ENGINEERING DELIVERY VEH I CLES FOR SIRNA THERAPEUTICS By D aniel B urton V ocelle A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemical Engineering - D octor of P hilosophy Quantitative Biology Dual Major 20 20 ABSTRACT ENGINEERING DELIVERY VEHICLES FOR SIRNA THERAPEUTICS By Daniel Burton Vocelle Small molecule and protein - based drugs, while critically important therap ies, cannot treat all diseases . As such, alternative treatment modalities must be developed to complement existing strategies. One potential alternative is small interfering RNA (siRNA) therapeutics, which are capable of specific inhibition o f a wide range of intracellular, membrane, and extracellular proteins . siRNAs are hydrophilic due to their anionic backbone and do not readily diffuse across cellular membranes. During systemic delivery, naked siRNA s are rapidly filtered by the kidneys o r degraded by serum nu cleases and can often initiate an immune response . Thus, for siRNAs to be useful as therapeutics, they must be complexed with delivery vehicles for protection during extracellular transport and cellular internalization. Once delivere d to the cytoplasm , siRNAs act through RNA interference (RNAi) to degrade messenger RNAs (mRNAs) in a sequence - specific manner, thereby reducing target protein expression . Despite the r ecent clinical success, development of siRNA therapeutics is limited du e to the inefficiency, toxicity, and immunogenicity of current delivery vehicles. To overcome these hurdles, this research aimed to understand the role of delivery vehicle characteristics in influencing the cellular uptake and processing of siRNA - containin g complexes. While many types of delivery vehicles have been developed for siRNAs, the characteristics that are essential for success are still not well understood. To address this issue, we synthesized a variety of silica nanoparticles (sNPs), and assess ed their ability to effectively deliver siRNAs to human lung carcinoma cells (H1299). By varying the concentration of amines and dextran during sNP synthesis, we defined chemical/physical characteristics important for active siRNA delivery. Another roadbl ock in the development of siRNA therapeutics is a limited understanding of the intracellular processing of siRNA - containing complexes leading to initiation of RNAi. With recent evidence showing that the intracellular fate of endocytosed material was influe nced by the endocytic pathway used for internalization , we developed a novel assay capable of differentiating uptake among the different endocytic pathways and assessing their functionality in initiating RNAi. Our results showed that Lipofectamine 2000 ( LF 2K ) wa s internalized by Graf1 - , Arf6 - , or flotillin - mediated endocytosis for the initiation of RNAi, depending on cell type. Additionally, our study identified functional differences among endocytic pathways in a cell, indicating that uptake alone was not sufficient to initiate silencing. In a mixed cell population, we found that targeted inhibition of the non - functional pathways in some cells enhanced silencing in the uninhibited cells. These findings suggest that designing delivery vehicles for specific e ndocytic pathways may enhance the activity of the delivered siRNAs by directing them preferentially to the intended target cells. Finally, due to the limitations of current techniques, the intracellular pathways used in processing siRNA - containing complexes are not well defined. As a result, it is unclear how delivery vehicle characteristics affect the intracellular trafficking of siRNAs. To address this issue, we developed a novel microscopy - based assay that uses automated multi - well li ve - cell imaging to track the intracellular location of siRNAs over time. Through this assay we determined the intracellular pathways utilized in sNP - mediated siRNA delivery and identified how dextran functionalization of sNPs altered the intracellular traf ficking of siRNAs. This assay provides a new analytical technique to assess intracellular pathways and could aid in the development of more efficient siRNA delivery vehicles. iv TABLE OF CONTENTS LIST OF TABLES vi LIST OF FIGURES vii KEY TO ABBREVIATIONS ix CHAPTER 1: INTRODUCTION 1 1.1 Significance 1 1.2 Background 1 1.3 Delivery Vehicle Design 2 1.4 Endocytosis 5 1.5 Intracellular Trafficking 9 1.6 Clinical Challenges and Successes 10 1.7 Approach and Specific Aims 13 CHAPTER 2: SILICA NANOPARTICLE CHARACTERISTICS ASSOCIATED WITH ACTIVE SIRNA DELIVERY 15 2.1 Abstract 15 2.2 Introduction 15 2.3 Results 17 2.3.1 Effect of Amine and Dextran Content on siRNA Silencing Efficiency 17 2.3.2 Inhibition of siRNA Endocytosis and Silencing 20 2.3.3 Intracellular Trafficking of sNPs 23 2.3.4 Acidic Degradation of sNPs 24 2.3.5 siRNA Binding under Acidic Conditions 25 2.4 Discussion 26 CHAPTER 3: ENDOCYTOSIS CONTROLS SIRNA EFFICIENCY: IMPLICATIONS FOR SIRNA THERAPEUTIC DESIGN AND CELL SPECIFIC TARGETING 31 3.1 Abstract 31 3.2 Introduction 31 3.3 Results 33 3.3.1 Silencing Efficiency in Different Cell Lines 33 3.3.2 Inhibition of siRNA Accumulation and Silencing 34 3.3.3 Overexpression of Endocytic Proteins 38 3.3.4 Targeted Inhibition in a Co - cultured Population 40 3.4 Discussion 43 CHAPTER 4: KINETIC ANALYSIS OF THE INTRACELLULAR PROCESSING OF siRNAs BY CONFOCAL MICROSCOPY 49 4.1 Abstract 49 4.2 Introd uction 49 4.3 Discussion 56 CHAPTER 5: CONCLUSIONS AND FUTURE WORK 58 v 5.1 Conclusions 58 5.2 Future Work 59 5.2.1 Silica Nanoparticle Optimization 59 5.2.2 Predicting Optimal Endocytic Pathways 60 5.2.3 Additional Intracellular Pathways 60 APPENDICES 62 APPENDIX A: Materials and Methods for Chapter 2 63 APPENDIX B: Materials and Methods for Chapter 3 77 APPENDIX C: Materials and Methods for Chapter 4 91 BIBLIOGRAPHY 94 vi LIST OF TABLES Table 1 - 1 siRNA therapeutics in clinical trial 12 Table 2 - 1 Target and mechanism of action for endocytosis inhibitors [53,195]. 22 Table 3 - 1 Chemical inhibitors of endocytic proteins 35 Table 3 - 2 Chemical inhibitor vs endocytic pathway matrix 35 Table A - 1 Statistical analysis for Figu res 2 - 2 and 2 - 3 75 Table A - 2 Statistical a nalysis for Figure A - 6 76 Table A - 3 Stati stical analysis for Figure 2 - 7 76 Table A - 4 Inhibitor toxicity 83 Table A - 5 Statist ical analysis for Figures 3 - 2, 3 - 3, and 3 - 4 90 Table A - 6 Gene expression 90 vii LIST OF FIGURES Figure 1 - 1 RNAi pathway 2 Figure 1 - 2 Endocytic pathways 6 Figure 2 - 1 Effect of dextran and amine content on silencing 19 Figure 2 - 2 Role of sNP zeta potential (mV) on silencing 20 Figure 2 - 3 Influence of endocytotic inh ibitors on the uptake of siRNAs 22 Figure 2 - 4 EGFP silencing in the presence of endocytotic inhibitors 23 Figure 2 - 5 TEM analysis of sNP - siRNA complex endocytosis and trafficking 24 Figure 2 - 6 sNP degradation under acidic conditions 25 Figure 2 - 7 Nucleic acid release under acidic conditions 26 Figure 3 - 1 EGFP silencing and siRNA accumulation 34 Figure 3 - 2 Influence of endocytic inhibitors on EGFP silencing and si RNA accumulation 36 Figure 3 - 3 Influence of endocytic protein overexpression on the intracellular accumulation of siRNAs 39 Figure 3 - 4 Influence of endocytic inhibitors on EGFP silencing and siRNA accumulation in co - cultured and mono - cultured populations 42 Figure 4 - 1 Intracellular trafficking flowchart 51 Figure 4 - 2 Intracellular trafficking pathways in eukaryotic cells 53 Figure 4 - 3 Kinetic colocalization profi les of siRNA with Rab4, Rab5, and Rab7 54 Figure 4 - 4 Kinetic colocalization profiles of siRNA with Rab11, Lysosome, and ER 55 Figure 4 - 5 Kinetic colocalization heat maps 56 Figure A - 1 Plasmid transfection efficiency of sNPs 70 Figure A - 2 Confocal microscopy of non - inhibited silencing 24 h post - transfection 71 Figure A - 3 Confocal micro scopy of inhibited silencing - 4 h post - transfection 72 viii Figure A - 4 Confocal microscopy of inhibited silencing 24 h post - transfection 73 Figure A - 5 EGFP silencing in the presence of endocytotic inhibitors (HeLa) 74 Figure A - 6 Inhibitor dose response 84 Figure A - 7 Inhibitor microscopy experiments 87 Figure A - 8 Flow cytometry fluorescence distribution 88 Figure A - 9 Flow cytometry fluorescence distribution 88 Figure A - 10 Overexpression microscopy experiment 89 ix KEY TO ABBREVIATIONS AAV adeno - associated virus ADE Arf6 - dependent endocytosis Ago2 argonaute 2 AP2 AP2 adapter complex APTES 3 - (triethoxysilyl) - propyl amine AS antisense strand ASGPR asialoglycoprotein receptors CCIE clathrin/caveolin - independent endocytosis CDC42 cell division cycle 42 CME clathrin - mediated endocytosis CNS c entral nerve system CNT carbon nanotubes CvME caveolin - mediated endocytosis DNM1 dynamin - 1 DNM2 dynamin - 2 EDS energy - dispersive X - ray spectroscopy EE early endosome EGF epidermal growth factor EGFP enhanced green fluorescent protein ER endoplasmic reticulum FACS fluorescence - activated cell sorting x FME flotillin - mediated endocytosis FWHM full width at half max GEEC GPI - e nriched e ndocytic c ompartments GFP green fluorescent protein GME Graf1 - mediated endocytosis H1299 human lung carcinoma HEK293 human embryonic kidney HepG2 hepatocellular carcinoma Hsc70 heat shock cognate 71 kDa protein HSPG heparan sulfate proteoglycan HSV herpes simplex virus LAMP1 lysosomal associated membrane protein 1 LDL low density lipoprotein LE late endosome LF2K Lipofectamine 2000 MHCI major histocompatibility complex class I proteins miRNA microRNA MP macropinocytosis mRNA messenger RNA MWCNT multi - wall ed carbon nanotube methyl - - cyclodextrin NSCLC non - small cell lung cancers PAK1 P21 Activated Kinase 1 xi PFS perfect focus system PI3K phosphatidylinositol - 3 - kinase PIP3K phosphoinositide 3 - kinase PM plasma membrane RAC1 Rac family small GTPase 1 RE recycling endosome RISC RNA induced silencing complex RLC RISC loading complex RNAi RNA interference shRNA short hairpin RNA siRNA small interfering RNA sNP silica nanoparticle SS sense strand SWCNT single wall ed carbon nanotube TEM transmission electron microscopy TRBP TAR RNA binding protein 1 CHAPTER 1: INTRODUCTION Note: This chapter has been m odified from previously published work [1] 1.1 Significance Small molecule and protein - based drugs, whi le critically important therapies, have limited therapeutic potential [2] . In some cases, the drugs cannot access or interact with proteins that are causing the disease phenotype. As such, alternative treatment modalities must be developed t o complement existing strategies. One potential alternative is siRNA therapeutics, that are capable of targeted inhibition for a wide range of intracellular, membrane, and extracellular proteins [3] . siRNA therapeutics are being developed as treatments for a variety of targets , including cancers and infectious diseases, with one therapeutic recently approved for clinical use [4 6] . Despite th e r ecent clinical success, the continued developme nt of siRNA therapeutics is limited by poor delivery efficiency that stems from an incomplete understanding of the intracellular pathways associated with RNAi [7 9] . 1.2 Background RNAi is a native pathway in eukaryotic cells that regulates cellula r functions through miRNA s and can be induced ex ogenously by siRNAs (Figure 1 - 1) [10] . Once delivered to the cytoplasm, siRNAs are identified by the RISC loading complex (RLC), a ribonucleoprotein complex minimally composed of Argonaute 2 (Ago2), Dicer, and TAR RNA binding prot ein (TRBP) [11 14] . The RLC preferentially selects one of the siRNA strands as the guide stra nd (anti sense stra nd) loading it into Ago2, forming the active RNA induced silencing complex (RISC) [11,12,14,15] . The other strand , the passenger str and (sense strand), is subsequently removed from the pathway and degraded [12] . Active RISC cleaves target mRNA at the center 2 region compl e mentary to the guide strand , causing the mRNA to be deg raded and halting production of the protein it encodes [12,16,17] . 1.3 Delivery Vehicle Design Previously, siRNA therapeutics were li mited by the ability to design and predict active siRNA sequences but are currently hindered by poor delivery [ 18,19] . siRNAs are hydrophilic due to their anionic backbone, and do not readily diffuse across cellular membranes [3] . During systemic delivery, naked s iRNAs are rapidly filtered by the kidneys, degraded by serum nucleases, and stimulate an immune response [20,21] . Thus, siRNAs require delivery vehicles for protection/concealment during transport until deliver ed to the cytoplas m of a target cell. Figure 1 - 1 RNAi pathway RNA Interference (RNAi) is a native pathway in eukaryotic cells that regulates cellular functions and can be induced exogenously by siRNA s . With the assistance of target - specific delivery vehicles, siRNAs are transported from the extracellula r environment into the cytoplasm of eukaryotic cells. Utilizing the RNAi pathway, siRNA s degrade messenger RNA (mRNA) in a sequence - specific manner, thereby reduc ing target protein expression. 3 siRNA delivery vehicles are classified as being either viral or non - viral. Viral approaches loaded genetic material into inactive viral envelopes, capitalizing upon a highly efficient natural mechanism. However, these viral approaches were generally limited to a single treatment due to an adaptive immune response [22] . Viral vectors are now primarily used in the treatment of chronic disorders, where a short hairpin RNA (shRNA) is incorporated into the genome of a target cell resulting in the constitutive express ion of siRNAs [23] . N on - viral vehicles were initially inefficient at delivering active siRNA s , but were considered non - immunogenic . In the last decade, considerable strides have been made at improving non - viral delivery efficiencies making them the preferred choice for development of siRNA therapeutic s [24] . There are many sub - types of non - viral delivery vehicles used by researchers, each categorized according to their composition. Lipid - based delivery vehicles are commonly referred to as lipoplexes and in general, have achieved the greatest clinical and commercial success among types of siRNA delivery vehicles [24,25] . Lipoplexes are principally comprised of cationic lipids roughly 100 nm in diameter , and during self - assembly form spherical lipid bilayers that encapsulate siRNAs [26] . While effective at delivering siRNAs, lipoplexes are generally toxic at concentrations required for therapeutic effect [27] . Lipoplexes have been shown to use multiple endocytic pathways depending on cell type and lipid compos ition [28,29] . I n vivo , lipoplexes are primarily used to target diseases in the liver due to the high concentration of lipoprotein receptors [30] . Polyplexes are comprised of biocompatible cationic polymers derived from both natural and synthetic sources [31,32] . Depending on the polymer formulation, polyplexes can range from 10 - 400 nm in size [33] . Additionally, chemical modification to the polymer backbone can produce spherical, rod, or globular particles [33] . Given their geometric variability, polyplexes can be 4 used to either encapsulate siRNAs in a micelle - like structure, or bind them electrostatically to a cationic surface [34] . The mechanism of e ndocytosis used by polyplexes varies given the diversity in their physical/chemical characteristics [35 37] . Polyplexes have been used to target the widest variety of diseases, most commonly targeting the liver, kidney, and lungs. Polyplexes are considered non - toxic and bio - degradable, but have poor delivery efficiency due to aggregation and inefficient siRNA r elease in the cytoplasm [38,39] . Carbon nanotubes (CNTs) are hollow gra phene based nano - cylinders containing a single wall (SWCNTs) or multiple walls (MWCNTs) [40] . Their length varies between 50 and 100 nm with a diameter of 0.4 - 2 nm for SWCNTs and 2 - 100 nm for MWCNTs [8,41] . Using surface functionalized cations; CNTs use electrostatics to bind siRNAs to their surface [42] . Instead of being endocytosed, CNTs pass through the cell membrane through a spontaneous and non - destructive mechanism [41 ] . While this process makes CNTs ideal for treating delivery resistant cells and bypassing biological barriers, it poses significant challenges for systemic delivery. In vivo , CNTs are rapidly filtered by the kidney due to their small diameter, sequestered by phagocytic cells due to their rod shape, or indiscriminately deliver siRNAs [43] . While short - term data suggests CNTs are safe, many researchers have expressed concerns over their accumulation and long - term toxicity [44] . Ceramic nanoparticles are porous inorganic spheres roughly , 50 nm in diameter, commonly composed of silica, titania, or aumina due to their biological inert ness [45] . Additionally, silver oxides have been used for their anti - bacterial properties, as CeO 2 and Y 2 O 3 have for their antioxidant properties . Generally, ceramic nanoparticles use amine functionalized surfaces to bind siRNAs through electrostatics [46] . No specific endocytic pathway has been id entified for ceramic nanoparticles or their various compositions [47] . While ceramic nanoparticles have been 5 used to target filtering organs in vivo , they are considered inefficient at delivery and toxic due to long - term accumulation [48,49] . Metallic nanoparticles are highly modifiable solid core particles most commonly synthesized using colloidal gold [50] . Typically 1 - 150 nm in size, metallic nanoparticles can form rods and spheres that electrostatically bind siRNAs to their surface via functionalized amines [50] . Metallic nanoparticles have been reported to use multiple endocytic pathways to successfully deliver siRNA s . In vivo , metallic nanoparticles have been used to target filtering organs with limited success due to rapid opsonization and toxic accumulation in the liver and spleen [51,52] . 