The use of silk-elastinlike proteins and semi-synthetic glycosaminoglycans for a prophylactic approach to treat radiation-induced proctitis

Radiation-induced proctitis is an injury of the rectum that is a common adverse event occurring from radiotherapy to the lower abdominal region. This inflammatory event results in acute and chronic presentation reducing patient quality of life. Currently treatment strategies are limited with very few prophylactic approaches in the clinic. To address this unmet need I have investigated rectal controlled delivery of an antiinflammatory agent. Semisynthetic glycosaminoglycan ethers (SAGEs) are a family of glycosaminoglycan ethers consisting of short, sulfated hyaluronans. The short length and chemical sulfation of these SAGEs gives them novel therapeutic properties. These include anti-inflammatory properties such as inhibition of pattern recognition receptors, inflammatory transcription factors, and of growth of gram-negative bacteria. The global hypothesis of this dissertation is that silk-elastinlike protein polymers (SELP) improve the bioaccumulation of SAGE in the rectum and increase its radioprotective behavior. I investigated several SELP analogs for development of a liquid to semisolid drug delivery depot of SAGE. In vitro screenings focused on mechanical properties, gel formation, and release kinetics. In a prophylactic radiation-induced proctitis (RIP) mouse model, the selected SELP-SAGE combination was capable of protecting against radiation after 3 and 7 days. This combination increased the survival of mice, as determined by weight. The molecular iv mechanisms of this combination were evaluated using transcriptomics with further indications of pattern recognition receptors playing a role in the pathology of RIP.

THE USE OF SILK-ELASTINLIKE PROTEINS AND SEMI-SYNTHETIC GLYCOSAMINOGLYCANS FOR A PROPHYLACTIC APPROACH TO TREAT RADIATION-INDUCED PROCTITIS by Douglas Steinhauff A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Biomedical Engineering The University of Utah May 2022 Copyright © Douglas Steinhauff 2022 All Rights Reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Douglas Steinhauff has been approved by the following supervisory committee members: Hamidreza S. Ghandehari , Chair 02/28/2022 Siam Oottamasathien , Member 02/21/2022 Jindrich Kopecek , Member 02/25/2022 Mingnan Chen , Member 02/24/2022 Michael Seungju Yu , Member and by the Department/College/School of David Grainger Date Approved Date Approved Date Approved Date Approved Date Approved , Chair/Dean of Biomedical Engineering and by David B. Kieda, Dean of The Graduate School. ABSTRACT Radiation-induced proctitis is an injury of the rectum that is a common adverse event occurring from radiotherapy to the lower abdominal region. This inflammatory event results in acute and chronic presentation reducing patient quality of life. Currently treatment strategies are limited with very few prophylactic approaches in the clinic. To address this unmet need I have investigated rectal controlled delivery of an antiinflammatory agent. Semisynthetic glycosaminoglycan ethers (SAGEs) are a family of glycosaminoglycan ethers consisting of short, sulfated hyaluronans. The short length and chemical sulfation of these SAGEs gives them novel therapeutic properties. These include anti-inflammatory properties such as inhibition of pattern recognition receptors, inflammatory transcription factors, and of growth of gram-negative bacteria. The global hypothesis of this dissertation is that silk-elastinlike protein polymers (SELP) improve the bioaccumulation of SAGE in the rectum and increase its radioprotective behavior. I investigated several SELP analogs for development of a liquid to semisolid drug delivery depot of SAGE. In vitro screenings focused on mechanical properties, gel formation, and release kinetics. In a prophylactic radiation-induced proctitis (RIP) mouse model, the selected SELP-SAGE combination was capable of protecting against radiation after 3 and 7 days. This combination increased the survival of mice, as determined by weight. The molecular mechanisms of this combination were evaluated using transcriptomics with further indications of pattern recognition receptors playing a role in the pathology of RIP. iv I dedicate this work to my friends, family, colleagues, and young scientists in the world who want to learn. We all must start somewhere. TABLE OF CONTENTS ABSTRACT....................................................................................................................... iii LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ............................................................................................................ x LIST OF ABBREVIATIONS ........................................................................................... xii ACKNOWLEDGMENTS ............................................................................................... xvi Chapters 1. INTRODUCTION .......................................................................................................... 1 1.1 Radiation-Induced Proctitis .................................................................................. 1 1.2 Semi-Synthetic Glycosaminoglycan Ethers......................................................... 3 1.3 Controlled Delivery with Silk-Elastinlike-Based Hydrogels............................... 6 1.4 Global Hypothesis................................................................................................ 9 1.5 Dissertation Specific Aims .................................................................................. 9 1.5.1 Specific Aim 1: To Determine an Optimal SAGE Polymer Composition for a Prophylactic Approach to Radiation-Induced Proctitis...... 9 1.5.2 Specific Aim 2: To Evaluate the Combination of Oligomeric Sulfated Hyaluronans and Silk-Elastinlike Protein Polymers for Protection of Early and Developed Radiation-Induced Proctitis .................................................... 11 1.6 Scope and Organization ..................................................................................... 11 1.7 References .......................................................................................................... 13 2. LITERATURE REVIEW ............................................................................................. 17 2.1 Radiation-Induced Proctitis ................................................................................ 17 2.2 Sulfated Hyaluronans ......................................................................................... 25 2.3 Silk-Elastinlike Protein Polymers ...................................................................... 31 2.4 Conclusions ........................................................................................................ 44 2.5 References .......................................................................................................... 45 3. SILK-ELASTINLIKE COPOLYMERS ENHANCE BIOACCUMULATION OF SEMISYNTHETIC GLYCOSAMINOGLYCAN ETHERS FOR PREVENTION OF RADIATION-INDUCED PROCTITIS ............................................ 58 3.1 Abstract .............................................................................................................. 58 3.2 Introduction ........................................................................................................ 59 3.3 Materials and Methods....................................................................................... 63 3.3.1 Materials ................................................................................................ 63 3.3.2 Macroscopic Phase Separation .............................................................. 64 3.3.3 Rheology ................................................................................................ 64 3.3.4 In Vitro Release ..................................................................................... 65 3.3.5 In Vivo Accumulation............................................................................ 66 3.3.6 In Vivo Efficacy ..................................................................................... 67 3.3.7 Pain Assessment..................................................................................... 68 3.3.8 Tissue Collection and Histological Assessment .................................... 68 3.3.9 Statistical Analysis ................................................................................. 69 3.4 Results ................................................................................................................ 69 3.4.1 Macroscopic Phase Separation .............................................................. 69 3.4.2 Rheological Analyses of Control Polymers and Polymers Formulated with GM-0111................................................................................................. 70 3.4.3 In Vitro Release ..................................................................................... 74 3.4.4 GM-0111 Bioaccumulation in the Rectum of BDF-1 Mice .................. 76 3.4.5 Behavioral Pain Responses .................................................................... 80 3.4.6 Animal Health ........................................................................................ 82 3.4.7 Histology ................................................................................................ 84 3.5 Discussion .......................................................................................................... 86 3.6 Conclusion ......................................................................................................... 94 3.7 References .......................................................................................................... 95 4. AN OLIGOMERIC SULFATED HYALURONAN AND SILK-ELASTINLIKE POLYMER COMBINATION PROTECTS AGAINST MURINE RADATIONINDUCED PROCTITIS ................................................................................................. 104 4.1 Abstract ............................................................................................................ 104 4.2 Introduction ...................................................................................................... 105 4.3 Materials and Methods..................................................................................... 108 4.3.1 Materials .............................................................................................. 108 4.3.2 Mouse Treatment and Irradiation......................................................... 108 4.3.3 Behavioral Pain Testing ....................................................................... 109 4.3.4 Histology .............................................................................................. 110 4.3.5 RNA Sequencing ................................................................................. 111 4.3.6 Statistical Analysis ............................................................................... 112 4.4 Results .............................................................................................................. 112 4.4.1 3-Day Behavioral Pain Responses ....................................................... 112 4.4.2 3-Day Histological Outcomes .............................................................. 114 4.4.3 Animal Survival Curves....................................................................... 115 4.4.4 Behavioral Pain Responses at Survival Endpoint ................................ 118 4.4.5 Histology at Survival Endpoint............................................................ 120 4.4.6 RNA Sequencing of Rectal Tissues ..................................................... 120 4.5 Discussion ........................................................................................................ 127 4.6 Conclusion ....................................................................................................... 131 4.7 References ........................................................................................................ 132 vii 5. CONCLUSION ........................................................................................................... 138 5.1 Overview .......................................................................................................... 138 5.2 Therapeutic Mechanisms of SAGEs ................................................................ 139 5.3 Rectal Delivery via SELPs............................................................................... 140 5.4 Advancing SAGE-SELP Combinations to Treat RIP...................................... 141 5.5 Summary .......................................................................................................... 143 5.6 References ........................................................................................................ 143 Appendices A. CHAPTER 3 SUPPLEMENTAL INFORMATION ................................................. 146 B. CHAPTER 4 SUPPLEMENTAL INFORMATION ................................................. 150 C. MATRIX-MEDIATED VIRAL GENE DELIVERY: A REVIEW .......................... 196 D. DEVELOPMENT OF A THERMORESPONSIVE PROTEIN COMPLEX FOR TARGETING CD20 RECEPTORS....................................................................... 247 viii LIST OF TABLES Tables 4.1: Canonical pathways identified from protection of SELP-415K/GM-0111 in a RIP model ....................................................................................................................... 123 4.2: Upstream regulators identified from protection with SELP-415K/GM-0111 in RIP model ....................................................................................................................... 125 B.1. Differentially expressed genes ................................................................................ 155 B.2 Identified canonical pathways .................................................................................. 162 B.3. Identified upstream regulators ................................................................................. 165 B.4. Identified gene ontology terms ................................................................................ 186 C.1:Various viral vectors used in gene therapy and respective physiochemical properties with the desired duration of transgene expression need to be considered ..... 199 C.2: Particulate matrix systems used to encapsulate viral vectors.................................. 203 C.3: Polymeric matrices used in matrix-mediated viral delivery ................................... 208 LIST OF FIGURES Figures 1.1: Structure of GM-0111, a semi-synthetic glycosaminoglycan ether ............................ 5 1.2: Primary structures of silk-elastinlike protein polymers ............................................... 8 1.3: Graphical abstract illustrating the enhancement of SAGEs provided by thermoresponsive SELPs .................................................................................................. 10 3.1: Structures of therapeutic and polymers ..................................................................... 62 3.2: Viscosity of candidate formulations .......................................................................... 71 3.3: Storage moduli and gelation behavior of candidate formulations ............................. 73 3.4: Controlled release of glycosaminoglycan .................................................................. 75 3.5: Assessment of rectal bioaccumulation ....................................................................... 77 3.6: Analysis of GM-0111 rectal bioaccumulation via confocal microscopy .................. 79 3.7: Behavioral response rates of irradiated BDF-1 mice ................................................. 81 3.8: Assessment of animal mass and fecal matter............................................................. 83 3.9: Histological analysis of rectal tissues ........................................................................ 85 4.1: Behavioral pain responses 3-day post irradiation .................................................... 113 4.2: Histological analysis of tissues 3 days following irradiation .................................. 116 4.3: Animal body weights and survival .......................................................................... 117 4.4: Behavioral pain responses at the time of sacrifice ................................................... 119 4.5: Time of sacrifice histological evaluation of rectal tissues ....................................... 121 4.6: EnrichR analysis of differentially expressed genes (p adjusted < 0.05) with an absolute log2 fold change greater than 1 ........................................................................ 126 A.1: Macroscopic phase separation of polymer SAGE combinations ............................ 148 A.2: Confocal microscopy of polymer SAGE combinations instilled in the rectums ............................................................................................................................ 149 B.1: Scoring of histological observations 3 days after treatment and irradiation ........... 152 B.2: Scoring of histological observations 3 days after treatment and irradiation ........... 154 C.1: Several polymer systems discussed in this review ................................................. 201 C.2: Poloxamers form micelles at low concentrations and gels of intertangled micelles at higher concentrations .................................................................................... 210 C.3: Enzymatic matrix degradation can increase target cell transduction ...................... 216 C.4: Silk-elastinlike protein polymers for gene delivery ................................................ 221 D.1: Protein subunits and approach ................................................................................ 250 D.2: Confirmation of ELP-CCE protein subunits ........................................................... 252 D.3: Characterization of ELP-CCE subunits .................................................................. 254 D.4: Generation of Rit-CCK subunit .............................................................................. 257 D.5: Analysis of protein complex binding to CD20 ....................................................... 259 xi LIST OF ABBREVIATIONS αCD α-cyclodextrin 5-ASA 5-aminosalicylic acid 633GM-0111 Fluorescently labeled GM-0111 8,1-ANS 8-Anilinonaphthalene-1-sulfonic acid AAV Adeno-associated virus AD Adenovirus BMP Bone morphogenetic protein CAM Chorioallantoic membrane CCE-V192 CCE ELP fusion protein with 192 repeats CCE-V48 CCE ELP fusion protein with 48 repeats CFC Critical formation concentration CS Chondroitin sulfate D Dose DMSO Dimethyl sulfoxide EDC N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide EDTA Ethylenediaminetetraacetic acid ELP Elastinlike polypeptide FAK Focal adhesion kinase G Guluronate G’ Storage modulus G’’ Loss modulus GDEPT Gene-directed enzyme-prodrug therapy PBS/GM-0111 GM-0111 in PBS SELP-415K/GM-0111 GM-0111 in SELP-415K GFP Green fluorescent protein H&E Hematoxylin and eosin HA Hyaluronic acid hMSCs Human mesenchymal stem cells HPLCs Human periodontal ligament cells IVIS In vitro imaging system LCST Lower critical solution temperature LV Lentivirus M Mannonate MM Matrix-mediated MW Molecular Weight MMP Matrix Metalloproteinase na Non-allodynic NF-KB Nuclear factor kappa beta NRF2 Nuclear factor erythroid 2-related factor 2 oAD Oncolytic adenovirus PAMPs Pathogen associated molecular pattern PBS Phosphate buffer saline xiii PDGFB Platelet derived growth factor beta PDGF-C Platelet derived growth factor C PEG poly(ethylene glycol) (PEG-PPO-PEG) poly(ethylene glycol)-block- poly(propylene oxide)-blockpoly(ethylene glycol) PEGmp Macroporous PEG hydrogels PEEUU Polyester ether urethane urea PEUU Polyester urethane urea PF-Alg Pluronics and alginate matrices PLGA Poly(lactic-co-glycolic acid) PLGA-PEG-PLGA (Poly(D,L-lactide-co-glycolide)-block-(poly(ethylene glycol)-block-poly(D,L-lactide-co-glycolide) PPDs Polypsuedorotaxane PRR Pattern recognition receptor RAGE Receptor against glycation end products rAAV Recombinant adeno-associated virus RIP Radiation-induced proctitis Rit-CCK Rituximab CCK conjugate RS-SELPs Responsive SELPs RS1 Responsive SELP 1 RS2 Responsive SELP 2 RS5 Responsive SELP 5 SAGE Semisynthetic glycosaminoglycan ether SELP Silk-elastinlike protein polymers sHA Sulfated hyaluronic acid xiv SF Surviving Fraction Standard single letter amino acid abbreviations TLR Toll like receptors TROMS Total recirculation one-machine system VEGF Vascular endothelial growth factor VSV-G Vesicular stomatitis virus glycoprotein G X Guest residue xv ACKNOWLEDGMENTS I would like to thank my family for their continued support and encouragement through my career in graduate school. Thank you to all my friends that I have made in the Biomedical Engineering Department and in Utah. Together, we explored life outside of work and maintained a healthy work life balance. These friends challenged me beyond the lab, to learn new things, and think differently. Without this group of people, I may have never made it through the entirety of my degree. They have largely helped shape me over the last few years and I am happy to say they will be close friends for life. Thank you to all the outstanding people within the Ghandehari Lab. Martin Jensen and Kyle Isaacson were very welcoming of me to the SELP team and pushed me to be the best I could in very different ways. Thank you to all my students that I have had over the years, of which I have lost count but not forgotten. Our time learning together was fun AND fruitful. You challenged me to become an effective mentor, a skill that I can now appreciate in detail as I move on. You kept me going some days with your eagerness and curiosity. It is my own reward to get to witness each of your own successes. A special thank you to all of my mentors. Without them I would be on a very different path. My mentors at Arizona State, Michael Caplan and Sarah Stabenfeldt, convinced me to pursue graduate school through much of my own reluctance. Michael was the one that pushed me to apply for this degree. He managed to overcome my stubbornness and for that I am very very thankful. Thank you to Martin Jensen, as he served as my mentor through much of my early years. Without him, my experimental and scientific knowledge would be far less than it is today. Thank you to my PI, Hamid Ghandehari, for his guidance and wisdom. Thank you to Siam Oottamasathien for guidance on my project and dissertation. And thank you to all committee members and coauthors. This would not have been possible without you. xvii CHAPTER 1 INTRODUCTION 1.1 Radiation-Induced Proctitis Lower abdominal cancers are commonly treated with regimens of radiotherapy. A common side effect of this therapy is radiation-induced proctitis (RIP). Of the patients receiving lower abdominal therapy, up to 20% will develop acute symptomology and potentially will develop late, chronic symptomology [1]. The exact prevalence of RIP is slightly obscured due to difficulties in diagnosing and other conditions that may present similar symptomology [2]. Acute RIP is presented with symptoms of diarrhea, abdominal pain, stool incontinence or urgency, rectal bleeding, and more. Chronic injury is presented with more severity of the aforementioned symptomology, along with more severe pathophysiology including fistulas, strictures, bowel obstruction, and perforations [3]. While treatment of acute RIP can typically be self-managed through cessation of radiotherapy schedules, chronic RIP necessitates further intervention for management. Chronic RIP can develop early following radiation exposure, or much later (months to years) [3]. While the pathological forces of RIP are not fully understood, they include mucosal injury, ischemia, and inflammatory responses [4]. Endoscopically, chronic RIP is commonly confirmed with presentation of pallor, mucosal friability, and edema [2]. To 2 address these tissue damages a range of non-invasive and invasive treatment options may be available to patients. Non-invasive treatment options largely surround the use of oral, gaseous, or rectal administration of therapeutic agents. These include antioxidants, anti-inflammatories, sucralfate, short chain fatty acids and more. Invasive interventions include ablative approaches and surgery. Ablative approaches are used for refractory cases and injury management. Surgical intervention is considered to be a last resort and is associated with poor outcomes and high rates of complications [4]. Preventative measures of RIP include, but is not limited to, prophylactic medication approaches and rectal spacers. The goal of varying radiotherapy techniques is to limit rectal doses while maximizing target doses. This can be accomplished to some degree with intensity modulation and image guided therapy [3]. Several newer techniques utilize heavy radio particles, which have shown improved outcomes and lower toxicities. However, the long-term effects of these systems are still being evaluated [5]. Several prophylactic strategies are also being explored. The use of amifostine, sucralfate, 5aminosalicylic acid, and sulphasalazine have only minimal effect in preventative strategies [3]. Properly controlled clinical trials of sucralfate have shown no benefit when administered orally or topically [6,7]. Rectal spacers are devices which have the goal of sparing rectal structures during radiotherapy. Endorectal balloons are inserted prior to each dose and increase the space between rectal walls and target organs. Rectal spacers are inserted surgically and remain throughout the duration of the radiotherapy schedule. These are typically biodegradable (within 6 months) and reduce the rectal dose by maximizing distances between target organs and the rectal wall [3]. 