of RNA could be extracted from the microparticles. To confirm whether the released RNA is intact and functional, these experiments need to be repeated with a significantly higher number of platelets, allowing for increased microparticle generation and increased yields of RNA.  In the assay, the effects of donor variation were minimized, as microparticles were generated from plasma concentrates pooled from three to four whole blood donations. However, there were discrepancies between flow cytometry and qPCR data for platelets prepared with icLNPs. Cy5 was only detected in microparticles generated from icLNP-treated platelets stored in buffer, but RNA was detected by qPCR from icLNP-treated platelets stored in all conditions. For flow cytometry experiments, platelet microparticles were identified by staining with a platelet membrane marker, CD42b, which is also commonly found on platelet microparticles 107  [295]. In comparison, qPCR analysis was performed on the entire population of microparticles, and it may have allowed for increased detection of microparticles containing the released mRNA. Alternatively, the larger samples size in the qPCR data led to a higher statistical power in the assay compared to the flow cytometry data. However, no decrease in biotin content was detected for icLNP-treated platelets stored under any condition. This suggests the majority of the materials delivered by icLNPs remained within platelets. Therefore, platelets prepared with icLNPs may be more suited for use in developing modified platelets capable of synthesizing exogenous proteins, while platelets prepared with cLNPs have potential to be used as delivery vehicles for delivered mRNA. Our experiments focused on microparticles 100 nm to 400 nm in diameter, based on multiple studies demonstrating transfer of mRNA and miRNA by platelet microparticles of this size. Sizing was confirmed by dynamic light scattering, which also indicated the presence of particles several microns in diameter. As the platelet releasate was pre-cleared to remove platelet debris prior to isolating microparticles, these large particles are likely due to aggregation of the platelet microparticles, induced by centrifugation and freeze-thawing [380]. Platelets also generate exosomes, 30 nm to 100 nm extracellular vesicles derived from the endosome rather than budding of the plasma membrane [380]. Exosomes are also enriched for specific platelet miRNAs and can also mediate miRNA transfer to recipient cells [381]. Future studies assessing release of LNP-delivered mRNA in platelet exosomes might conform the potential of LNP-treated platelets as delivery vehicles for nucleic acid therapies.  While platelet activation is required for microparticle formation, platelet microparticles are heterogenous and their composition varies depending on the agonist used to activate platelets [69]. During storage GPIIb/IIIa signalling leads to destabilization of the actin cytoskeleton and 108  microparticle formation [382]. These microparticles are pro-coagulant, while microparticles generated when platelets are activated with pro-coagulant agonists are not all pro-coagulant [291, 295]. If LNP-treated platelets were to be used in the clinic, further characterization of RNA-positive microparticles would be required. Production of pro-coagulant microparticles may reduce the safety of the LNP-treated platelets following transfusion. Alternatively, they may be used to improve the hemostatic efficacy of platelets that are transfused during severe bleeding, for instance during trauma. Since translation of the delivered mRNA was not observed, additional characterization of the RNA following uptake into the platelets is needed. It is unclear whether the absence of translation was due to degradation of the mRNA, differences in how translation is regulated in platelets compared to other mammalian cells, or whether the mRNA was not delivered to the appropriate location in the platelet. In newly formed platelets, mRNA is rapidly degraded, although the mechanisms regulating mRNA decay in platelets are not fully understood [162].. Here we amplified a fragment of mRNA that was 127 base pairs long, but we were not able to determine how much intact, functional mRNA was present in platelets and microparticles. While we attempted to determine if there was intact FLuc mRNA present in the platelets using qPCR, this requires amplifying about a 1,900 base pair fragment, compared to the recommended length of 50 to 150 base pairs. Longer templates can lower the efficiency of PCR, reducing the sensitivity of this assay [383].  Based on qPCR analysis, it was estimated about 1,000-fold increase in platelets would be required, equivalent to about one and a half units of platelet concentrate.  