1.4 Endocytosis To reach the cytoplasm, vehicle - siRNA complexes must first be endocytos ed . Until recently, cellular endocytic path ways were classified as macropinocytosis (MP), clathrin - mediated endocytosis (CME), caveolin - mediated endocytosis (CvME), or clathrin/caveolin - independent endocytosis (CCIE) [53] . Researchers have since characterized three distinct types of CCIE, flotillin - mediated endocytosis (FME), Arf6 - dependent endocytosis (ADE), and Graf1 - mediated endocytosis (GME) (Figure 1 - 2) [54 56] . Currently, there is no consensus regarding the optimal endocytic pathway for active siRNA delivery, as multiple endocytic pathways have been foun d to result in successful delivery of siRNAs and initiation of silencing [57] . Further, it is difficult to correlate the characteristics of a delivery vehicle with a specific pathway as most studies are limited to a single cell type or c annot distinguish among the current endocytic pathways. 6 MP is traditionally associated with the bulk uptake of extracellular fluid and nutrients. Unlike the other endocytic pathways, macropinocytosis is characterized by membrane ruffles at the cell surface that envelop extracellular fluid [58] . This process is driven by actin polymer ization, and regulated at the plasma membrane by phosphatidylinositol - 3 - kinase (PI3K) and the GTPases r ac f amily s mall GTPase 1 ( Rac1 ) , c ell d ivision c ycle 42 ( Cdc42 ) , and p 21 a ctivated k inase 1 ( PAK1 ) [5 9] . Additionally, while other endocytic pathways are initiated by ligand binding with surface receptors, macropinocytosis is transiently induced by growth factors such as epidermal growth factor (EGF) [60] . Macropinocytosis leads to the intracellular formation of macrop inosomes, that range from 150 - 5000 nm in diameter, and undergo acidification during vesicular transport to the lysosome [59] . The use of macropinocytosis by delivery vehicles is Figure 1 - 2 Endocytic p athways Different endocytic pathways used by mammalian cells for the internalization of cargo. At present the following pathways have been identified: macropinocytosis (actin, Cdc42, and Rac1 dependent), clathrin - mediated (clathrin, actin, AP2, Arf6, and dynamin dependent), caveolin - mediated (caveolin, actin, Src, dynamin, and lipid - raft dependent), Arf6 - dependent (Arf6, actin, PIP3K, and lipid - raft dependent), flotillin - mediated (flotillin, dynamin, and lipid - raft dependent), and Graf1 - mediated (Graf1, dynamin, Cdc42, actin, and lipid - raft d ependent). 7 primarily associated with the non - specific uptake of macro particles and nanoparticle aggregates [61] . CME is the pathway most often associated with receptor mediated endocytosis, and is [62] . CME is characterized by the formation of a clathrin coated pit at the plasm a membrane that internalizes receptor bound cargo. This process is dependent on both dynamin and actin, but regulated by the AP2 adapter complex (AP2) that recruits clathrin to the plasma membrane and catalyzes formation of the clathrin triskelion lattice. In the cell, heat shock cognate 71 kDa protein ( Hsc70 ) mediates disassembly of the clathrin coat, allowing the endocytic vesicle to fuse with the early endosome [63] . E ndocytic vesicles formed during CME are typically 100 150 nm in diameter, although they have been shown to encapsulate larger cargo. It has been reported that CME is used by cells to internalize a variety of drug delivery complexes (polyplexes, lipoplexes, metallic, etc) with varying degrees of success [35,64] . Using endocytic targeting motifs to target specific receptors a ssociated with CME, such as the transferrin and low density lipoprotein (LDL) receptor, researchers have enhance d both target specificity and delivery efficiency [65,66] . All other forms of endocytosis are differentiated from MP and CME by their dependence on lipid - rafts, hydrophobic subdomains of the plasma membrane rich in cholesterol and glycosphingolipids [67] . The first of these is CvME , which upon activation of a surface receptor, forms caveolar - coated pits through the recruitment of caveolin proteins to lipid rafts. Src - dependent phosphorylation of the caveolins initiates coat disassembly and dynamin/actin - dependent vesiculation [68] . The resulting vesicles, roughly 50 - 60 nm in diameter, are trafficked to the early endosome thr ough a process that inhibits vesicle acidification [69] . Similar to CME, 8 multiple delivery platforms have reported using CvME, with varying degrees of success [35,61] . It is unclear what receptors and cargo utilize CvME, as those previously assigned to other pathways ( e.g., albumin and cholera toxin B) , are now known to use other lipid - raft dependent pathways [70,71] . ADE is a relatively recently - identified type of endocytosis regulated by the GTP cycle of Arf6 [55] . Internalization though ADE leads to the format ion of A rf6 - containing endosomes that are either recycled to the plasma membrane or trafficked to early endosomes , a process dependent upon the hydrolysis of Arf6 - GTP, actin polymerization, and activation of Phosphoinositide 3 - kinase ( PIP3K ) . Vesicles that result from ADE have been shown to form intermediate endosomal compartments, that are capable of sorting cargo before reaching the early endosome [55] . It also has been shown that compare d to CME, vesicles from ADE take roughly 6 times longer to reach the early endosome [55] . ADE has currently been implicated in the internalization of IL - 2 receptor subunit Tac [72] , major histocompatibility complex class I proteins (MHCI) [73] - integrin [74] , and the herpes simplex virus (HSV) [75] . As Arf6 has been shown to regulate AP2, it is likely that some uptake by ADE has been mistaken for CME , which is also depend ent on AP2 . GME is commonly characterized by tubulovesicular invaginations rich in Graf1 and an intracellular association with GPI - Enriched Endocytic Compartments (GEECs). In GME, Graf1 and dynamin form a stable complex that regulates the scission and stability of th e tubulovesicular structures, through a process also dependent on actin and Cdc42. Interestingly, Graf1 has a higher affinity for dynamin - 1 (DNM1), thought to be exclusive to neurons, than dynamin - 2 (DNM2), which has ubiquitous expression [56,76] . Similar to ADE, GME forms unique endosomal compartments capable of sorting cargo before reaching the early endosome s . Since its 9 discovery, GME has been implicated in the uptake of GPI - linked proteins [56] , a deno - associated virus (AAV) [77] , dextran [78] , and extracellular fluid [56] . It is likely that uptake by GME has been mistaken for macropinocytosis, as they both facilitate uptake of dextran and are dependent upon Cdc42. FME was first characteri zed as the endocytic pathway associated with CD59 [54] and cholera toxin B [54] . It h as since been implicated in the uptake of lipids [79] , silica nanoparticles [80] , and cationic polyplexes [79] . In FME, flotillin - 1 and flotillin - 2 co - assemble into plasma membrane microdomains in lipid - rafts and are internalized after phosphorylation by FYN [81] . Interestingly, the role of dynamin in this process is, as of yet, undefined and possibly dependent on cell type or cargo [82] . Upon internalization, flotillin endosomal vesicles are trafficked directly to the G olgi, bypassing the early endosome. At present, multiple endocytic pathways have been found to result in successful delivery of siRNAs and initiation of silencing. As a result, researchers have focused on the intracellular hurdles of siRNA delivery instead of exploring alternate endocytic pathways. However, due to the discovery and characterization of the CCI E pathways, it has become evident that the pathway of endocytosis greatly impacts the subsequent trafficking of cargo [66,83] . This suggests that the role of endocytosis in siR NA delivery is worth reinvestigating, and may provide an alternative means to enhancing the effectiveness of siRNA s . 1.5 Intracellular Trafficking For siRNA to be incorporated into the RNAi pathway they must reach the cytoplasm of a target cell, however the o ptimal intracellular pathway for achieving this goal is unclear [84] . S tudies have show n that only a small portion of internalized siRNAs reach the cytoplasm, whereas the majority are exocytosed through endosomal recycling or ret ained within endolysosomal vesicles [85] . While the exact mechanism for cytoplasmic delivery is unknown, 10 the prevailing t heories suggest membrane fusion or the proton sponge effect [86,87] . In membrane fusion, amphipathic functional groups on the surface of a delivery vehicle interact with the cholesterol rich regions of the endosomal membrane; destabilizing the integrity of the [88] . According to the proton sponge effect, once particles are internalized via endocytosis, the endosome begins to acidify. However, because nucleic acid delivery vehicles are often polycationic to all ow for self - assembly with the polyanionic nucleic acids, they possess a basic pKa and significant buffering capacity [24] . As such, the cell continues to acidify the vesicle, resulting in an accumulation of Cl - . The excess of Cl - causes osmotic swelling to the point that the endos ome eventually bursts, releasing its contents into the cytoplasm [89] . 1.6 Clinical Challenges and Successes siRNA therapeutics in clinical trials have predominately used variations of lipoplexes and polyplexes - conjugated siRNAs now represent ~50% of RNAi drugs in clinical trials (Table 1 - 1) . In lieu of a delivery vehicle, siRNA conjugates use a chemically modified siRNA backbone to enhance their overall potency and attenuate activation of the immune system. By conjugating these siRNA s to a receptor targeting ligand, they achieve cell specific targeting. So far the success of siRNA conjugates has been limited to GalNAc, a sugar derivative of galactose, that targets the asialoglycoprotein receptors (ASGPRs) found in the liver [6] . While a promising delivery method for siRNA therapeutics, the development of additional ligands that are non - toxic, metabolically stable, and potent has proved challenging [90] . In addition to a greater diversity among the delivery platforms represented in clinical trials, the number of disease targets has also expanded in recent years. Presently there are 11 promising siRNA therapies that target ocular [91] , renal [92] , central nerve system (CNS) [93] , and solid tumor [84] diseases. Despite these successes, there is considerable room for further advancement of siRNA therapeutics. I n vivo , delivery methods favor subcutane ous injection over IV infusions to limit systemic toxicity [94,95] . On a cellular level, siRNA therapeutics are limited by endolyso so mal retention and rapid recycling to the plasma membrane [85] . Additionally, studies have shown a passive escape rate o f <0.01% for internalized siRNAs [96] . R esearchers are actively exploring the use of endosomolytic motifs [97] and retrograde intracellular transport [98 ] to overcome these hurdles, however progress is limited due to an incomplete understanding of the biological pathways involved in cargo transport. 12 Table 1 - 1 siRNA t herapeutics in c linical t rial Delivery system Disease T arget NCT ID Cardio metabolic and endocrinological disease Conjugated siRNAs Primary Hyperoxaluria NCT03681184 Conjugated siRNAs Amyloidosis NCT03759379 Conjugated siRNAs Acute hepatic porphyrias NCT03338816 Conjugated siRNAs Hypercholesterolemia NCT03814187 Conjugated siRNAs Amyloidosis NCT02319005 Conjugated siRNAs Alpha 1 liver disease NCT03767829 Conjugated siRNAs Cardiovascular disease NCT03626662 Conjugated siRNAs Primary Hyperoxaluria NCT03392896 Conjugated siRNAs Hypertriglyceridemia NCT03747224 Conjugated siRNAs Hypertriglyceridemia NCT03783377 Conjugated siRNAs Alpha 1 antitrypsin deficiency NCT03362242 Lipoplex Amyloidosis NCT01960348 Lipoplex Hypercholesterolemia NCT00927459 Lipoplex Primary Hyperoxaluria NCT02795325 Polyplex Alpha 1 antitrypsin deficiency NCT02900183 Infectious disease Conjugated siRNAs Hepatitis B NCT03365947 Conjugated siRNAs Hepatitis B NCT03772249 Conjugated siRNAs Hepatitis B NCT02826018 Lipoplex Hepatitis B NCT02631096 Lipoplex Ebola NCT02041715 Polyplex Hepatitis B NCT02535416 Polyplex Hepatitis B NCT02797522 Cancer Exosome Pancreatic cancer NCT03608631 Gold nanoparticle Gliosarcoma NCT03020017 Lipoplex Hepatocellular carcinoma NCT02191878 Lipoplex Carcinoma, pancreatic ductal NCT01808638 Lipoplex Hepatocellular carcinoma NCT02314052 Lipoplex Solid tumors NCT00882180 Lipoplex Advanced cancers NCT01591356 Polyplex Pancreatic cancer NCT01676259 Polyplex Cancer, solid tumor NCT00689065 Viral Vector Chronic myeloid leukemia NCT00257647 Others Conjugated siRNAs Hemophilia NCT03549871 Conjugated siRNAs Hemolytic uremic syndrome NCT03303313 Conjugated siRNAs Hypertrophic scar NCT03133130 Conjugated siRNAs Hypertrophic cicatrix NCT03569267 Lipoplex Idiopathic pulmonary fibrosis NCT03538301 13 1.7 Approach and Specific Aims The work described here aimed to improve the design criteria for siRNA delivery vehicles with the ultimate goal of improving their therapeutic efficacy. This research explored the characteristics of delivery vehicles that optimized active siRNA delivery an d developed assays to assess the impacts of these characteristics on endocytosis and intracellular trafficking. This dissertation details three approaches taken towards understanding characteristics and intracellular pathways used by delivery vehicle s for active siRNA delivery. The specific aims of this dissertation were to: 1. Identify vehicle characteristics that promote active siRNA delivery While many types of delivery vehicles have been developed for siRNA delivery, there is relatively little information to guide the design of a delivery system de novo . Here, we used silica nanoparticles (sNPs) that varied by charge and functionalized surfac e, to assess the role of each characteristic among the following design criteria: siRNA binding, membrane translocation, and silencing. Our results suggest an optimal binding affinity facilitates active siRNA delivery. Additionally, we show ed that dextran functionalization enhance d the efficacy of sNPs for delivering siRNAs, by facilitating their uptake through a scavenger receptor - mediated endocytic pathway that is clathrin/caveol in - independent. Going forward, the generality of these findings can be furthe r evaluated in other delivery systems . 