3 While several preventative options have been explored, many of these approaches suffer from small samples sizes, minimal improvement in outcomes, non-blinded trials and/or short follow up periods [4]. Clinically, a prophylactic approach to prevent RIP is needed with novel delivery of anti-inflammatory agents. This approach needs to address clinically observed declines in patient quality of life including symptoms such as pain, bleeding, and diarrhea. A successful preventative measure will be able to address presented symptoms as well as tissue associated damages due to radiation. An approach of this type could allow for improvement of patient outcomes and even schedules of radiotherapy in the treatment of cancer. 1.2 Semi-Synthetic Glycosaminoglycan Ethers Naturally occurring biomacromolecules are attractive candidates as therapeutics due to their biocompatibility and natural programming. Glycosaminoglycans, such as heparin, hyaluronic acid, and chondroitin sulfate, are abundantly present within biological systems and serve as important structural and anti-inflammatory mediators [8]. Further chemical and structural programming of several glycosaminoglycans have resulted in the creation of semi-synthetic glycosaminoglycans (SAGEs). These SAGEs combine natural and synthetic biological programming to achieve therapeutics with novel and hybrid properties [9]. One type of SAGE is derived from the fermentation of hyaluronic acid, chemically alkylated, digested with hyaluronidase, and chemically sulfated. The combination of a short hyaluronic acid backbone with sulfated chemical groups instilled novel properties within this molecule [10]. For instance, SAGE contains a higher stability 4 due to its resistance to hyaluronidases and weak inhibition of chondroitinases and heparinases. The fermented nature of SAGE eliminates the risk of potential inflammatory contamination found within animal products [11]. This process can be adjusted to produce a variety of SAGEs, for example GM-1111 and GM-0111 (Figure 1.1). One of the proposed mechanisms of these SAGEs centers around the inhibition of the Receptor Against Glycation End Products (RAGE). The V domain of RAGE consists of a hydrophobic pocket ringed with basic residues and a cationic center. This may provide an ionic interaction site for the sulfated groups on SAGEs [10]. RAGE is considered an amplifier of inflammation through the major inflammatory transcription factor nuclear factor-κβ [12]. The blocking of toll-like receptors (TLRs) -2 and -4 have also been noted as a mechanism of SAGEs, limiting downstream inflammatory cytokine release [9]. Recruitment of neutrophils and leukocytes has also been decreased in disease models treated with SAGEs [10]. These various mechanisms have made SAGEs an attractive therapeutic for antiinflammatory diseases. Reduction in cutaneous inflammation was observed in a murine model [10]. Protection against sinonasal inflammation was increased with the use of SAGE verses its derivative, hyaluronic acid, and controls as indicated by mast cell infiltration, lamina propria thickening, and myeloperoxidase levels [11]. Treatment of interstitial cystitis, also known as painful bladder syndrome, has benefited from intravesical delivery of SAGE both in soluble form and with thermoresponsive polymers [13,14]. Polymer formulations were capable of having an increased analgesic and anti-inflammatory effect compared to non-polymer formulations [14]. Protection and treatment against radiation-induced inflammation has also shown promise in the settings 5 Figure 1.1: Structure of GM-0111, a semi-synthetic glycosaminoglycan ether. 6 of RIP and radiation-induced mucositis [15,16]. Studies of mucositis suggest targeting of pattern recognition receptors and the inhibition of the nlrp3 inflammasome [16].The novel and extensive anti-inflammatory mechanisms provided by SAGE merit its investigation in further anti-inflammatory diseases. While RIP has previously been explored with SAGEs, we believe we can further improve this treatment regimen through carefully tailored drug delivery strategies. Currently, approaches in the clinic use enema systems consisting of liquid states of varying viscosities. This may limit exposures of SAGEs to the rectal tissues due to limited residence time in the rectal cavity. The development of a liquid to semisolid enema could maximize the drug accumulation in the tissue leading to enhanced therapeutic outcomes. 1.3 Controlled Delivery with Silk-Elastinlike-Based Hydrogels Recombinant silk-elastinlike proteins (SELPs) consist of repeats of silk (G-A-GA-G-S) and elastin (V-P-G-V-G) motifs [17]. The combination of these motifs renders these protein-based polymers with the strength of silk and thermoresponsive nature of elastin. The genetic design of these proteins results in block copolymer molecules capable of being produced by Escherichia coli [17]. Following fermentation, bioprocessing of SELPs can result in a pure, endotoxin free product. Further processing under high shearing conditions can result in further variation, optimization, and normalization of batch to batch material properties [18]. Depending on polymer sequence and concentration, SELPs can exist in a soluble state at cooler temperatures and, upon heating, form gel like structures. The formation of these structures center around the thermoresponsiveness of elastin to bring polymer 7 strands together, upon which silk motifs begin to form antiparallel beta-sheet crosslinks with one another [19]. This leads to a robust hydrogel structure. The mechanics and conditions that support this sol to gel transition vary upon the specific SELP analog being discussed. In our lab we have investigated the use of SELP-815K, -415K, and -47K for controlled release applications [14,15,27–30,19–26]. The primary structure of these polymers (Figure 1.2) informs each of their microscopic assemblies, macroscopic gel formations, and their corresponding physiochemical properties [31,32]. The variation in primary polymer structure and resulting assemblies have allowed us to study varying SELPs for application in gene and drug delivery [30,31]. The incorporation of SAGEs into SELPs has also been investigated. The cumulative release rates of SAGEs from SELP-815K increase with increasing concentration of SAGEs, suggesting ionic retention in matrices. This drug-polymer interaction is also indicated by varying mechanical properties upon drug loading [14,15]. Delivery of SAGE via SELP matrices in vivo resulted in improved treatment of interstitial cystitis and protection against RIP, compared to free SAGE. This proof of concept study illustrated the therapeutic enhancement provided by SELP mediated delivery, specifically the sol to gel transition that SELPs use to create a local drug depot. However, only a single SELP polymer was investigated. Further development of this liquid to semi-solid enema system could benefit from the investigation of polymer systems with varying mechanical properties, liquid viscosities, and gelation rates. The various structures of SELP that have been generated (Figure 1.2) provide us with a set of thermoresponsive polymer carriers with a variation of mechanical properties. SELP-815K is more stiff and gels more quickly than its SELP-415K 8 Figure 1.2: Primary structures of silk-elastinlike protein polymers. 9 counterpart [31]. SELP-415K and -815K viscosities are both below 10 PaS and considered to be injectable [14]. By evaluating in vitro properties and in vivo accumulation we can correlate the differences between each polymer and rectal bioaccumulation. This provides an opportunity to delineate the relevant mechanical properties of SELPs, such as mechanical stiffness, gelation rate, or viscosities which are crucial for rectal bioaccumulation of SAGEs. 1.4 Global Hypothesis By utilizing thermoresponsive polymer vehicles, we can enhance SAGE bioaccumulation through controlled release within the rectum. This enhanced accumulation will result in a higher degree of radioprotection against radiation-induced proctitis in a murine model. 1.5 Dissertation Specific Aims 1.5.1 Specific Aim 1: To Determine an Optimal SAGE Polymer Composition for a Prophylactic Approach to Radiation-Induced Proctitis We hypothesize that thermoresponsive polymers could be utilized for enhancing rectal bioaccumulation of SAGE (Figure 1.3). Several polymers were explored in combination with SAGE. These combinations were evaluated for mechanical properties and release kinetics before assessing in vivo rectal accumulation. The lead candidate from these combinations was then used to assess a prophylactic approach for RIP using a murine model. This model assessed for protection against behavioral pain and rectal 10 Figure 1.3: Graphical abstract illustrating the enhancement of SAGEs provided by thermoresponsive SELPs. 11 tissue toxicities. 1.5.2 Specific Aim 2: To Evaluate the Combination of Oligomeric Sulfated Hyaluronans and Silk-Elastinlike Protein Polymers for Protection of Early and Developed Radiation-Induced Proctitis We hypothesize that our lead SAGE SELP candidate is capable of ameliorating early pathophysiology and symptomology of RIP in acute conditions and increasing survival compared to control groups. SELP-415K SAGE compositions were able to extend the survival of mice following radiation exposure. This combination also illustrated 3-day endpoint protection against pain and developing toxicities within rectal tissues. 1.6 Scope and Organization In this dissertation we aimed to develop an effective prophylactic strategy to address acute radiation-induced proctitis, and the symptomology it presents. We utilized several different thermoresponsive polymers, with primary and secondary structural components, to identify a polymer formulation that could best deliver a SAGE into rectal tissue. This was accomplished by first assessing in vitro properties, including macroscopic phase separation, rheology, and release kinetics. Careful characterization of this nature informed us of important formulation physiochemical properties, as well as potential polymer, drug, and solvent interactions. These initial studies explored pertinent properties for use in the clinic and efficacy in vivo. Identified formulations were then assessed for drug bioaccumulation, with the lead candidate being evaluated in vivo for 12 endpoints of behavioral pain and tissue analysis in a murine RIP model. Subsequent in vivo investigations are centered around behavior pain responses, histological evaluations, and mechanisms of SAGEs in rectal protection of RIP The following Chapters are organized to address two aims of this dissertation as well as the future directions for this work. In Chapter 2, RIP is discussed along with gaps in treatment options for this injury. Semi-synthetic glycosaminoglycans are reviewed, with an emphasis on the importance of material properties of sulfated hyaluronans. SELPs are discussed in terms of their material properties and ability to control spatiotemporal release of therapeutics. In Chapter 3, Aim 1 is addressed regarding the development of a thermoresponsive polymer enema system for RIP from a panel of polymers [33]. Chapter 4, addresses Aim 2 in which early and later stage treatment of RIP is assessed with our treatment strategy [34]. Conclusion and the future directions of this project are discussed in Chapter 5. Appendices A and B include relevant supplemental information with regards to Chapters 3 and 4, respectively. In addition to the main scope of this dissertation which involves the delivery of SAGE by SELP hydrogels additional works were performed that are included in Appendices C and D. Appendix C contains a review of matrix-mediated viral gene delivery systems [35]. Appendix D discusses the development of an intrinsically disordered protein subunit, focusing on the development of a novel, foldable elastinlike polypeptide fusion protein. The subunit properties and capabilities for protein complexes to target cell surfaces is explored. 13 1.7 References (1) McCaughan, H.; Boyle, S.; McGoran, J. J. Update on the Management of the Gastrointestinal Effects of Radiation. World J. Gastrointest. Oncol. 2021, 13 (5), 400. https://doi.org/10.4251/WJGO.V13.I5.400. (2) Lee, J. K.; Agrawal, D.; Thosani, N.; Al-Haddad, M.; Buxbaum, J. L.; Calderwood, A. H.; Fishman, D. S.; Fujii-Lau, L. 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In Vitro and In Vivo Evaluation of Recombinant Silk-Elastinlike Hydrogels for Cancer Gene Therapy. J. Controlled Release 2004, 94 (2–3), 433–445. https://doi.org/10.1016/j.jconrel.2003.10.027. (27) Hwang, D.; Moolchandani, V.; Dandu, R.; Haider, M.; Cappello, J.; Ghandehari, H. Influence of Polymer Structure and Biodegradation on DNA Release from SilkElastinlike Protein Polymer Hydrogels. Int. J. Pharm. 2009, 368 (1–2), 215–219. https://doi.org/10.1016/j.ijpharm.2008.10.021. (28) Dinerman, A. A.; Cappello, J.; Ghandehari, H.; Hoag, S. W. Solute Diffusion in Genetically Engineered Silk-Elastinlike Protein Polymer Hydrogels. 2002, 82, 277–287. https://doi.org/10.1016/S0168-3659(02)00134-7 (29) Megeed, Z.; Cappello, J.; Ghandehari, H. Controlled Release of Plasmid DNA from a Genetically Engineered Silk-Elastinlike Hydrogel. Pharm. Res. 2002, 19 (7), 954–959. https://doi.org/10.1023/a:1016406120288 16 (30) Price, R.; Poursaid, A.; Ghandehari, H. Controlled Release from Recombinant Polymers. J. Controlled Release 2014, 190, 304–313. https://doi.org/10.1016/j.jconrel.2014.06.016. (31) Gustafson, J. A.; Ghandehari, H. Silk-Elastinlike Protein Polymers for MatrixMediated Cancer Gene Therapy. Adv. Drug Delivery Rev. 2010, 62 (15), 1509– 1523. https://doi.org/10.1016/j.addr.2010.04.006. (32) Isaacson, K. J.; Jensen, M. M.; Watanabe, A. H.; Green, B. E.; Correa, M. A.; Cappello, J.; Ghandehari, H. Self-Assembly of Thermoresponsive Recombinant Silk-Elastinlike Nanogels. Macromol. Biosci. 2018, 18 (1), 1–11. https://doi.org/10.1002/mabi.201700192. (33) Steinhauff, D.; Jensen, M.; Talbot, M.; Jia, W.; Isaacson, K.; Jedrzkiewicz, J.; Cappello, J.; Oottamasathien, S.; Ghandehari, H. Silk-Elastinlike Copolymers Enhance Bioaccumulation of Semisynthetic Glycosaminoglycan Ethers for Prevention of Radiation Induced Proctitis. J. Controlled Release 2021, 332 (August 2020), 503–515. https://doi.org/10.1016/j.jconrel.2021.03.001. (34) Steinhauff, D.; Jensen, M. M.; Griswold, E.; Jedrzkiewicz, J.; Cappello, J.; Oottamasathien, S.; Ghandehari, H. An Oligomeric Sulfated Hyaluronan and SilkElastinlike Polymer Combination Protects against Murine Radiation Induced Proctitis. Pharmaceutics 2022, 14 (1), 175. https://doi.org/10.3390/pharmaceutics14010175. (35) Steinhauff, D.; Ghandehari, H. Matrix Mediated Viral Gene Delivery: A Review. Bioconjugate Chem. 2019, 30 (2), 384–399. https://doi.org/10.1021/acs.bioconjchem.8b00853. CHAPTER 2 LITERATURE REVIEW 2.1 Radiation-Induced Proctitis Radiotherapy is a common treatment modality for patients with lower abdominal caners including, but not limited to, prostate, ovarian, rectal, and bladder cancers. This treatment modality utilizes ionizing radiation to target tumors selectively over healthy tissues. However, off target radiotherapy effects still exist, and are considered to be one of the main limitations of this treatment. The anatomical positioning of the rectal cavity, along with its rapidly dividing cell population, makes this organ especially susceptible. Upon irradiation, rectal inflammation, also known as radiation-induced proctitis (RIP), can occur. While the exact prevalence of RIP is difficult to elucidate, it is estimated that 5-20% of radiotherapy patients will develop RIP, and others will develop chronic, or late developing conditions [1]. Due to the clinical prevalence of this injury, several grading schemes to describe proctitis severity have been developed, for example by the Radiation Therapy Oncology Group [2] and the European Organization for Research and Treatment of Cancer [3]. Acute RIP can be presented with varying symptoms ranging from increased stool frequency and discomfort (Grade 1) to obstruction, fistulas, or perforation (Grade 4). Other symptoms include, but are not limited to, diarrhea and rectal bleeding. Chronic RIP may present with more severe symptoms. These range from mild diarrhea, 18 bleeding, and rectal discharges (Grade 1) to necrosis, perforations, or fistulas (Grade 4) [4–6]. Presentation of late developing chronic RIP increases nearly 5-fold in prostate cancer patients receiving external beam therapy when they are first presented with acute RIP [7]. By managing patient quality of life at the early stages, or by prevention, proctitis can be controlled to improve outcomes in the clinic. RIP remains an unaddressed need despite advances in radiotherapy. The likelihood of developing RIP is correlated with the volume of irradiated rectum, total dose, technique of administration, and administered dose per fraction [6]. Other factors may also play a role, including comorbidities and concomitant treatments. Concurrent treatment of adjuvant or chemotherapy with radiotherapy is a popular course of treatment. The use of chemotherapy with radiotherapy, or chemoradiotherapy, can improve outcomes. Chemoradiotherapy may increase radiation sensitization, target off site metastases, or eliminate radioresistant cells. However, the observed additive or supraadditive effects of this concurrent strategy may increase local tissue toxicities and adverse events, including RIP [8]. Modern radiotherapy techniques aim to limit these adverse events. Current state of the art techniques includes intensity modulation, stereotactic body radiotherapy, and particle beam therapy. Intensity modulated radiotherapy is based upon three-dimensional radiotherapy and allows for dose customization based on variation of tissue characteristics as identified with functional imaging. Stereotactic body radiotherapy utilizes steep dose gradients to minimize damages across a broad range of anatomical positions. Particle beam therapy uses particles, for example protons, to improve distributions and offsite tissue damages [9]. Simple alterations, such as supine positioning, can further improve outcomes [10]. 19 Typical radiotherapy schedules use fractionated doses to achieve a total cumulative dose over the entire schedule. Fractionation is employed to increase the therapeutic effectiveness while decreasing off site toxicities. These may include hypofractionated or hyperfractionated doses, depending on cancer cell sensitivities to radiation. Prostate cancer regimens for example can be treated with up to 79 Gy over a 7to 9-week period. Cervical cancer treatment on the other hand is treated by a total dose of up to 85 Gy throughout the radiotherapy schedule. Rectal cancers can be treated with a short or long course treatment typically summing up to 25 and 50 Gy respective total doses. The total tolerated dose by the colon is estimated to be 80 – 90 Gy [4]. Rectal areas receiving a total of ≥60 Gy have been associated with higher risks of rectal toxicities and bleeding. Clinically, hypofractionated doses are used to decrease toxicities with dose volume histograms suggested to be 1.8 or 2 Gy fractions [11]. The additional combination of neoadjuvant or chemotherapy with radiotherapy increases rectal toxicity risks [4]. The rational for dose fractionation is provided by the radiobiological response of certain tissue and cell types. The linear quadratic model is used to predict the effects of fractionation in radiotherapy. This model predicts the surviving fraction (SF) as a function of dose (D): !"($) = ' !"∗$!%∗$ ! (2.1) The parameters, α and β, represent the natural radiosensitivity of specific cell types. The higher the values of α and β, the more sensitive. The ratio of α/β is further described as the fractionation sensitivity of those cells. The higher the ratio, the less sensitive cells are to the beneficial effects of fractionation [12]. These ratios vary from between tumor and tissue types, allowing some to benefit more from the sparing effects of fractionation in 20 radiotherapy schedules. In the clinic, most tumors are thought to have an α/β of about 10 Gy and are therefore treated with a standard 2 Gy/fraction. On the other hand, some prostate cancers have an increased α/β (1.5 Gy) indicating increased sensitivity to higher doses per fraction. Healthy tissues such as the rectum and bladder have respective α/β ratios of about 3 and 5- 10 Gy [13]. This indicates that prostate cancers are more sensitive to higher fractions than the rectum. However, many other tumor types are less sensitive to higher doses per fraction. Ionizing radiotherapy results in double stranded DNA breaks, alteration in protein signaling, and damages to the cell membrane [14]. Nuclear chromatin can be damaged via inter- and intra-strand breaks, linkages, and mutations. Natural repair mechanisms have evolved to repair DNA damages of this nature leading to successful resolution at low doses of radiation. However, with increasing radiation these repair mechanisms become exhausted, leading to cell death. Further cellular damages occur with alterations of cellular membrane, specifically alterations in the rigidity and ionic gradient [10]. Radiotherapy is most effective in tissues with a high turnover rate as cellular repair processes occur within certain stages of the cell cycle. Typically, the most radiosensitive stage of the cycle is the G2-M phase [10]. In just two weeks from beginning a radiotherapy schedule, histological changes associated with acute RIP can be observed. These changes include all levels of the mucosa (lamina propria, epithelium, and glandular elements) and include elements of cryptitis, crypt abscesses, inflammatory infiltrates, and eosinophilic granulocytes. Rectal glands can also exhibit luminal migration of nuclei, atypical mitosis, distortion, loss of goblet cells, and potentially complete loss of entire glands [15]. Interestingly, early onset of RIP in radiotherapy schedules exhibits rapid 21 mucosal injury followed by progressive tissue healing throughout radioschedules [15]. The early stages of rectal injury could be attributed to loss or damage of intestinal stem cells and inflammatory infiltrates in response to damage [16–18]. The inflammatory mediators of this process include leukocytes, eosinophils, and macrophages among others [15]. A growing body of literature is devoted to the understanding of the role of mast cells in RIP pathophysiology. Rectal tissues show activated mast cells in cases of patients with RIP [19–21]. When investigated, mast cell deficient mice developed less acute and chronic RIP disease states than controls [20]. This suggests that acute mechanisms and resolution may be responsible for the long-term effects exhibited by chronic RIP. The more homeostatic chronic state is a result of progressive epithelial atrophy and fibrosis. Indications of obliterative endarteritis, mucosal ischemia, and new blood vessel formation are also included in the pathological identity of chronic RIP [6]. A large clinical study focused on the investigation of histological changes of 70 patients undergoing radiotherapy. Initial tissue changes following irradiation included cells associated with acute inflammation and an abundance of eosinophils in the mucosa. Unlike studies which lack either large sample populations and/or long follow up periods, these investigations point to a significant association between loss of crypts and late presenting proctitis in the anterior rectum. This suggests that initial loss or damage to intestinal crypts can be an indicator for late/chronic RIP [22]. Techniques to reduce occurrence of RIP have been employed with varying successes. Preventative measures typically include alteration of radiation techniques, rectal spacers, and several prophylactic approaches [6]. Varying radiation techniques attempt to minimize doses that the rectum receives as the probability of developing RIP 22 correlates with the volume of rectum irradiated, total dose, and dose per fraction [6]. Image guided radiotherapy can assist with this treatment modality. Newer techniques using heavy particles, such as protons or carbon ions, can further reduce toxicities. However, long-term outcomes are unclear [9]. Rectal spacers can anatomically distance target organs, providing a lower probability of radiating the rectum. Endorectal balloons are inserted prior to each procedure to spare the dorsal rectal wall from receiving a radiodose. Other rectal spacers in development can be implanted with the aim to remain for the entirety of the radiotherapy schedule. Several of these spacers have been developed including hyaluronic acid and a collagen-based rectal spacer [6]. Prophylactic approaches include the use of sucralfate, 5-aminosalicylic acid, or sulfasalazine. However, only minimal benefits have been recorded with a phase III trial of sucralfate showing no improvement through either topical or oral delivery [6,23–26]. The lack of prophylactic approaches and minimal effects observed in those approaches that have been studied necessitates the development of an effective prevention strategy. There are many treatment routes for RIP, although there is no widely accepted regimen in the field. Typically, acute RIP can be self-managed by ceasing current radiotherapy schedule, antidiarrheals, and hydration [4]. A variety of different approaches have been explored for the treatment of chronic RIP. These approaches include medicinal agents, endoscopic therapy, and surgical therapy [4]. Typically, surgery is reserved for the most severe of cases due to its high rates of morbidity of 30-65% with postoperative mortality estimated to fall within 6-25% [4]. Most surgical procedures focus on fecal diversion, local