Alternatively, the functionality of the RNA isolated from LNP-treated platelets could be tested in a cell-free translation system, such as rabbit reticulocyte lysate. However, as with 109  qPCR, this technique only tells us if the mRNA is intact, and not whether it is present in the cytoplasm of the platelet. The half-life of unmodified GFP mRNA is around 3 to 4 hours [333], and translation of GFP in platelets was assessed 16 hours after removal of excess LNPs by the platelets. While this timeline was selected to allow sufficient time for protein translation, it may have also limited the amount of protein translation that could be detected if the mRNA was degraded in the platelets prior to translation. To improve the sensitivity of the assay, GFP mRNA containing 5-methoxyuridine was used, as this enhances both the stability of the mRNA, and its ability to be translated in mammalian cells [333]. In our initial studies, 250 ng of mRNA-LNP complex was delivered to the platelets, as this was sufficient for protein expression in HEK293 cells and minimized unwanted activation of platelets. Preliminary experiments indicated that increased concentrations of free RNA, Lf, nLNPs, or icLNPs led to an increased number of RNA-positive platelets, without causing further platelet activation. Additional studies would be required to determine whether the RNA is internalized for all the different classes of LNPs, and whether platelet function is still not compromised with higher concentrations of mRNA. However, these experiments suggest increased delivery of mRNA to platelets is feasible, perhaps through use of icLNPs. As icLNPs are similar in composition to siRNA-icLNPs that have already progressed through clinical trials with promising results, mRNA-icLNPs would be expected to have good biocompatibility, making this class of lipids ideal for generating transfusable, modified platelets capable of de novo protein synthesis.  Efficient protein expression in other transfected cells is often limited by the release of transfected RNA from endosomes. In siRNA-LNP treated HeLa cells, about 70% of the nanoparticles are recycled back out of the cell [21], and only 1-2% of the total siRNA is released 110  into the cytosol [20]. To address this, platelets were prepared with modified cLNPs containing DOPE, a fusogenic lipid which enhances endosomal release of RNA, and lacking a PEGylated lipid, which impairs uptake. While slightly larger then cLNPs, possibly impaired uptake at the lowest concentration of mRNA tested, modified cLNPs associated with platelets at higher mRNA concentrations. This was also observed in platelets treated with Lf, which also contains DOPE. However, no translation was observed in platelets prepared with modified cLNPs, so it is unclear if poor endosomal escape is the reason no translation occurred in platelets. In our studies, platelets were only stained with a membrane marker or actin. In mammalian cells, subcellular location has been determined using electron microscopy along with gold nanoparticle-conjugated siRNA [21]. Endocytosis of protein-coated gold nanoparticles has been studied in platelets using electron microscopy [258], suggesting this is a viable approach. Alternatively, co-localization between fluorescently labelled mRNA and markers of organelles can be used to study in vitro trafficking [21], as has been down for tracking of fibrinogen in platelets [263].  Even if the mRNA escapes from the endosome, it is unclear whether the mRNA would be bound by the platelet ribosome. Polyribosome isolation could be used to capture platelet ribosomes and determine if the delivered mRNA is present, however, the smaller number of ribosomes present in platelets relative to other cells makes this technically challenging. Furthermore, while platelets express proteins during storage and activation, our understanding of the factors that regulate translation in platelets is incomplete [162, 198]. Ribosome profiling indicates recycling of ribosome complexes may be a limiting factor in protein synthesis. It is also unknown treatment with LNPs or exogenous RNA effects protein translation in platelets. In mammalian cells, foreign RNA is recognized by TLR3, TLR7 and TLR9 present in endosomes or cell surface [19]. This can lead to activation of caspases, which cleave RNA-dependent 111  protein kinase (PKR) and allow PKR to phosphorylate eIF-2α, repressing translation [384]. To prevent this from occurring experiments were performed with mRNA containing 5-methyloxyuridine, a modified nucleotide that enables high protein expression in vitro [333]. Caspases are also activated during apoptosis, where they also cleave eIF4G, a protein necessary for cap-dependent translation [384]. Cationic liposomes can induce apoptosis in macrophages and hepatocytes, even altering the transcriptome of hepatocytes in vitro [321, 385]. While LNPs did not impair platelet activation, we did not determine if global and signal-dependent translation can still occur in platelets following treatment with LNPs. This may be done using pulse-chase labelling of newly synthesized proteins with radiolabelled amino acids, as has been done to study protein synthesis in platelets in the past [152]. These experiments are necessary to determine whether LNPs have any potential for generating modified platelets capable of synthesizing exogenous proteins.  