2. Determine the preferential endocytic pathway for active siRNA delivery The discovery of new clathrin/caveolin - independent endocytic pathways has resulted in the reclassification of the endocytic pathways associated with many species. As a result of these findings, it has also become evident that the intracellular trafficking of cargo is highly influence d by the endocytic pathway used during internalization. In this study, our work explores 14 endocytosis (whether by cla thrin, caveolin, Arf6, Graf1, flotillin, or macropinocytosis) across multiple cell types ( human cervical cancer (HeLa) , human lung carcinoma (H1299) (lung), human embryonic kidney ( HEK293 ) , and hepatocellular carcinoma ( HepG2 ) ). Our results showed that act ive siRNA delivery occurs via Graf1 ( GME) - , Arf6 (ADE) , or flotillin - mediated (FME) endocytosis depending on cell type. Additionally, we determined that a portion of siRNA - containing complexes are internalized by pathways that do not initiate silencing. In a mixed cell population, we found that inhibition of a cell specific endocytic pathway enhanced siRNA delivery in the remaining cell populations. In the field of siRNA therapeutics, these findings suggest that delivery vehicles should be designed to uti lize specific endocytic pathways when targeting a particular cell type. 3. Characterize the intracellular pathways associated with siRNA delivery While understanding the intercellular pathways associated with cargo trafficking is critical to the design of a siRNA delivery vehicle, it is unclear how the specific characteristics of a nanoparticle affect intracellular trafficking. In part, the techniques used to track the intracellular location of cargo limit additional study in the field due to high operating c osts and low throughput associated with data acquisition. Herein, we developed a new method that uses automated multi - well imaging of stable cell lines, to detect the intracellular pathways used by target molecules and is capable of measuring kinetic varia tions in target localization as a result of external stimuli. Using this assay, we characterize d how dextran functionalization of sNPs affected the intracellular trafficking of siRNA s . Our results indicate that dextran enhances s iRNA retention in the early endosome, reduces fast endosomal recycling, and enhances association with the ER. 15 CHAPTER 2: SILICA NANOPARTICLE CHARACTERISTICS ASSOCIATED WITH ACTIVE SIRNA DELIVERY. Note: This chapter is adapted from previously published work [1] 2.1 Abstract Understanding the endocytosis and intracellular trafficking of siRNA - delivery vehicle complexes remains a critical bott leneck in designing siRNA delivery vehicles for highly - active RNAi - based therapeutics. In this study, we show that dextran functionalization of silica nanoparticles enhanced uptake and intracellular delivery of siRNAs in cultured cells. Using pharmacologic al inhibitors for endocytotic pathways, we determined that our complexes are endocytosed via a previously unreported mechanism for siRNA delivery in which dextran initiates scavenger receptor - mediated endocytosis through a clathrin/caveolin - independent pro cess. Our findings suggest that siRNA delivery efficiency could be enhanced by incorporating dextran into existing delivery platforms to activate scavenger receptor activity across a variety of target cell types. 2.2 Introduction New therapeutic approaches are continually needed for targeting disease - associated proteins. Short interfering RNA (siRNA) therapeutics are a potential approach capable of highly specific targeting of a wide range of proteins through the activation of RNA interference (RNAi) [2] . With the assistance of delivery vehicles, siRNAs are transported from the extracellular environment into the cytoplasm of eukaryotic cells. After processi ng by the RNAi pathway proteins [11 14,99] , siRNAs guide degradation of target messenger RNAs (mRNAs) in a sequence - specific manner, resulting in a decrease in target protein levels. siRNA therapeutics are being developed for the treatment of cancers, genetic disorders, and infectious diseases [4] . 16 While siRNA technology is well - established in the laboratory environment, continued progress on the in vivo use of siRNAs will depend on the development of improved delivery vehicles. Delivery vehicles are required to prevent degradation of siRNAs by serum nucleases, rapid filtration of siRNAs by the kidneys, or siRNA - initiated immunogenic responses [100 102] . Moreover, if designed correctly, delivery vehicles can maximize delivery of the siRNAs to the target cells/tissues of inter est [3] . Currently, lipoplexes (complexes of siRNA s with lipids) and polyplexes (complexes of siRNAs with polymers) are the most prevalent among ongoing clinical trials with some in vivo successes [2,4,7,18,37,48,103] . However, most existing delivery vehicles cannot be used clinically due to in vivo toxicity, immunogenicity, or inactivity [7 9,49,50] . One means to address these limitations is through functional modifications [104 106] . One modification, dextran, has demonstrated success in enhancing the activity of multiple del ivery vehicles [37,39,107 109] . Functionally, dextran has been used to reduce toxicity, prevent opsonization, and block non - specific binding [110 112] . Furthermore, functionalization with dextran polymers has been used for targeted delivery to various tissues [113 115] . While progress has been made on enhancing the systemic and extracellular trafficking of delivery vehicles, transfection efficiencies among delivery vehicles remain low relative to viral vect ors, due in part to an incomplete understanding of siRNA - vehicle complex endocytosis and intracellular trafficking [4,116] . It remains unclear if RNAi is associated with a particular mechanism of endocytosis or if the mechanism of cytoplasmic delivery is specific to a certain cell type or delivery vehicle. Lipid and polymer based vehicles have been reported to use clathrin - mediated endocytosis, caveol in - mediated endocytosis, macropinocytosis, and phagocytosis [35,53,117] , though endocytosed material typically remains in endosomal compartments rather than entering the cytoplasm [87,116] . siRNAs that cannot escape early 17 endosomes are exocytosed or degraded [118] . It has been postulated that siRNAs and complexes escape the endos omes via lipid fusion with the membrane, endosomal swelling (proton sponge effect), leaky membranes, or, for vehicles with the appropriate functional groups, photochemical disruption [87] . However , these escape mechanisms are not used in all cases [119,120] . Recent reports suggest that activation of RNAi m ay not even require endosomal escape [121] , as the RNAi machinery has been found to be associated with early endosomes. Developing a better u nderstanding of the intracellular trafficking events associated with RNAi remains a significant hurdle towards improving the efficacy of siRNA delivery vehicles. The aim of this study was to investigate the impacts of chemical characteristics of delivery v ehicles, specifically the inclusion of dextran, in influencing the endocytosis and intracellular trafficking of siRNA - silica nanoparticle ( sNP ) complexes. sNP s were chosen as a delivery platform for their highly tunable synthesis, consistent physical confo rmation, low cytotoxicity, and delivery efficacy [48,122,123] . Our results showed that dextran significantly enhanced the utility of sNP s for delivering siRNAs to cultur ed cells, by initiating their uptake through a scavenger receptor - mediated endocytosis mechanism that is clathrin/caveol in - independent. Subsequent degradation of the sNP s, attributed in part to the acidic conditions of intracellular vesicles, suggested a m eans for activating release of the siRNAs from the sNP s and initiation of the silencing cascade. 2.3 Results 2.3.1 Effect of Amine and Dextran Content on siRNA Silencing Efficiency s NPs were synthesized with varying concentrations of the primary amine - containing moiety 3 - (triethoxysilyl) - propyl amine ( APTES ) , with and without dextran, to determine if these variables (amine content and the presence of dextran) influenced the delivery or s ilencing of the siRNA cargo (Figure 2 - 1). At 24 h post transfection, seven of the nine dextran - containing s NPs 18 achieved significant silencing compared to the nanoparticle only controls. Six achieved > 50% reduction in enhanced green fluorescent protein ( EG FP ) levels and were statistically equivalent to LF2K. Increasing amines resulted in increased silencing, with maximal silencing achieved at 40% APTES, with little gain and perhaps some loss of activity at higher amine contents. Only one non - dextran s NP ach ieved statistically significant reduction in EG FP levels, again at 40% APTES. s NPs were characterized for their potential to bind siRN A s using zeta potential (Figure 2 - here may be an ideal amine content/charge density for siRNA delivery vehicles, though further characterization would be required to establish this concretely. As our objective was to understand better the uptake and trafficking mechanism for s NPs that yiel d active silencing, we focused our subsequent a nalyses on our best performing s NP (40% APTES with dextran). 19 Figure 2 - 1 Effect of dextran and amine content on silencing Relative fluorescence of EGFP - expressing H1299 cells transfected for 24 h with siRNA complexes and normalized to the EGFP fluorescence of vehicle - rying percentages of APTES and either 0 mole% (hatched) or 1 mole% dextran (filled), and 100 nM of siRNAs. Error bars represent + 1 standard deviation; n = 3. Statistical analysis was performed using two - - hoc analysis . *Significant difference (p < 0.05) as compared to vector - only treatments. **Significant difference (p < 0.05) of non - dextran sNPs to dextran - containing sNPs . 20 2.3.2 Inhibition of siRNA Endocytosis and Silencing Having confirmed the ability of our sNPs to deliver siRNAs that then silence the EGFP target, we wanted to evaluate the mechanism of uptake for our sNP - siRNA complexes as compared to LF2K lipoplexes and naked siRNAs. From the literature, we selected a number of chemical inhibitors for individual endocytosis pathways ( Table 2 - 1) and investigated the quantity of siRNA s delivered (Figure 2 - 3 ) and the silencing achieved (Figure 2 - 4 ) following delivery of siRNAs using our most effective sNP (40% APTES +dextran ), LF2K, or no vehicle. As expected, intracellular levels of siR NA were significantly increased using LF2K (5x increase) and sNPs (40x increase), compared to naked siRNA (Figure 2 - 2, see insets). Delivery Figure 2 - 2 Role of sNP zeta potential (mV) on silencing Zeta Potentials were determined using 1 mg/mL of sNP s in HEPES buffer. sNP s were functionalized with varying percentages of APTES and either 0 mole% (hatched) or 1 mole% dextran (filled). Results were correlated to silencing of the siRNAs af ter sNP delivery (Figure 2 - 1). sNP s containing 0.05% APTES +dextran ( - 40 mV) and 0.05% APTES - dextran ( - 20 mV) are not shown for clarity of the plot. 21 by LF2K was significantly reduced by the combination of chlorpromazine and filipin, low temperature (4°C), and dext ran sulfate. For sNP delivery, the active inhibitors were chlorpromazine, cytochalasin D, low temperatures (4°C), and dextran sulfate. The differential effects of the inhibitors indicate that the sNPs were delivered through a mechanism distinct from that o f LF2K. Moreover, plasmid DNA delivered with our sNPs (Figure A - 1 ) did not result in significant overexpression whereas plasmid delivery with LF2K resulted in significant gene overexpression, further suggesting that sNPs and LF2K utilize different delivery pathways. However, the strong impact of dextran sulfate suggests that both sNP and LF2K complexes used scavenger receptor - mediated uptake in delivering siRNAs. Our Fluorescence - activated cell sorting ( FACS ) analyses were corroborated using confocal micros copy at 4 h and 24 h after transfection (Figures A - 2 , A - 3 , and A - 4 ) . Likewise, to examine the generality of our results across cell types, replicate experiments were performed in HeLa cells, producing similar results (Figure A - 5 ). In examining the resulti ng silencing in the presence of inhibitors (Figure 2 - 4), low temperature and dextran sulfate significantly reduced silencing following delivery by both LF2K and our sNP. This is a direct reflection of the inhibition observed for siRNA delivery (Figure 2 - 3) . This suggests that both sNP - siRNA complexes and LF2K - siRNA lipoplexes are effectively endocytosed and processed by the scavenger receptor pathway. However, there are discrepancies between the inhibition of delivery and the reduction in silencing activity . Cytochalasin D also significantly inhibited silencing by sNP delivery, indicating that the processing of endocytotic vesicles on actin networks may be essential for silencing, regardless of the pathway of endocytosis. 22 Table 2 - 1 Target and mechanism of ac tion for endocytosis inhibitors [53,195] . Figure 2 - 3 Influence of endocytotic inhibitors on the uptake of siRNAs Relative fluorescence of labeled siRNA complexes delivered to EGFP expressing H1299 cells. Cells were pre - treated with endocytosis inhibi tors and assayed 24 h post - transfection using flow cytometry. Results are normalized to uptake of siRNA only controls. Error bars represent + 1 standard deviation; n = 3. Statistical analysis was performed using two - - hoc analysis. *Significant difference (p < 0.05) as compared to delivery in the absence of an inhibitor. 23 2.3.3 Intracellular Traff icking of sNPs Using TEM, we confirmed the uptake of our sNP - siRNA complexes and identified the subcellular locations that sNPs wer e trafficked (Figure 2 - 5 ). In all cases, we confirmed the presence of our sNPs using energy - dispersive X - ray spectroscopy ( ED S ) line scans to detect silicon, that should not be present at significan t levels endogenously (Figures 2 - 5 C - F). The images showed sNPs localized in membrane - bound endocytotic vesicles. Visual inspection of the signal intensity for the endocytosed particle s suggested that the density of the interiors of the particles was decreased relative to particles before endocytosis. This observation was further supported by the appearance of degraded sNPs adjacent to the cell membrane, suggesting that the y had been re cen tly exocytosed from the cells. Figure 2 - 4 EGFP silencing in the presence of endocytotic inhibitors Relative fluorescence of EGFP - expressing H1299 cells using fluorescently labeled siRNA complexes. Cells were pre - treated with endocytosis inhibitors and assayed 24 h post - transfection in flow cytometry. Results were normalized to particle only controls within corresponding inhibit or. Error bars represent + 1 standard deviation; n = 3. Statistical analysis was performed using two - post - hoc analysis. *Significant difference (p < 0.05) as compared to conditions without inhibitors. 24 2.3.4 Acidic Degradation of sNPs To determine if the observed intracellular degradation of our sNPs could be attributed to the acidic environments of some vesicles, we tested wh ether acidic pH would result in similar degradation patterns in vitro (Figure 2 - 6 ). Both visual inspection (compare signal intensities for Figures 2 - 6 A and B) and quantification of the signals from multiple treated and untreated particles (Figure 2 - 6 C) ind icate that acidic conditions promote sNP degradation with maximum degradation, roughly 40% of the transmission electron microscopy ( TEM ) signal, in the centers of the particles. The particles appear rough after exposure to acid, again reflecting what was o bserved in the cellular experiments, suggesting some surface degradation. However, the average diameter of the particles did not change significantly during the in vi tro acid exposure. Figure 2 - 5 TEM analysis of sNP - siRNA comp lex endocytosis and trafficking TEM images show internalization of sNP s (40% APTES + dextran ) in EGFP - expressing H1299 cells 24 h post - transfection. A) Intracellular sNP s are contained in vesicles and show varying degrees of degradation. B) sNP s with internal degradation are observed in the extracellular environment. EDS line scan analysis for silico n on non - transfected (C,D), intracellular (E,F), and extracellular (G,H) particles. 