repair/reconstruction, or proctectomy [4]. Endoscopic therapies are used mainly to control rectal bleeding, but may require multiple procedures. They are 23 associated with very high risk of adverse events [6]. Endoscopic therapies include argon plasma coagulation, topical formalin, laser, bipolar heater probe, and more [4,6]. Since surgical intervention and endoscopic therapies have high rates of adverse events, medical intervention is typically used as a first line of defense. Typical medical interventions center around the use of anti-inflammatory agents such as sulfasalazine or 5-amino-sailicylic acid (5-ASA) [4]. These can be utilized individually or in conjunction with steroids. The results surrounding these approaches have mixed outcomes with very little consensus in clinical improvement of symptomology. For instance, the use of 5-ASA in a trial of 20 patients revealed improved endoscopic scoring and rectal bleeding, but no improvements in pain reduction and stool frequency were observed [27]. The use of steroids alone has not been well studied. Sucralfate, a sulfated disaccharide, is another commonly used medical therapy. This polyanionic molecule adheres to mucosal cells, exhibiting a cryoprotective effect. In addition it can increase local epidermal growth factor, increase prostaglandin synthesis, and promote local blood flow [4]. In a controlled study sucralfate exhibited superiority over sulfasalazine, and improved bleeding in 92% of patients 16 weeks following treatment [4]. Other popular medical approaches include the use of antioxidants, short chain fatty acids, antibiotics, loperamide, prostaglandins, hyperbaric oxygen, and formalin [4]. The broad investigations of treatment modalities for RIP have only yielded limited successes, emphasizing the need to develop novel treatments, while aiming to further our understanding of the pathophysiology of RIP. Preclinical investigations surrounding RIP are aiming to understand the pathological mechanisms behind RIP, utilizing these as therapeutic targets, and to 24 enhance therapeutic delivery to the rectum. Within the clinic vascular endothelial growth factor has been found to correlate with the clinical symptoms and mucosal changes that are associated with chronic RIP patients [28]. Ex vivo analysis of chronic RIP patients implicates dysregulated blood vessel development via angiostatin, suggesting successful restoration of normal vasculature as a valuable therapeutic mechanism [29]. The expression of Focal Adhesion Kinase (FAK), a protein promoting cell migration, proliferation, adhesion, and survival, inversely correlates with radiosensitivity. This was illustrated by increased radiation damage in mice with FAK knockdown [30]. Utilizing knockout mice, nuclear factor erythroid 2-related factor (NRF2), a potent regulator of cellular antioxidant activity, has been determined to contain crucial radioprotective effects through the inhibition of necrosis and necrotic associated proteins [31]. The loss of intestinal stem cells has been identified as a major driver of RIP pathophysiology. Knockout of NRF2 can result in the protection of radiation-induced intestinal injuries via proliferation and differentiation of Lgr5+ stem cells [32]. However, this protection via NRF2 is not completely understood with some variation in findings [31]. Protection provided by stem cells is evidenced in the depletion of WNT ligands provided by macrophage extracellular vesicles. Depletion rescued intestinal stem cells from radiation damage in a regenerative manner [16]. Recently, Lu et al. discovered the upregulation of platelet-derived growth factor C (PDGF-C) in patients with radiation proctitis and in a murine RIP model. Knock out of PDFG-C resulted in dampened RIP injuries through decreased activation of PDGF receptors and chemokine receptors. Crenolanib, an inhibitor of PDGF receptors, was able to reduce RIP in mice through this proposed mechanism [33]. Another strategy aims to target damaged endothelial cells which can 25 recruit immune cells through adhesive molecules. Pravastatin is able to inhibit this interaction through anti-inflammatory and anti-thrombotic effects [34]. The variety of pathological mechanisms and therapies being explored suggests there is no single approach that will address all symptoms or demographics. RIP remains an under addressed injury in the clinic. Few prophylactic approaches have shown promising results in the clinic and current strategies are largely aimed at responding and managing clinically presented symptoms. There are a plethora of pathological drivers, and the exact development of RIP may vary and/or remains to be elucidated. Promising preclinical therapies targeting biological mechanisms can be expanded upon. Novel approaches will further our understanding and therapeutic regiment for RIP. 2.2 Sulfated Hyaluronans Glycosaminoglycans are a family of polysaccharides consisting amino sugars, typically a uronic sugar and an amino sugar disaccharide repeat. These polysaccharides are naturally occurring and play important roles in the physiological environment including, but not limited to, mucus membranes, cell structural integrity, and inflammatory processes. There are several different classes of glycosaminoglycans including keratin sulfate, heparin/heparan sulfate, chondroitin sulfate/dermatan sulfate, and hyaluronan [35]. The unique structures of each glycosaminoglycan program them with a variety of biological properties, allowing them to be used for treatment in biological and inflammatory settings. Glycosaminoglycans are strongly implicated within settings of cancer, allowing 26 for growth, progression, and metastasis with several glycosaminoglycans having been found to even inhibit tumor progression. In the presence of inflammation glycosaminoglycans play a crucial role, especially in the setting of immune cells, including regulation of leukocytes along endothelial cells, chemokine migration/activation, and trans-endothelial migration of leukocytes. These roles have led the investigation of glycosaminoglycans as anti-inflammatory agents. Specifically, sulfated glycosaminoglycans have been investigated for their role as anti-inflammatory agents. Heparin has been investigated for use in asthma, colitis, and burns. However, its anticoagulant property has limited its clinical translation due to potential for increased risks of bleeding [35]. Modification of naturally occurring glycosaminoglycan structures could provide improved properties useful for therapeutics. Natural polysaccharides can be improved chemically to generate semi-synthetic glycosaminoglycans. This chemical programming can improve undesirable material properties, program additional properties, or both. For instance, the anticoagulant properties of heparin were reduced in such mimetics by generation of a 2-O,3-Odesulfated heparin. This heparin mimetic was capable of reducing inflammation derived from neutrophil elastases within the airway [36]. Another non-anticoagulant heparin can be isolated from shrimp with capabilities to reduce inflammatory infiltrates in several inflammation models [37]. On the other hand, super-sulfation of heparin disaccharides can inhibit allergic responses within an airway model of asthma [38]. The size of glycosaminoglycans plays an important role in their biological properties. Keratin sulfate has demonstrated anti-inflammatory properties in the setting of interleuikin-1α driven pathology, arthritis, and within the cornea [39,40]. Specifically, low molecular weight 27 keratin sulfate can disrupt the binding of keratin proteoglycans to the chemokine CXCL1, which can prevent stromal migration within the cornea [41]. The choice of glycosaminoglycan and synthetic modification provides the material programming for biological properties. Hyaluronic acid (HA) has been utilized in a variety of therapeutic applications due to its endogenous biological presence and inherent roles including but not limited to angiogenesis, wound healing, and immune regulation [42]. The properties of endogenous HA largely rely on the biopolymer location and size. Typically, HA exists at a high molecular weight HA (>1000 kDa) biopolymer. Under conditions of fragmentation, such as release of reactive oxygen or nitrogen species, smaller HA fragments (~100-500 kDa) are produced and have been shown to induce inflammatory responses [42]. Shorter oligomeric HA fragments contain further diversity in biological properties. Some of these include activation of dendritic cells via TLR4[43], pro-angiogenic capabilities [44], and migration/proliferation of fibroblasts [45]. However, differences in biological properties can be detected between small variations in the number of HA repeats. For example, HA disaccharides competitively block TLR4 mediated LPS signaling [46]. Chemical sulfation of HA can yet yield varying properties including differential interactions with membrane receptors or soluble extracellular proteins [42]. The chemical sulfation of HA has been explored in numerous molecular and biological systems as a potential therapeutic. Sulfated hyaluronans (sHA) are an emerging class of semi-synthetic glycosaminoglycans and can be generated via chemical sulfation of hydroxyl groups. Analogs of these sHAs have been shown to interact with a number of proteins, including 28 but not limited to VEGF [47–49], TGF-β1[50–52], p-selectin [53], fibronectin[54], bone morphogenetic proteins (BMPs) [55,56], and heparin[47,57]. The location and degree of this sulfation can program critical properties on sHAs. HA is composed of D-glucuronic acid and N-acetyl-D-glucosamine. Sulfation of the C6 N-acetyl-D-glucosamine can result in binding with VEGF165 and inhibition of its interactions with heparin. Dependence of sulfate location plays a more crucial role than overall sulfate content [47]. This suggests the unique disaccharide shape and location of sulfation is an important material characteristic to consider for downstream biological properties. Dependence of properties on size is also observed with sHA. Tetrasacharrides were determined to be the minimum length required for sHA binding to VEGF165, however overall polysaccharide length is not correlative with VEGFF165 interactions and does have an upper limit. Interactions such as these reduce the biological activity of VEGF-A and can be implicated in reducing phosphorylation of VEGFR-2 upon binding [48] and altered sprouting of endothelial cells [47]. Despite these anti-angiogenic effects via VEGF-A, sulfated hyaluronans exhibited pro-angiogenic effects independent of VEGF [47]. These properties illustrate the angiogenic modulation properties of sHAs. Sulfated hyaluronan has shown osteogenic capabilities via human mesenchymal stromal cells and bone marrow stromal cells [54,58]. Culturing mesenchymal stromal cells with an artificial extracellular matrix containing sHA resulted in osteogenic differentiation. This included increased expression of FAK, RhoA, and Ras, which promote osteogenic differentiation. In addition, sulfated hyaluronan resulted in increased cell-matrix interactions, cell signaling, and endocytosis. Specifically, cell proteins interacting with integrins, such as β1 and α5, were found to be upregulated [58]. Together, 29 these imply the involvement of sHA in the structural organization of cells and extracellular matrices. Fibronectin, a common extracellular matrix component in bone, has several binding sites for sulfated glycosaminoglycans. SHAs have been found to increase expression of and to alter the assembly of fibronectin matrices. These instances were compared with heparin sulfate and HA. Inclusion of sHA in synthetic matrices resulted in thinner and more extended fibronectin fibrils. This altered assembly may expose cryptic fibronectin domains. In addition to this altered assembly, bone marrow stromal cells expressed tissue non-specific alkaline phosphatase, an early osteogenic marker [54]. Indications of other osteogenic activity has been exhibited by sulfated hyaluronans. Increased degrees of sulfation result in stronger interactions with recombinant human BMP-4 (KD 13 – 20 pM). Similar interactions between sulfated glycosaminoglycans and BMP-2, BMP-4, and BMP-7 have also been reported [56,59,60]. It has become evident that sulfated hyaluronans are able to modulate the differentiation or proliferation of a plethora of cell types. Wound healing is also modulated by sulfated glycosaminoglycans, making it an attractive candidate for sHA intervention. TGF-β is an essential mediator of wound resolution by modulating the transition of dermal fibroblasts to myofibroblasts. Highly sulfated HA can reduce the ability of TGF-β to modulate this transition and the development of myofibroblasts, as assessed with qRT-PCR and immunofluorescence. This decreased bioactivity of TGF-β is mediated through the TGβRI binding site and could be used to modify fibrotic processes driven by TGFβ signaling [51]. This effect was also observed with TGβRII. However, when a sulfated hyaluronan is added to an already formed TβR-II/TGF-β1 complex, then the further recruitment of TβR-1 was 30 enhanced. In the presence of sulfated hyaluronans, TβR-I expression and Smad2 phosphorylation were decreased indicating impaired signaling mechanisms that could be utilized in applications to modulate dysregulated fibrosis [50]. A family of sHAs, semi-synthetic glycosaminoglycan ethers (SAGEs), have been extensively explored, utilizing the native biological functions of HA, in addition to programmed properties provided by sulfation. The parent hyaluronic acid is created via fermentation, digested with hyaluronidases, and chemically sulfated [53]. This results in a small, sulfated, polyanionic molecule. Sulfation increases its stability compared to other glycosaminoglycans due to resistance of degradation by hyaluronidases, and to some degree, chondroitinases and heparinases [53,61]. The hybrid properties of SAGEs give them a variety of anti-inflammatory functions. These properties include inhibiting release of inflammatory cytokines by blocking TLR2 and TLR4 activation [53,61]. Additionally, it acts as a competitive inhibitor for the receptor for advanced glycation end products (RAGE), P-selectin, and polymorphonuclear leukocyte proteases [53]. Inhibition of RAGE is thought to occur from interference of RAGE-ligand interactions. RAGE consists of a V protein domain that contains a basic, hydrophobic cavity. This cavity could offer interaction sites for acidic polyanionic molecules with alkyl groups such as SAGEs. This would prevent activated glycation end products from binding to RAGE, dampening the positive inflammatory feedback loop, subsequent activation of nuclear factor kappa beta (NF-KB), increased production of RAGE, and further release of inflammatory signals [53]. SAGE also reduces mast cell and neutrophil infiltration into inflamed sinonasal epithelium and submucosa. The mechanism behind this is postulated to be the formation of a protective layer against inflammatory and toxic mediators [62]. 31 When compared to naturally occurring glycosaminoglycans (hyaluronic acid, heparin), SAGE has enhanced anti-inflammatory properties in sinonasal inflammation. The mechanism of this action is hypothesized to be protection of the epithelium from cell death, reduced leukocyte infiltration, and limited signaling from proinflammatory mediators. Additionally, SAGE has exhibited increased suppression of bacteria, Porphyyromonas gingivalis and Aggretgatibacter actinomycetemcomitans, compared to hyaluronic acid. These novel structural and biological properties of SAGE have led to their evaluation in several inflammatory disorders with animals including, interstitial cysititis, RIP, radiation-induced oral mucositis, periodontitis, and rosacea [53,61,63–67]. The combination of the natural disaccharide repeat unit, provided by HA, and synthetic sulfation allow unique properties for semi-synthetic glycosaminoglycans. Tailoring of hyaluronan length, degree of sulfation, and use of other glycosaminoglycan templates will lead to future semi-synthetic glycosaminoglycans. Meanwhile, the molecular investigations of sHA will continue to yield novel material and biological properties. Their use in biomedical applications may produce efficacious treatments in a variety of biological environments. 2.3 Silk-Elastinlike Protein Polymers Recombinant polymers are proteins designed at the genetic level and produced using cellular machinery. Typically, depending on the desired properties, this can be accomplished within E. coli and provides monodisperse, reproducible products. The natural amino acids have historically been used as the monomers for these polymers, and recently non-canonical amino acids have been used to increase the functionality of 32 recombinant polymers [68]. In many cases, peptide motifs from naturally occurring proteins are identified and used to generate protein-based polymers with a desired property in mind. For example, elastinlike polypeptides (ELPs) are inspired from tropoelastin, a 72 kDa protein. Within tropoelastin there are numerous glycine and proline rich hydrophobic domains. These hydrophobic domains contain an abundance of valine and alanine residues [69]. The consensus of glycine, valine, and proline resulted in the development of ELPs (V-P-G-X-G) which contained well characterized stimuli responsive behaviors, similar to that or tropoelastin [69,70]. Motifs derived from Bombyx mori, or silkworms, are yet another example. Natural spider silk contains a high level of strength, provided from its molecular structure. It is currently thought that the molecular properties of spider silk result from the fibroin heavy chain (350 kDa) which, through molecular analysis, shows a tendency of (GA)NGX repeat motifs. These repeats have a high degree of crystallinity or semi-crystallinity, likely providing the observed strength of silk [71]. These are just two motifs used in rational design of protein polymers, others may include collagen mimetic peptides, resilin motifs, and more. The incorporation of various motifs into block copolymers can further provide novel properties unobtainable from single motifs alone. Silk-elastinlike protein-based polymers (SELPs) consist of both silk and elastinlike motifs. The first SELPs to be constructed were made by random concatimerization. The genes encoded for monomer repeats were assembled into a plasmid, which was subsequently transformed into E coli. Bacterial fermentation produces SELPs and subsequent bioprocessing produces a pure product [72,73]. Genetic design of these polymers has resulted in structures with a molecular weight of 33 approximately 65- 85 kDa and a variety of properties [73–75]. Post-purification shearing can enhance and normalize material properties between batches. This shear processing is hypothesized to linearize molecular strands from globule states. This would allow for increased intermolecular rather than intramolecular polymer interactions to occur [76]. This step may limit the translatability, due to the need for cold chain storage, but has been found beneficial for the numerous applications discussed below. Elastinlike polypeptides, derived from human tropoelastin, are renowned for their thermoresponsive and unique solubility properties [70]. The combination of silk and elastinlike motifs provides SELPs the ability to undergo thermoresponsive crosslinking assemblies. The cooperativity of these motifs informs the SELP protein primary structure with capability of a thermoresponsive sol to gel transition, forming stable hydrogels with unique properties. The macroscopic and microscopic assemblies of SELPs have been studied extensively for structure - function relationships, injectable therapeutic delivery, applications for liquid embolics, and more [63,64,76–88]. As with any protein, the primary amino acid structure and environment informs overall protein properties. In the case of SELPs this has largely been investigated in the context of macroscopic gelation with a few investigations of microscopic protein assemblies [76,80,85,86]. At cold or room temperature, elastinlike motifs provide solubility to SELPs and allow them to be easily manipulated and injected as a liquid state. Upon heating elastinlike motifs become insoluble. SELPs then undergo nucleation events and subsequent fiber assembly through the silk region [75]. These regions form antiparallel beta sheets mediated by hydrogen bonding [73]. Above a minimum gelation concentration of SELPs a sol to gel transition occurs resulting in the formation of 34 macroscopic hydrogels from a previous liquid solution [76]. This transition depends upon several factors, including, but not limited to, the concentration of SELPs in solution, the silk to elastin ratio within the primary sequence, ionic strength of the solution, cure time, solutes present, and other factors [74,75,80,89]. Several analogs of SELPs have been synthesized, including SELP-47K (4 silk, 7 elastin, and one elastin containing lysine), SELP-815K (8 silk, 15 elastin, and one elastin containing lysine), and SELP- 415K (4 silk, 15 elastin, and one elastin containing lysine) [73–75]. SELPs with increased ratios of silk to elastin motifs (47K, 815K) have increased storage moduli, gelation kinetics, and decreased concentrations required for gelation compared to those with lower silk to elastin ratios, such as SELP-415K [80]. In all these constructs the SELP concentration correlates with gelation rates and final gel strength. The amount of water uptake, or swelling ratio, of SELP gels also depends heavily upon the sequence [80]. Constructs with increased elastin content (415K, 815K) retain more water content due to the relative hydrophilicity of elastin compared to silk. The soluble fraction of SELP hydrogels, or the amount of SELP molecules not incorporated into hydrogel networks, depends on the number of silk junctions possible. As the number of possible junctions increases (415K, 47K) there are more opportunities for polymers to be incorporated within the hydrogel. Elastin units are believed to play a secondary role as well [80]. The post purification shearing exhibited in some SELP preparations allow for increased gelation kinetics and mechanical strengths of SELPs, owing to the increase in intermolecular interactions [76]. In vivo, SELPs exhibit similar gelation properties as demonstrated in a variety of properties. Upon subcutaneous injection into guinea pigs, SELP-47K showed no signs of inflammatory responses, toxicities, or allergic reactions [73]. The injected material 35 appeared to penetrate dermal collagen networks or remain isolated in pockets. After 28 days there were several signs of cellular infiltration into the SELP gel with some foreign body giant cells around the periphery of the gel. Besides this there were no evident reactions to the material [73,80]. In physiological conditions, SELPs are responsive to elastases, with cleavage occurring within the ELP motif regions. Elastases are not typically expressed at relevant concentrations within the body, and intratumoral administrations of SELPs have revealed the persistence of SELP depots over a 12-week period with development of a fibrotic layer at the tissue-gel interface [81]. These observations helped prompt the development of SELPs capable of enzymatic degradation in the presence of matrix metalloproteases. These responsive SELPs (RS-SELPs) were synthesized genetically as previously described [76,90]. Initial monomer sequences were programmed to contain an enzymatically cleavable site. This site, programmed as GPQGIFGQ, is cleavable via matrix metalloproteinases (MMP) -2 and -9[76,90]. MMPs are a family of proteinases naturally found in vivo, and breakdown the extracellular matrix. They are commonly observed in sites of inflammation, making them an attractive method to adjust spatiotemporal release of therapeutics [91]. The family of RS-SELPs was derived from SELP-815K and resulted in 3 new constructs with MMP cleavable sites at three different locations with each monomer sequence. These locations include between silk and elastin blocks (RS1), within the elastin block (RS2), and within the silk block (RS5). The inclusion of this MMP cleavable sequence and its location within the monomer both have an impact on macroscopic SELP assembly properties [76,90]. The RS5-SELP has increased swelling ratios, likely due to the loss of crystalline silk, as it is interrupted from 36 the MMP cleavable sequence. This interruption also likely contributes to the increased soluble fraction of RS5-SELP assemblies. The remaining two RS-SELPs were able to maintain properties acceptable for injectable SELP depots, despite slight changes in swelling ratio, soluble fractions, and storage moduli compared to the parent SELP-815K [76]. The careful analysis of this sol to gel transition across a variety of SELPs has informed us on how to use these as drug delivery vehicles and for other therapeutic approaches. SELPs have also been studied for their capabilities to form nano- or microassemblies. Investigations of this nature have yielded supramolecular structures including nanogels, nanoparticles, and micellar-like structures [85,86,92,93]. Nano-assemblies of SELP-415K, -47K, and -815K have been studied at concentrations below the minimum gelation concentration to understand polymer-polymer interactions and potentially to develop a novel nanocarrier. Dilute concentrations of SELPs (≤2 mg/mL) were found to interact with and form globular like assemblies that are responsive to temperature. Critical formation concentrations of these nanogels (CFCs) were determined using a hydrophobic reporter dye, 8-anilino-1-naphthale-nesulfonic acid (8,1-ANS). All studied structures formed CFCs below 2 mg/mL. Interestingly, the observed CFCs corresponded with trends observed in soluble fraction of polymers from macroscopic assemblies [80,85]. For example, SELP-415K has increased soluble fraction and CFCs compared to SELP-47K and SELP-815K. This suggests the importance of silk cross-linking sites, and the length of each crosslinking site. These nanogel structures presented dilution stability as well, in contrast to similar structures such as micelles, providing rationale for stability upon in vivo administration. This stability is postulated to occur from the silk β-sheets. 