Here, we show that the contents of icLNPs and cLNPs are stable in resting platelets under specific storage conditions, and mRNA delivered using cLNPs can be released in platelet microparticles following activation. However, further characterization and optimization is still needed before LNPs can be used as an experimental or clinical tool for de novo protein synthesis in platelets. To determine whether intact, functional mRNA is delivered to platelets and released in microparticles, methods for increasing the yield of delivered mRNA are required. Preliminary experiments here suggest this may be feasible with increased concentrations of icLNPs. Studies characterizing the intracellular location and translational activity of LNP-treated platelets are also necessary to see whether de novo protein synthesis is feasible. If endosomal escape is not occurring, it may be necessary to first optimize the LNP formula and screen for intracellular location using co-localization analysis. Once endosomal escape is confirmed, the ability of the 112  platelets to translate the delivered mRNA can be tested using cell-free lysates. Modifications to the mRNA, described in chapter 5, may be necessary to facilitate translation in platelets. If platelets cannot translate exogenous mRNA, the LNP-treated platelets may instead be used as a delivery vehicle for therapeutic nucleic acids. The characterization performed here suggests icLNPs may be more suited as a transfection agent if de novo protein synthesis in platelets is the desired outcome, while cLNPs are more suited as a transfection agent if mRNA release is the long-term goal.  113  Chapter 5: Conclusion and Future Directions 5.1 Summary Platelets are small but complex cells that are frequently transfused to prevent or stop bleeding in patients. Physiologically, they regulate inflammation, innate and acquired immunity, angiogenesis, and tumor progression, as well as their primary role in hemostasis. A method for genetically modifying platelets has the potential to improve their hemostatic potential in situations where there is platelet dysfunction or coagulopathy, address concerns regarding platelet storage, and extend their therapeutic applications beyond bleeding. Genetically modified platelets do exist, but are created by modifying the precursor cells, and this approach currently cannot be used to generate transfusable platelets ex vivo. No method exists for directly transfecting platelets. As a first step towards addressing this problem, a method for delivering mRNA to platelets was identified in this thesis.  In chapter 2, the ability of four classes of LNPs to deliver purified, IVT mRNA to platelets were compared. LNPs containing cationic or ionizable cationic lipids, were able to deliver the highest amount of RNA compared to neutral LNPs or the commercial reagent Lf. As Lf also contains a cationic lipid, this suggests that efficient mRNA delivery to platelets requires not just a cationic lipid, as seen in other mammalian cells, but also small nanoparticles less than 200 nm in diameter. In addition, a method for synthesizing RNA in platelets was developed, optimizing protocell technology to create controllable, RNA-synthesizing LNPs suitable for cell delivery. For all formulas, uptake of LNPs occurred in an energy and dynamin-dependent manner. Uptake of icLNPs, but not cLNPs, also required actin polymerization, suggesting that the two classes of LNPs can be endocytosed by separate pathways. This effects the trafficking and intracellular location of the LNP, which is a determining factor in transfection, and suggests 114  that icLNPs and cLNPs may not be equally effective for de novo protein synthesis in platelets. While protein synthesis has not yet been achieved, this work describes the first examples of direct mRNA delivery to platelets as well as exogenous mRNA synthesis in an anucleate cell. While delivery of IVT mRNA was chosen for further optimization, as it was a more efficient and simpler approach, RNA-synthesizing LNPs provided proof-of-principle that a variety of biological macromolecules, including enzymes