25 2.3.5 siRNA Binding under Acidic Conditions We hypothesized that the degradation of the sNPs would contribute to the release of siRNAs from the complexes, resulting in a more rapid activation of silen cing than would be achieved by purely diffusive release. To examine whether acidic conditions promoted nucleic acid release from sNPs, we incubated complexes of sNPs with siRNAs under neutral and acidic conditions. The amount of nucleic acid retained on th e particles after exposure to acid was assayed using gel electrophoresis. Acidic conditions resulted in only 4% of the original nucleic acid being retained in complexes with the sNPs (Figures 2 - 7 A and B). The relatively minimal degradation of the naked siR NAs in acid suggested that the reduction in retained material was not due to degradation of the siRNAs , but to a reduced ability of the particles to bind t hem (Figures 2 - 7 C and D). Figure 2 - 6 sNP degr adation under acidic conditions Relative intensity (I) of sNP s (40% APTES +dextran ) exposed to neutral or acidic conditions. sNP s were suspended in a (A) pH 7.00 or (B) pH 4.75 solution for 16 h at RT and imaged using TEM. (C) Percent degradation was determined by comparing the difference in relative intensity at pH 4.75 to that at pH 7.00, using a normalized particle diameter. Average diameter of pre - acid particles was 381 nm + 32nm; after acid, the average diameter was 375 nm + 28 nm. Error bars repr esent + 1 standard deviation; n = 10. Scale bars are 200 nm. 26 2.4 Discussion Designing effective, non - toxic siRNA delivery vehicles remains a critical challenge in the development of siRNA therapeutics. Here, we used sNP s as a means of identifying chemical characteristics of delivery vehicles that correlate with high activity of the delivered siRNA cargo. The sNP system is convenient as it allows for changes in the chemical functionality of the vehicle without altering its physical conformation. We plan to use the sNP system as a platform for further evaluation of othe r chemical functionalities (e.g., biodegradable disulfide linkages, PEGylation) that may enable high activity of the delivered siRNAs, with the goal of identifying characteristics that apply to any siRNA delivery vehicle. Moreover, by modifying the synthes is protocol, we will examine these characteristics on particles of multiple sizes. Our approach differs from purely combinatorial efforts that have been undertaken [124] , where both the Figure 2 - 7 Nucleic acid release under acidic conditions Relative intensity of siRNA s exposed to acidic conditions (4.75 pH). (A,B) Complexes were prepared in sNP 40% APTES + dextran ) and then incubated for 16 h at RT in an acidic solu tion (4.75 pH). Results are normalized to siRNA release at 7.00 pH. (C,D) siRNA (200 nM) were incubated for 16 h in an acidic solution (4.75 pH) without delivery vehicles. Results are normalized to siRNA release at 7.00 pH. Error bars represent + 1 standar d deviation; n = 3. Statistical analysis was performed using one - - hoc analysis. Significant difference (p < 0.05) between acidic and neutral conditions was established for siRNA binding (A,B) but not degradation (C,D) . 27 chemical fun ctionality and the molecular structure/size of the vehicle can change simultaneously, potentially confounding why some vehicles result in higher siRNA activity than others. Our data suggest that the majority of silencing that occurs results from uptake of either LF2K lipoplexes or sNP - siRNA complexes through a clathrin/caveol in - independent, energy - dependent process mediated by scavenger receptors. While nucleic acids and gold nanoparticles have been shown to be taken up via scavenger receptor - mediated endoc ytosis [125,126] , this uptake mechanism was limited to macrophage s and was either clathrin - or caveol in - dependent. Utilization of a clathrin/caveol in - independent, scavenger receptor - mediated pathway in non - macrophage cells distinguishes this mechanism of uptake from those previously reported. For both vehicles, there were cases where changes in siRNA uptake and silencing were not correlated. For LF2K, where uptake inhibition by chlorpromazine and filipin did not result in a reduction in silencing, this may be because the amount of reduction in siRNA levels was small de spite it being statistically significant. Additionally, it has been shown that lipoplexes enter cells through multiple pathways [29,85] , making the inhibition of any one or two pathways, especially if those are not primary pathways to silencing, less likely to affect silencing. For sNP s, the results are more complex. Inhibition by chlorpromazine, either in the presence or absence of filipin, resulted in a significant reduction in uptake with no concomitant reduction in silencing. This s uggests inhibition of a non - productive uptake pathway. In contrast, cytochalasin D resulted in comparable levels of inhibition of siRNA uptake and a significant reduction in silencing. This indicates that sNP - siRNA complexes associate with the actin networ k, either directly or while in vesicles, and that this association is essential for delivering siRNAs in a manner (e.g., to a specific subcellular location) that eventually results in silencing. It 28 is also worth noting that uptake, trafficking, and silenci ng are dynamic processes, and measurements at a single time point do not necessarily reflect a steady - state . It is important to note that our sNP s deliver considerably more siRNA to cells than LF2K (~8 - fold, Figure 2). This indicates that our sNP s deliver more siRNA than many delivery vehicles (using LF2K delivery as a reference) [127 129] . However, the degree of silencing achieved by the delivered siRNAs was only comparable to LF2K. This may indicate that a large fraction of the siRNAs delivered by sNP s is inactive due t o sequestration, either by being retained on the sNP s or by being trapped in vesicles [8 5] . These internalized siRNAs may then be degraded prior to achieving silencing, mitigating any improvement in function that would result from delivery of higher quantities of siRNA. If this is the case, our sNP s may be valuable for delivering chemicall y - modified siRNAs, in particular those designed for resistance to nucleases or enhanced endosomal escape, potentially providing a facile approach for increasing or extending the persistence of maximal silencing [130] . Among the various classes of scavenger receptors, dextran sulfate is a known inhibitor of acetyl - LDL scavenger receptors, that are found among class A (SCARA1/SR - AI/II, SCARA2/MARCO) and class H (FEEL 1/stabilin - 1/CLEVER - 1, FEEL - 2/stabilin - 2/HARE) [131,132] . These receptors recognize targets with a high density of negative charges, common among bacterial polysaccharides [131] . While previously considered to be macrophage specific, scavenger receptors have been identified across mul tiple cell types including endothelial, smooth muscle, dendritic, fibroblast, and epithelial cells [132] . Scavenger receptors are known to induce phagocytosis and macropinocytosis, although the exact signaling mechanism remains unknown [133] . Scavenger receptors can enact a variety of functional responses due to their association w ith various co - receptors (SRC family kinases, toll - - integrins, and tetraspanins) 29 [133] . Our results suggest that the scavenger receptors used by our sNP s may associate with dynamin - independent GTPases of the Rho family, given their association with other scavenger receptors that had a reported role in actin polymer ization and clathrin/caveol in - independent endocytosis [134] . While the trafficking of high amine - content particles such as our s NPs within acidic endosomes su ggests that siRNA release into the cytoplasm is due to the proton sponge effect [135] , our results do not support this mechanism for endosomal releas e. From our TEM images, s NPs were only observed in membrane - bound vesicles and never observed in the nucleus or cytoplasm. These observations, in concert with the inability of sNP s to deliver plasmid DNA, are inconsistent with release mechanisms that requi re the endosomal membrane to rupture. Rather, it seems that only the siRNAs escape the endosomes in our system and that escape occurs after the sNP - siRNA complex has dissociated. This is further substantiated by our findings that acidic conditions inhibit the binding of siRNAs to sNP s. Formation of endosomal membrane pores would enable siRNA release into the cytoplasm. However, our sNP s lack any specific functionality designed to generate pores [136] , making this unlikely. It may also be that siRNAs do not need to escape the endosomes to activate RNAi. Recent evidence has shown that RNAi pathway proteins, specifically Dicer and Ago2, are associated with vesicle and ER membranes [121,137] . It is possible that siRNAs are recognized in the endosomes after release from the s NPs, with the RNAi proteins shuttling them across the endosomal membrane. Based on our current results, however, the exact mechanism by which siRNAs achieve endosomal escap e and initiate silencing remains unclear . The design of siRNA delivery vehicles remains a somewhat haphazard process, without clear rules for which chemical and physical features provide the greatest probability of high siRNA 30 activity. In this work, we have demonstrated that dextran associates with scavenger receptors to initiate clathrin/caveol in - independent endocytosis, and that internalization by this pathway results in active siRNA delivery. In doing so, we have identified both a vehicle design varia ble (presence of dextran) and a biological mechanism (clathrin/caveol in - independent, scavenger receptor - mediated endocytosis) that warrant further examination for their contributions to the high activity of delivered siRNAs. Going forward, we hope to exami ne the generality of these rules for siRNA delivery vehicles based on lipids, polymers, and nanoparticles. 31 CHAPTER 3: ENDOCYTOSIS CONTROLS SI RNA EFFICIENCY: IMPLICATIONS FOR SI RNA THERAPEUTIC DESIGN AND CELL SPECIFIC TARGETING Note: This chapter is adap ted from previously published work [138] 3.1 Abstract While siRNAs are commonly used for laboratory studies, development of siRNA therapeutics has been slower than expected, due in part t o a still limited understanding of the endocytosis and intracellular trafficking of siRNA - containing complexes. With the recent characterization of multiple clathrin/caveolin - independent endocytic pathways, i.e., those mediated by Graf1, Arf6, and flotilli n, it has become clear that the endocytic mechanism influences subsequent intracellular processing of the internalized cargo. To explore siRNA delivery in light of these findings, we developed a novel assay that differentiates uptake by each of the endocyt ic pathways and can be used to determine whether endocytosis by a pathway leads to the initiation of RNA interference (RNAi). Using LF2K , we determined the endocytosis pathway leading to active silencing (whether by clathrin, caveolin, Arf6, Graf1, flotill in, or macropinocytosis) across multiple cell types (HeLa, H1299, HEK293, and HepG2). We showed that LF2K is internalized by Graf1 - , Arf6 - , or flotillin - mediated endocytosis for the initiation of RNAi, depending on cell type. Additionally, we found that a portion of siRNA - containing complexes are internalized by pathways that do not lead to initiation of silencing. Inhibition of these pathways enhanced intracellular levels of siRNAs with concomitant enhancement of silencing. 3.2 Introduction Small molecule an d protein - based drugs, while critically important therapies, cannot treat all diseases [2] . In some cases, the drugs cannot access or interact with proteins that are causing the disease phenotype. As such, alternative treatment modalities mu st be developed to complement existing strategies. One potential alternative is small interfering RNA (siRNA) therapeutics, that 32 are capable of specific inhibition of a wide range of intracellular, membrane, and extracellular proteins [3] . To function, siRNAs must be transpo rted from the extracellular environment into the cytoplasm of the targeted eukaryotic cells. Once there, siRNAs act through RNA interference (RNAi) to degrade messenger RNAs (mRNAs) in a sequence - specific manner, thereby reducing target protein expression [11 14,99] . siRNA therapeutics are being developed as treatments for a variety of diseases, including cancers and infectious diseases, with one therapeutic approved for clinical use [4 6] . Despite the recent clinical success, development of siRNA therapeutics has been hindered by multiple technical challenges, including poor delivery efficiency [7 9 ] . One limitation to delivery is efficient endocytosis of delivered siRNAs to the cells of interest . Until recently, cellular endocytic pathways were classified as macropinocytosis (MP), clathrin - mediated endocytosis (CME), caveolin - mediated endocytosis (CvME), or clathrin/caveolin - independent endocytosis (CCIE) [53] . Researchers have since characterized three distinct types of CCIE, flotillin - mediated endocytosis (FME), Arf6 - dependent endoc ytosis (ADE), and Graf1 - mediated endocytosis (GME) [54 56] . The identification of these pathways has resulted in the reclassification of the uptake mechan isms of many species [58,139,140] . For instance, adeno - associated viruses and ~50% of fluid - phase uptake, including uptake of dextrans, are now attributed to GME, though they were previously thought to occur via other pathways [55,77] . Likewise, cholera toxin B is taken up by FME but was previously thought to enter cells by MP [54] . Currently, there is no consensus regarding the optimal endocytic pathway for active siRNA delivery, as multiple endocytic pathways have been found to result in successful delivery of siRNAs and initiation of silencing. It is difficult to generalize which pathways are optimal as most studies are limited to a single cell type or did not distinguish among FME, ADE, and GME. 33 Howe ver, it has also recently become evident that the endocytic mechanism influences the molecular composition of the endosomes, their intracellular trafficking, and the processing of their cargo [66,83] . Thus, we hypothesized that the mechanism used by cells to endocytose siRNA - containing complexes could significantly impact the ability of the siRNAs to initiate RNAi . In this study, we used chemical inhibitors and endocytic protein overexpression to investigate the endocytic pathways used to internalize and process siRNA - containing complexes in four cell lines. Our results show that while the complexes are internalized through multiple endocytic pathways, active delivery occurs primarily through a single pathway that varies according to cell type. The res ults suggest that both cell specificity and siRNA delivery efficiency can be enhanced by designing delivery vehicles to favor the preferred endocytic pathway . 3.3 Results 3.3.1 Silencing Efficiency in Different Cell L ines To assess the role of endocytosis in siRNA accumulation and EGFP silencing, we tested the ability of LF2K to deliver siRNAs and achieve active silencing in four common human cell lines stably expressing EGFP: H1299 (lung), HeLa (cervical), HEK293 (kidney), and HepG2 (liver) (Figure 3 - 1). At 24 hour s post - transfection, EGFP silencing and siRNA accumulation were measured in all cell lines. Silencing was greatest in H1299 cells (Figure 3 - 1A ), yet levels of intracellular siRNAs were highest in HEK293 cells (Figure 3 - 1B), and the most efficient use of si RNAs (silencing/siRNA accumulation) was seen in HeLa cells (Figure 3 - 1C). These differences suggested that the internalization and processing of LF2K - siRNA complexes differ among cell types, possibly due to the predominance of different endocytic pathways across the different cell types. 34 3.3.2 Inhibition of siRNA Accumulation and Silencing It is known that drug complexes are taken into cells via multiple endocytic pathways. However, in most circumstances, it is unclear whether the mechanism of uptake influences downstream function of the complexes. To differentiate among the types of endocytosis, we used a minimal set of chemical inhibitors, w hich, when evaluated collectively, result in unique patterns of inhibition for each endocytic mechanism (Table 3 - 1) . Using data from the literature, a logic matrix was constructed for each inhibitor and its effect on each type of endocytosis (Table 3 - 2 ). U sing this logic matrix, we identified the type of endocytosis used by LF2K for active siRNA delivery across each of the cell lines tested. By measuring the effect of inhibitors on both intracellular levels of siRNAs and EGFP silencing, we classified endocy tic pathways according to their role in facilitating siRNA function. Results were normalized against siRNA accumulation and silencing in the absence of inhibitor, allowing the relative position of a data point to indicate the degree to which an inhibitor a ffected siRNA accumulation and silencing (Figure 3 - 2, also see Materials and Methods Chapter 3 information for equations). Figure 3 - 1 EGFP silencing and siRNA accumulation EGFP silencing and siRNA accumulation in H1299, HeLa, HEK293, and HepG2 cells. Cells were transfected with 100 nM fluores cently - - transfection using flow cytometry (10,000 events). A) c ontrol value = 0 and B) control value = 1. In each panel, values for each cell line were statistically different from all others, p < . 05; error bars represent ±1 standard deviation; n = 4. Statistical analysis was performed using one - HSD post - hoc analysis. 