37 Furthermore, the thermoresponsive properties of the elastin components was conserved in these SELP structures, observed through temperature-dependent size collapses. Aspects of assembly, size-collapse, aggregation, and precipitation were all observed over temperature window of 25-67°C [85]. Similar observations were achieved with RSSELPs, again with properties depending on the location of the MMP sequence. The RS5SELP failed to assemble into nanogel structures, likely due to its impaired ability for silk crosslinking [76,86]. These SELP nanogels were then explored for drug delivery capabilities. Doxorubicin was loaded into several SELP and SELP-RS analogs, achieving loading efficiencies below 15%. Cumulative release was then studied over 4 days with each polymer formulation releasing somewhere between 40-80% of its cargo. These nanocarriers are capable of protecting against hemolysis, compared to free doxorubicin and did not result in significant increases in complement activation as assessed via iC3b [85,86]. Altogether, these systems show promise for systemic administrative routes with potential for local accumulation and retention. Other SELP micro-assemblies have been developed [92,93]. Micellar like SELP nanoparticles have been generated with different SELP analogs. These include SELP28Y (two silk, 8 elastin, one elastin-substituted with tyrosine), SELP-18Y, and SELP48Y [93]. Assembly of these block copolymers resulted in a hydrophobic core and a hydrophilic corona consisting of the silk and elastin motifs respectively. SELP-18Y and SELP-28Y demonstrated a two phase, reversible transition dependent on temperature. These assemblies took place at lower concentrations than the CFCs of the previously described nanogel structures. These micellar like SELP structures were also evaluated for doxorubicin loading achieving a maximum loading efficiency of 6.5% in the SELP-18Y 38 [93]. Proteomics modeling is consistent with this experimental data, illustrating that shorter silk blocks have more conformational flexibility over a range of temperatures [94]. Some of these nanoparticle structures have been further modified to contain mucoadhesive properties, largely achieved through modification of the elastin guest residue to consist of either a lysine or cysteine residue [95]. The unique structures, assembly, thermoresponsive nature, and biocompatibility of SELPs have allowed them to be used for a plethora of biomedical uses. One of the first explored applications of SELPs was in matrix-mediated gene delivery [80]. Loading of these delicate biologics within SELP can be achieved by simple mixing of therapeutic and polymer. The physical crosslinking that occurs upon heating preserves biological structures, unlike other crosslinking mechanisms such as chemical or ultraviolet light. The release of plasmid DNA from SELP matrices has been explored. Plasmid release from SELP-47K illustrates a strong dependence between release and media ionic strength, due to the ionic interaction between plasmid phosphates and SELP lysine residues. By increasing the ionic strength of the present media, plasmids release more quickly likely due to charge shielding of the lysine and arginine residues within the studied polymer (SELP-47K). This ionic interaction between the negative DNA phosphates and the positive lysine and arginine residues could be one possible explanation for enhanced bioactivity observed in studies. SELP-47K matrices were able to achieve over 28 days of sustained release of the pRL-CMV plasmid [96]. The controlled release can further be tuned based upon the plasmid sizes. Plasmid sizes ranging from 2.8 kbp – 11 kb have established a positive correlation between size and sustained release from SELP matrices. Release kinetics also depend upon plasmid 39 conformation [97]. Release from SELP-47K matrices resulted in bioactive plasmids up to 28 days that were capable of transfecting breast tumor cells in vitro [97]. Plasmid delivery suffers from poor transfection rates and short transgene expressions, prompting the investigation of viral gene delivery from SELP matrices. Matrix-mediated viral release can benefit by providing sustained release and shielding viral vectors from potential immune neutralization. Matrix-mediated release of adenovectors from SELP matrices was shown to depend on SELP concentration and primary sequence [98]. Increased concentrations of SELPs inhibit viral vector release likely due to physical entrapment of vectors [97,99,100]. Comparison between viral release from SELP-415K and SELP-47K at 11.7 wt/wt% show a dependence on the length of elastin blocks, with SELP-415K achieving a higher cumulative release over 28 days [99]. Release of viral vectors from SELP results in increased bioactivity, prolonged expression, and sustained release [82,101,102]. The observed increases in bioactivity may result from polymer-capsid interactions. Hexon, a major adenoviral capsid protein, contains numerous glutamate and aspartate residues. These anionic components have the potential to interact positive residues within the SELP backbone. This interaction could also explain some of the sustained retention of adenovectors within SELP matrices [103]. In vivo, SELP mediated viral release has several beneficial effects. The safety of viral therapy with SELP vehicles has shown improved mice weight maintenance, decreased levels of white blood cells, maintenance of alanine aminotransferase levels, and decreased liver dissemination compared to saline controls [80,100]. SELPs have been utilized for localized gene therapy for head and neck cancers, with a 4 wt/wt% solution resulting in the highest transfection efficiency and sustained gene expression lasting 40 through 21 days [100]. Matrix-mediated adenoviral prodrug therapy with SELPs exhibited an approximate 5-fold reduction in head and neck tumor sizes obtained from SELP-815K matrices [79]. Increased cumulative release from SELP matrices necessitates a low wt/wt percentage solution, due to physical entrapment of vectors. This hinders capabilities to tune spatiotemporal release rates by adjusting SELP concentrations and was addressed with the development of the RS-SELPs, as they could slowly degrade in inflammatory environments with upregulated MMP activity [76,90,104]. When evaluated in a solid tumor xenograft, RS-SELPs at 8 wt/wt % increased survival in mice compared to virus in saline controls. Histologically, observed tissue-gel interfaces appeared tortuous with cell infiltrates, indicating in vivo MMP degradation of gels [104]. Several other approaches of SELP directed gene therapy include the delivery of vaccina viruses and oncolytic adenoviruses [101,102]. The biological nature and passive crosslinking of SELPs make them ideal for encapsulation of biological candidates and as potential biomedical devices. Understanding release mechanisms from SELP matrices is vital for understanding in vivo spatiotemporal release and for the design of new approaches. Release of small molecule drugs and biologics have also been explored. The drug delivery potential of SELPs prompted careful investigations of solute diffusion throughout formed matrices of 12 wt% SELP-47K. Diffusion of molecules ranging from 180-12,384 Da and 0.38 -1.88 nm in radius were explored. Release kinetics were diffusion-controlled in a Fickian manner dependent on size. However, partitioning of solutes suggests other factors in play as an intermediate compound (Vitamin B12) was preferred by SELP matrices. This may be due to a lower isoelectric point exhibited by Vitamin B12 [105]. Incorporation of 41 doxorubicin, sorafenib, and sorafenib tosylate have also been investigated in several different forms including soluble dimethyl sulfoxide (DMSO), powdered hydrochloric salt, and powdered base. The impact of mixing these drugs led to an increased viscosity in almost all cases with the exception of base powders. The mechanical strength following gelation was also impacted with decreased storage moduli exhibited in half of the explored formulations. Scanning electron microscopy revealed that DMSO formulations resulted in a consistent polymeric coating of doxorubicin and sorafenib crystals. On the other hand, incorporation of powdered drugs exhibited drug clusters entrapped within the polymer matrix. Cumulative release of these drugs was explored over two weeks with doxorubicin achieving near complete release, while sorafenib formulations reached only 50% or lower cumulative release. The swelling ratio of each formulation was further studied, and in all cases decreased compared to a 9 wt % SELP control [88]. The release of SAGEs has been explored with SELPs for amelioration of inflammatory conditions [63,64]. A SELP-815K and 100 mg/mL SAGE formulation was capable of achieving a sustained release of 24 hours. While the initial burst release was dependent on SELP-815K weight percent (4% > 11%), cumulative release was independent of polymer concentration [64]. Release of a 10 mg/mL SAGE cargo from SELP-815K at 12 wt/wt% only achieved 50% cumulative release after 24 hours. This suggests an ionic interaction between sulfate groups and lysine within SAGEs and SELP respectively, specifically as lower loading concentrations (10 mg/mL) will have a greater percentage of SAGEs interacting with lysine residues in contrast to higher loading concentrations (100 mg/mL) [63]. The addition of SAGE to SELPs varied viscosities at a 42 range of temperatures and storage moduli up to 3 hours at 37 °C, corroborating this hypothesis [63,64]. A chosen SELP-815K 11 wt% SELP was found to enhance rectal accumulation of SAGEs compared to PBS formulations. This increased rectal accumulation was investigated for a prophylactic approach in a murine radiation-induced proctitis model and compared to control formulations. The drug-polymer combination was capable of reducing behavioral pain responses after 3, 7, and 21 days following radiation insult to the lower abdominal area. Histologically, tissues were able to maintain similar characteristics to that of healthy tissue, with maintenance of the crypts of Lieberkühn and lamina propria [64]. Similar SELP SAGE (10 mg/mL) formulations were utilized to treat interstitial cystitis in mice. SELP-415K and SELP-815K both enhanced accumulation in the bladder of fluorescently labeled SAGE. However, SELP-415K outperformed SELP-815K which may have blocked the urethra of mice, leading to incontinence. The SELP-415K formulation was able to reduce behavioral pain responses and restore some physiological function [63]. The observed differences among SELP carriers exhibited in these studies illustrate the need to carefully evaluate and assess polymer properties for in vivo applications. These include mechanical properties and the sol to gel transition for each polymer. The sol to gel properties of SELPs make them ideal candidates for space filling applications. Customization of the gelation rate, viscosity, and mechanical strengths of utilized polymers is vital for appropriate disease and physiology specifications. SELPs have been explored as liquid embolic devices for aneurysms, blockage of hepatic vessels, and surgical management [83,87,106]. Liquid embolics have the potential to fill spaces of varying size in a single administration, which may not be true of calibrated microspheres 43 and/or drug eluting beads [107]. In addition, the passive sol to gel transition of SELPs make them unique in their ability to encapsulate therapeutics and delicate biological cargo. Careful rheological analysis was used to evaluate SELP-815K and SELP-47K for potential applications as liquid embolics. A sheared SELP-815K 12 wt/wt % formulation was selected based upon several key parameters. It maintained an injectable viscosity (<150 cP) at 25 °C, contained a rapid gelation, and achieved a 5 minute log stiffness of 2.48 ± 0.07 Pa. This formulation illustrated its ability to occlude microfluidic devices under flow conditions similar to that of the hepatic vasculature. In New Zealand white rabbits, stasis was achieved by administering the SELP formulation within the hepatic artery. No off-target emboli were observed, along with minimal signs of toxicities in the administered hepatic region [106]. The initial successes of this SELP-815K formulation have led to its further uses as an embolic. Surgical management of hypervascular head and neck tumors has also been explored. SELP-815K’s embolic and sustained release properties were combined in order to mark the tumor margins for surgeons while controlling interoperative bleeding. An FDA approved dye, indocyanine green, was utilized with sustained release of greater than 24 hours. This formulation maintained injectability, assessed by viscosity, and appropriate gelation times. In vitro microfluidic investigations support the in vivo assessment of this dual acting system, for future investigations [87]. Preliminary investigation of SELP-815K as a liquid embolic agent for cerebral aneurysms has also been explored [83]. The crucial sol to gel transition of SELPs can be used and optimized for further indication in embolics. Inclusion of therapeutics and bioactive agents can result in future dual acting systems as well. The protein nature of SELPs provide them excellent biocompatibility and 44 potential to serve as bioactive delivery depots. Several investigations have explored the use of SELPs to deliver cells or to serve as a cell scaffold. Encapsulation of human mesenchymal stem cells (hMSCs) in SELP-47K can result in the differentiation into chondrocytes. When exposed to chondrogenic medium, hMSCs generated chondrogenic extracellular matrix including sulfated glycosaminoglycans and collagen [108]. A photoresponsive SELP has been developed and used to incorporate L929 fibroblasts for facile release. Upon exposure to white light, SELPs formed gel indicated by an increase in storage moduli. Incorporated cells maintained viability above 90% as assessed with Live/Dead staining, indicating potential for use in cell culture systems [109]. Electrospinning of SELP-47K has been investigated for fibroblast scaffolds, achieving elastic moduli of 3.4-13.2 MPa with ultimate tensile strengths of 5.7-13.5 MPa. These scaffolds incorporated 3T3 fibroblasts and illustrated cell-cell and cell-scaffold interactions [110]. These studies support the use of SELPs as cell delivery vehicles and tissue scaffolds for several in vivo environments. The use of scaffold preparative techniques, such as electrospinning, can be used to further improve material properties. Genetic modifications of SELPs can also be employed to generate more cytocompatible matrices. Approaches of this nature are being explored as illustrated by the insertion of cell attachment sites (AVTGRGDSPASSV) into a SELP recombinant-based polymer [111]. 2.4 Conclusions The generation of novel biomaterials can be inspired largely from nature. However, the combination of naturally occurring characteristics can expand our 45 biological toolset. Semi-synthetic glycosaminoglycans, such as sulfated hyaluronans, utilizes sulfation dependent properties from naturally occurring glycosaminoglycans and properties from the unique disaccharide repeat unit of hyaluronans. SELPs utilize the crosslink ability of silks and solubility of elastin to generate a polymer capable of a passive sol to gel transition upon heating. The combination of SELPs and sHA can provide a unique therapeutic approach. SELPs can and have been used to deliver therapeutics in applications requiring specific spatiotemporal release to achieve a desired outcome. The incorporation of sHA into SELPs can allow for controlled delivery to sites of inflammation to enhance accumulation and biological effects. The combination of natural motifs, molecular properties, and vehicle-drug combinations allows for unique therapeutic outcomes. 2.5 References (1) McCaughan, H.; Boyle, S.; McGoran, J. J. Update on the Management of the Gastrointestinal Effects of Radiation. World J. Gastrointest. Oncol. 2021, 13 (5), 400. https://doi.org/10.4251/WJGO.V13.I5.400. (2) Radiation Therapy Oncology Foundation. https://www.rtog.org/ (accessed 202112-12). (3) European Organisation for Research and Treatment of Cancer. www.eortc.org (accessed 2021-12-12). (4) Tang, S. J.; Bhaijee, F. Chronic Radiation Proctopathy and Colopathy. Video J. Encycl. 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G. de; Sierra, L. Q.; Rodrigo, M. A.; Kock, L.; Rodriguez-Cabello, J. C. Cartilage Regeneration in Preannealed Silk Elastin-Like Co-Recombinamers Injectable Hydrogel Embedded with Mature Chondrocytes in an Ex Vivo Culture Platform. Biomacromolecules 2018, 19 (11), 4333–4347. https://doi.org/10.1021/ACS.BIOMAC.8B01211. CHAPTER 3 SILK-ELASTINLIKE COPOLYMERS ENHANCE BIOACCUMULATION OF SEMISYNTHETIC GLYCOSAMINOGLYCAN ETHERS FOR PREVENTION OF RADIATION-INDUCED PROCTITIS 3.1 Abstract Radiation-induced proctitis (RIP) is a debilitating adverse event that occurs commonly during lower abdominal radiotherapy. The lack of prophylactic treatment strategies leads to diminished patient quality of life, disruption of radiotherapy schedules, and limitation of radiotherapy efficacy due to dose-limiting toxicities. Semisynthetic glycosaminoglycan ethers (SAGE) demonstrate protective effects from RIP. However, low residence time in the rectal tissue limits their utility. We investigated controlled delivery of GM-0111, a SAGE analogue with demonstrated efficacy against RIP, using a series of temperature-responsive polymers to compare how distinct phase change behaviors, mechanical properties and release kinetics influence rectal bioaccumulation. Poly(lactic acid)-co-(glycolic acid)-block-poly(ethylene glycol)-block-poly(lactic acid)co-(glycolic acid) copolymers underwent macroscopic phase separation, expelling >50% of drug during gelation. Poloxamer compositions released GM-0111 cargo within 1 hour, Steinhauff, D.; Jensen, M.; Talbot, M.; Jia, W.; Isaacson, K.; Jedrzkiewicz, J.; Cappello, J.; Oottamasathien, S.; Ghandehari, H. Silk-Elastinlike Copolymers Enhance Bioaccumulation of Semisynthetic Glycosaminoglycan Ethers for Prevention of Radiation Induced Proctitis. J. Controlled Release 2021, 332 (August 2020), 503–515. https://doi.org/10.1016/j.jconrel.2021.03.001. 59 while silk-elastinlike copolymers (SELPs) enabled controlled release over a period of 12 hours. Bioaccumulation was evaluated using fluorescence imaging and confocal microscopy. SELP-415K, a SELP analogue with 4 silk units, 15 elastin units, and one elastin unit with lysine residues in the monomer repeats, resulted in the highest rectal bioaccumulation. SELP-415K GM-0111 compositions were then used to provide localized protection from radiation-induced tissue damage in a murine model of RIP. Rectal delivery of SAGE using SELP-415K significantly reduced behavioral pain responses, and reduced animal mass loss compared to irradiated controls or treatment with traditional delivery approaches. Histological scoring showed RIP injury was ameliorated for animals treated with GM-0111 delivered by SELP-415K. The enhanced bioaccumulation provided by thermoresponsive SELPs via a liquid to semisolid transition improved rectal delivery of GM-0111 to mice and radioprotection in a RIP model. 3.2 Introduction Radiotherapy is a common treatment for lower abdominal cancers, including prostate, ovarian, cervical, bladder, and colonic cancers. More than 300,000 patients receive radiotherapy for lower abdominal malignancies annually, and many will develop radiation-induced proctitis (RIP), a radiotoxic, inflammatory injury of the rectum [1,2]. An estimated 30-75% of all patients will develop acute RIP, up to 20% will develop chronic RIP, and 5% of all patients are at risk for developing more debilitating disorders including fistulas, rectal/anal stenosis, and/or fecal incontinence [1,3]. Patients with acute RIP typically present with symptoms of lower abdominal pain, rectal bleeding, fecal urgency, and diarrhea [1]. Patients that develop chronic symptoms have persistent and 60 more severe symptomology along with increased occurrences of severe bleeding, perforations, intestinal obstructions, strictures, and increased risk of other debilitating conditions (anemia, sepsis, fistulas) [4–6]. Most radiotherapy patients experience an undisclosed loss in quality of life both during and after their treatment. New therapeutic strategies are needed to preserve the quality of life of these patients. Radiotoxicity and RIP remain prevalent despite advances in administration of radiotherapy, including supine positioning, intensity modulation, image guidance, conformal radiation, and endorectal balloons. Acute presentation of RIP is typically selfmanaged and, in many cases, causes cessation or alteration of radiotherapy schedules [4]. Endoscopic laser ablation and surgical intervention is reserved for the most severe presentations of RIP. Surgical associated morbidity occurs in 30-65% of instances and postoperative mortality is estimated at 6-25% [3,7–12]. Pharmacological treatments include, but are not limited to sucralfate [13–15], 5-aminosalicylic acid [16], balsalazide [17], Vitamin E [18], Vitamin C [18], steroids [15], formalin [19–21], sodium butyrate [22], pentoxifylline [23], rebamipide [24], Vitamin A [25], short chain fatty acids [26], estrogen/progesterone [27], sodium pentosan polysulphate [28], and misoprostol [29]. While some of these approaches have shown beneficial outcomes, the effects are only moderate and reactionary. None of these treatments, with the exception of sucralfate, are regarded as current practice [6,23,27,30–32]. However, preventative strategies utilizing sucralfate have failed to show differences when compared to placebo treatments in late stage clinical trials, in some cases increasing occurrences of diarrhea and rectal bleeding [14,32,33]. Current management strategies focus on treating pathophysiology and symptomology only once presented. Preventative measures are largely underexplored with small double-blinded trials of misoprostol or sucralfate failing to have an effect 61 [4,32,34–36]. Management strategies to address and prevent the development of acute and subsequent progression to chronic RIP are needed. Previously our lab explored the use of a semi-synthetic glycosaminoglycan ether (SAGE) to prevent RIP [37]. SAGEs are modified glycosaminoglycans, containing inherent biological properties enhanced and altered through chemical modification. One such SAGE, GM-0111 (Figure 3.1A), is the product of hyaluronic acid digestion and chemical sulfation [38]. GM-0111 has been used to treat a variety of mucosal inflammatory diseases, including RIP, interstitial cystitis, and periodontitis [37–40]. GM-0111 is considered as a broad anti-inflammatory agent, with many purported mechanisms of action ranging from ligand blocking, barrier formation, minimization of immune cell migration, reducing cytokine release, inhibition of mast cell infiltration into inflamed tissues, and inhibition of bacterial growth [38,40,41]. GM-0111 in combination with a recombinant thermoresponsive silk-elastinlike protein polymer (SELP) was shown to prophylactically protect mice from RIP [37]. Utilizing the transition from a liquid state to a semisolid gel, SELPs improved rectal delivery and increased GM-0111’s therapeutic effects. The recombinant nature of SELPs allows for precise genetic tuning of structure and resulting function. The combination of Bombyx mori silk (G-A-G-A-G-S) with human tropoelastin (V-P-G-V-G) provides polymer strength and thermoresponsivity, respectively [42]. A wide variation of mechanical, structural, and functional properties can be achieved through variation of the silk to elastin motif ratio [43]. For instance, SELP-815K (8 silk, 15 elastin, 1 lysine substituted elastin motif) has a higher stiffness than SELP-415K (4 silk, 15 elastin, 1 lysine substituted elastin motif), due to a higher silk to elastin ratio (Figure 3.1B, 3.1C) [44,45]. Additional commercially available polymers, 62 Figure 3.1: Structures of therapeutic and polymers. A) Structure of the semisynthetic glycosaminoglycan ether, GM-0111. B) & C) Schematics of silk-elastinlike protein polymers 815K and 415K. Red and blue motifs represent silk and elastin portions, respectively. SELP-815K contains repeats of 8 silk units, 15 elastin units, and one lysine substituted elastin unit. SELP-415K contains repeats of 4 silk units, 15 elastin units, and one lysine substituted elastin unit. Silk motifs hydrogen bond to form beta sheets. Elastin units provide elasticity and form a porous structure. One lysine unit occurs in every 15 elastin blocks giving the overall polymer a net positive charge at pH 7.4. D) Structure of Poloxamer 407. E) Structure of poly(D,L-lactide-co-glycolide)-block-(poly(ethylene glycol)-block-poly(D,L-lactide-co-glycolide). 63 such as Poloxamer 407 (Figure 3.1D) and poly(D,L-lactide-co-glycolide)-block(poly(ethylene glycol)-block-poly(D,L-lactide-co-glycolide) (PLGA-PEG-PLGA) (Figure 3.1E) have also served as rectal delivery systems and used in the clinic [46–49]. SELPs undergo sol to gel transitions by hydrogen bonding and subsequent formation of β-sheets between intermolecular silk blocks, while Poloxamers and PLGA-PEG-PLGA utilize entanglement of micelles and Van der Waals interactions for formation of gel structures. Based on previous success in prophylactically treating RIP, in this study we performed systematic correlation of polymer structure with function in SAGE delivery to inform the appropriate selection of polymer formulations for rectal delivery of SAGE in the treatment of RIP. We hypothesized that primary polymer structure, varying mechanical properties, and ultimate gel architecture will result in differences in GM-0111 rectal bioaccumulation and protection by employing a RIP animal model. In this manuscript, we compare how variation of polymer structures (Figure 3.1, B-E) influences rectal bioaccumulation and efficacy of GM-0111 (Figure 3.1A). 