and nucleic acids, can be loaded into platelets and remain intact and active. In chapter 3, the effect of pH, plasma, and platelet activation on the uptake of the four classes of LNPs were compared using confocal microscopy and flow cytometry. While this approach did not identify whether mRNA delivered is intact and can be translated, delivery of mRNA to the cell interior is the first step for protein translation. Identifying conditions under which mRNA is internalized was necessary before further characterization of the mRNA could be performed. In buffered systems, only the contents of cLNPs were internalized by platelets at physiological pH 7.4 and when platelets were activated. At pH 6.5, both the contents of icLNPs and cLNPs were internalized. This confirmed the importance of a cationic lipid for RNA delivery to platelets. In plasma, uptake was not observed for any formulation. Ideally, a transfection agent for platelets would work in platelet concentrates, which may contain plasma and/or PAS, so testing uptake in various percentages of plasma, supplemented with PAS, would be an important future direction once an optimal LNP formulation is identified. As ex vivo storage leads to platelet activation, reducing the hemostatic ability following transfusion, the effect of storage with mRNA-LNPs was assessed for all LNP classes. None of the LNPs impaired platelet aggregation or spreading, and only cLNPs led to granule release and faster thrombin generation. These in vitro changes were minor, suggesting mRNA-LNP treated 115  platelets can still function in vivo following transfusion. The ability to still respond to natural platelet agonists would be required in genetically modified platelets whether they are intended to treat hemorrhage or inflammatory diseases, as either application would involve activation-dependent release of the newly-synthesized proteins.  In chapter 4, the stability, translation, and release of mRNA delivered using icLNPs and cLNPs were compared. Using qPCR, a 127 base pair fragment of the reported mRNA could be detected in icLNP- and cLNP-treated platelets, suggesting intact mRNA remains following delivery. Using flow cytometry and confocal microscopy, the amount of fluorescent marker from the delivered RNA in icLNP-treated platelets did not change with any storage condition, while the amount of fluorescent marker from the delivered RNA in cLNP-treated platelets did not change in resting platelets stored at either pH 6.5 or pH 7.4. However, no protein translation could be detected in LNP-treated platelets. Further experiments are required to determine why this was the case, as it could a problem with the delivery vehicle, the mRNA, or platelets’ inherent translational capabilities. The stability of the mRNA was poor in platelets stored in plasma, whether it was delivered using icLNPs or cLNPs. This would have to be addressed if LNP-treated platelets were used in the clinic, as it would require LNP-treated platelets to be produced in the clinic immediately prior to use, rather than at blood banks, and limit the amount of time the treated platelets could produce exogenous protein once transfused. While translation was not observed, release of mRNA delivered using cLNP-treated platelets was detected using flow cytometry and qPCR. This suggests cLNP-treated platelets could potentially be used to deliver therapeutic mRNA to sites of vascular damage, as platelets release RNA-enriched microparticles that can be endocytosed by nearby cells at these sites. 116  In this thesis, optimal classes of LNPs for mRNA delivery to platelets and conditions for preparing and storing the LNP-treated platelets were identified. The release and translation of the delivered mRNA was also characterized. While the end goal of creating genetically-modified platelets was not achieved, only four classes of LNPs were compared. There are libraries of hundreds of lipid and lipid-like materials that have been screened to identify the most potent LNPs for in vivo use, and there is large variation in the helper lipids that are used in these formulas. The work in this thesis suggests that with further optimization, LNPs may be used to synthesize exogenous proteins in platelets. This may enable the creation of genetically-modified platelets with enhanced hemostatic abilities, longer lifespans, or new therapeutic abilities, leading to transfused platelets with increased efficacy and safety. 