35 Table 3 - 1 Chemical inhibitors of endocytic proteins F or general information about in hibitors, see Ivanov, et al [157] . Table 3 - 2 Chemical i nhibitor vs endocytic pathway matrix Logic matrix illustrating the effects of chemical inhibitors on different endocytic pathways. An X indicates decreased endocytic function as a result of the chemical inhibitor at the concentration used in our experiments, whereas empty spaces indicate that there is no known effect. It should be noted that there are con flicting reports in the literature regarding the role of dynamin in flotillin - mediated endocytosis (See Discussion 3.4 for details). 36 Figure 3 - 2 Influence of endocytic inhibitors on EGFP s ilencing and siRNA accumulation EGFP - expressing cells were pre - treated with endocytic inhibitors and assayed 24 hours after siRNA transfection using flow cytometry (10,000 events). x - axis: - 100% (inhibited silencing) vs 100% (enhanced silencing). y - axis: - 100% (inhibi ted siRNA accumulation) vs 100% (enhanced siRNA accumulation). Error bars represent ±1 standard deviation; n = 3. Statistical analysis was performed using one - way ANOVA, - ed to delivery in the absence of an inhibitor. 37 In comparing the effects of the different inhibitors among the four cell lines, the strongest, most consistent inhibition of silencing (and siRNA accumulation) was from methyl - - cyclodextrin 3 - 2 - endocytic pathways ( Table 3 - 2 inclusion complexes with cholesterol in the cell membrane, principally destabilizing lipid rafts [141] - siRNA comple xes by lipid - raft dependent pathways is critical for the initiation of RNAi in each of these 3 - 2 - blue), the critical pathways in these cells involve one or more of the foll owing: FME, ADE, and GME. Cytochalasin D significantly inhibited siRNA accumulation and EGFP silencing in all but H1299 cells (Figures 3 - 2B - D vs. Figure 3 - 2A - green). Cytochalasin D, a mycotoxin that binds to F - actin and blocks its polymerization, prevents the formation of endocytic vesicles as they bud from the plasma membrane [142] . FME, however, forms endocytic vesicles through actin - independent tubular invaginations and is unaffected by cytochalasin D [143,144] . Thus, we concluded that, in H1299 cells, FME of LF2K - siRNA complexes result in the initiation of RNAi. Dynasore also reduced EGFP silencing, but only in HEK293 and H1299 cells (Figure 3 - 2A & B - red). Dynasore, a noncompetitive inhibitor of dynamin, prevents endocytic vesicle fission from the cell membrane [145] . Among the lipid - raft depe ndent endocytic pathways, only ADE is considered dynamin - independent [146] . Because EGFP silencing in HeLa and HepG2 initiated following ADE of LF2K - siRNA complexes in these cel l lines . Amiloride was the only other inhibitor to reduce EGFP silencing but only in HEK293 cells (Figure 3 - 2B - black). Amiloride, a derivative of a guanidinium - containing pyrazine, increases 38 submembranous pH by inhibiting Na+/H+ exchangers [147] . Because EGFP silencing in that GME is the principal RNAi - initiating pathway in HEK293 cells . While inhibition of RNAi - initiating pathways is evide nt from reductions in EGFP silencing, inhibition of other pathways may also alter siRNA accumulation without a concomitant decrease in silencing. In H1299 cells, chlorpromazine significantly reduced siRNA accumulation without affecting EGFP silencing (Figu re 3 - 2A - purple). Chlorpromazine, which translocates clathrin and AP2 from the plasma membrane to intracellular vesicles, inhibits the formation of clathrin - coated pits used in CME [148] . Therefore, we concluded that, in H1299 cells, CME internalizes siRNAs but does not allow them to i nitiate silencing. In HeLa and HepG2 cells, chlorpromazine enhanced siRNA accumulation and EGFP silencing (Figure 3 - 2C&D - purple), suggesting inhibition of CME in these cells results in additional siRNAs entering ADE and initiating RNAi. However, the inhibitor data does not allow us to determine whether CME is also capable of internalizing siRNAs or if the enhancement of ADE results from an intracellular connection between ADE and CME. In HEK293 cells, amiloride inhibited uptake via GME, reducing silen cing. The data show that this also resulted in additional siRNAs accumulating via an uninhibited pathway. As multiple endocytic pathways are unaffected by amiloride, these data alone were insufficient to identify the pathway(s) responsible for the enhanced siRNA accumulation. 3.3.3 Overexpression of Endocytic Proteins To validate the findings from our inhibitor experiments and make additional distinctions between pathways, we overexpressed individual endocytic proteins and measured the effects on siRNA accumulati on (Figure 3 - 3 ). Green fluorescent protein ( GFP ) - labeled proteins were used 39 so that localization of the overexpressed protein could be confirmed to match that of endogenous protein ( Figure A - 9 ), and to ensure that siRNA accumulation was only measured for c ells overexpressing the protein. It is critical to note that, as in the inhibitor experiments, the effects of protein overexpression are cell - specific (compare Figures 3 - 3 A - D). In H1299 cells, we found that siRNA accumulation was enhanced by overexpression of flotillin - 1 and AP2, though reduced by clathri n overexpression (Figure 3 - 3 A). These findings indicate that both FME and CME are capable of internalizing siRNA s , supporting the findings from our inhibitor data. Figure 3 - 3 Influence of en docytic protein overexpression on the intracellular accumulation of siRNAs - labeled siRNAs delivered to cells transiently expressing EGFP - labeled endocytic proteins. Plasmid transfection did not alter siRNA accumulation. Cells were assayed 4 hours post - transfection using flow cytometry (10,000 events). Error bars represent ±1 standard deviation; n = 3. Statistical analysis was performed using one - way ANOVA, - ho accumulation in cells transfected with EGFP - only plasmid. 40 siRNA accumulation in HEK293 cells was enhanced by the overexpression of Graf1 and Arf6, th ough reduced by dynamin, clathrin, and caveolin overexpression (Figure 3 - 3 B). This would suggest that both GME and ADE are capable of internalizing siRNA - LF2K complexes. This supports our inhibitor results for GME in these cells. It also demonstrates that ADE can internalize siRNAs, though without leading to RNAi, and is likely responsible for the enhanced siRNA accumulation that occurred in the presence of amiloride (Figure 3 - 2B). Similarly, our results suggest that CME, CvME, and GME share common regulato ry elements, where overexpression of clathrin or caveolin dilutes the availability of these common elements for GME, resulting in reduced siRNA accumulation. In HeLa and HepG2 cells, the accumulation of siRNAs was enhanced by overexpression of Arf6 and AP 2 but reduced by overexpression of clathrin (Figures 3 - 3 C and 3 - 3D). These findings confirm that internalization of siRNA - LF2K complexes occurs through both ADE and CME, as in our inhibitor data. It is interesting that the cell lines show different respons es to the overexpression of actin (Figures 3 - 3C and 3 - 3D). This difference may partially explain why siRNA - LF2K complexes accumulate to a lesser degree and are considerably less efficient at initiating RNAi in HepG2 cells (Figure 3 - 1C), though a direct mechanistic link is not currently known (see 3.4 Discussion). 3.3.4 Targeted Inhibition in a Co - cultured Population Having demonstrated that the pathways that are important for internalizing siRNAs and initiating RNAi vary by cell type, we t heorized that inhibitors could be employed in a mixed cell population to enhance cell specific delivery by reducing uptake by untargeted cell types. To test this, we repeated our inhibitor assay using a co - culture consisting of H1299, HEK293, HeLa, and Hep G2 cells and assessed the effect of inhibitors on siRNA accumulation and EGFP silencing 41 (Figure 3 - 4). In general, the effects of the inhibitors in co - culture were the same as the effects on mono - cultures (Figure 3 - from the mono - culture results, treatment with cytochalasin D and chlorpromazine in H1299 cells (Figure 3 - 4A green, purple) and dynasore in HeLa cells (Figure 3 - 4C - red). For two of these cases, endocytosis of siRNA - complexes by a specific cell type was enhanced by inhibition of endocytosis by other cell types. 42 Figure 3 - 4 Influence of endocytic inhibitors on EGFP silencing and siRNA accumulation in co - cultured and mono - cultured populations Co - cultured populations consisted of H1299, HEK293, HeLa, and HepG2 cells. EGFP - expressing cells (named in the header for each panel) were pre - treated with endocytic inhibitors and assayed 24 hours post - transfection using flow cytometry (5,000 events). x - a xis: - 100% (inhibited silencing) vs 100% (enhanced silencing). y - axis: - 100% (inhibited siRNA accumulation) vs 100% (enhanced siRNA accumulation). Error bars represent ±1 standard deviation; n = 3. Statistical analysis was performed using one - way ANOVA, fo - the absence of an inhibitor. 43 3.4 Discussion Using inhibition and overexpression of endocytic proteins, we showed that LF2K - siRNA complexes are internalized t hrough multiple endocytic pathways. Moreover, the pathways used for endocytosis of LF2K - siRNA complexes were found to vary across cell types. The functional roles of these pathways were further characterized according to whether they facilitated LF2K - media ted RNAi. We also demonstrated that understanding the endocytic pathways of cells allowed targeting of specific cells in a mixed population and a resulting enhancement of siRNA accumulation and RNAi i n the targeted cell populations. We recognize that LF2K is not an option for future clinical applications and that delivery vehicle development has progressed since LF2K first became available. Nonetheless, we chose LF2K for these studies for two principal reasons. First, we have considerable prior experience u sing this vehicle [1,10,149] . Second, there is extensive prior literature on the use of this vehicle [150,151] , allowing our results to be compared to the extant lite rature. We are not suggesting that the pathways used by LF2K are those that will be preferred by other vehicles. Rather, as our results show, the same vehicle works differently depending on the cell type, and uptake alone is not sufficient to achieve activ ity. These lessons can be applied to the development of any vehicle. Previous studies regarding the cellular uptake of lipoplexes have reported that internalization occurs by CME or through direct fusion with the plasma membrane [152 155] . The differences in our conclus ions relative to these prior studies may be a result of differences in the concentrations of inhibitors used, the presence of serum in the treatment media, wash procedures, or inhibitor exposure time. It may also be that the inhibitors chosen for this stud y, and an improved understanding of their impacts on cell function, allowed us to identify endocytic pathways with more clarity than was possible previously. We and others have shown that 44 transfection at low temperature (4°C) reduces silencing [1,152] , supporting our current conclusions that the best pathways for endocytosis of siRNA - containing complexes in the cell types tested are energy - dependent. Chemical inhibitors, siRNAs, and protein overexp ression are commonly used to characterize the function of endocytic pathways [140] . Our inhibitor logic matrix was derived from the current understanding of the proteins targeted by the inhibitors and their associations with each endocytic pathway, including any known side effects at the concentrations used in our experiments (Table 3 - 2). We chose to use inhibitors , as they work more quickly than siRNAs and overexpression, and result in a shorter - term reversible disruption of native cell function. However, among the many chemical inhibitors used to evaluate endocytosis, none possesses absolute specificity for a sing le endocytic pathway [156] . In many cases, the molecular target of an inhibitor is utilized by multiple endocytic pathways. In addition, experimental conditions (high concentrations, prolonged incubation, and serum protein interactions) can cause unintended side effects [157] - raft dependent endocytosis, can also inhibit CME when used at concentrations > 10mM [158] . Fluorescent endocytic markers are generally used to determine the effective concentration of an inhibitor. To date, howeve r, none has been established that is specific for GME, ADE, or FME, and those traditionally associated with CvME (albumin) [70,71] and MP (dextran) [56] have been shown to be endocytosed via multiple pathways. It is still unclear what factors impact whether pathways are used for endocytosis of siRNA - containi ng complexes or which pathways lead to initiation of RNAi. Intracellular trafficking of endocytic vesicles varies across cell type and disease state [159] . Many of these variations are observed in relation to processing through the early endosome (EE), a common node among 45 intracellular trafficking pathways. In HeLa cells, the time for cargo to reach the EE was 5 - 10 minutes via CME and 30 - 60 minutes for ADE [55] . FME is capable of retrograde transport directly to the Golgi, bypassing the EE [160] . ADE and GME have been shown to form intermediate endosomal compartments capable of sorting cargo before joining the EE [55,72,161] . The se differences alone could explain the differences in siRNA accumulation and silencing across cell types. In addition, the pH and composition of the endosomal vesicles differ among endocytic pathways [87,116] , which could alter endosomal escape, depending on the mechanism (e.g., formation of membrane pores, pH buffering, or membrane fusion) [87] . Thus, differences in the endosomal release kinetics for each endocytic pathway, in addition to uptake, may result in the differences in siRNA activity we observed among the different endocyt ic pathways and cell types. Differences in release kinetics may also explain why the active endocytic pathway for uptake of drugs and other molecules differs depending on the cell type [152,162 165] . By measuring both the intracellular accumulation of siRNA and its functional activity in silencing EGFP, we identified multiple endocytic pathways u sed to internalize siRNA - LF2K complexes. In three cases, we observed a significant increase in siRNA accumulation (see Figure 3 - 2B - black and Figures 3 - 2C&D - purple). In each case we identified a regulatory protein common to both the inhibited and enhanced pat hway (Cdc42 for amiloride and AP 2 for chlorpromazine), that was also directly affected by the inhibitor. Given the duration of incubation with inhibitor, it is unlikely that the increase in endocytic activity is caused by increased protein levels. It is more likely a reallocation of cellular resources. AP2, which regulates CME, is in turn regulated by Arf6. Sequestration of AP2 to intracellular compartments by chlorpromazine would, in theory , increase the availability of Arf6 for ADE. In this way, the 46 relative activities of endocytic pathways are affected by competition for common resources. Indeed, if this is the case, the relative expression of endocytic and regulatory proteins in a cell ma y control the relative activities of the respective endocytosis pathways. We concluded that FME facilitates LF2K - mediated RNAi in H1299 cells. In FME, flotillin - 1 and flotillin - 2 co - assemble into plasma membrane microdomains in lipid rafts and are internal ized after phosphorylation by FYN [81] . Previously, FME has been implicated in the uptake of CD59 [54] , cholera toxin B [54] , silica nanoparticles [80] , and cationic polyplexes [79] . The role of dynamin in this process, however, is still undefined and possibly dependent on cell type or cargo [82] . Based on our inhibitor data with dynasore, we concluded that FME is dynamin - dependent in H1299 cells. Interestingly, the progressi on of malignancy in non - small cell lung cancers (NSCLC), like H1299s, is characterized by increased expression of flotillin - 2, and decreased expression of flotillin - 1 and caveolin - 1 [166] . This aligns with our findings where siRNA accumulation was unaffected by overexpression of flotillin - 2 but enhanced by the overexpression of flotillin - 1. Expression profiles of the mRNAs for the flotillins and caveolin - 1 correlate across tis sue samples, with the highest expression levels in heart, lung, and skeletal muscle tissue [167] . Using gene expression data, we found that