3.3 Materials and Methods 3.3.1 Materials GM-0111 was purchased from GlycoMira Therapeutics, Inc. (Salt Lake City, UT). Poloxamer 407 was purchased from Spectrum Chemical Mfg. Corp. (New Brunswick, NJ). Poly(lactic acid)-co-(glycolic acid)-block-poly(ethylene glycol)-blockpoly(lactic acid)-co-(glycolic acid) (PLGA-PEG-PLGA) was purchased from Sigma Aldrich (St. Louis, MO). CFTM633 was purchased from Biotium (Fremont, CA). SELP815K and SELP-415K were produced and characterized as previously described [45,50– 64 52]. Post purification shear processing was performed on SELPs to normalize and enhance material properties [51]. 3.3.2 Macroscopic Phase Separation Prior to testing all polymer formulations were prepared fresh. Poloxamer 407 and PLGA-PEG-PLGA were solubilized in phosphate buffered saline (PBS) to 10 and 20 wt% solutions with GM-0111 (100 mg/mL). Sheared SELP-815K and -415K 12 wt% were mixed with GM-0111 in PBS, yielding 11 and 4 wt% polymer solutions with 100 mg/mL GM-0111. Formulations were distributed into scintillation vials and sealed. They were then immersed in a 37 °C water bath. At various timepoints macroscopic phase separation was observed by horizontally tilting the vial and observing separated fluid. Solutions exhibiting gelation and phase separation were then replicated at smaller volumes (100 µL), and the mass of GM-0111 unloaded was quantified using an Azure A colorimetric assay. Briefly, 10 µL of GM-0111 sample were mixed with 190 µL of a 0.025 mg/mL Azure A solution. Absorbance was observed at 650 nm on a SpectraMax M2 spectrophotometer (Molecular Devices, Sunnyvale, CA). Calibration curves were utilized with every sample reading to experimentally derive concentrations of GM-0111. 3.3.3 Rheology Prior to testing all polymer formulations (Poloxamer 407 20 wt%; SELP-815K 11 wt%; SELP-415K 11 wt%) were freshly prepared with GM-0111 (100 mg/mL). Rheological testing was performed on a Malvern Kinexus Ultra+ Rheometer (Malvern Panalytical, Malvern, United Kingdom) with a 2°, 20 mm steel cone. The environment 65 was kept humidified using an environmental chamber. Viscosity was measured using an oscillatory test over temperatures 4-37 °C (5.76 °C/min) at an angular frequency of 6.283 rad/s. This was followed by a 3-hour oscillatory sweep at 37 °C, 0.01% strain, and 6.283 rad/s. Storage (G’) and loss (G’’) moduli were monitored throughout. Conditions of gelation were defined as the crossover point between storage and loss moduli. All rheology experiments were conducted in at least triplicate. 3.3.4 In Vitro Release Prior to testing, all polymer formulations (Poloxamer 407 20 wt%; SELP-815K 11 wt%; SELP-415K 11 wt%) were freshly prepared with GM-0111 (100 mg/mL). Release rates were investigated in vitro using previous methodology [37]. Briefly, polymers and GM-0111 were mixed yielding a 100 mg/mL final concentration of GM0111. Immediately following, each formulation was loaded into a tuberculin syringe, sealed with Parafilm, and allowed to gel in a humidified incubator at 37°C overnight. The next day a razor blade was used to cleave the needle end of the syringe, producing a uniform cylindrical geometry. Gel formulation was then extruded using the plunger and cut into 20 µL discs. Each disc was placed in an Eppendorf tube and massed for normalization. Then 1 mL of simulated intestinal fluid was added to each tube. At various timepoints (5 min., 15 min., 30 min., 1 hr., 3 hrs., 6 hrs., 12 hrs.) 100 µL of release media was collected and replaced. This was stored at 4°C until evaluated using an Azure A colorimetric assay. All groups were run in triplicate. 66 3.3.5 In Vivo Accumulation GM-0111 was fluorescently labeled as previously described [40,41]. Briefly, 800 mg of GM-0111, 126 mg of N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), and 113 mg of N-hydroxysuccinimide, were suspended in 20 mL of water. This solution was stirred at room temperature for 15 minutes. CF633 (9 mg) was thawed, solubilized in 0.9 mL of water, and added to the reaction. The reaction proceeded overnight before dialysis to remove remaining starting materials and the final product was lyophilized (633GM-0111). In all steps, care was taken to protect sensitive materials from light. BDF-1 mice (7-9 weeks, Strain: 099, 50% male, 50% female) (Charles River, Wilmington, MA) were purchased and allowed to acclimate for one week. Two days prior to experimentation mouse diet was switched to AIN-76A (Research Diets, New Brunswick, NJ), an alfalfa free feed with the goal to limit tissue autofluorescence. The night prior to experimentation food and bedding were removed to fast mice and empty digestive tract. Following this, experimentation proceeded, and the alfalfa free diet continued. Formulations were prepared on ice immediately prior to testing by mixing 2parts GM-0111 and 1-part 633GM-0111 with thermoresponsive polymers (Poloxamer 407 20 wt%; SELP-815K 11 wt%; SELP-415K 11 wt%). Animals were then anesthetized using 3% isoflurane and instilled as previously described [37]. Each animal received approximately 100 µL of the appropriate formulations (n=10). At various timepoints (6 hrs., 12 hrs., 24 hrs., 48 hrs.,) animals were sacrificed and rectums collected. The rectums were then cut down the longitudinal axis to expose the inner lumen. This tissue was then pinned down on a foam backing and imaged using a Spectrum In Vivo Imaging System 67 (IVIS) (Caliper Life Sciences, MA). Tissues were then fixed overnight in 4% neutral buffered formalin at 4°C followed by storage in 70% ethanol. Sectioning was performed by ARUP (Salt Lake City, UT) for a tissue thickness of 2 µm. Tissues were then mounted with ProLong Diamond Antifade Mountant with DAPI (Invitrogen, Carlsbad, CA) and imaged using a Nikon A1 confocal microscope. Images obtained from IVIS were quantified for a radiant efficiency using Living Image Software (PerkinElmer, Waltham, MA). Acquired confocal images were analyzed via ImageJ. Semiquantitative analysis was performed by normalizing fluorescent signaling to tissue area using DAPI staining. 3.3.6 In Vivo Efficacy BDF-1 female mice (7 weeks, Stock no. 100006) (The Jackson Laboratory, Bar Harbor, ME) were purchased and allowed to acclimate for at least one week. The night prior to experimentation mice were massed and fasted by removing food and bedding. Animals were assigned into groups at random, anesthetized, and subsequently instilled with 100 µL of appropriated formulation (n=6). This followed established methodology as described[37]. Immediately following rectal administration, anesthetized mice were transferred to a wood plate in a supine position. Targeted irradiation to the lower abdominal area was applied utilizing 1 x 4 cm2 aperture, with the remainder of the mouse protected by 16.35 mm thick lead plate. A total radio dose of 37.12 ± 0.95 Gy was achieved using a RS 2000 X-ray irradiator (RAD SOURCE Technologies, GA, USA) set to level 4 for 716 s. Following this procedure, mice were monitored during recovery and returned to cages with feed. Mice were sacrificed and tissue collected 7 days following treatment and irradiation. All animal study protocols in this manuscript were reviewed 68 and approved by the Institutional Animal Care and Use Committee at the University of Utah. 3.3.7 Pain Assessment Behavioral pain was evaluated immediately prior to fasting and prior to sacrifice on day 7. Briefly, mice were placed in an enclosure with a mesh bottom and allowed to acclimate for no less than 10 minutes. The degree of pain in the lower abdominal area was assessed with von Frey filaments of varying stiffnesses (0.04, 0.16, 0.40, 1.00, and 4.00 g). The lower abdominal area was stimulated ten times with each stiffness and positive responses (sharp abdominal retraction, jumping, or licking/scratching of stimulated area) were recorded (n=6) [37,53]. 3.3.8 Tissue Collection and Histological Assessment Following animal sacrifice, 1 – 2 cm sections of the rectum were necropsied and placed in 4% neutral buffered formalin overnight at 4 °C for fixation. Tissue was then transferred to a 70% ethanol solution and submitted to the ARUP core facility for processing and embedded in paraffin blocks. 2 µm sections were obtained, and stained with hematoxylin and eosin stain (n=6). Slides were analyzed microscopically and representative images were obtained using an Olympus U-MDOB3 with an Olympus DP27 camera. Histological scoring was performed to assess for surface cell flattening, luminal migration of nuclei, cryptitis, edema, inflammation in the surface epithelium and within the lamina propria, eosinophilic crypt abscesses, loss of glands, and loss of goblet cells as previously described [54,55]. A corresponding overall histologic grade was 69 assigned based on the severity of identified mucosal damage including crypt atrophy and crypt loss and assigned as follows: normal (grade 0), mild (grade 1), moderate (grade 2), marked (grade 3), and severe (grade 4). Analysis was performed in a blinded fashion as the pathologist was unaware of assigned treatment group at the time of evaluation. 3.3.9 Statistical Analysis A Grubb’s test was used for assessment of outliers within data sets. A two-way, unpaired student’s t-test was used to assess statistical significance between experimental and control groups. A two-way ANOVA was utilized to determine significance of data comparing more than two groups. Bonferroni post-tests assigned significance between specific groups. Graphs and charts were created in GraphPad Prism (GraphPad Software, Inc. La Jolla, CA) and were edited in Adobe Illustrator (Adobe, San Jose, CA). All data are represented as a mean ± standard deviation, with the exception of computed area under the curve represented as mean ± propagated error. Significance was assigned as p ≤ 0.05 (*), p ≤ 0.01 (**), or p ≤ 0.001 (***). 3.4 Results 3.4.1 Macroscopic Phase Separation To evaluate thermoresponsive behavior of polymers for uniform rectal delivery, we evaluated gels for formation and homogeneity. The PLGA-PEG-PLGA (10 and 20 wt %) compositions resulted in macroscopic phase separation (Figure A.1A). This separation was collected and the percentage of unloaded GM-0111 was calculated to be 76.30 ± 2.09 % and 54.77 ± 34.29% for 10 and 20 wt% matrices, respectively (Figure A.2B). 70 Macroscopic phase separation of PLGA-PEG-PLGA copolymers exhibited a large degree of GM-0111 unloading upon gelation and were deemed unsuitable for further investigation. The addition of GM-0111 to low wt % Poloxamer 407 and SELP-415K solutions resulted in non-gelling solutions. All other formulations formed an observable gel within one hour. SELP-815K (4 and 11 wt %), SELP-415K (11 wt %), and Poloxamer 407 (20 wt %) did not result in macroscopic phase separation. The macroscopic separation exhibited by PLGA-PEG-PLGA formulations and subsequent GM-0111 retention may have led to nonuniform coating of the rectum lumen. 3.4.2 Rheological Analyses of Control Polymers and Polymers Formulated with GM-0111 Rheological evaluation of each formulation was conducted to determine if they would be injectable within the clinical environment. To determine aspects of injectability, gelation kinetics, and potential drug-polymer interactions, rheological experiments were performed. For formulations that produced gels within one hour and no indications of macroscopic phase separation, we evaluated the viscosity from 4-37 °C to determine injectability (Figure 3.2A). Viscosities were then analyzed at 4 °C, 23 °C, 37 °C, representing refrigeration, ambient, and body temperatures (Figure 3.2B). A clear shift in the thickening temperatures (p <0.05). Upon GM-0111 addition, the viscosity of SELP415K increased from 0.064 ± 0.026 PaS to 0.423 ± 0.042 PaS (p < 0.001) at 23 °C and from 0.090 ± 0.009 PaS to 0.720 ± 0.389 PaS (p = 0.049) at 37 °C. Increased viscosity due to the addition of GM-0111 to SELPs was more than simply additive, indicating intermolecular interactions between SELP and GM-0111. Poloxamer/GM-0111 71 Figure 3.2: Viscosity of candidate formulations. A) Viscosity trace of control polymers and polymers formulated with GM-0111 (100 mg/mL). B) Viscosity values at various temperatures of control polymers and polymers formulated with GM-0111 (100 mg/mL) (*:p<0.05, **:p<0.01, ***:p<0.001). 72 interactions were not as apparent. SELP-415K and SELP-815K formulations reached respective maximum viscosities of 0.72 ± 0.39 PaS and 12.015 ± 14.912 PaS at 37 °C. However, all formulations had viscosities at 4 °C that would allow them to be administered through standard clinical approaches for rectal delivery. To monitor the sol to gel transition of polymers alone and polymer/GM-0111 formulations, we conducted a 3-hour oscillatory time sweep (Figure 3.3A). At specific timepoints, 1, 5, 30, and 180 minutes, we quantified the storage modulus, also known as the elastic modulus (Figure 3.3B), for statistical analysis. The incorporation of GM-0111 in SELP-415K significantly increased the storage modulus from 0.81 ± 0.43 Pa to 8.51 ± 3.77 Pa after 5 minutes (p = 0.024) and from 137.58 ± 50.88 Pa to 1890.00 ± 840.54 Pa after 30 minutes (p = 0.022). The increased storage modulus of SELP-415K/GM-0111 formulations at early timepoints suggests a hygroscopic property of GM-0111, which would dehydrate polymer strands, increasing effective concentration. Interestingly, the addition of GM-0111 to SELP-815K significantly decreased the storage modulus, at timepoints after its gelation time (Figure 3.3C), from 136.50 ± 10.57 kPa to 35.42 ± 20.33 kPa after 30 minutes (p = 0.001) and from 302.60 ± 23.72 kPa to 46.64 ± 26.32 kPa after 180 minutes (p < 0.001). GM-0111 interaction with SELP-815K may interrupt the formation of longer silk blocks, decreasing the storage modulus. The increased storage modulus of the SELP-815K/GM-0111 formulation is also observed at 1 minute, reinforcing the possible hygroscopic property of GM-0111. Poloxamer 407/GM-0111 storage modulus was significantly lower than Poloxamer 407 alone after 180 minutes (p < 0.001), exhibiting a gradual decrease in modulus over the three-hour measuring period (Figure 3.3A). This indicates that GM-0111 interfered with the formation of the micellar 73 Figure 3.3: Storage moduli and gelation behavior of candidate formulations. A) Storage moduli trace of control polymers and polymers formulated with GM-0111 (100 mg/mL). B) Storage moduli after 1 min., 5 min., 30 min., and 3 hrs. of testing. (*:p<0.05, **:p<0.01, ***:p<0.001) C) Gelation time of control polymers and polymers formulated with GM-0111 (100 mg/mL) (*:p<0.05, **:p<0.01, ***:p<0.001) 74 network in poloxamer gels. Incorporation of GM-0111 significantly slowed the gelation time of Poloxamer 407 from near instantaneous (3.33 ± 2.88 s) gelation up to several minutes (186.67 ± 319.00 s) (Figure 3.3C). SELP-815K and SELP-815K/GM-0111 gelation times were not statistically different. SELP-415K/GM-0111 formulations formed a gel significantly faster (330.00 ± 18.03 s) than the SELP-415K polymer only (636 ± 132.88 s). Additionally, the SELP-415K/GM-0111 composition underwent gelation significantly more slowly than Poloxamer/GM-0111 (p = 0.006) and SELP-815K/GM0111 (p = 0.036). Inclusion of GM-0111 alters the rate of gel formation and final gel strengths, indicating modification of network formation likely due to existing polymerdrug and drug-solvent interactions. 3.4.3 In Vitro Release Rectal delivery must be carefully balanced for locally active therapeutics to deliver their payload prior to defecation yet provide prolonged delivery to maximize therapeutic effect. In vitro testing was performed using gels produced from Poloxamer 407 20 wt%, SELP-815K 11 wt%, and SELP-415K 11 wt%, all with 100 mg/mL GM0111 (Figure 3.4A). Analysis of the initiating one hour of release revealed significant differences among Poloxamer and SELP formulations (Figure 3.4B). Poloxamer 407 gels released 7.89 ± 1.92% of GM-0111 cargo after 5 minutes, completing cumulative release within 1 hour. SELP-815K and SELP-415K gels resulted in similar release kinetics with respective release of 1.55 ± 2.12% and 4.3 ± 3.95% after 5 minutes. SELP gels resulted in sustained release for 12 hours in vitro. Compared to SELP gels, Poloxamer gels released GM-0111 significantly faster after 15, 30, and 60 minutes of release (p < 0.001) 75 Figure 3.4: Controlled release of glycosaminoglycan. A) Release kinetics of GM-0111 (100 mg/mL) from Poloxamer 407, SELP-815K, and SELP-415K. B) Burst release quantified at various times (***: p<0.001). 76 (Figure 3.4B). There were no statistical differences between SELP-815K and SELP-415K formulations. This similarity suggests a pore mediated release mechanism, likely dominated by the length of elastin domains. SELP gels provided a controlled release profile over a 12-hour period, compared to only 1 hour for Poloxamer. The sustained release of SELP matrices may provide an increased duration of therapeutic window, as rapid mucosal turnover clears accumulated GM-0111 promptly. 3.4.4 GM-0111 Bioaccumulation in the Rectum of BDF-1 Mice To understand how formulations impacted local bioaccumulation in the rectum we used an IVIS to observe the luminal surface excised rectal tissue (Figure 3.5A) and confocal microscopy. During testing all formulations were easily capable of being administered through 0.51 mm diameter (1.5 Fr) catheters. Macroscopic evaluation via an IVIS showed semi-quantitative enhancement of accumulation in rectal tissue with all polymeric groups (Figure 3.5B). Bioaccumulation persisted past 24 hours following rectal administration, especially within the SELP-415K and Poloxamer 407 groups. After 6 hours of administration, SELP-415K copolymers resulted in significantly higher degrees of GM-0111 accumulation when compared to all other experimental groups (7.393 x 107 ± 6.427 x 107 p/sec/cm2/sr; p < 0.001) (Figure 3.5C). Poloxamer 407 (4.587 x 107 ± 3.159 x 107 p/sec/cm2/sr) resulted in significantly higher accumulation compared to the PBS (1.425 x 107 ± 8.179 x 106 p/sec/cm2/sr) and SELP-815K (1.637 x 107 ± 6.151 x 106 p/sec/cm2/sr), after 6 hours post administration (p < 0.05). SELP-415K showed the greatest degree of enhancement at the 6-hour time point having 5 times the fluorescent signal as the PBS group and nearly twice as much as the Poloxamer 407. The maximum 77 Figure 3.5: Assessment of rectal bioaccumulation. A) Schematic of tissue imaging. Tissue was dissected from sacrificed animals, and rectums were cut down the longitudinal axis. Luminal tissues were then pinned face up and imaged using IVIS. B) Representative images of processed rectums receiving fluorescently labeled GM-0111 via SELP-415K, Poloxamer 407, SELP-815K, of PBS. C) Normalized radiant efficiency of mice receiving respective compositions. (ꝉ: SAGE delivered by 415K bioaccumulate significantly higher than all other compositions (p<0.05); ⱡ: Delivery by Poloxamer 407 results in significantly higher bioaccumulation than SELP-815K and PBS (p,0.05). D) Maximum normalized radiant efficiency occurred at 6 hours. (**: p<0.01; ***:p<0.001). E) Calculated total area under the curve of GM-0111 for each delivery system (**: p<0.01; ***:p<0.001). 78 radiant efficiency for all groups was observed 6 hours following rectal administration (Figure 3.5D). The area under the curve was then calculated for each polymer composition to determine overall exposure of the tissue to GM-0111 over the study period (Figure 3.5E). SELP-415K again showed the greatest enhancement of GM-0111 delivery compared to the other compositions evaluated over the studied period. Microscopic evaluation via confocal microscopy (Figure A.2) confirmed SELP-415K significantly increased GM-0111 accumulation as analyzed, compared to all other groups after 6 hours (p < 0.001) and compared to PBS after 12 hours (p < 0.01) (Figure 3.6A). After 6 hours, SELP-415K increased the relative fluorescence signal 4.8-, 8.0-, and 82.9fold compared to Poloxamer 407, SELP-815K, and PBS, respectively. Calculated area under the curve (Figure 3.6B, 3.6C) further illustrates the enhancement of utilizing SELP-415K as a polymeric carrier to deliver GM-0111. In both IVIS and confocal analysis, the majority of tissue fluorescence was lost 24 hours after administration, likely resulting from rapid tissue turnover found in the mucosal compartment. SELP-415K retains a larger portion of fluorescence after 12 hours. Confocal analysis revealed absorption into the muscular mucosae, sub mucosa, and even interactions with endothelial cells. The increase of fluorescent signal from SELP-415K matrices could be a factor of improved rectal spreading and sustained release across the mucosal layer. The corroboration between the macroscopic (IVIS) and microscopic (confocal microscopy) analyses led us to proceed with SELP-415K as our polymeric carrier of choice in our subsequent investigations. 79 Figure 3.6: Analysis of GM-0111 rectal bioaccumulation via confocal microscopy. A) Fold increase in tissue fluorescence from polymers delivering fluorescently labeled GM0111 per tissue area (ꝉ: 415K significantly higher than all other polymers, p<0.001; ⱡ: SELP415K significantly higher than PBS, p<0.01). B) Calculated GM-0111 area under curve (ꝉ: 415K significantly more than all other systems, p<0.01). C) Total calculated area under the curve for polymeric systems delivering GM-0111 (***: p<0.001). 80 3.4.5 Behavioral Pain Responses To understand how GM-0111 formulations impacted radiation-induced pain in mice we assessed behavioral pain responses. Irradiated animals (n=6) showed a dramatic increase in their sensitivity to mechanical stimulation (Figure 3.7A). All animals showed a typical log shaped response increasing stimulation levels. Allodynia, or the sensitization to previously nonpainful stimuli, was assessed by calculating the stimulation level required to increase response rates by 30% from baseline measurements (Figure 3.7B). Non-irradiated healthy mice (n=5) had no meaningful change in their pain response, indicating that radiation and no other environmental factors were responsible for changes to the pain response. The SELP-415K/GM-0111 enema group needed a stimulus threshold of 2.76 ± 1.92 g, exhibiting improved allodynia compared to all other irradiated mice which had stimulus thresholds less than 0.15 g (p<0.001) (Figure 3.7B). The greatest resolution of behavioral responses was observed at the 0.16 g and 0.40 g stimulus levels (Figure 3.7C). At these forces, the nonirradiated baseline response rates (n=30) were 16.33 ± 11.8 % and 19.66 ± 10.66 %, respectively. Upon irradiation the animal response rates increased across all groups. The PBS only group had response rates of 70.00 ± 25.29 % and 70.00 ± 17.89 % at 0.16 and 0.40 g stimulus respectively. At the 0.16 g, 0.40 g, and 1.0 g stimuli the nonirradiated baseline had a significantly lower response rate (p < 0.001) than the irradiated PBS, PBS/GM-0111, and SELP-415K compositions (Figure 3.7C). Where traditional saline-based rectal delivery of GM-0111 failed to provide meaningful protection to radiation-induced pain, delivery from thermoresponsive polymers, particularly SELP-415K, showed improved responses. There was no significant difference between the non-irradiated baseline response rate and 81 Figure 3.7: Behavioral response rates of irradiated BDF-1 mice. A) Response rate of BDF-1 mice 7 days following treatment. Mice were assessed with stimulation of 0.04, 0.16, 0.4, 1.0, and 4.0 g filaments to the lower abdomen. B) Threshold of stimulus needed to increase response rate 30% from baseline measurements. (ꝉ: threshold exceeded 4.0 g (***: p <0.001). C) Response rates for 0.16, 0.40, and 1.0 g stimulus (***: p<0.001, *:p<0.05). 82 SELP-415K/GM-0111 composition at stimuli of 0.16 g, 0.40 g, and 1.0 g (p >0.05). The SELP-415K/GM-0111 treatment significantly lowered response rates compared to PBS, PBS/GM-0111, and SELP-415K groups at the 0.16 g and 0.40 g stimulus levels (Figure 3.7C). Behavioral outcomes indicate the necessity of SELP-415K to deliver GM-0111 for a protective capability in this RIP animal model. The retention and increased bioaccumulation afforded by this formulation likely increases rectal bioavailability and more easily achieves a therapeutic window for pain reduction. 3.4.6 Animal Health The ability of GM-0111 to protect animal health in response to radiation-induced injury was evaluated by measuring animal weight change, fecal mass, and fecal water content. Healthy control mice maintained their initial mass, whereas radiation caused a statistically significant drop in weight (Figure 3.8A). The irradiated animals receiving the SELP-415K/GM-0111 enema maintained 96.98 ± 13.28% of their body weight, similar to the non-irradiated control group (99.84 ± 2.59 %). Mice receiving the SELP-415K/GM0111 composition exhibited significantly better weight maintenance than mice that received no treatment, or PBS/GM-0111. SELP-415K alone was insufficient to provide any protection. Both SELP-415K and GM-0111 were required in combination to have a meaningful therapeutic effect. During final animal assessment and sacrifice, we collected animal fecal matter for further analysis. The mass of fecal content (Figure 3.8B) was insignificantly higher in both healthy and irradiated animals receiving SELP-415K/GM0111. This increase was not associated with a decreased water content in the fecal material (Figure 3.8C). Together, these data indicate an indirect increase in animal health 83 Figure 3.8: Assessment of animal mass and fecal matter. A) Animal mass reported as a percentage of starting mass prior to irradiation. B) Mass of collected feces from animals. C) Percentage of water content in fecal matter of mice (*:p<0.05, **:p<0.01, ***:p<0.001). 