5.2 Future Directions 5.2.1 Optimizing LNP and mRNA delivery To achieve protein expression in platelets, it may be necessary to optimize both the mRNA and the LNP transfection agent in the future. Large lipid libraries have been used to identify potent ionizable lipids and lipid-like materials for transfection of siRNA and mRNA in the past. This includes libraries of ionizable lipids, which could potentially be used to improve the delivery of icLNPs to platelets [308, 315], as well as libraries of lipidoids [378, 386], lipid-like materials which may be used to produce another class of LNPs to be tested with platelets. In silico screening may also be used to identify the helper lipids and lipid ratios that lead to the highest transfection efficiency [337]. While we performed preliminary experiments using DOPE, high-throughput methods are needed to compare the full range of lipid to mRNA weight ratios and lipid molar concentrations possible in LNPs composed of four different lipids, particularly when the saturation and head group of the helper lipids can also alter transfection efficiency 117  [339]. Both icLNPs and cLNPs delivered mRNA to platelets, although their stability and release in microparticles differed. This suggests that their intracellular trafficking occurs through different pathways and only one class of LNP may allow for delivery of intact, functional mRNA to the cytosol. As there are numerous cationic lipids and ionizable cationic lipids, it would useful to first determine the intracellular location of the mRNA and whether it is intact, as discussed in chapter 4, before performing high-throughput screening with lipid libraries.  If mRNA delivered by platelets is intact and in the cytosol there are several ways in which mRNA could be modified to enhance translation. In these experiments, we used capped, polyadenylated mRNA that contained modified nucleotides that reduce the immune response and enhance translation and stability in other mammalian cells [306, 335, 336]. While we used mRNA containing 5-methoxyuridine, modified mRNA containing 5-methylcytidine and pseudouridine or 1-methylpseudouridine has also shown increased efficacy in vivo [335, 336]. It is unknown whether these modified nucleotides would enhance translation in platelets, and which combination would be most suitable. Transcripts containing different modifications or quantities of modified nucleotide may lead to detectable protein translation in platelets. Protein expression can also be optimized by modifying the mRNA sequence, particularly the 5’ or 3’ UTRs [336]. Platelets synthesize several proteins upon activation. While it is not known how these transcripts are selectively translated upon activation, cloning the 5’UTR or 3’UTR of mRNA that is translated upon activation onto the mRNA with a coding sequence for a reporter enzyme, such as GFP, may allow for activation-dependent synthesis of exogenous proteins. Platelets may also be able to utilize internal ribosome entry sites (IRESs), allowing for cap-independent translation [201]. These have already been cloned onto mRNA for reporter genes and could be tested in platelets. Platelets can also translate the Dengue virus genome, which uses 118  an unique cap-dependent, IRES-independent mechanism relying on secondary structure in its 5’ and 3’ UTRs [208, 387]. This presents another strategy for altering the platelet mRNA. In each approach, immunofluorescence confocal microscopy or polyribosome profiling might be used to assess co-localization or binding, respectively, to platelet ribosomes, while reporter mRNA can be used to assess translation. 5.2.2 Assessing the in vivo safety and efficacy of mRNA-LNPs Once a suitable transfection agent was identified, in vivo safety and efficacy testing would be required following transfusion of platelets prepared with mRNA-LNPs. Vitals, blood chemistry, complement activation, and histological analysis have been used to assess the safety of synthetic platelets and other hemostatic agents in murine and porcine models in the past [243, 244, 388]. Blood loss following tail transection in mice or liver puncture in pigs as well as blood clotting properties measured by rotational thromboelastometry can be used to determine whether mRNA-LNPs impair the hemostatic properties of platelets [243, 244, 388-390]. While there is potential for creating a transfection agent that could target platelets in vivo, perhaps by modifying LNPs with fibrinogen-mimicking peptides [246, 273, 276], a more feasible approach would be to develop an agent that can transfect platelets during storage as platelet concentrates. This should maximize delivery to platelets and minimize off-target effects by reducing the concentration of LNPs available for uptake by other cells once transfused into the patient. In the agents tested here, we found that uptake was significantly reduced in the presence of plasma. Platelet concentrates are 20% to 50% plasma, supplemented with PAS, and next-generation PAS may allow for use of even less plasma [145, 146]. Modifying LNPs to contain fibrinogen-mimicking peptides may potentially improve uptake in plasma. Alternatively, uptake could be performed in buffer, as performed here, and platelets reconstituted in plasma. Storage conditions 119  can affect platelet recovery and function following transfusion, as demonstrated by the differences observed between cold-stored and room temperature-stored platelets [147], and therefore the in vivo recovery of mRNA-LNP modified platelets should be tested. While platelet recovery typically uses radioisotopes or chromophores [143], involving time-consuming and expensive studies, the delivery of Cy5-containing mRNA may provide an simpler method for monitoring in vivo recovery. 