ETS1, a transcription factor for both flotillins and caveolin - 1, was 9.6x higher in H1299 cells than HeLa, HEK293, and HepG2 cells ( Table A - 6 ). This suggests that elevated expression of the flotillins, caveolin - 1, or ETS - 1 may facilitate uptake by FME and initiation of RNAi. In HEK29 3 cells, we concluded that the cells use GME to initiate RNAi. Since its discovery, GME has been implicated in the uptake of GPI - linked proteins [56] , adeno - associated virus [77] , and dextran [78] . It was also identified as a major source of uptake of extracellular fluid [56] . In GME, Graf1 and dynamin form a stable complex that regulates the scission and stability of the 47 tubulovesicul ar structures [56] . Interes tingly, Graf1 in this complex has a higher affinity for dynamin - 1 (DNM1), thought to be exclusive to neurons, than dynamin - 2 (DNM2), which has ubiquitous expression [56,76] . Comparing gene expression data for Graf1, DNM1, and DNM2 among the four cell lines, we found that similar expression levels of DNM1, DNM2, a nd Graf1 only occurred in HEK293 cells (Table A - 6 ). It is possible then that the relative expression levels of DNM1, DNM2, and GRAF1 determine the prominence of GME in a given cell type. Additionally, mRNA expression levels of proteins associated with GME (Graf1, Cdc42, and Arf1) were significantly higher in HEK293 cells relative to the other cell lines tested ( Table A - 6 ). ADE is regulated by the GTP cycle of Arf6 [55] . Internalization though ADE leads to the formation of Arf6 - containing endosomes that are either recycled to the plasma membrane or trafficked to the EE, a process dependent upon the h ydrolysis of Arf6 - GTP [168] . ADE has currently been suggested as the route of internalization of T ac [72] , major histocompatibility complex class I proteins (MHCI) [73] - integrin [74] , and the herpes simplex virus [75] . We found that both HeLa and HepG2 use ADE to initiate RNAi, albeit with different efficiency. In ADE, localization and phosphorylation of Arf6 is d ependent upon actin polymerization [55] . Overexpression of actin in HeLa cells reduced siRNA accumulation, whereas in HepG2 cells, actin overexpression enhanced siRNA accumulation. Basal H eLa cell expression of actin mRNA is 2.3 - fold higher than in HepG2 cells ( Table A - 6 ). Given the different responses of the cell types to actin overexpression, it may be that there is an optimal amount of actin to support ADE, with too much or too little be ing inhibitory. We also showed that endocytic inhibitors could be used in a co - cultured population of cells to enhance silencing in multiple cell types or achieve preferential uptake in a given cell type 48 (Figure 3 - 4). This was principally observed through treatment with chlorpromazine in H1299, HeLa, and HepG2 cells (Figure 3 - 4 purple), cytochalasin D in H1299 cells (Figure 3 - 4A vs Figure 3 - 4B - D green), and dynasore in HeLa cells (Figure 3 - 4C vs Figure 3 - 4A, B, & D - red). Given our results, we believe that controlling the design of siRNA delivery vehicles and accounting for the variability in endocytic pathways when delivering siRNAs could allow improved cell specificity in vivo, thereby enhancing the overall de livery efficiency and efficacy of siRNA - based therapeutics. Although the specific pathways utilized by LF2K are, almost certainly, not ubiquitous among delivery systems, our findings demonstrate that 1) uptake alone is not sufficient to achieve silencing and 2) the role of CCIE endocytosis in siRNA therapeutics warrant additional study. Overall, these findings also support a growing body of evidence that the endocytic pathway used for internalization is dependent on cell type in addition to the characteris tics of the cargo. In the field of siRNA therapeutics, these findings suggest that delivery vehicles should be designed to utilize specific endocytic pathways when targeting a particular cell type. By simultaneously enhancing uptake through pathways that i nitiate RNAi and avoiding uptake through pathways that do not, the efficacy and specificity of siRNA - based therapeutics could be markedly enhanced. 49 CHAPTER 4: KINETIC ANALYSIS OF THE INTRACELLULAR PROCESSING OF siRNAs BY CONFOCAL MICROSCOPY 4.1 Abstract Here, we describe a method for tracking intracellular processing of siRNA - containing complexes using automated microscopy controls and image acquisition to minimize user effort and time. This technique uses fluorescence colocalization to monitor dual - label ed fluorescent siRNAs delivered by silica nanoparticles (sNPs) in different intracellular locations, including the early/late endosomes, fast/slow recycling endosomes, lysosomes, and the endoplasmic reticulum. Combining the temporal association of siRNAs w ith each intracellular location, we reconstructed the intracellular pathways used in siRNA processing, and demonstrate how these pathways vary based on the chemical composition of the delivery vehicle. 4.2 Introduction Understanding the intracellular process ing of siRNA - containing complexes is critical to the design of siRNA delivery vehicles. While siRNAs trafficked to the cytoplasm can be actively incorporated into the RNA interference (RNAi) pathway, endosomal recycling and endolysosomal retention can resu lt in siRNAs being exocytosed or degraded [84,85] . It is estimated that <1% of internalized siRNAs reach the cytoplasm [85] . Thus, to maximize siRNA activity, it is useful to design delivery vehicles to enhance the trafficking of siRNAs to the cytoplasm. However, it is unclear how to optimize delivery vehicle characteristics for optimal intracellular processing [90] . Confocal microscopy is the preferred method to study intracellular trafficking, as fluorescent colocalization analysis can quantify spatiotemporal biological interactions. However, it is currently considered labor in tensive, requiring constant operator supervision to maintain well position, focal plane, and cell viability over the duration of the experiment [169,170] . Here, we 50 describe a method that uses automated multi - well fluorescence imaging of stable cell lines to increase the throughput of live - cell imaging and decrease the labor associated with image collectio n, while not sacrificing data quantity or quality. We applied our method to characterize the intracellular processing of siRNA - containing complexes and measure kinetic variations that arise from delivery by silica nanoparticles (sNPs) with different chemic al compositions. During the development of our assay, several points of automation were included to reduce operator intervention and improve the throughput of live - cell imaging. Long - term cell viability and function were maintained with a stage - top incuba tor equipped with temperature, humidity, and CO 2 control. Automated stage controls were used to record and recall the exact X/Y coordinate of an image position, allowing multiple wells to be imaged in a single live - cell stem (PFS) was used to prevent axial drift in the focal plane during long term and multi - well imaging [171] . Finally, a dry objective was used to collect images across multiple wells/positions without continual application of liquid immersion media. Fluorophores used in live - cell imaging are susceptible to photobleaching, depending on the sensitivity of the fl uorophore and the frequency of image acquisition [172] . For our assay, intracellular organelles were labeled through the constitutive expression of fluorescent chimeric proteins, thereby minimizing photobleaching through the continual supply of new fluorophores. Photobleaching of both the siRNA str ands and organelles was further minimized by collecting images at multiple positions in each well. This allowed wells to be imaged at short intervals (~30 minutes) while the specific positions in each well were imaged at longer intervals (~1.5 hours) (Figu re 4 - 1). 51 Figure 4 - 1 Intracellular trafficking flowchart A flow chart for tracking the intracellular localization of endocytosed molecules. Shown is the protocol for generating live - cell, kinetic colocalization profiles between the molecules and multiple intracellular locations. 52 To characterize the kinetic association of siRNAs with intracellular locations common to endocytosis and intrace llular trafficking, we engineered HeLa cells to express chimeric EGFP - labeled proteins associated with intracellular trafficking: Rab4, Rab5, Rab7, Rab11, Lysosomal Associated Membrane Protein 1 (LAMP1), and Calreticulin (Endoplasmic Reticulum ER) (Figure 4 - 2). Ras - like GTPases (Rab) proteins, of which over 60+ members have been identified in humans, are associated with membrane trafficking [173] . Each Rab protein has distinct intracellular localization and trafficking through their association with motor, membrane, and SNARE proteins [173] . Endosomal membranes often contain multiple Rab proteins that promote sorting of contents to distinct regions of the membrane and trafficking to different intracellular destinations [174] . Rab5 is associated with the recycling endosome and endosomal maturation, but the majority of activated Rab5 is localized to the early endosome (EE) [175] . Rab4, also localized in the EE, regulates fast endosomal recycling from the EE to the plasma membrane (PM) [176] . Rab11 mediates slow endosomal recycling from the EE to an inte rmediate recycling endosome (RE) before trafficking to the PM [177] . Rab7 directs trafficking and fusion of the LE to the lysosome [178] . Endosomal maturation from EE to LE is characterized by a simultaneous increase in Rab7 and decrease in Rab5 [179] . Lamp1 is a t ransmembrane protein primarily residing in the lysosome [180] . Calreticulin is a calcium binder that resides in storage compartments of the ER [181] . 53 I n our previous work, we demonstrated that the activity of siRNAs was altered by the presence of dextran in the sNP delivery vehicles [1] . As both formulations of sNPs (with and without dextran) were capable of delivering siRNAs to c ells (as quantified through flow cytometry), we hypothesized that the addition of dextran significantly altered the intracellular processing of the siRNAs. Here, we have investigated the kinetics of siRNA intracellular trafficking associated with delivery by sNPs +/ - dextran over a ~24 hour period, with data collected at ~ 30 minute intervals (Figures 4 - 3 & 4 - 4). The kinetic association of each siRNA strand (guide and passenger) with Rab4 - , Rab5 - , and Rab7 - containing vesicles was similar, with Figure 4 - 2 Intracellular trafficking pathways in eukaryotic cells To track the intracellular location of fluorescent molecules, HeLa cells were engineering to express EGFP - labeled proteins. Rab5 (5) facilitates receptor mediated endocytosis and vesicle fusion with the early endosome (EE). Rab4 (4) regula tes fast endosomal recycling from the EE to the plasma membrane (PM), and Rab11 (11) mediates slow endosomal recycling through the recycling endosome (RE). Rab7 (7) directs trafficking and fusion of the late endosome (LE) with the lysosome. Lysosomal Assoc iated Membrane Protein 1 (LAMP1) is used as a marker for the Lysosome, and Calreticulin as a marker for the endoplasmic reticulum (ER). 54 rapid accumula tion, retention, and decay for siRNAs delivered by either sNP (Figure 4 - 3). While siRNAs trafficked to Rab7 were unaffected by dextran functionalization, siRNAs delivered by the - dextran sNP had greater retention of the passenger strands in Rab4 - and Rab5 - containing vesicles than the guide strands. This would suggest that functionalizing sNPs with dextran alters the way siRNAs strands are initially processed in fast/early endosomes (Rab4/5) but does not affect their subsequent trafficking to late endosomes (Rab7). In Rab11 - containing vesicles, the accumulation of passenger/guide strands was similar when delivered by sNPs - dextran, whereas siRNAs delivered by sNPs +dextran had greater accumulation of passenger strands than guide strands. In the lysosome, there was little difference between the traffi cking of siRNA strands when delivered by either sNP, however the rate of Figure 4 - 3 Kinetic colocalization profiles of siRNA with Rab4, Rab5, and Rab7 Colocalization of siRNA strands, either guide (magenta) or passenger (cyan), with EGFP - labeled proteins. Following s iRNA transfection by sNPs with (+) or without ( - ) dextran, live cell images were collected at ~ correlation coefficient. 55 siRNA accumulation in the lysosome decreased over the duration of the experiment for sNPs +dextran, but increased for sNPs - dextran. The greatest difference in siRNA trafficking betw een sNPs was observed in the ER where siRNA strands delivered by sNPs - dextran were only briefly localized, whereas siRNA strands delivered by sNPs +dextran steadily accumulate over time. Using the combined data sets of intracellular localization, we determined the intracellular pathway used by each siRNA strand and compared the differences between delivery by the different sNPs (Figure 4 - 5). Initially, siRNAs rapidly accumulate in the EE and colocalize to r egions associated with fast endosomal recycling (Rab4) and endosomal maturation (Rab5/7). Variations in siRNA strand trafficking are observed, with greater retention of the passenger strand than the guide strand in Rab5 vesicles. After the EE, siRNAs began to accumulate in Figure 4 - 4 Kinetic colocalization profiles of siRNA with Rab11, Lysosome, and ER Colocalization of siRNA strands, either guide (Magenta) or passenger (Cyan), with EGFP - labeled proteins stably expressed in HeLa cells. Following siRNA transfection, using sNPs functiona lized either with (+) or without ( - ) dextran, live cell images were collected using a confocal microscope at ~ 30 minute intervals for 56 lysosomes, recycling endosomes (Rab11), and the ER. The primary distinction in siRNA trafficking between sNPs +/ - dextran occurred in the ER, where siRNA colocalization diminishes over time for sNPs - dextran but increases for sNPs +dextran . Further, the accumulation and retention of the passenger strand was biased towards fast endosomal recycling when delivered using sNPs - dextran but slow endosomal recycling when using sNPs +dextran. 4.3 Discussion Here, we have described an automated method that increases the throughput of confocal microscopy for analyzing the trafficking of endocytosed material. Comparing the kinetic colocalization profiles of guide and passenger siRNAs, this assay was capable of d etecting changes in colocalization across multiple intracellular locations and methods of delivery. This assay is beneficial to studying of the impact of the characteristics of delivery vehicles on siRNA trafficking and activity. However, there is consider able potential for further optimization, by expanding the scope of the assay to include additional intracellular pathways and organelles. Figure 4 - 5 Kinetic colocalization heat maps Heat maps generated from the colocalization profiles between siRNA strands and the corresponding organelles (Figures 4 - 3 and 4 - 4). 57 Further, we believe that the findings presented demonstrate the potential applications of this assay to a variety of c ellular processes involving the intracellular transport of therapeutic cargo, such as DNA, mRNA, small molecules, and peptides. 58 CHAPTER 5: CONCLUSIONS AND FUTURE WORK 5.1 Conclusions The purpose of these studies was to better understand how the characte ristics of delivery vehicles impact the active delivery of siRNAs . The use of sNPs allowed changes to be made in the chemical functionality of the particles while maintaining relatively constant physical characteristics. By varying amine content, it was sh own that optimizing siRNA binding affinity enhances silencing. Additionally, the utility of sNPs was enhanced through dextran functionalization, which facilitated uptake by a clathrin/caveolin - independent endocytic pathway. The combination of these finding s resulted in a sNP with no observable cytotoxicity and silencing comparable to LF2K . These findings could be applied to additional delivery systems, such as lipids or polymers, to further enhance the efficiency of siRNA delivery vehicles. In light of rec ently discovered CCIE pathways, we developed a novel assay that differentiates uptake by each of the endocytic pathways and can be used to determine the functional role of a pathway in initiating RNAi. Our results are the first to demonstrate that LF2K uti lizes GME, ADE, or FME for the initiation of RNAi, depending on the cell type. We also showed that, in each cell type, a portion of the siRNA - containing complexes is internalized by endocytic mechanisms that do not lead to silencing. Moreover, we demonst rate d that understanding the endocytic pathways that are important for uptake of siRNA - containing complexes allow enhancement of cell - specific uptake in a mixed cell population. These findings suggest delivery vehicles should be designed to utilize spe cific endocytic pathways . While siRNA therapeutics have been approved for clinical use, their continued development is hindered by a lack of information regarding the intracellular pathways used by endocytosed siRNAs . To address these shortcomings, we developed a confocal based assay that uses live - cell 59 automated image acquisition to assess the intracellular trafficking of siRNAs . Here we identified the intracellular pathways used by siRNAs and correlated difference s in siRNA trafficking to specific del ivery vehicle characteristics, i.e., dextran functionalization enhanced siRNA accumulation and retention in the ER. These findings suggest that delivery vehicle characteristics can be used to optimize the intracellular trafficking of siRNAs. 