84 and that the SELP-415K/GM-0111 formulation did not substantially interfere with voiding. 3.4.7 Histology To understand the SELP-415K/GM-0111 system’s local radioprotective effects, we performed histological evaluation of rectal tissue. Evidence of radiation-induced damage was identified in irradiated animals (Figure 3.9A). Sections show variable amounts of inflammation within the lamina propria and surface epithelium along with cryptitis. There was loss of glands (crypt dropout) and goblet cells, eosinophilic crypt abscesses, cell flattening in the surface epithelium, and luminal migration of epithelial nuclei. These findings were comparable with RIP injury histologically as described by Hovdenak et al [54]. Epithelial alterations attributed to irradiation were identified in 21 of 24 samples. All healthy animals (n=5) showed tissue with intact crypts and no significant microscopic alterations. Overall, animals receiving the SELP-415K/GM-0111 showed moderate amount of mucosal damage, with all other irradiated groups exhibiting more severe changes. When scored in a blinded manner, SELP-415K/GM-0111 reduced cell flattening, luminal migration, gland loss, and goblet cell loss within the rectal tissue. Both treatments utilizing GM-0111 recorded histological scorings of 0 for inflammatory infiltrates, suggesting that GM-0111 may have an immunomodulatory effect. The overall injury grade was further calculated with the SELP-415K/GM-0111 treatment recording the lowest overall grade of injury, compared to all other irradiated groups (Figure 3.9B). Together these findings indicate a protective effect from delivery of GM-0111 by SELP415K copolymers. 85 Figure 3.9: Histological analysis of rectal tissues. A) Representative tissue images stained with hematoxylin and eosin (H & E). Healthy tissue did not exhibit any abnormalities. All other irradiated tissues showed crypt abscesses (arrows) or dropping of crypts altogether (arrowheads). (Scalebar = 50 µm) B) Blinded scoring of injury grade for each tissue (***: p < 0.001). 86 3.5 Discussion RIP is a debilitating disease and radiotoxic event, in need of effective prophylactic therapeutic strategies. However, current pharmaceutical interventions have been largely ineffective or carry significant side effects that prevent their usage until after symptoms emerge. GM-0111 has prophylactic efficacy in treating radiation-induced disease but requires the use of drug delivery technologies to achieve meaningful results in the rectum. Previously, we showed the capability of SELPs to effectively and efficiently enhance delivery of GM-0111 to the rectum for protection in a RIP model [37]. These positive outcomes and advantages of rectal administration (noninvasive nature, localized delivery, limited systemic exposure, natural clearance via defecation, and rapid effect) led us to further investigate other thermoresponsive formulations [56]. In this study, we demonstrated the utility of temperature responsive polymer systems for enhancing rectal delivery of GM-0111 and showed that primary polymer structure and physicochemical properties have dramatic impact on function. Initial compositions were chosen from studied material properties. The concentration of drug chosen was based on careful evaluation of drug/material interactions. Previously, incorporation of less GM-0111 (10 mg/mL) into these polymers yielded compositions with incomplete release by 24 hours [39]. This sustained release likely results from drug-polymer ionic interactions. Increased loading of SELP-815K to 100 mg/mL GM-0111 increased the percent released cargo after 24 hours and maintained mechanical properties sufficient for rectal gelation. We hypothesized this increased GM-0111 concentration would lead to increased bioavailability. All polymeric systems used had injectable viscosities at 4 °C, with thickening, or gelation events following elevation to body temperature after 87 administration. Controlled release from SELP-415K and SELP-815K provided a sustained release profile lasting significantly longer than poloxamer-based delivery. Upon rectal administration, GM-0111 delivery via SELP-415K resulted in the highest degree of bioaccumulation. This formulation showed the ability for prophylactic reduction of radiation-induced tissue damage and amelioration of RIP in an animal model. The combination of a thermoresponsive polymer-drug delivery system significantly reduced pain response and substantially ameliorated local radiotoxicity. Mechanical properties of rectal formulations influence drug bioaccumulation. Typically, administration of liquid enemas allows for rapid absorption due to circumvention of matrix-mediated drug release [57], however these commonly suffer from poor retention time and leakage [58]. The viscosity properties of a liquid enema largely influences the degree of rectal spreading [56], with low viscosity formulations capable of spreading more. Thus, the transition of a phase-changing enema from a liquid to semisolid provides a mechanism to ensure uniform coverage and increased bioaccumulation by improving retention. SELP-415K and SELP-815K obtained similar release kinetics, but the use of SELP-415K drastically increased the bioaccumulation of GM-0111 when compared to SELP-815K. We hypothesize this is due to the decreased gelation rate of SELP-415K, the resulting increased duration of a liquid state may allow for increased penetration of the mucus coating, followed by retention as a semisolid formulation. SELP-815K due to is faster gelation, did not penetrate as deeply into the mucus prior to gelling, resulting in reduced accumulation. Poloxamer 407 also lead to a high amount of GM-0111 accumulation, likely due to its native gel architecture of intertangled micellular arms, non-physical crosslinking through Van der Waals 88 interactions, and highly viscous gel nature [59]. This would lead to luminal spreading due to the slow flow aspects of Poloxamer formulations. Additionally, both Poloxamer and SELP analogs have exhibited mucoadhesive properties [59,60], which also influence rectal spreading [56]. After 6 hours, the 7.9-fold increase in relative fluorescence signal from SELP-815K to SELP-415K illustrates the importance of the liquid to semisolid enema transition. The duration of SELP-415K in the liquid state enhanced bioaccumulation compared to SELP-815K formulations, while the retention provided by semisolid enema formation increased accumulation as seen in an 82.9-fold increase of fluorescence signal compared to the liquid PBS enema. Many formulations utilize Poloxamer to deliver and accumulate therapeutics in the gastrointestinal space [61–64]. Our SELP-415K formulation outperformed the Poloxamer 407 formulation in terms of rectal bioaccumulation of GM-0111. Liquid enemas are commonly employed for rectal drug delivery and have shown efficacy in treating a variety of diseases. Utilization of a thermoresponsive enema, such as SELP-415K, could further improve these treatments with increased retention, bioaccumulation, and rectal or systemic bioavailability. Upon rectal administration of the selected formulations, GM-0111 must overcome the mucosal barrier to enter the columnar epithelial layer prior to defecation and resulting enema expulsion. Due to this the swift controlled release, Poloxamer 407 (1 hour) and SELPs (12 hours) are advantageous for luminal bioaccumulation. The rectal mucosal barrier has a turnover time of 3-4 hours [56,65]. This turnover time can be affected by disease states and mucoadhesive materials, such as the selected formulations. The sustained release exhibited by SELP-415K is advantageous, as it would span the time of several mucosal turnovers. Additional mucosal-SELP interactions may lengthen this 89 mucosal turnover time [56,60] and, hypothetically, lengthen the duration that the therapeutic window is achieved within patients. A Poloxamer formulation would be cleared in a single mucosal turnover and decrease overall bioavailability. Once the mucosal barrier is crossed, GM-0111 will likely undergo absorption via the paracellular pathway due to its ionicity, water solubility, and relatively larger molecular weight [66], yet this is still to be determined experimentally. Increased accumulation within the rectal epithelium then potentially increases therapeutic effects. Once absorbed, GM-0111 may exert its immunomodulatory effects locally in the mucosa, submucosa, muscularis propria, or enter systemic circulation [56]. Rectal radiation injury can lead to the breakdown of the protective epithelial layer, progressive endothelial dysfunction, and an inflammatory state resulting from tissue and pathogen associated damages [2,67,68]. Breakdown of the columnar epithelial layer may lead to bacterial invasion from native flora. GM-0111 inhibits growth of gram negative bacteria (Porphyyromonas gingivalis and Aggretgatibacter actinomycetemcomitans) as well as inhibit pathogen recognition receptors (PRRs) such as toll like receptors 2 and 4 (TLR2, TLR4) [41]. TLR-2/4 recognize lipopolysacharrides, lipoproteins, and bacterial toxins which exacerbate radiation damage by entering compromised tissues. Receptor signaling leads to nuclear translocation of nuclear factor kappa beta and subsequent transcription of inflammatory mediators [69,70]. Analogs of GM-0111 have also been shown to block toxicities associated with Clostridium difficile toxin A in colonic epithelium. Clostridium difficile has been classified as one of three “urgent threats” by the Centers for Disease Control and Prevention in the United States [71]. Additional GM-0111 inhibition of receptor against glycation endproducts, P-selectin, and polymorphonuclear leukocytes 90 can protect from endogenous tissue damage associated molecular patterns that may be associated with radiation challenge [41]. This multimechanism action presented by GM0111 was ineffective in liquid enema form, likely stemming from poor retention, and subsequent loss of rectal bioavailability. The combination of this broad anti-inflammatory drug and an enema to semisolid formulation resulted in improved outcomes. Behavioral pain responses were decreased in animals receiving SELP-415K/GM-0111 formulations, with indications of decreased allodynia. Animal biometrics indicated healthier animals that were producing a higher amount of non-diarrhea fecal content, and capable of maintaining body mass. Histologically, animals receiving SELP-415K/GM-0111 formulations had a decreased grade of injury, with increased retention of goblet cells and glands. Goblet cells are responsible for mucus secretion, and retention may improve future outcomes and regeneration of the mucosal barrier. The healthy rectal mucosa shows uniform arrangement of glands, inconspicuous mitotic activity and very little inflammatory cell infiltrate. Rectal mucosa obtained from irradiated animals show variable amounts of epithelial injury and resulting inflammatory infiltrates. This includes increased mitosis (SELP-415K/GM-0111), crypt drop out (arrow heads), and crypt abscesses (arrows). Additionally, the observed epithelial cells have nuclear pleomorphism (PBS/GM-0111), loss of goblet cells (PBS), migration of epithelial nuclei towards the lumen (PBS) and flattening of cells on the surface (SELP-415K). There was also some degree of acute neutrophilic inflammation within the mucosa adjacent to the injured epithelium and lymphoplasmacytic cell infiltrate around damaged crypts (SELP415K/GM-0111). Preservation or regeneration of crypts seem evident in SELP415K/GM-0111 compositions. Previously, GM-0111 has been shown to protect urothelial 91 cells from LL-37 induced apoptosis [72]. Distinction between preservation and/or regeneration of radiation damage will need to be investigated at earlier timepoints. These improved outcomes illustrate the ability of SELP-415K to retain and improve rectal bioavailability of GM-0111, which can inhibit molecular inflammatory processes from developing. Sucralfate is another commonly used treatment in the clinic for RIP [32]. Most uses of sucralfate stem from the adhesive properties to inflamed epithelium, protecting the sterile submucosa from inherent luminal flora [73]. Additional antioxidant properties of sucralfate allows for scavenging of free radicals and protection of mucosa from lipid peroxidation [73–75]. Production of epidermal growth factor and prostaglandin E2 are also enhanced with sucralfate. The latter increases mucus secretion by gastrointestinal epithelium goblet cells [76] and may prove problematic for rectal drug delivery. Despite these mechanisms, sucralfate has failed prophylactic clinical trials for RIP, and in some cases increased rectal bleeding and diarrhea [14,32,33]. The key aspect of sucralfate is the formation of a protective barrier; sucralfate treatment following RIP pathophysiology development needs to address pathogen driven inflammation in the submucosa. Both sucralfate and GM-0111 are inherently anti-inflammatory, however in numerous studies GM-0111 has proven to inhibit molecular signaling leading to mucosal inflammatory disorders [37,39,41]. This molecular inhibition of inflammatory events could prevent inflammatory mechanisms from ever occurring. Typical clinical doses of radiation to the pelvic region range from 45 to 50 Gy in total for neoadjuvant therapies, and up to 85 or 90 Gy for adjuvant treatment prostate and curative gynecological cancers, respectively [4,67]. Neoadjuvant and adjuvant therapies 92 occur respectively before and after primary treatment courses, typically surgical intervention. However, these doses are typically administered in a fractionated manner to maximize therapeutic efficacy and minimize toxicities in other tissues. Upon presentation of radiotoxicities, cessation of tumor radio-treatment is a common strategy [4]. Therapeutic efficacy, or curvature of cell killing, is described by the Linear Quadratic Mode. In this mode the cell killing curve is described as the α/β ratio. As the α/β ratio decreases, the tumors respond more to larger doses of radiation. Prostate cancers, for example have a low α/β ratio estimated to be ~1.5 Gy. With this low ratio prostate cancers are more responsive to larger doses of radiation than other nearby tissues such as the rectum (α/β = 3-5 G). This could lead to a therapeutic gain that is not proportional to increased toxicities [77]. The radioprotection of SELP-415K/GM-0111 in this high dose mouse model illustrates potential in preventing RIP resulting from cases such as severe hypofractionation stereotactic body radiotherapy of prostate cancer patients. By utilizing this preventative approach additional dose escalation may be achievable without proportional increases in toxicities. This is valuable as dose escalation is commonly performed for prostate cancers and is associated with improved outcomes [78]. Further investigation of SELP-415K/GM-0111 utility in moderate and extreme hypofractionation approaches is needed. Other tumor types with α/β ratio (>10 Gy) are best treated using smaller fractions to minimize toxicity. Prophylactic intervention for RIP may be of less utility for tumors with a high α/β ratio treated with hyper-fractionated treatment paradigms. Tumors and the rectum are less responsive to increasing fraction size than normal tissues [77]. Our current model which delivered just over 37 Gy likely affects healthy, non-rectal tissue, to a much higher degree than that seen in the clinic. This is 93 difficult to ascertain as rodents have varying sensitivity to ionizing radiation depending on species and strain. The 1x4 cm aperture may have also resulted in irradiation of the small intestine in some animals. These model limitations may obscure results and efficacy, especially in those groups with high behavioral response rates. Additionally, this may challenge efficacy of treatments in later timepoints, as late developing effects may occur. These late developing effects were previously observed in the prior proof of concept study [37]. The chosen radio dose of 37 Gy was similar to a total dose routinely used for prostate cancer patients receiving hypofractionation stereotactic body therapy (36.25, 37.5, 38, etc. Gy) [78]. This dose represents an extremely high, one-time exposure which would not be used in the clinic. However, its purpose to establish severe radiation-induced damage is evident as presented by the histology and behavioral pain responses. The observed positive animal outcomes will likely continue in settings with fractionated radiation challenge in tandem with multiple protective SELP-415K/GM0111 administrations. This investigation evaluated multiple thermoresponsive polymers to form a liquid to semisolid system for rectal delivery of an anti-inflammatory SAGE. Initial indications place emphasis on the importance of gelation rate in bioaccumulation of GM-0111, with more slowly gelling materials capable of providing a higher degree of accumulation. This phenomenon was not directly investigated but could be confirmed or disproven with further controlled experimentation. The efficacy of using SELP-415K/GM-0111 delivery systems for prophylactic protection has shown merit during the 7 day follow up study. However, evaluation at earlier and later timepoints should be conducted to establish time dependence of outcomes, as these may vary from late developing tissue responses. For 94 further preclinical advancement, rectal GM-0111 delivery from SELP-415K needs to be assessed for potential systemic absorption and subsequent biodistribution. Further research needs to be conducted to investigate the utility of SAGE for investigating chronic RIP pathophysiology including development of fibrosis and animal survival curves. This includes alteration of total dose, fractionation, and field of irradiation. Future investigations will include radiation fractionating, dose escalation, and posttreatment rather than pretreatment strategies. In future studies translational differences between rodents and humans need to be addressed including pharmacokinetics and toxicology. Rodents have a much narrower rectal passage, tend to defecate involuntarily, more frequently, and have a faster mucus turnover rate than humans [10,79–81]. Despite these limitations, this work has shown that polymer structure for drug delivery in the rectum can have profound results in potential therapeutic outcomes and that SELPs show promise for rectal delivery of radioprotective agents in treatment of RIP. 3.6 Conclusion Thermoresponsive polymer systems are an effective strategy for increasing the rectal accumulation of glycosaminoglycan-based therapeutics. Slower gelation of thermoresponsive polymer solutions likely improves delivery through increased mucus penetration and enhanced surface contact. In situ gelling polymers improved local retention and bioaccumulation with SELP-415K>Poloxamer>SELP-815K. 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Sci. 2010, 60 (1), 75–79. https://doi.org/10.1007/s12576-009-0060-8. CHAPTER 4 AN OLIGOMERIC SULFATED HYALURONAN AND SILK-ELASTINLIKE POLYMER COMBINATION PROTECTS AGAINST MURINE RADATION-INDUCED PROCTITIS 4.1 Abstract Semisynthetic glycosaminoglycan ethers (SAGEs) are short, sulfated hyaluronans which combine the natural properties of hyaluronan with chemical sulfation. In a murine model, SAGEs provide protection against radiation-induced proctitis (RIP), a side effect of lower abdominal radiotherapy for cancer. The anti-inflammatory effects of SAGE have been studied in inflammatory diseases at mucosal barrier sites, however few mechanisms have been uncovered necessitating high throughput methods. SAGEs were combined with silk-elastinlike polymers (SELPs) to enhance rectal accumulation in mice. After high radiation exposure of the lower abdominal area, mice were followed for 3-days or until they met humane endpoints, before evaluation of behavioral pain responses and histological assessment of rectal inflammation. RNA sequencing was conducted on tissues from the 3-day cohort to determine molecular mechanisms of SAGE-SELP. After 3 days, mice receiving the SAGE-SELP combination yielded significantly lowered pain Steinhauff, D.; Jensen, M. M.; Griswold, E.; Jedrzkiewicz, J.; Cappello, J.; Oottamasathien, S.; Ghandehari, H. An Oligomeric Sulfated Hyaluronan and Silk-Elastinlike Polymer Combination Protects against Murine Radiation Induced Proctitis. Pharmaceutics 2022, 14 (1), 175. https://doi.org/10.3390/pharmaceutics14010175. 105 responses and amelioration of radiation-induced rectal inflammation. Mice receiving the drug-polymer combination survived 60% longer than other irradiated mice, with a fraction exhibiting long term survival. Sequencing reveals varied regulation of toll like receptors, antioxidant activities, T-cell signaling, and pathways associated with pain. This investigation elucidates several molecular mechanisms of SAGEs and exhibits promising measures for prevention of RIP. 4.2 Introduction Radiation-induced proctitis (RIP) is a common side effect of pelvic radiotherapies which aim to treat lower abdominal cancers such as prostate, uterine, vaginal, and cervical cancers [1]. The anatomical fixed position makes the rectum especially susceptible to exposure from ionizing radiation, commonly resulting in inflammation. Of the patients receiving lower abdominal radiotherapy, it is estimated that 5-20% will develop some form of acute and/or chronic RIP [2]. Acute RIP occurs within days to months following irradiation and can result in common symptomology of abdominal pain/cramps, diarrhea, hematochezia, and other adverse effects [2,3]. The occurrence of acute RIP can lead to cessation of radiotherapy schedules which are needed for cancer treatment. Chronic RIP typically develops months to years following irradiation, and is met with more debilitating symptoms of acute RIP along with possibilities of fistulas, incontinence, strictures and adverse effects [2]. The full pathological development of RIP is still being elucidated. Upon irradiation, double stranded DNA breaks and cell membrane damage could lead to cell and tissue injury. Radiolysis of intracellular water leads to free radical generation and further tissue damages [4,5]. After just two weeks of initiating radiotherapy, histological changes have been observed in the clinic. These 106 changes include inflammatory infiltrates, migration of nuclei, atypical mitoses, loss of intestinal stem cells, loss of glands, etc. [3]. Presentation of acute RIP increases the risk of late developing chronic proctitis by more than 5-fold in patients following external beam radiotherapy for prostate carcinoma [6]. The resolution of acute RIP can result in characteristics of chronic proctopathy which include fibrosis and epithelial atrophy. Currently, there are limited therapeutic prophylactics which aim to modulate the presentation of acute inflammation found within RIP. Semi-synthetic glycosaminoglycans are derived from naturally occurring glycosaminoglycans with additional molecular programming provided by synthetic modifications. Chemically sulfated hyaluronic acid-based molecules are one such example. Some of the programmed properties of sulfated hyaluronans include inhibition of hyaluronidase [7], blocking of P- and L-selectin [8], and increased interactions with BMP-4 and TGF-β1[9,10]. Semi-synthetic glycosaminoglycan ethers (SAGEs), consisting of oligomeric sulfated hyaluronans, exhibit a variety of therapeutic mechanisms in dampening inflammatory injuries. Some of these mechanisms include blocking of pathogen associated molecular patterns (PAMPs) [11], release of extracellular ATP from urothelial cells [12], inhibition of the receptor for advanced glycation end products (RAGE) [13], and reduced inflammatory infiltrates into inflamed sinonasal epithelium[14]. A particular SAGE, GM-0111, has exhibited anti-inflammatory properties in mucosal settings such as interstitial cystitis [12,15], periodontitis [11], and RIP [16,17]. In a murine RIP model, rectal administration and accumulation resulted in a radioprotective effect against RIP, illustrated by decreased histological injury and symptomology as assessed via behavioral pain responses. This protection was made possible by delivery via a liquid to semi solid enema system, mediated by delivery via 107 hydrogels made of silk-elastinlike protein polymers (SELPs) [16,17]. SELPs have been utilized to enhance the rectal bioaccumulation and efficacy of GM-0111 in a prophylactic murine RIP model [16,17]. SELPs are composed of motifs derived from Bombyx mori silk (GAGAGS) and human tropoelastin (GVGVP). The combination of these silk and elastin motifs provides SELPs with the ability for in situ crosslinking and thermoresponsive behavior [18]. Upon heating to body temperature, and depending on structure and concentration, SELPs undergo a rapid sol to gel transition, resulting in a robust crosslinked hydrogel. These crosslinks, mediated by silk motifs, are composed of antiparallel beta sheets held together through hydrophobic interactions and hydrogen bonding [19,20]. This passive crosslinking mechanism provides SELPs with injectability and biocompatibility, capable for use in local gene delivery [21,22], drug delivery [23], embolic applications [24], and cellular scaffolds[25]. Several analogs of SELPs have been produced with variation in silk and elastin sequences, providing opportunity to tune SELP gel mechanical properties, matrix characteristics, and control over spatiotemporal release kinetics in vivo [21]. In the context of RIP, SELP-415K (4 silk units, 15 elastin units with one lysine substituted elastin unit per repeat) enhanced rectal accumulation of GM-0111 compared to phosphate buffered saline, Poloxamer 407, and another SELP analogue. This enhanced accumulation was attributed to sustained release over 12 hours and slower gelation compared to other investigated polymers. In vivo this translates to GM-0111 accumulation through several mucus turnovers and a larger gel-tissue interface, respectively [16]. When mice were exposed to high doses of irradiation, this drug-polymer composition provided rectal protection after 7 days by limiting rectal injury, decreasing behavioral pain responses, and reducing radiotoxicities 108 [16]. The aim of this study is to expand upon the protection provided by matrix-mediated delivery of GM-0111 via SELP-415K hydrogels. Due to the enhanced delivery provided by SELPs, we hypothesize that delivered GM-0111 will: 1) provide early protection against RIP, 2) sustain animal health, and 3) modulate the early pathological development of RIP. 4.3 Materials and Methods 4.3.1 Materials SELP-415K was synthesized and processed as previously described [26–29]. GM0111 was purchased from GlycoMira, Inc. (Salt Lake City, UT). Phosphate buffered saline (PBS) was purchased from Sigma Aldrich (St. Louis, MO, USA). Female (8 week old) BDF-1 mice (stock no: 100006) were obtained from The Jackson Laboratory (Bar Harbor, ME, State). All animal care and procedures were conducted in accordance with The University of Utah Institutional Animal Care and Use Committee policies [approval number: 20-03015 (2020)]. 