5.2.3 Potential uses of modified platelets in the clinic Modified platelets may also have potential uses in blood banks to improve platelet storage. The main limit for storing platelets is the need to reduce bacterial contamination, while preventing platelet storage lesion is a concern with longer storage periods [134]. Targeting the apoptotic pathway, through overexpression of anti-apoptotic proteins or caspase inhibitors, may be a strategy for improving platelet function and reducing the demand for platelet concentrates. Platelet apoptosis during storage can contribute platelet storage lesion, which may reduce platelet survival and recovery in vivo [137]. Inhibition of the anti-apoptotic protein Bcl-xL leads to reduced platelet survival [30]. Overexpression of Bcl-xl may be a useful tool for extending platelet survival, reducing the number of platelet transfusions required when used prophylactically to treat thrombocytopenia. Apoptosis has also been linked to immune thrombocytopenia [391], suggesting this strategy would be particularly useful when platelets are transfused for this indication. Targeting the proinflammatory activities of platelets may be an alternative strategy for reduced adverse transfusion reactions.  During TRALI, platelets induce NET formation, which is associated with lung injury [131]. DNase I treatment protected mice against TRALI. Platelets engineered to produce DNase I might be an alternative approach to reducing mortality from TRALI following transfusion.  120   Modified platelets have the potential to have improved hemostatic potential and extend the therapeutic functions of platelets. Platelets loaded with thrombin have improved coagulability ex vivo, suggesting they may be useful for controlling active bleeding during trauma, which is often associated with platelet dysfunction [366]. De novo synthesis of the A-subunit of FXIII would be another approach for improving the hemostatic function of platelets, as this would potentially lead to improved clot stability [392]. While FXIIIA is protected from degradation in plasma by the B-subunit of FXIII, which is in excess in plasma. FXIIIA released by platelets may be bound by the B-subunit, protecting it from degradation until FXIIIA is activated at the site of clot formation [392]. Beyond their role in hemostasis, platelets mediate cancer progression and inflammatory reactions. Delivery of pro-apoptotic or anti-angiogenic proteins to tumors may be a method for extending the therapeutic potential of platelets. This would be useful as many cancer patients already receive platelet transfusions due to disease or therapeutic-induced thrombocytopenia [393]. Platelet rich plasma is used to treat ulcers, burns, muscle repair, bone diseases, and tissue recovery following surgery [105]. PDGF and TGF-β1 are key components of platelet rich plasma and inducing platelets to express increased amounts of these growth factors prior to applying the platelet rich plasma may help improve its efficacy [394]. Exosomes have already been used for siRNA delivery in vitro and in vivo. Electroporation has been used to load siRNA into exosomes targeted to the brain, leading to protein knockdown of a therapeutic target in mouse models of Alzheimer’s disease [395]. Cholesterol-conjugated siRNA has been used to load exosomes ex vivo, and treatment of HEK293 cells with these exosomes led to silencing of a therapeutic target in cancer [396]. Effective loading of exogenous siRNA into exosomes as well as targeting of exosomes to specific cells are limiting factors in translating this technology to the clinic [303]. Ex vivo 121  derived platelet microparticles might overcome these limitation, and with improvements in ex vivo culturing of platelets it may be possible to generate sufficient platelet microparticles for therapeutic uses. For example, a platelet microparticles which contain P-selectin could be used for targeting leukocytes at the sites of vessel injury, inflammation, or tumorigenesis [11]. During arthritis, platelet microparticles are shed in the synovial fluids through GPVI and FcR-mediated signalling, causing release of inflammatory cytokines at the joint [97]. The use of specific platelet agonists may be a means by which targeted microparticles are produced ex vivo. This could enable delivery of anti-inflammatory proteins to treat arthritis, extending the therapeutic range of platelets. Platelet microparticles can also infiltrate solid tumors in mice and humans [302]. Delivery of mRNA for pro-apoptotic or antiangiogenic factors to tumors would be an alternative method for targeting cancer using modified platelets, even if de novo protein translation in platelets is not achieved.  