5.2 Future Work Th e results of these studies, while beneficial to the design of delivery vehicles, also present new questions for the field of siRNA therapeutics. Outlined below are possible future directions resulting from the studies presented in this dissertation. 5.2.1 Silic a Nanoparticle Optimization Given the success of the dextran sNPs and the well - established synthesis methods using the modified S [182] , additional experiments could be performed on a variety of delivery vehicle characteristic s and functional groups. While some initial experiments were conducted on sNPs of different sizes and types of dextran, optimizi ng the reproducibility of nanoparticle synthesis would aid in the discovery of characteristics essential for siRNA delivery. In a ddition to the current methods used to characterize the sNPs ( dynamic light scattering ( DLS ) , zeta - potential, and TEM), it would be beneficial to further characterize the sNPs for their respective molecular weight [183] , concentration of accessible amines/dextran [184,185] , and their stability in solution [186] , to ensure consistency between syntheses . Once fully characterized, the silencing capacity of different delivery vehicle characteristics could be assessed across multiple cell types to generate cell specific desi gn criteria. Delivery vehicle characteristics could also be assessed for the ir specific role in endocytosis and intracellular trafficking using the assays outlined in chapter s 3 and 4 of this dissertation . 60 5.2.2 Predicti ng Optimal Endocytic Pathways The work presented in chapter 3 suggest ed that endocytic pathways compete for shared resources. The relative expression of these regulatory proteins in a cell may predict which endocytic pathway is optimal for a siRNA delivery vehicle. The first st ep would be determining the best endocytic pathway for siRNAs in multiple cell types and , then identifying trends in proteins expression that correlate with uptake by a particular endocytic pathway s . As discussed in chapter 3, the oncogenesis of NSCLC is c haracterized by changes in the expression of flotillin and caveolin proteins. C omparing the optimal endocytic pathway for siRNA delivery in both healthy lung cells and in H1299s would make possible the study of the effect of disease states on endocytosis . Further, i f a disease state altered the expression of regulatory proteins to the extent that the optimal endocytic pathway for siRNA delivery is changed , then inhibition of the original endocytic pathway would limit siRNA delivery to diseased cells. 5.2.3 Add itional Intracellular Pathways As stated in previous chapters, there is a lack of information regarding the intracellular events associated with siRNA delivery [90] . While the as say detailed in chapter 4 incorporates the basic intracellular locations associated with cargo transport, the overall scope of the assay would be enhanced by additional cell lines . In addition to the those presented in chapter 4, stable E GFP constructs w ere also generated in HeLa cells for the following endocytosis - related proteins , clathrin, caveolin, Arf6, Graf1, flotillin - 1, flotillin - 2, actin, and dynamin ; RNAi - related proteins, TRBP, Dicer, and Ago2 ; and t rafficking - related proteins , TPST2 (Golgi) a nd Rab9. Generating stable constructs for the following Rab proteins would allow the intracellular trafficking assay to encompass retrograde transport and exocytosis of siRNAs [173] 61 62 APPENDICES 63 APPENDIX A: Materials and Methods for Chapter 2 Materials 4 - Well Confocal Plate (LabTek, #155383) 96 - Well Plate (Costar, #3610) Acetic Acid (J.T. Baker, #15500760) Ammonium Hydroxide (Sigma, #320145 - 500ML) APTES: (3 - Aminopropyl) triethoxysilane (Sigma, #A3648 - 100ML) Chlorpromazine hydrochloride (Sigma, #C8138 - 5G) Copper Grids, 200 Mesh (Electron Microscopy Sciences, #G200 - Cu) Cytochalasin D (Sigma, #C2618 - 200uL) - Diamidino - 2 - phenyli ndole dihydrochloride) (Sigma, #10236276001) Dextran Sulfate, Mw 500k (Sigma, #D6001) Dextran, Mw 10k (Sigma, #D9260 - 10G) DMEM (Life Technologies, #11965092) Ethanol (VWR, #89125 - 164) Fetal Bovine Ser um (Life Technologies, #16000044) Filipin Complex III (Sigma, #F4767 - 1MG) Formaldehyde/Glutaraldehyde, 2.5% each in 0.1M Sodium Cacodylate Buffer, pH 7.4 (Electron Microscopy Sciences, #15949) Formvar Solution in Ethylene Dichloride (Electron Microscopy Sc iences, #RT 15820) Geneticin (Life Technologies, #10131 - 035) Heparin (Sigma, #H3393 - 25KU) Lead Citrate (Electron Microscopy Sciences, #512 - 26 - 5) Lipofectamine 2000 (Life Technologies, #11668019) Milli - Opti - MEM (Life Technologies, #11058021) Osmium Tetroxide, 1% (Electron Microscopy Sciences, #19152) pDNA (pd2EGFP - N1, clontech #6009 - 1) Penicillin/Streptomycin (Life Technologies, #15140122) Round - Bottom Tubes, 5 ml (BD Falcon, #352063) - GCUGACCCUGAAGUUCAU C - - GAUGAACUUCAGGGUCAGC - Fluorescent siRNA: Sense DY547 - - GCUGACCCUGAAGUUCAUC - - GAUGAACUUCAGGGUCAGC - Sodium Cacodylate Buffer (Electron Microscopy Sciences, #11653) Sphero Rainbow Calibration Parti cles (Spherotech) Spurr Resin (Electron Microscopy Sciences, #14300) SYBR gold staining (Life Technologies, #S - 11494) TEOS: Tetraethyl Orthosilicate (Sigma, #86578 - 250ML) Trypsin (Life Technologies, #25200056) Ultracel Regenerated Cellulose Membrane, 30 kD a NMWL, 47 mm (Millipore, #PLTK04710) Uranyl Acetate (Electron Microscopy Sciences, #22400) 64 Cell Culture H1299 cells constitutively expressing a 2 h half - life EGFP were generously provided by Dr. J. Kjems (University of Aarhus, Denmark). H1299 and HeLa cel ls were maintained in DMEM High Glucose, 10% fetal bovine serum, and 1% penicillin/streptomycin. 1% Geneticin was included in the H1299 culture medium to maintain EGFP expression. Cells were incubated at 37°C in 5% CO2, at 100% relative humidity, and subcu ltured every 4 5 days by trypsinization. Synthesis of Silicon Nanoparticles A 500 mL round bottom Schlenk flask was charged with 150 mL of absolute ethanol and 50 mL of Milli - Q water with constant stirring. Dextran (9 - 11 kDa, 2.4 x 10 - 6 mol, 24 mg) was di ssolved in 10 mL of Milli - Q water and added, followed by 10 mL of NH4OH (~30% as NH3). Tetraethyl orthosilicate (TEOS) (2.4 mmol, 0.53 mL) was added dropwise via syringe. The mixture was stirred for 10 minutes at room temperature (RT) under nitrogen follow ed by addition of (3 - Aminopropyl) triethoxysilane (APTES) (concentration varied as mole percentage of TEOS; e.g., 40% APTES used 0.96 mmol, 0.224 mL). The reaction mixture was stirred for 24 h at RT under nitrogen atmosphere and purified by pressure filtra tion using an Ultracel regenerated cellulose membrane (Millipore) at 40 psi and rinsed three times with Milli - Q water - Q water and sonicated until well dispersed. Zeta Potential A Malvern Zetasizer Nano ZS was used to determine the zeta potential (mV) of sNP s. Measurements were collected using 1 mg/mL of sNP in HEPES buffer. 65 EGFP Silencing Analysis H1299 - EGFP cells were seeded in 96 - growth media without antibiotics. Cells were treated 24 h post - solution containing Opti - MEM, siRNA, and delivery vehicle that was mixed for 30 min prior to addition to the cells. Final concentrations were maintained at 100 nM siRNA in either 2.3 µg/ml sNP . Cells were incubated in the transfection solutions at 37°C, 5% CO2, and 100% humidity. At 24 h after transfection, cells were washed s quantified with a Gemini EM fluorescent plate reader (Molecular Devices) at 480 nm excitation and 525 nm emission. Fluorescence intensity was normalized to control wells treated with a delivery vehicle but no siRNA. Cell morphology and E GFP expression as a measure of cytotoxicity was assessed by microscopy and was not observed in any of the treatments (Figure 2 - 6 ). HeLa cells were seeded in 96 - antibiotic - free growth media. Cells were treated 24 h p ost - solution containing Opti - MEM, 20 ng pd2EGFP - N1, and 2.3 µg/ml LF2K. Cells were then - MEM, siRNA, and delivery vehicle that was mixed for 30 min prior to additio n to the cells. Final concentrations were maintained at sNP . Cells were incubated in the transfection solutions at 37°C, 5% CO2, and 100% humidity. Cells were washed 4 h post - transfection with antibiotic f ree growth media. At 24 h after transfection, cells were washed twice with DPBS, and EGFP fluorescence was quantified with a Gemini EM fluorescent plate at 480 nm excitation and 525 nm emission. Fluorescence intensity was normalized to control wells treate d with a delivery vehicle but no siRNA (Figure 2 - 5 ). Cell morphology and E GFP 66 expression as a measure of cytotoxicity was assessed by microscopy and was not observed in any of the treatments. Inhibition experiments EGFP - expressing H1299 cells were seeded in 12 - well plates at a density of 150,000 cells/well and cultured in antibiotic free growth media. Immediately prior to transfection, cells were washed with media and replaced with inhibitor containing media for the appropriate pre - - treatment, cells were treate solutions in Opti - sNP ) and incubated at 37°C, 5% CO2, and 100% relative humidity. Cells were washed 4 h post - for 15 minutes at 37°C to remove extracellularly bound complexes. Antibiotic - free media was then added to the cells. For FACS analysis, cells were trypsinized 24 h post - transfection, pelleted by centrifugation (200 g) at 4°C, and re - suspended in DPBS. The cells were then transferred into 5 mL round bottom tubes. Immediately prior to analysis, cells were treated with DAPI at a final concentration Cytometer to detect DAPI (355/460), EGFP (488/530), and Dy547 tagged siRNA (561/585), gated to include 10,000 events/sample. For comparison across experiments, the instrument was calibrated using Sphero Rainbow Calibration particles. Geometric mean was used to calculate fluor escence intensity values among samples. EGFP fluorescence was normalized to particle 67 only controls treated with the corresponding inhibitor. Dy547 fluorescence was normalized to the uptake of siRNA only (no vehicle) controls. HeLa cells were seeded in 96 - w ell plate in antibiotic - free growth media. Cells were transiently transfected 24 h post - seeding with pd2EGFP - N1 and LF2K. Immediately prior to transfection, cells were washed with media and replaced with inhibitor containing media for the appropriate pre - t reatment time and incubated at 37 °C, 5% CO2, and 100% humidity. Cells were washed 4 h post - transfection with antibiotic free growth media. Cells were washed twice with DPBS 24 h post - transfection and EGFP fluorescence was quantified using a Gemini EM fluo rescent plate reader at 480 nm excitation and 525 nm emission. Fluorescence intensity was normalized to control wells treated with a delivery vehicle but no siRNA (Figure 2 - 5 ). Cell morphology and E GFP expression as a measure of cytotoxicity was assessed b y microscopy and was not observed in any of the treatments. Transmission Electron Microscopy/Energy Dispersive X - ray Spectroscopy (EDS) Intracellular TEM: EGFP - expressing H1299 cells were seeded in 6 - well plates at a density of 400,000 cells/well and cult ured in antibiotic - free media. Cells were treated 24 h post - seeding - sNP ) and incubated at 37°C, 5% CO2, and 100% relative humidity. Cells were trypsinized 24 h post - transfection and pelleted by centrifugation (200 RCF) at 4°C. Samples were fixed using 2.5% formaldehyde/glutaraldehyde in DPBS, stained with 1% osmium tetroxide in DPBS, dehydrated through a graded series of ethanol concentrations, and embedded in Spurr resin. Samples wer e sectioned to a thickness of ~90 nm using an RMC MYX ultramicrotome and placed onto a 200 mesh formvar coated copper grid. Samples were additionally stained with uranyl acetate and lead 68 citrate. Images were acquired using a JEOL 100CXII transmission elect ron microscope operating at an accelerating voltage of 100 keV and equipped with an Olympus MegaView III digital camera. EDS analysis was performed on JEOL 2200FS transmission electron microscope operating at an accelerating voltage of 200 keV. C onfocal M icroscopy EGFP - expressing H1299 cells were seeded in 4 - well plates at a density of 75,000 cells/well and cultured in antibiotic free growth media. Immediately prior to transfection, cells were washed with media and replaced with inhibitor - containing media for the appropriate pre - pre - arious transfection solutions in Opti - MEM sNP ) and incubated at 37°C, 5% CO2, and 100% relative humidity. Cells were washed 4 h post - transfection with Opti - MEM to remove extracellularly bound complexes a nd imaged 4 h and 2 4 h post - transfection (Figures 2 - 7 and 2 - 8 ). Confocal images were taken using an Olympus FluoView 1000 Spectral - based Laser Scanning Confocal Microscope. An Olympus PLAPON 60x/1.42 oil objective was used to acquire all images. EGFP (488 /530) fluorescence was measured using an excitation of 488 nm with a multi - line Argon laser, and displayed as green (LUT). Dy547 (559/568) fluorescence (siRNA) was excited at 559 nm by a HeNe laser, and displayed as red (LUT). The focal plane for each imag e was chosen based on the highest intensity EGFP fluorescence. All images were collected sequentially as single XY images and used 2 count Line Kalman averaging. 69 Acidic Degradation sNP s were dispersed in acetic acid (pH 4.75) at a concentration of 0.5 mg/m l and incubated at RT. After 16 h, the samples were centrifuged and washed three times with DPBS (Invitrogen). copper grid and air dried overnight. Images were acqui red using a JEOL 100CXII transmission electron microscope operating at an accelerating voltage of 100 keV and equipped with an Olympus MegaView III digital camera. Polyacrylamide Binding Gels /ml sNP , and allowed to incubate for 30 min. Milli - Q water (pH 7, control) or acetic acid (pH 4.75) was added to the sample and incubated at RT. After 16 h, the samples were centrifuged to pellet the sNP s, washed with Milli - Q water, and suspended in DPBS. (Sigma) for 3 min to elute the siRNA s from the sNP s and then resolved on a 12% polyacrylamide gel. In lieu of centrifugation and washing, siRNA samples without sNP were diluted with Milli - Q water. Nucleic acid d etection was performed with SYBR gold staining, imaging was performed with the Molecular Imager ChemiDoc XRS System, and analysis was performed using ImageJ [187] . Statistical analyses Multiple comparisons were performed with two - HSD post hoc analysis (Tables 2 - 3, 2 - 3, & 2 - 4). Analyses were performed using OriginPro 8 and Microsoft Excel. 70 Figure A - 1 Plasmid transfection efficiency of sNPs The relative fluorescence of EGFP in HeLa cells 24 h post - transfection with pd2EGFP - N1 vehicle complexes. Results are normalized to the EGFP fluorescence of vehicle - only control cells at 0 nM pDNA. l of 40% APTES +dextran sNP , and either 0, 3, 6, or 9 nM of pDNA. Error bars represent + 1 standard deviation; n = 3. 71 Figure A - 2 Confocal microscopy of non - inhibited silencing 24 h post - transfection Confocal images of various siRNA - vehicle complexes 24 h post transfection into EGFP - expressing H1299 cells. Cyan fluorescence represents the E GFP - expressing cells. Gray scale images were obtained from a phase contrast objective. A,B) Control cells with no delivery vehicle. C +dextran 72 Figure A - 3 Confocal microscopy of inhibited sil encing - 4 h post - transfection Confocal images of EGFP - expressing H1299 cells ( Cyan ) using 100 nM fluorescently labeled siRNA ( Magenta - +dextran sNP . Images (C - F) were pre - treated with endocytosis inhibitors and imaged 4 h post - transfection. Inhibited pathway (inhibitor): C) Clathrin (Chlorpromazine), D) Caveolae (Filipin), E) Actin (Cytochalasin D), and F) Scavenger Receptors (Dextran Sulfate). 