4.3.2 Mouse Treatment and Irradiation Mice were treated as previously described [17]. Prior to procedures, mice were weighed and fasted overnight by removing all food and bedding from cages. The next morning mice were assigned randomly to experimental groups (n = 6). These included PBS, GM-0111 (100 mg/mL) in PBS (PBS/GM-0111), SELP-415K 11 wt. %, and GM0111 (100 mg/mL) in SELP-415K 11 wt. % (SELP-415K/GM-0111). Mice were anesthetized with 2 % isoflurane. A catheter was inserted into rectums and up to 100 µL 109 of each formulation was slowly instilled into the rectum of each mouse as the catheter was slowly retrieved from the cavity. Mice were then immobilized on an irradiation platform in a supine position. A lead plate was placed above the mice with 4x1 cm apertures to restrict irradiation to the lower abdominal region over the rectum. The anus of each mouse was aligned to the bottom of the aperture or immediately below it. Mice then received 37 Gy of irradiation using a RS 2000 X-ray irradiator (RAD SOURCE Technologies, Buford GA, USA). Immediately following, mice were monitored for a recovery period before being placed back into cages with food and bedding. Mice were then monitored for signs of distress and loss of body weight. Mice were sacrificed at a 3day timepoint or after loss of >20% of body weight. Animals not exhibiting excess body weight loss were sacrificed at a 14-day endpoint. 4.3.3 Behavioral Pain Testing Before overnight fasting, mice were assessed for behavioral pain responses to obtain baseline response rates. Mice were placed in a mesh enclosure for at least 10 minutes prior to stimulation of the suprapubic area with 0.04, 0.16, 0.40, 1.0, and 4.0 g Von Frey filaments, as previously described [30]. To avoid wind up effects, each stimulation trial occurred at least 10 seconds apart. A total of 10 replicates for each filament strength were recorded. Positive responses included sharp abdominal retractions, jumping, vocalization, and/or immediate scratching/licking of the stimulated area. Behavioral pain responses were also evaluated immediately prior to sacrifice to assess the sensitivity of each mouse to mechanical stimulation (n=6). 110 4.3.4 Histology The tissue sampling was performed on deceased animals 3 days after irradiation and at the survival endpoint determined by animal weight loss of >20% after irradiation (variable survival length). Rectal tissues were dissected grossly during necropsy and subsequently fixed in 10% neutral buffered formalin, before soaking in 70% ethanol for at least 24 hours. Specimens were then submitted to Associated Regional and University Pathologists, Inc. (Salt Lake City, UT) for sectioning, processing, and embedding into the paraffin blocks. 2 µm sections were obtained and placed into glass slides and stained with hematoxylin and eosin (H&E) (n=6). All slides were evaluated histologically according to the previously described scoring method [3,31]. Evaluation included assessment of surface epithelium such as loss of cellular height and flattening of cells and cellular inflammatory infiltrates (in the form of neutrophils). Glandular composition was also assessed particularly the luminal migration of epithelial nuclei, loss of goblet cells, mitotic activity, cryptitis, eosinophilic crypt abscesses, loss of glands, atrophy of glands and gland distortion. The lamina propria assessment included evaluation of inflammatory infiltrates, edema, and congestion of vasculature. The mitotic activity was scored as normal, increased, or decreased. The eosinophilic crypt abscesses, gland atrophy, and distortion were marked as absent or present. The remaining histologic abnormalities were scored on a scale of 0 to 4 (0 = normal, 1 = mildly abnormal, 2 = moderately abnormal, 3 = markedly abnormal, 4 = severely abnormal). Additionally, an overall microscopic damage score was assigned based on histologic alterations identified with low power magnification (mostly based on architectural abnormalities of glands and inflammatory infiltrates) and scored on a scale 111 of 0 to 4 (0 = normal, 1 = mildly abnormal, 2 = moderately abnormal, 3 = markedly abnormal, 4 = severely abnormal). Histologic assessment was performed in a blinded manner, as the pathologist was not aware of the treatment groups at the time of assessment [3,31]. Data was tabulated in Microsoft Excel spreadsheets (2111, Arlington, VA, USA). 4.3.5 RNA Sequencing Upon animal sacrifice, at least 10 mg of rectal tissue was collected and flash frozen (n=6). Samples were then sent to GENEWIZ (South Plainfield, NJ) for processing. Counts were collected with an Illumina HiSeq (Illumina, San Diego, CA, USA), 2x150 bp configuration, single index, per lane sequence configuration. Received counts were then processed as follows. A mouse genome (GRCm38) and gene feature files were obtained from the Ensembl release 102[32]. A reference database was generated using STAR (2.7.6a, Cold Spring Harbor, NY, USA) with optimized splice junctions of 150 base pair reads. Reads were trimmed using cutadapt (1.16, Dortmund, Germany) [33] and aligned using STAR in two pass mode to the reference database. Reads were assigned to genes using featureCounts (1.6.3, Parkville, Australia) [34]. Output files were summarized with MultiQC (1.11 Stockholm, Sweden) to assess for outliers [35]. Differentially expressed genes were identified using a false discovery rate of 5% with DESeq2 (1.30.1, Heidelberg, Germany) [36] and pathways analyzed with Ingenuity Pathway Analysis (70750971, Qiagen, Hilden, Germany) [37]. Gene Ontology was assessed using EnrichR (March 2021 Version, New York, NY, USA) [38–40]. 112 4.3.6 Statistical Analysis Behavioral response rates were analyzed with a 2-way ANOVA with a Bonferroni post hoc test. Nonparametric histological data was assessed using a Kruskal-Wallis test with a Dunn’s Multiple Comparison Test for group-to-group comparison. Survival trends were analyzed using a Mantel-Cox test and corrected for multiple comparisons [41,42]. Data is represented as mean ± standard deviation. Gene ontology p-values were computed using the Fisher exact test, assuming binomial distribution and independence for probability of any gene belonging to any set. Data was organized using Microsoft Excel, graphs prepared in GraphPad Prism (5.01, GraphPad, San Diego, CA, USA), and figures adapted for publication in Adobe Illustrator (23.01, Adobe, San Jose, CA, USA). 4.4 Results 4.4.1 3-Day Behavioral Pain Responses To evaluate the development of nociceptive response during the acute phase of RIP development, we assessed mechanical sensitivity 3 days after irradiation. All irradiated animals had signs of increased pain as assessed with Von Frey filaments (Figure 4.1A). At a stimulus of 0.4 g, the animals having received the PBS and PBS/GM0111 compositions yielded significantly increased positive response rates (61.7 ± 22.3 and 56.7 ± 19.7 %) compared to healthy control animals (27.1 ± 22.8 %). At this stimulus force of 0.4 g, the animals receiving the SELP-415K/GM-0111 composition only had a response rate of 33.3 ± 8.2 % (Figure 4.1B). At 0.16 g stimulus, animals receiving SELP415K/GM-0111 (+3.3 ± 20.7 %) had a significantly lower change in response rate compared to animals receiving PBS (+40.0 ± 20.0 %). Groups receiving PBS/GM-0111 113 Figure 4.1: Behavioral pain responses 3-day post irradiation. A) Response rates of irradiated animals receiving selected treatments and healthy controls. B) Response rates with the 0.4 g filament. C) Change in response rates from baseline measurements with the 0.16 g filament. D) Threshold required to elicit an allodynic response as measured by an increase in 30% from the baseline. Red box indicates animals with no allodynic response (na) (**: p < 0.01, *: p < 0.05) 114 (+20.0 ± 16.7 %) and SELP (+16.7 ± 15.1 %) had insignificant changes to their sensitivity (Figure 4.1C). Analysis in this manner emphasizes the individual animal basis, as it normalizes to using baseline responses prior to treatment and irradiation. Interestingly, SELP alone exhibited some effect in reduction of behavioral pain responses. This has been observed in prior studies and may arise from maintenance of the mucus layer [16,17]. The degree of allodynia, or painful response to a normally not painful stimulus, was determined by the lowest level of stimulus required to achieve a 30 % increase in response rate from baseline. Allodynia was observed in 20 out of 24 irradiated animals. Of the mice receiving SELP-415K/GM-0111, 3 out of 6 did not experience allodynia within the constraints of the Von Frey filament testing (0.04 – 4.0 g). Out of 6 mice, a single mouse receiving PBS also did not experience allodynia (Figure 4.1D). Of those mice experiencing typically non-normal pain, all groups receiving either PBS/GM-0111 (0.2 ± 0.16 g, n = 6), SELP (0.82 ± 1.57 g, n = 6), or SELP-415K/GM-0111 (0.2 ± 0.18 g, n = 3) required higher thresholds for painful responses than mice receiving PBS (0.11 ± 0.7 g, n = 5). These outcomes indicate there was a prophylactic effect of GM-0111 after 3 days, especially in the context of enhanced bioaccumulation provided by SELP. 4.4.2 3-Day Histological Outcomes The local early radioprotective effect provided by the SELP-415K/GM-0111 combination was evaluated histologically 3 days after irradiation of rectal tissues in 24 animals. Microscopic assessment showed increased mitotic activity (n = 11), luminal migration of nuclei within epithelium (n = 14), and increased crypt apoptosis (n = 22) 115 (Figure 4.2A). Additionally, there were variable inflammatory infiltrates within the lamina propria in a subset of samples (Supplemental Figure B.1). All irradiated groups had increased injury scores compared to healthy controls (n = 5). The injury score of SELP-415K/GM-0111 (0.4 ± 0.5) was reduced compared to all other irradiated animal groups (PBS: 0.7 ± 0.5, PBS/GM-0111: 0.6 ± 0.5, SELP: 0.8 ± 0.4) when scored in a blinded manner (Figure 4.2B). The overall tissue damage identified histologically 3 days after irritation was mild and averaged 0.6 ± 0.5 (on 0-4 scale). 4.4.3 Animal Survival Curves To understand the protective benefits of SELP-mediated delivery at later time points, an additional cohort of animals was evaluated for long term survival following treatment and subsequent exposure to high doses of irradiation to the pelvic region. Animals were followed throughout the study for signs of radiotoxicities and declining health. The primary endpoint was >20% loss in body weight and animals were sacrificed if they crossed this threshold. The >20% weight loss was observed in nearly all irradiated animals, except for a single animal receiving the protective GM—SELP combination (Figure 4.3A). Over the study period, the SELP-415K/GM-0111 combination resulted in the least amount of weight loss per day, compared to the other irradiated groups (PBS, PBS/GM-0111, SELP) (Figure 4.3B), indicating a protective effect against radiotoxicities. Decreased weight loss per day resulted in an increased SELP-415K/GM0111 animal survival time. The median survival times of animals receiving PBS (6 days), PBS/GM-0111 (6.5 days), and SELP (5 days) were less than animals treated with SELP415K/GM-0111, which survived 8 days (Figure 4.3C). SELP-415K/GM-0111 improved mean survival time by 60%. A fraction of these animals (1/6) exhibited signs of long- 116 Figure 4.2: Histological analysis of tissues 3 days following irradiation. A) Histological sections of healthy and irradiated animals receiving selected treatments. Black arrows indicate apoptosis, arrowheads indicate epithelial cell pleomorphism, red arrows indicate mitoses. B) Blinded scoring of histological sections to determine injury score. 117 Figure 4.3: Animal body weights and survival. A) Change in mice body weight as a percentage of baseline weights. Each line represents the body weight of a single animal. B) Mean percentage of body weight loss per day. C) Survival curves of irradiated animals receiving prophylactic treatments. (**: p < 0.01, *: p < 0.05) 118 term survival, maintaining body weight past the 14-day study period. The use of SELP415K/GM-0111 as a prophylactic resulted in significantly increased survival times when compared to SELP (p < 0.01) alone or the PBS (p < 0.05) sham treatment group (Figure 4.3C), suggesting protective effect against early onset of RIP may be imperative for improved quality of life and delayed morbidity. 4.4.4 Behavioral Pain Responses at Survival Endpoint Upon a >20 % loss in body weight, mice in the survival group were tested for behavioral pain responses prior to sacrifice. At the time of sacrifice, response rates vary in the exact time following irradiation due to the nature of animal survival and defining a humane endpoint. Increases in response rates, for all irradiated animals, were observed compared to the healthy control group (Figure 4.4A). At a filament stiffness of 0.16 g, animals receiving SELP-415K/GM-0111 (31.7 ± 7.5 %) had the smallest increase in response rates compared to all other irradiated groups (Figure 4.4B). Animals receiving PBS (56.7 ± 17.5 %) and SELP (51.7 ± 16.1 %) had significantly higher response rates, at 0.16 g stimulus, than healthy animals (20.41 ± 21.03 %) at the time of sacrifice (Figure 4.4B). Allodynia was also evaluated as described above (30% increase from baseline responses). Of these animals, 1/6 and 3/6 mice receiving SELP or SELP-415K/GM-0111 did not exhibit allodynic responses respectively. The remaining 20/24 animals exhibited allodynia as assessed with von Frey filaments. The stimulus threshold required to exhibit a 30% increase in response rate was much smaller in animals receiving PBS (0.08 ± 0.06 g, n = 6) compared to those animals receiving PBS/GM-0111 (0.36 ± 0.50 g, n = 6), SELP (0.86 ± 1.75 g, n = 5) or SELP-415K/GM-0111 (0.40 ± 0.52 g, n = 3). This behavioral pain assessment at the time of sacrifice further illustrates the implications of 119 Figure 4.4: Behavioral pain responses at the time of sacrifice. A) Response rates of irradiated animals receiving selected treatments and healthy controls. B) Response rates with the 0.16 g filament. C) Threshold required to elicit an allodynic response as measured by an increase in 30% from the baseline. Red box indicates animals with no allodynic response (na). (**: p < 0.01, *: p < 0.05) 120 the early stages of RIP in this murine model. The modulation of inflammation, pain pathways, or both before or at RIP onset has a sustained effect in this model as illustrated by reduced behavioral pain responses. 4.4.5 Histology at Survival Endpoint Histologic evaluation at the survival endpoint showed increased luminal migration of epithelial nuclei and variable inflammation in all irradiated groups. Additional alterations included cell flattening in the surface epithelium (n = 11), inflammatory infiltrates within the epithelium (n = 5), loss of goblet cells (n = 11), cryptitis (n = 6), loss of glands (n = 12) and edema (n = 9). Histologic alterations appeared more prominent at the survival endpoint as compared to the findings seen in samples evaluated three days after irradiation (Supplementary Figures B.1 and B.2) as illustrated by loss of crypts (crypt drop out) and epithelial erosion(s) (Figure 4.5A). The injury score of SELP-415K/GM-0111 (1.5 ± 1.7) was reduced compared to all other irradiated animal groups (PBS: 2.0 ± 1.7, PBS/GM-0111: 2.2 ± 1.8, SELP: 2.2 ± 1.3) when scored in a blinded manner (Figure 4.5B). 4.4.6 RNA Sequencing of Rectal Tissues Tissue samples from irradiated mice receiving either PBS or the SELP-415K/GM0111 combination were collected at the 3-day time point and evaluated for gene expression using RNA sequencing. Differential gene expression and pathway analysis were conducted to understand the therapeutic mechanisms of GM-0111 in the protective approach to our RIP model. The use of the SELP-415K/GM-0111 prophylactic in this RIP model resulted in 263 differentially expressed genes (adjusted p < 0.1) compared to 121 Figure 4.5: Time of sacrifice histological evaluation of rectal tissues. A) Histological sections of healthy and irradiated animals receiving selected treatments. Arrows indicate epithelial erosion and arrowheads indicate crypt dropout. B) Blinded scoring of histological sections to determine injury scores. 122 mice receiving only PBS. Of these differentially expressed genes, 10 genes had a 2-fold change greater than 1, and 67 had a 2-fold change less than -1 (Supplementary Table B.1). Indications of dampened immune activation are evident by decreased expression of chemoattractants, cytokines, interleukins, and associated receptors (CD83, IL27RA, IL9R, CD6, CCR7, IL2RG, CXCL13, CD52, CD4, CD84, IL16, etc.) (Supplementary Table B.1). Pathway analysis of SELP-415K/GM-0111 protection in mice revealed 91 significantly (-log(p) ≥ 1.3) enriched canonical pathways (Supplementary Table B.2). Of these enriched pathways a total of 5 activated (Z-score ≥ 2) and 6 deactivated (Z-score ≤ 2) pathways were identified. The top 10 pathways with the highest absolute Z score revealed pathways involved with T lymphocytes, immune-based modulation, stress, and inflammation (Table 4.1). Immune pathways of T cell signaling (T-cell Receptor, PKCΘ, ICOS, Th1) were deactivated compared to mice only receiving PBS. Increased antioxidant activities similar to Vitamin C are predicted, possibly owing to the polysulfated nature of GM-0111. Pathways specific to non-rectal tissues were excluded from this table. These and all other identified pathways can be found in Supplementary Table B.2. These activated or deactivated pathways reflect potential therapeutic mechanisms and pathological results of the protective SELP-415K/GM-0111 strategy. The differentially expressed genes were further used to identify key upstream regulators of the protective benefits of GM-0111 delivery via SELP. A total of 697 potential upstream regulators were identified to be significant (p-value of overlap < 0.05) (Supplementary Table B.3). Of these significant upstream regulators, 46 contained absolute Z-scores greater than 2 indicating differential activation status from mice 123 Table 4.1: Canonical pathways identified from protection of SELP-415K/GM-0111 in a RIP model. Ingenuity Canonical Pathways PKCθ Signaling in T Lymphocytes Crosstalk between Dendritic Cells and Natural Killer Cells -log(p-value) 1.65 5.94 Ratio 0.0179 0.0879 z-score Molecules -2.646 CACNA1I, CACNG1, CARD11, CD4, HLA-A, HLA-DMB, HLA-DRA, HLADRB5, NFKBIA, Trbc1 -2.449 CCR7, CD83, CSF2RB, HLAA, HLA-DRA, HLA-DRB5, IL2RG, ITGAL Th1 Pathway 3.19 0.0492 -2.449 CD4, HLA-A, HLA-DMB, HLA-DRA, HLA-DRB5, IL27RA PD-1, PD-L1 cancer immunotherapy pathway 2.66 0.0472 2.236 HLA-A, HLA-DMB, HLADRA, HLA-DRB5, IL2RG Senescence Pathway 1.37 0.0202 2.236 CACNG1, CAPN9, HBP1, PDHA1, PDHB, PPP2R5A Corticotropin Releasing Hormone Signaling 2.03 0.0336 2 ADCY9, CACNA1I, CACNG1, SLC39A7, SMO Antioxidant Action of Vitamin C 1.82 0.036 2 CSF2RB, NFKBIA, PLA2G2D, PLCB2 Insulin Receptor Signaling 1.49 0.0286 2 GAB1, PPP1CB, RHOQ, SHC1 -1.941 CARD11, CD4, CD8B, HLAA, HLA-DMB, HLA-DRA, HLA-DRB5, ITGAL, NFKBIA, PTPN6, PTPN7, SKAP1, Trbc1 -1.89 CD4, HLA-A, HLA-DMB, HLA-DRA, HLA-DRB5, IL2RG, NFKBIA, SHC1, Trbc1 T Cell Receptor Signaling ICOS-ICOSL Signaling in T Helper Cells 2.6 1.51 0.0212 0.0178 124 receiving PBS only. Specifically, there were 33 deactivated (Z-score ≤ -2) and 13 activated upstream regulators (Z-score ≥ 2) when compared to mice only receiving PBS prior to irradiation. The top 10 most activated/deactivated upstream regulators, as determined by Z-score, include those associated with tumor necrosis factor, interferon gamma, aryl hydrocarbon receptors, lipopolysaccharides, colony stimulating factor 2, interleukin 10 receptor alpha (IL10RA), toll-like receptor 7 (TLR7), and CD28 (Table 4.2). Non-endogenous regulators were omitted from this table (Supplementary Table B.3). Of these top 10 upstream regulators only IL10RA was activated, while all others were deactivated. Reduced involvement of pattern recognition receptors such as TLR1,3,9,4 was predicted by IPA. Upstream immune regulators of both Th1 (TNF, IFNG, CSF2, IL2) and Th2 (ILL6, IL1, IL18) were all predicted to be deactivated (Z-score ≤ -2) via casual analysis (Supplementary Table B.3). Together, these activation/deactivation statuses reflect the ameliorated immune response exhibited by delivered GM-0111. To determine significantly enriched biological processes (p < 0.05), significant differentially expressed genes were then analyzed using EnrichR. Analysis of the protective action of the SELP-415K/GM-0111 prophylactic revealed 185 significant biological processes (Supplementary Table B.4). The top 10 gene ontology terms included regulation of cell adhesion, cytoskeletal reorganization, actin filament polymerization, and several immune cell signaling pathways (Figure 4.6). Of these enriched pathways, upstream regulators, and biological processes, a notable number were related to immune responses, emphasizing the immunomodulation capacity of GM-0111. 