5.2.4 Potential uses of modified platelets for studying platelet function  If modified platelets cannot be translated to the clinic, either due to poor safety or efficacy, they may still have potential uses as experimental tools for studying platelet function. Genetic manipulation is an important tool for studying protein function but is challenging in platelets. While transfection of platelets with siRNA has been used to reduce protein expression in some cases, overexpression of proteins or complete gene knockout requires modifying the platelet precursors. However, this method is limited to proteins that do not prevent platelet production or megakaryocyte survival. A method to directly overexpress proteins in platelets could overcome this, and allow new questions about platelet biology to be asked and answered. For example, overexpressing a ribosome recycling factor in megakaryocytes reduced the amount of ribosomes accumulated on the 3’UTR of platelet transcripts, which should lead to enhanced 122  protein translation. It would be worthwhile to see what the effect this has on platelet survival and hemostatic function, as enhancing either survival or coagulability would have a large therapeutic potential, as discussed above. LNP-modified platelets could also be used to study mRNA regulation in platelets, using modified mRNA transcripts for translation. As previously discussed, the exact mechanisms regulating protein expression in platelets are not fully understood. While platelet mRNA contains binding sites for proteins that regulate mRNA stability and translation, such as HuR and TIA-I, the physiological role of these regulatory pathways are not clear. Transcripts containing modified 5’ or 3’UTRs would be useful in testing if these proteins are sufficient to alter transcript stability during platelet storage and response to platelet agonists, and whether the length of the 3’UTR is important. Transcripts containing different types of IRESes, which require different combinations of eIF’s to be initial translation, may be used to probe the relative importance of canonical eIFs in regulating protein translation in platelets [201]. Even if de novo protein synthesis cannot be achieved, LNP-modified platelets have the potential to be used for studying RNA release by platelets. The factors regulating packaging of miRNA and mRNA into platelet microparticles are unknown. Several sorting pathways have been identified in other mammalian cells, either involving binding with specific proteins, including AGO2 from the RISC complex, or specific modifications to the RNAs. In human primary glioblastoma cells, a 25-nucleotide sequence which served as a binding site for miR-1289 was identified as a ‘zipcode’-like sequence in the 3’UTR of mRNAs, leading to enrichment in microparticles [397]. In peripheral blood mononuclear cells, binding of heterogenous nuclear ribonucleoproteins to specific sequences led to enrichment of specific miRNAs in microparticles [398], while adenylation or uridylation of the 3’ ends of miRNAs led to preferential sorting of 123  miRNAs between B cells or urine [399]. miRNAs or mRNAs containing these modifications could be loaded into cLNPs, and their release in platelet microparticles compared to determine whether these sorting pathways are functional in platelets. The ability to genetically modify a cell is a powerful tool that has furthered our understanding of many physiological and pathological processes. Our understanding of platelet biology has already been improved by creating modified platelets using precursor cells, but the ability to directly modify platelets would provide new insights into the molecular pathways that regulate platelets. While several examples are provided above, numerous other possibilities exist, depending on the pathway of interest. Furthermore, a better understanding of mechanisms regulating intracellular trafficking and protein synthesis in platelets may help increase the efficiency of a platelet transfection agent once an initial formula is identified. 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