73 Figure A - 4 Confocal microscopy of inhibited silencing 24 h p ost - transfection Confocal images of EGFP - expressing H1299 cells ( Cyan ) using 100 nM fluorescently labeled siRNA ( Magenta - +dextran sNP . Images (C - F) were pre - treated with endocytosis inhibitors and imaged 24 h post - transfection. Inhibited pathway (inhibitor): C) Clathrin (Chlorpromazine), D) Caveolae (Filipin), E) Actin (Cytochalasin D), and F) Scavenger Receptors (Dextran Sulfate). 74 Figure A - 5 EGFP silencing in the pre sence of endocytotic inhibitors (HeLa) Relative fluorescence of EGFP - expressing HeLa cells transfected with siRNA complexes. Cells were pre - treated with endocytosis inhibitors and assayed 24 h post - transfection by flow cytometry. Results were normalized to particle - only controls within corresponding inhibitors. Error bars represent + 1 standard deviation; n = 3. Statistical analysis was performed using two - post - hoc analysis. *Significant differenc e (p < 0.05) as compared to conditions without inhibitors. 75 Table A - 1 Statistical analysis for Figures 2 - 2 and 2 - 3 Analyses were performed using two - - hoc analysis in Origin 8. DF - Degrees of Freedom, Sig Flag - Significance flag, where 0 indicates no significance (p > 0.05) level and 1 indicates significance (p < 0.05) . 76 Table A - 2 Statistical analysis for Figure A - 6 Analyses were performed using two - - hoc analysis in Origin 8. DF - Degrees of Freedom, Sig Flag - Significance flag, where 0 indicates no significance (p > 0.05) level and 1 indicates significance (p < 0.05). Table 6 - 3 Statistical analysis for Figure 2 - 7 Analyses were performed using one - - hoc analysis in Origin 8. Sig Flag - Significance flag, where 0 indicates no significance (p > 0.05) level and 1 indicates si gnificance (p < 0.05) . 77 APPENDIX B: M aterials and M ethods for C hapter 3 Materials Cell Culture 96 - Well Plate (Costar, #3610) 24 - Well Plate (Costar, #3513) 24 - Well Confocal Plate (Ibidi, #82406) DMEM (Life Technologies, #11965092) Fetal Bovine Serum (Atlanta Biological, #S11550) Paraformaldehyde (Sigma, #P6148 - 500g) - Mg/Ca) (Sigma, #D8537) Trypsin (Life Technologies, #25200056) Opti - MEM (Life Technologies, #11058021) Heparin Sul fate (Sigma, #H3393 - 25KU) Lipofectamine 2000 (Life Technologies, #11668019) Fluorescent siRNA: Sense DY547 - - GGCUACGUCCAGGAGCGCA - - UGCGCUCCUGGACGUAGCC - Inhibitors Chlorpromazine hydrochloride (Sigma, #C8138 - 5G) Cytochalasin D (Sigma, #C2618 - 200uL) Filipin Complex III (Sigma, #F4767 - 1MG) Dynasore hydrate (Sigma, #D7693 - 25MG 5 - (N,N - Dimethyl)amiloride (Sigma, #A4562 - 25MG Methyl - - Cyclodextrin (Sigma, #C4555 - 5G) Plasmids pd2EGFP - N1, (Clontech #6009 - 1) wt - dynamin - 2 - pEGFP, (Addgene #34686) EGFP - Actin - 7 (Addgene #56421) GFP - alpha - adaptin[1] GFP clathrin[2] Cav1 - GFP (Addgene #14433) pFlot - 1 - GFP - N1[3] pFlot - 2 - GFP - N1[3] pDEST47 - ARF6 - GFP (Addgene #67394) pEGFP - C3 - GRAF1[4] Solutions Paraformaldehyde Solutions: 2% Paraformaldehyde (w/v) in PBS 78 Media: 10% FBS (v/v) in DMEM Equations The following equations were used in the normalization of fluorescent data. Signals used in the equations below are labeled as Signal (raw fluorescence) ex perimental conditions : Cell Lines EGFP - expressing H1299 and HeLa cells were generously provided by Dr. Jørgen Kjems and Dr. Manfred Gossen, respectively [188,189] . HepG2 and HEK293 cells constitutively expressing EGFP (HepG2 - EGFP and HEK293 - EGFP) were generated using the meth ods outlined in Gossen et al [189] . Briefly, cells were seeded in 6 - well plates and transfected 24 hours post - seeding with Three days post - transfection, cells were sorted and re - plated according to their EGFP expression using a flow cytometer. This process was repeated at seven and fourteen days post - transfection. The average EGFP expression of the final population was analyz ed over several cell cycles and found to be stable. All cell lines were maintained in antibiotic - free DMEM supplemented with 10% fetal bovine serum (FBS). Cells ( Table A - 1 ) (Figures 3 - 1, 3 - 2, and A - 1) (Figures 3 - 1 and 3 - 2) (Figure 3 - 3 ) 79 were incubated at 37°C in 5% CO2, at 100% relative humidity, and subcultured every 4 5 days by trypsinization. EGFP Silencing EGFP - expressing cells were seeded in 24 - well plates at 200,000 cells/well (400,000 cells/well - free DMEM/FBS. Cells were treated 24 hours post - ion containing Opti - MEM, siRNA, and LF2K, yielding final concentrations of 100 nM siRNA and 2.3 µg/mL LF2K. Cells were washed 4 hours post - sulfate for 5 minutes to remove any extracellular siRNAs. The heparin sulfate solution was subsequently removed and replaced with DMEM/FBS. At 24 hours post - transfection, cells were trypsinized, fixed in 2% paraformaldehyde (v/v in DPBS ( - Mg/Ca)), and stored in DPBS ( - Mg/Ca) at 4°C unti l analysis (typically less than 3 days; results were stable 24 days post - fixation). Cells were analyzed by using a Becton Dickinson Influx Flow Cytometer to detect both EGFP (488/530), and Dy547 tagged siRNA (557/574) signal in each event. Samples were, ga ted to include 10,000 events/sample. EGFP fluorescence was measured using an excitation of 488 nm with a multi - line Argon laser. Dy547 - tagged siRNA fluorescence was excited at 552 nm by a HeNe laser. Geometric mean was used to calculate fluorescence intens ity values among samples. Incubations were conducted at 37°C, 5% CO2, and 100% humidity. Cell morphology and EGFP expression as a measure of cytotoxicity were assessed by microscopy and were not significant in any of the treatments. 80 Endocytic Inhibitors Endocytic inhibitors were used for 5 hours at concentrations based on the literature and our own toxicity and dose response experiments (Table 3 - 1, Table A - 1, and Figure A - 1 ). The specificity (or lack thereof) of the inhibitors was assessed from the liter ature yielding a logic matrix that allows for differentiation of the function of different endocytic pathways through comparison of the effects of multiple inhibitors ( Table 3 - 2 ). Inhibition Experiments EGFP - expressing cells were seeded in 24 - well plates at 200,000 cells/well (400,000 cells/well - free DMEM/FBS. After 23 hours, cells were washed with DMEM and incubated for 1 hour in DMEM containing inhibitors (Table 3 - 1). Cells were then transfected with siRNA s as abo ve. Cells were washed 4 hours post - transfection with antibiotic - free DMEM/FBS and incubated in heparin sulfate solution for 5 minutes to remove extracellular siRNAs. The heparin sulfate solution was subsequently removed and replaced with antibiotic - free DM EM/FBS. At 24 hours post - transfection, cells were trypsinized, fixed using a 2% paraformaldehyde solution, and stored in DPBS ( - Mg/Ca) at 4°C until analysis. All incubations were conducted at 37°C, 5% CO2, and 100% humidity. Cells were then analyzed by flo w cytometry and microscopy ( Figure A - 2 and A - 3 ) as above. Co - culture Inhibitor Experiments HeLa, H1299, HEK293, and HepG2 cell lines, only one expressing EGFP, were mixed and seeded into 24 - well plates at a density of 50,000 cells/well for HeLa, H1299, an d HEK293 and 81 DMEM/FBS. Cells were treated with siRNAs, fixed, and analyzed by flow cytometry as above. Endocytic Protein Overexpression Experiments Cells were seede d in 24 - well plates at a density of 150,000 cells/well (300,000 cells/well for - free DMEM/FBS. After 24 hours, cells were transfected - MEM, Lipofectamine 3000 (LF3K), an d one of the following plasmids: pd2EGFP - N1 (EGFP; control), wt dynamin 2 pEGFP (Dynamin), EGFP - Actin - 7 (Actin), GFP - alpha - adaptin (AP2) (kin dly provided by J. Rappoport [190] ), GFP clathrin (Clathrin) (kindly provided by J. Keen [191] ), Cav1 - GFP (Caveolin), pFlot - 1 - GFP - N1 (Flot 1) (ki ndly provided by R. Tikkanen [192] ), pFlot - 2 - GFP - N1 (Flot 2) (ki ndly provi ded by R. Tikkanen [192] ), pDEST47 - ARF6 - GFP (Arf6), and pEGFP - C3 - GRAF1 (Graf1) (ki ndly provided by R. Lundmark [56] ). Concentrations (after addition to the growth med ia) were optimized for both toxicity and expression level: HeLa and H1299 (150 ng plasmid, 0.55 µg LF3K), HEK293 (600 ng plasmid, 2.2 µg LF3K), and HepG2 (800 ng plasmid, 4.4 µg LF3K). Cells were washed 6 hours after plasmid transfection with antibiotic - fr ee DMEM/FBS. 24 hours after plasmid transfection, cells were transfected with siRNAs, as above. Cells were then fixed and analyzed by flow cytometry and microscopy ( Figure A - 4 ) as above. Statistics Statistical analyses were performed using one - way ANOVA, - hoc analysis (Table A - 2) . 82 Endocytic Inhib itor Toxicity and Dose Response Each endocytic inhibitor was evaluated over a range of concentrations for each cell line to assess both toxicity and dose response. EGFP - expressing cell s were seeded in 96 - well plates at - free DMEM/FBS. After 23 hours, cells were washed with DMEM and incubated for 1 hour in DMEM containing inhibitors. For toxicity assessment, cel - MEM ( Table A - 1 ). - MEM, siRNA, and LF2K, yielding final concentrations of 100 nM siRNA and 2.3 µg/mL LF2K (Figure A - 1). Cells were washed 4 hours post - transfection with antibiotic - free DMEM/FBS and incubated in heparin sulfate solution for 5 minutes to remove extracellular siRNAs. The heparin sulfate solution was subsequently removed and replaced with antibiotic - free DM EM/FBS. At 24 hours post - transfection, cells were washed with DPBS (+Mg/Ca) and analyzed using a BioTek Synergy H1 plate reader. All incubations were conducted at 37°C, 5% CO2, and 100% humidity. Confocal Microscopy For the cellular images of the inhibitor experiments, cells were fixed 24 hours post - transfection, using a 2% paraformaldehyde solution, and stored in DPBS (+Mg/Ca) at 4°C. Confocal images were taken using a Nikon A1 laser scanning confocal microscope. Nik on Plan Apo 20×/.75NA and Apo 60x/1.4NA objectives were used to acquire all images. EGFP (488/530) fluorescence was measured using an excitation of 488 nm with a multi - line Argon laser and displayed as green (LUT). Dy547 - tagged siRNA (557/574) fluorescence was excited at 560 nm by a HeNe laser and displayed as red (LUT). The focal plane for each image was chosen to include the highest intensity EGFP fluorescence and maintained using the Nikon Perfect 83 Focus System. All images were collected sequentially as s ingle XY images and used 2 count Line Kalman averaging. For overexpression images (Figure A - 2), cells were fixed 1 hour after siRNA transfection using a 2% paraformaldehyde solution and stored in DPBS (+Mg/Ca) at 4°C. Confocal images were taken as above. Table A - 4 Inhibitor toxicity Inhibitor toxicity was assessed in each cell line by measuring EGFP fluorescence over a range of inhibitor M) (Figure 3 - 5). 84 Figure A - 6 Inhibitor dose response The effect of each inhibitor on EGFP silencing was assessed over a range of inhibitor concentrations. The value listed below each cell line represents the inhibitor concentration at which 5% toxicity was observed ( Table A - 1 ). Data points that exceed the 5% toxicity dose are indicated with open symbols. The working concentration chosen for each inhibitor is indica ted with a vertical black line. 85 Figure A - 6 86 Figure A - 6 87 Figure A - 7 Inhibitor microscopy experiments Confocal microscopy images of EGFP - expressing cells ( Cyan ) transfected with 100 nM fluorescently labeled siRNA ( Magenta Cells were pre - treated with endocytosis inhibitors and imaged 24 hours post - 88 Figure A - 8 Flow cytometry f luorescence d istribution Fluoresce nce distribution of siRNA and E GFP in un - inhibited cell lines. The fluorescent signal in the siRNA channel of un - transfected cell ( - LF2K, - siRNA) was to establish a population gate for siRNA positive cells. Figure 6 - 9 Flow cytometry f luorescence d istribution Fluorescence distribution of siRNA and GFP in un - inhibited cell lines. The fluorescent signal in the siRNA channel of un - transfected cell ( - LF2K, - siRNA) was to establish a population g ate for siRNA positive cells. 89 Figure A - 10 Overexpression microscopy experiment Confocal microscopy images of cells overexpressing EGFP - labeled endocytic proteins ( Cyan ) and transfected with 100 nM fluorescently labeled siRNA ( Magenta - LF2K complexes. Scale 90 Table A - 5 Statistical analysis for Figures 3 - 2, 3 - 3, and 3 - 4 p - values for Figures 3 - 2, 3 - 3, and 3 - 4. Analyses were performed using two - way ANOVA, followed by - hoc analysis in Origin 8. p < 0.05 was used to determine significance. Table A - 6 Gene e xpression Gene expre ssion data for endocytic proteins. Expression is listed in transcripts per million (TPM) from Affymetrix data and normalized in GENEVESTIGATOR®. The following experimental IDs were used: HS - 00859, HS - 01099, HS - 00217, HS - 01921, HS - 00048, and HS - 00856. 91 APPENDIX C: Materials and Methods for Chapter 4 Materials Cell Culture 6 - Well Plate (Costar, #3516) 15 - Well Confocal Plate (Ibidi, #81506) DMEM (Life Technologies, #11965092) Fetal Bovine Serum (Atlanta Biological, #S11550) Trypsin (Life Technologies, #25200056) Opti - MEM (Life Technologies, #11058021) Lipofectamine 2000 (Life Technologies, #11668019) - UGCGCUCCUGGACGUAGCCUU - - Q570 - (Sigma) - GGCUACGUCCAGGAGCGCAUU - 3 - (Sigma) Plasmids pEGFP - C1 RAB11A , ( Addgene # 12674 ) pEGFP - C1 - RAB4b, (Addgene #49468) pEGFP - ER - 14, (Addgene #56432) pEGFP - N3 - LAMP1, (Addgene #16290) Cell Lines HeLa cells constitutively expressing EGFP labeled proteins Rab5 and Rab7 were generously provided by Matthew Seaman (University of Cambridge). HeLa cells constitutively expressing EGFP labeled proteins (Rab4, Rab11, Lamp1, and Calreticulin) were generated using published methods [18] . Briefly, cells were seeded in 6 - well plates and transfected 24 hours post - seeding - C1 RAB11A (Rab11), pEGFP - C1 - RAB4b (Rab4), pEGFP - ER - 14 (Endoplasmic Reticulum), or pEGFP - N3 - LAMP1 (Lysosome). Three days post - transf ection, cells were sorted and re - plated according to their EGFP expression using a flow cytometer. This process was repeated at seven and fourteen days post - transfection. The average EGFP expression of the final population was analyzed over several cell cy cles and found to be stable. All cell lines were maintained in 92 antibiotic - free DMEM supplemented with 10% fetal bovine serum (FBS). Cells were incubated at 37°C in 5% CO 2 , at 100% relative humidity, and subcultured every 4 5 days by trypsinization. Intrac ellular Trafficking HeLa cells constitutively expressing an EGFP - labeled protein (Rab4, Rab5, Rab7, Rab11, Calreticulin, and Lamp1) were seeded in 15 - well confocal plates at a density of 1000 cells/well and cultured in antibiotic - free growth media (DMEM+FB S). 24 hours after seeding, cells were transferred to a stage - top incubator chamber and incubated at 37°C, 5% CO2 and 100% relative humidity. The X, Y, and Z positions were recorded for 3locations in each well. Cells were then ction solution containing Opti - MEM, siRNA, and sNPs, yielding positions in each well, images were collected at ~30 minute intervals over ~24 hours. Cell morphology was monitored for signs of cytotoxicity, which was not observed at any time. Image Acquisition and Analysis Images were acquired on a Nikon A1 confocal laser scanning microscope using a Nikon Plan Fluor 40×/.75 dry objective. EGFP (488/530) fluorescence was m easured using an excitation of 488 nm with a multi - line Argon laser. Q570 (560/595) fluorescence (siRNA guide strand) was excited at 560 nm by a HeNe laser. Q670 (647/700) fluorescence (siRNA passenger strand) was excited at 647 nm by a HeNe laser. The foc al plane for each image was chosen to maximize EGFP fluorescence intensity, which should be the focal plane through the middle of the cells, and maintained using the Nikon Perfect Focus System. 93 Images were acquired sequentially as single XY images using tw o - count Line Kalman averaging. Each well was imaged at its first position prior to returning to the first well and then imaging the second position in each well. After acquiring images at three positions in each well, the sequence began again at the first position in the first well. Using this imaging approach minimized photobleaching while also minimizing the time between images for a given well. 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