125 Table 4.2: Upstream regulators identified from protection with SELP-415K/GM-0111 in RIP model. Upstream Regulator Activation zscore p-value of overlap TNF -3.784 0.00334 IFNG -3.694 0.000314 AHR -3.268 0.000668 -3.193 0.000000174 IL6 E. coli B4 lipopolysaccharide -3.064 0.000261 -2.918 0.00022 CSF2 -2.905 0.00291 IL10RA 2.813 0.00667 TLR7 -2.764 0.00014 CD28 -2.621 0.0385 Lipopolysaccharide Molecules ACADM, BCL2A1, BTBD3, CAMP, CCL5, CCR7, CD4, CD83, CDK5R1, CSF1R, CSF2RB, CX3CR1, CXCL13, CXCL2, GAB1, Glycam1, HLA-A, HLA-DRA, IL16, IL27RA, ITGAL, KCNJ2, LAMP3, MUC2, NFKBIA, PLK3, SMO, VCL, ZNF750 ABCB1, BCL2A1, C1QB, CCL5, CCR7, CD4, CD83, CDK5R1, CSF1R, CSF2RB, CX3CR1, CXCL2, HCK, HLA-A, HLADMB, HLA-DRA, HLA-DRB5, Ighg3, ITGAL, Klrk1, LAMP3, LAT2, MUC2, NFKBIA, PDHA1, PTPN6, RAB27A C1QB, CARD11, CCL5, CCR7, CD4, CD8B, CSF2RB, CX3CR1, CXCL13, CXCL2, IL9R, VCL ABCB1, ACADM, BCL2A1, CAMP, CCL5, CCR7, CD4, Cd52, CD83, CDK5R1, CNNM2, CNST, CSF1R, CSF2RB, CX3CR1, CXCL13, CXCL2, FBN1, GAB1, GIMAP7, HACD2, HCK, HLA-A, HLADMB, HLA-DRB5, IER5, Ighg3, IKZF1, IL16, IL2RG, ITGAL, ITPKC, LAMP3, LYZ, MUC2, NFKBIA, PLA2G2D, PLK3, PPP1CB, PTPN7, TFDP2, TNFRSF13B, VCL BTC, CCL5, CCR7, CD83, CSF1R, CSF2RB, CX3CR1, CXCL13, CXCL2, HLA-A, HLADRB5, IL2RG, LYZ, NFKBIA, PLA2G2D, RAB27A, RNASE6, SMO C1QB, CCL5, CCR7, Cd52, CD83, CXCL2, HLA-A, LCP1, NFKBIA, PTPN6 BCL2A1, CARD11, CCR7, CD83, CSF1R, CSF2RB, CX3CR1, CXCL2, IL16, LAMP3, LCP1, NFKBIA ABCB1, CCL5, CCR7, FBN1, HLA-A, IL2RG, Klrk1, Treml4 ACAP1, BCL2A1, CCL5, CCR7, CD83, CXCL13, CXCL2, NFKBIA, PTPN6 BCL2A1, CXCL13, CXCL2, IL27RA, ITGAL, LCP1, NFKBIA 126 Figure 4.6: EnrichR analysis of differentially expressed genes (p adjusted < 0.05) with an absolute log2 fold change greater than 1. Gene ontology as determined via “GO Biological Process 2021”. P values are listed on figure. * Indicates adjusted p values below 0.05 127 4.5 Discussion RIP is an inadequately addressed form of rectal injury, resulting from lower abdominal radiotherapy for cancer. Radiation leads to damage and inflammation in the rectal tissue leading to a plethora of symptomology and tissue alterations. Previously, a SELP-415K/GM-0111 combination was utilized in a prophylactic approach to protect mice against RIP and was evaluated after 7 days [16]. This study aimed to expand the beneficial effects of the SELP-415K/GM-0111 prophylactic by studying short (3-day) and long (survival) term efficacy. Once in the mucosa, SAGE exhibits its therapeutic capabilities [16]. The enhanced spatiotemporal delivery provided by SELP results in increased rectal accumulation and therapeutic efficacy compared to saline formulations. By treating or protecting against acute RIP, further chronic complications can potentially be avoided. In this study, histological changes were evident within three days of exposure to a high dose of irradiation. Pathologically, these changes were mild and include migration of apical nuclei, increased mitoses, and crypt apoptosis [3]. The observed nuclear migration and mitoses necessitate the use of cellular machinery such as actin filaments, a common gene ontological term identified by RNA sequencing. The degree of rectal injury was diminished when mice were protected with the SELP-mediated delivery of GM-0111. The histologic alterations appeared more pronounced at the time of sacrifice as compared to changes observed 3 days after irradiation, illustrating the need to define a specific timeframe for evaluation of RIP models. Modulation of the cytoskeleton through the Rho/Rho kinase pathway has been associated with fibrogenic smooth muscle cells in intestinal radiation damage [43]. This initial injury and dysregulation of the cytoskeleton 128 precluded later pathological changes. In this instance at the time of sacrifice tissues exhibited a loss of goblet cells, cryptitis, eosinophilic crypt abscesses, inflammation, loss of glands, and edema (Supplemental Figure B.2). Upon irradiation, a plethora of changes lead to inflammatory events within the rectum. Damaged cells may release damage associated molecular signals. Pathogen associated molecular patterns may be presented due to loss of epithelial barrier and bacterial/viral invasion of native tissue. Previously, GM-0111 exhibited inhibition of TLR-2,4[11]. Within this study, we have further identified TLR-1,3,9 as deactivated through casual analysis of differentially expressed genes, which can result in decreased transcription via IRF-7, IRF-3, and NF-KB. TLR-1,9 rely on a TLR adaptor, myeloid differentiation primary response 88, for downstream activation of transcription factors [44]. This adaptor is predicted to decrease activation with SELP-415K/GM-0111 protection, as identified with casual analysis. Identification of upstream regulators also identifies liposaccharides as molecular mediators. These are common ligands for several TLRs. Mast cells have been implicated in the pathology of RIP [45]. IL-33, a potent mast cell activator, results in the upregulation of CCR7 [46]. Transcriptomics in this investigation identified CCR7 to have significantly decreased differential expression. This investigation’s primary goal was to evaluate the short-term efficacy and potential molecular mechanisms behind the SELP-415K/GM-0111 prophylactic effects for RIP. These mechanisms are largely due to the therapeutic activity of GM-0111 and are consistent with previously published studies regarding its anti-inflammatory properties. It is possible that SELP influenced the observed molecular mechanisms in this study as well. SELP hydrogels release soluble polymer fraction, however these amounts 129 are lower compared to release of GM-0111[21]. The pathology, as evaluated via histology, clearly develops beyond 3 days and up until sacrifice. Animals studied at the time of sacrifice provide an understanding of animal health when >20% loss of body weight was observed. The variation in sacrifice times makes it difficult to make direct comparisons and establish pathological development within a specific time frame. The rapid decline of animal health in this investigation illustrates the effects of extreme doses of radiation on mice and presenting effects in late responding tissues to ionizing radiation. Within the aperture and radiodose provided, it is likely that parts of the lower intestine were irradiated and damaged, although this was not directly observed. These non-rectal tissue effects make this model an unlikely candidate for evaluation of chronic RIP. Improvement of the current model could capitalize on animal positioning to limit intestinal exposure, optimization of total radiodose, and fractionation to limit slow developing tissue effects. Future studies will focus on: 1) validating the molecular mechanisms of SELP-415K/GM-0111 discovered in this investigation by microarray and proteomic analyses, 2) establishing a model capable of evaluating outputs in the context of chronic RIP, 3) evaluating the efficacy of SELP-415K/GM-0111 on treatment strategies following irradiation, 4) assessing the efficacy of SELP-415K/GM-0111 in improving pathologies and symptoms from established chronic RIP, and 5) determining the pharmacokinetics and biodistribution of rectally delivered GM-0111.Pharmacokinetic and biodistribution studies will inform off target and systemic exposures of GM-0111 and possibly SELP-415K. Once determined further safety and toxicology investigations should be conducted depending on the biodistribution of GM-0111. These could include pulmonary, cardiovascular, renal, hepatic, and more toxicological assessments. If 130 presented in the hepatic and renal regions, functionality tests should be assessed as well. While the SELP-415K/GM-0111 combination does not directly interact with vasculature, it is likely that GM-0111 may enter the blood stream. To this end, hemocompatibility should be assessed at relevant concentrations that may be ascertained during pharmacokinetic and biodistribution studies. A low concentration of SELP may be taken up into systemic circulation. SELPs have been used prior for liquid embolic applications, and illustrate minimal activation of the complement system, hepatoxicity, and hemolysis [24,47]. Obtaining complete blood profiles in future works could provide another indicator of safety and radiotoxicities. In the clinic, this SAGE SELP combination can be used as a protective strategy administered several hours or immediately prior to radiotherapy. This can take the form of a coating agent applied directly to the rectal tissue or as a liquid to semi-solid enema. The protective success of this combination within the current model provides an indication for prophylactic intervention in patients undergoing hypo-fractionated radiotherapy. This is commonly used for cancers with a low α/β ratio, such as prostate cancers [48]. Additionally, the protection provided by SELP-415K/GM-0111 may allow for dose escalation in other cancer types, further improving anti-tumor efficacies while minimizing rectal toxicities. Pain is a common symptom of RIP and clearly develops with the model utilized in this study. The exact drivers of this pain are not known but may be associated with inflammation, pain pathways, or both. A possible explanation of the observed decrease in pain could be attributed to a reduced injury score of the rectal tissue. However, enriched canonical pathways also suggest other mechanisms acting in parallel or downstream of 131 inflammation. The Insulin Receptor Signaling Pathway has also been implicated in neuropathic pain as evidenced in numerous studies of diabetic neuropathy [49]. Vitamin C has exhibited analgesic properties in the clinic, which is hypothesized to result from its antioxidant properties and subsequent scavenging of free radicals [50]. Similar antioxidant properties and scavenging of free radicals may be presented from GM-0111 owing to its sulfate group. This is especially pertinent in the setting of RIP due to the reactive species generated during radiolysis and inflammation. The Corticotropin Releasing Hormone Pathway, identified in this analysis, is also implicated in nociceptive pain. Upon activation in the hypothalamus, this pathway results in secretion of corticosteroids to peripheral sites for analgesic action [51]. Interestingly, analyses indicates an activated state of this pathway, raising the question of its involvement in RIP and the role GM-0111 plays in activating these potential analgesic capabilities. 4.6 Conclusion A drug-polymer combination (SELP-415K/GM-0111) can provide acute protection against pain and rectal inflammation in a RIP model utilizing high doses of radiation. Prophylactic protection with SELP-415K/GM-0111 translated into a 60% prolonged survival. Animals receiving the drug-polymer combination exhibited decreased pain and rectal inflammation. Assessment of the molecular basis of this protection includes, but not limited to, decreased activation of inflammatory pathways associated with pattern recognition receptors, lymphocyte signaling, antioxidant properties, and effects of lipopolysaccharides. 132 4.7 References (1) Lenz, L.; Rohr, R.; Nakao, F.; Libera, E.; Ferrari, A. Chronic Radiation Proctopathy: A Practical Review of Endoscopic Treatment. World J. Gastrointest. 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Mast Cells Are an Essential Component of Human Radiation Proctitis and Contribute to Experimental Colorectal Damage in Mice. Am. J. Pathol. 2011, 178 (2), 640–651. https://doi.org/10.1016/j.ajpath.2010.10.003. (46) Emi-Sugie, M.; Toyama, S.; Matsuda, A.; Saito, H.; Matsumoto, K. IL-33 Induces Functional CCR7 Expression in Human Mast Cells. J. Allergy Clin. Immunol. 2018, 142 (4), 1341–1344. https://doi.org/10.1016/j.jaci.2018.06.007. (47) Isaacson, K. J.; Jensen, M. M.; Steinhauff, D. B.; Kirklow, J. E.; Mohammadpour, R.; Grunberger, J. W.; Cappello, J.; Ghandehari, H. Location of StimuliResponsive Peptide Sequences within Silk-Elastinlike Protein-Based Polymers Affects Nanostructure Assembly and Drug-Polymer Interactions. J. Drug Target. 2020, 28 (7–8), 766–779. https://doi.org/10.1080/1061186X.2020.1757099. (48) Hegemann, N.-S.; Guckenberger, M.; Belka, C.; Ganswindt, U.; Manapov, F.; Li, M. Hypofractionated Radiotherapy for Prostate Cancer. Radiat. Oncol. 2014, 9 (4), 275. https://doi.org/10.1186/s13014-014-0275-6. (49) Grote, C. W.; Wright, D. E. A Role for Insulin in Diabetic Neuropathy. Front. Neurosci. 2016, 10, 581. https://doi.org/10.3389/FNINS.2016.00581. (50) Carr, A. C.; McCall, C. The Role of Vitamin C in the Treatment of Pain: New Insights. J. Transl. Med. 2017, 15 (1), 77. https://doi.org/10.1186/S12967-0171179-7. 137 (51) Zheng, H.; Lim, J. Y.; Seong, J. Y.; Hwang, S. W. The Role of CorticotropinReleasing Hormone at Peripheral Nociceptors: Implications for Pain Modulation. Biomedicines 2020, 8 (12), 623. https://doi.org/10.3390/BIOMEDICINES812062 CHAPTER 5 CONCLUSION 5.1 Overview This dissertation illustrates the utility of silk-elastinlike protein polymers (SELPs) for delivery of semi-synthetic glycosaminoglycans (SAGEs) for enhancing rectal delivery and bioaccumulation. This enhancement in bioaccumulation was then utilized for a radioprotective strategy to prevent radiation-induced proctitis (RIP) in a murine model. In Chapter 1 unmet clinical needs of RIP and limitations of SAGEs were discussed, with SELP-mediated delivery presented as a potential solution. In Chapter 2 a brief review is provided for RIP, sulfated hyaluronans, and SELPs. This review provides an overview of the pathology that occurs in RIP and recent advancements in addressing this pathology. The review of sulfated hyaluronans provides insight into mechanisms and applications being explored for therapeutics of this nature. Finally, the review of SELPs provides historical context of these recombinant polymers as delivery vehicles with an emphasis provided on polymer structure-function relationships. In Chapter 3 the combination of SAGEs with several thermoresponsive polymers is explored for enhancing rectal bioaccumulation. From these investigations a SELP415K demonstrated appropriate properties for a liquid to semi-solid enema and showed enhancement of bioaccumulation over other investigated polymers. We further illustrated that this combination was able to provide a radioprotective effect in mice following lower 139 abdominal radiation insult. In Chapter 4 the protective effects of a SELP-415K and SAGE combination were further explored and found to provide early protection against developing RIP and prolonging animal health. This approach of radioprotection shows promise and requires further investigation. 5.2 Therapeutic Mechanisms of SAGEs SAGEs are an emerging class of therapeutics derived from the properties of sulfated glycosaminoglycans and hyaluronic acids. These molecules, like others, depend upon molecular structure for their programed properties. These include, but are not limited to, number of disaccharide repeat units, degree of sulfation, and location of sulfate groups with respect to monomer units [1,2]. Variation in these properties will yield varying biological and therapeutic properties. In this work a SAGE bioaccumulation is achieved in the rectum. The accumulation of SAGE resulted in a radioprotective effect in mice [3]. This radioprotective effect likely results from modulation of immune cells as this is an established mechanism from prior studies [4–6]. In addition it is possible that SAGEs can alter the α/β ratio, which describes the sensitivity of cells to fractions of irradiation [7]. In vitro investigations of the cell killing efficiency of radiation, with and without SAGE, could yield cell types that are most impacted by this radioprotective strategy. From these experiments we could start to understand relevant concentrations for effective protection in varying cell populations, and the differences presented between murine and primary derived, human cell lines. The mechanism of SAGE synthesis provides a large degree of variation between 140 batches. This may result in slight variation of molecular properties. However, this also provides an opportunity to correlate molecular properties with biological and therapeutic responses. Through variation of molecular size and degree of sulfation, we can begin to delineate the effects of SAGE material properties on biological responses, specifically in the context of radiation damages. The clear differences seen amongst hyaluronic acid sizes and sulfated glycosaminoglycans indicates that the mechanisms of action of semisynthetic GAGS as a function of physicochemical properties need further elucidation [1]. Within this work, the mechanisms of this radioprotective effect were assessed with RNA sequencing and hypothesized to occur from dampening immune responses, inhibition of pattern recognition receptors, minimization of pathogen infiltrations, and more. In vitro confirmation of these capabilities will further the understanding of SAGE mediated mechanisms. RNA sequencing analysis of healthy mouse rectums, and comparison to irradiated rectums treated with and without the SELP SAGE combination will address current gaps in this approach and provide further therapeutic targets. 5.3 Rectal Delivery via SELPs SELP mediated delivery of therapeutics has been well studied ranging from small molecules to gene therapy agents [8,9]. This work has improved upon delivery via SELP hydrogels to the rectum from prior studies by providing a side by side comparison of multiple polymers, including SELPs [3,10]. The material properties, namely gelation rate and stiffness of the SELPs used, proved to be the most important factors studied for controlled rectal drug delivery via SELPs [3]. We hypothesized the slow gelation provided by SELP-415K enhance the gel tissue interface, allowing for more 141 accumulation to occur. Confirmation of this hypothesis could further the development of SELPs for rectal delivery. This work provides a basis for investigating rectal delivery of other therapeutics using SELP-415K. In the context of radioprotective agents, there are several investigative treatments currently in development, some of these include inhibitors of Platelet Derived Growth Factor Receptors, extracellular vesicles, and modulators of angiogenesis [11–13]. The passive sol to gels transition provided by SELPs has already proven useful for the delivery of delicate biotherapeutics and the investigation of other therapeutics using a SELP mediated delivery could enhance therapeutic outcomes [8]. Specifically, protective or regenerative approaches to intestinal and rectal stem cells should be explored, as evidence of radiodepletion of stem cells are a major driver of observed pathologies [14]. Other approaches could target the dysregulation of blood vessel development within proctitis [13]. Approaches of this individual nature may be beneficial in targeting specific drivers of pathology. However, the effects of radiation damage are vast and may require multiple approaches. The delivery of simultaneous biotherapeutics from SELPs can be achieved due to its sol to gel nature and may provide even more improved outcomes in the future. 5.4 Advancing SAGE-SELP Combinations to Treat RIP While there are always improvements to make upon existing systems, the existing approaches need to be developed towards the clinic to allow for improvements in patient quality of life. The current SELP SAGE combination discussed in this work shows promise in a radioprotective strategy, specifically maintenance of behavioral pain 142 responses and reduced signs of damaged rectal tissues. The current approach utilizes a single dose of radiation to generate an RIP model. This does not mimic the current fractionated approach and may produce a different biological response. High degrees of radiotoxicity were observed during this work as evidenced by animal survival curves [3]. Future investigations need to produce an animal model which utilizes a fractionated approach to generate a murine RIP model. This fractionated approach provides further opportunity to improve the protective strategy explored in this work. For instance, a high single dose of irradiation only allows a single opportunity to prophylactically treat mice, while 5 fractions provide 5 opportunities to protect mice prior to treatment. This model should allow for long term animal survival such that the treatment and protection against chronic RIP can be evaluated. Further expansion on model outcomes, such as endoscopic scoring, normalized behavioral pain responses, and immunohistochemistry can be included in model expansion. A successful model will provide symptomology and pathology of that which is seen in acute RIP and subsequent progression to chronic RIP. Upon successful model generation evaluation of posttreatment should be conducted for both acute and chronic RIP. Posttreatment of acute RIP needs to be evaluated at various endpoints to assess the developing pathology and treatment efficacy. An untreated pathological development should result in chronic RIP and the definitions of this state need to be defined. Once defined, prevention and treatment of chronic RIP should both be evaluated with the SAGE SELP combination. The ability to provide dose escalation to patients with either a preventative or treatment strategy will also be investigated, as this serves the potential of making lower abdominal radiotherapy more efficacious with fewer toxicities. The success of this study may be complemented with in 143 vitro studies investigating altered α/β ratios. Finally, investigations of in vivo mechanisms would greatly benefit the field. There are indications of mast cell involvement in the pathology of RIP [15,16]. This can be confirmed using mast cell deficient mice and comparing them to immunological control animals. Once confirmed, the use of the selected SELP SAGE combination can be used to evaluate differences in protection or treatment between immunologically altered or normal mice. 5.5 Summary This dissertation has illustrated the ability of SELP-415K to enhance rectal bioaccumulation of therapeutic SAGEs. This enhancement provides a radioprotective affect to the rectal tissue in a murine RIP model. Rectal protection extends to both the symptomology and tissue damage exhibited in instances of RIP. Mechanisms of this protection are explored with next generation sequencing, providing a basis for understanding the therapeutic properties of SAGE in this application. The combination of properties from nature can yield unique therapeutics and delivery strategies for such therapeutics. This is seen both with the synthetic nature of SAGEs and the unique properties presented by the combination of silk and elastinlike motifs into a SELP construct. Future work will continue to improve upon these properties and strategies. 5.6 References (1) Liang, J.; Jiang, D.; Noble, P. W. Hyaluronan as a Therapeutic Target in Human Diseases. Adv. Drug Delivery Rev. 2016, 97, 186–203. https://doi.org/10.1016/J.ADDR.2015.10.017. (2) Koehler, L.; Ruiz-Gómez, G.; Balamurugan, K.; Rother, S.; Freyse, J.; Möller, S.; Schnabelrauch, M.; Köhling, S.; Djordjevic, S.; Scharnweber, D.; et al. Dual Action of Sulfated Hyaluronan on Angiogenic Processes in Relation to Vascular 144 Endothelial Growth Factor-A. Sci. Reports 2019, 9 (1), 1–18. https://doi.org/10.1038/s41598-019-54211-0. (3) Steinhauff, D.; Jensen, M.; Talbot, M.; Jia, W.; Isaacson, K.; Jedrzkiewicz, J.; Cappello, J.; Oottamasathien, S.; Ghandehari, H. Silk-Elastinlike Copolymers Enhance Bioaccumulation of Semisynthetic Glycosaminoglycan Ethers for Prevention of Radiation Induced Proctitis. J. Controlled Release 2021, 332 (August 2020), 503–515. https://doi.org/10.1016/j.jconrel.2021.03.001. (4) Savage, J. R.; Pulsipher, A.; Rao, N. V.; Kennedy, T. P.; Prestwich, G. D.; Ryan, M. E.; Lee, W. Y. A Modified Glycosaminoglycan, GM-0111, Inhibits Molecular Signaling Involved in Periodontitis. PLoS One 2016, 11 (6), e0157310. https://doi.org/10.1371/journal.pone.0157310. (5) Pulsipher, A.; Savage, J. R.; Kennedy, T. P.; Gupta, K.; Cuiffo, B. G.; Sonis, S. T.; Lee, W. Y. GM-1111 Reduces Radiation-Induced Oral Mucositis in Mice by Targeting Pattern Recognition Receptor-Mediated Inflammatory Signaling. PLoS One 2021, 16 (3), e0249343. https://doi.org/10.1371/journal.pone.0249343. (6) Lee, W. Y.; Savage, J. R.; Zhang, J.; Jia, W.; Oottamasathien, S.; Prestwich, G. D. Prevention of Anti-Microbial Peptide LL-37-Induced Apoptosis and ATP Release in the Urinary Bladder by a Modified Glycosaminoglycan. PLoS One 2013, 8 (10), 1–13. https://doi.org/10.1371/journal.pone.0077854. (7) Leeuwen, C. M. van; Oei, A. L.; Crezee, J.; Bel, A.; Franken, N. A. P.; Stalpers, L. J. A.; Kok, H. P. The Alfa and Beta of Tumours: A Review of Parameters of the Linear-Quadratic Model, Derived from Clinical Radiotherapy Studies. Radiat. Oncol. 2018, 13 (1). https://doi.org/10.1186/S13014-018-1040-Z. (8) Gustafson, J. A.; Ghandehari, H. Silk-Elastinlike Protein Polymers for MatrixMediated Cancer Gene Therapy. Adv. Drug Delivery Rev. 2010, 62 (15), 1509– 1523. https://doi.org/10.1016/j.addr.2010.04.006. (9) Price, R.; Poursaid, A.; Ghandehari, H. Controlled Release from Recombinant Polymers. J. Controlled Release 2014, 190, 304–313. https://doi.org/10.1016/j.jconrel.2014.06.016. (10) Jensen, M. M.; Jia, W.; Isaacson, K. J.; Schults, A.; Cappello, J.; Prestwich, G. D.; Oottamasathien, S.; Ghandehari, H. Silk-Elastinlike Protein Polymers Enhance the Efficacy of a Therapeutic Glycosaminoglycan for Prophylactic Treatment of Radiation-Induced Proctitis. J. Controlled Release 2017, 263, 46–56. https://doi.org/10.1016/j.jconrel.2017.02.025. (11) Lu, W.; Xie, Y.; Huang, B.; Ma, T.; Wang, H.; Deng, B.; Zou, S.; Wang, W.; Tang, Q.; Yang, Z.; et al. Platelet-Derived Growth Factor C Signaling Is a Potential Therapeutic Target for Radiation Proctopathy. Sci. Transl. Med. 2021, 13 (582), 2344. https://doi.org/10.1126/SCITRANSLMED.ABC2344. 145 (12) Saha, S.; Aranda, E.; Hayakawa, Y.; Bhanja, P.; Atay, S.; Brodin, N. P.; Li, J.; Asfaha, S.; Liu, L.; Tailor, Y.; et al. Macrophage-Derived Extracellular VesiclePackaged WNTs Rescue Intestinal Stem Cells and Enhance Survival after Radiation Injury. Nat. Commun. 2016, 7. https://doi.org/10.1038/NCOMMS13096. (13) Wu, P.; Li, L.; Wang, H.; Ma, T.; Wu, H.; Fan, X.; Yang, Z.; Chen, D.; Wang, L. Role of Angiogenesis in Chronic Radiation Proctitis: New Evidence Favoring Inhibition of Angiogenesis Ex Vivo. Dig. Dis. Sci. 2017, 63 (1), 113–125. https://doi.org/10.1007/S10620-017-4818-1. (14) Yang, W.; Sun, Z.; Yang, B.; Wang, Q. Nrf2-Knockout Protects from Intestinal Injuries in C57BL/6J Mice Following Abdominal Irradiation with γ Rays. Int. J. Mol. Sci. 2017, 18 (8), 1656. https://doi.org/10.3390/IJMS18081656. (15) Albrecht, M.; Müller, K.; Köhn, F. M.; Meineke, V.; Mayerhofer, A. Ionizing Radiation Induces Degranulation of Human Mast Cells and Release of Tryptase. Int. J. Radiat. Biol. 2007, 83 (8), 535–541. https://doi.org/10.1080/09553000701444657. (16) Blirando, K.; Milliat, F.; Martelly, I.; Sabourin, J. C.; Benderitter, M.; François, A. Mast Cells Are an Essential Component of Human Radiation Proctitis and Contribute to Experimental Colorectal Damage in Mice. Am. J. Pathol. 2011, 178 (2), 640–651. https://doi.org/10.1016/j.ajpath.2010.10.003. APPENDIX A CHAPTER 3 SUPPLEMENTAL INFORMATION A.1 Methods A.1.1 Macroscopic Phase Separation SAGE combination with polymers was evaluated for macroscopic phase separation. Polymers (Poly(D,L-lactide-co-glycolide)-block-(poly(ethylene glycol)block-poly(D,L-lactide-co-glycolide) (PLGA-PEG-PLGA) 10 wt.%; PLGA-PEG-PLGA 20 wt.%; Poloxamer 407 10 wt.%; Poloxamer 407 20 wt.%; SELP-415K 4 wt.%; SELP415K 11 wt.%; SELP-815K 4 wt.%; SELP-815K 11 wt.%) were mixed with SAGE to yield 100 mg/mL. These were added to cold scintillation vials and placed into a 37 °C water bath. After 1 hour, vials were tilted and imaged to observe macroscopic phase separation, indicated by a present liquid phase. The liquid phase was then collected and quantified for excluded in SAGE from the gels. A.1.2 Confocal Microscopy SAGE combination with polymers was evaluated for rectal bioaccumulation. Polymers included Poloxamer 407 (20 wt. %), SELP-415K (11 wt. %), and SELP-815K (11 wt. %). Each formulation consisted of 100 mg/mL SAGE. BDF-1 mice were anesthetized with isoflurane and instilled with 100 µL of each formulation as previously 147 described[1]. Animals were then sacrificed after 6, 12, 24, and 48 hours. Rectal tissue was collected upon sacrificed and fixed in 4% neutral buffered formalin overnight before soaking in 95 % ethanol for at least 24 hours. Fixed tissues were then submitted to ARUP for processing and sectioning into 2 µm sections. Slides were then treated with a nuclear mounting stain and imaged using an Olympus DP27 camera. A.2 Results A.2.1 Macroscopic Phase Separation Both PLGA-PEG-PLGA SAGE combinations resulted in macroscopic phase separation (Figure A.1A). The 10 wt% and 20 wt% solutions resulted in greater than 50% exclusion of SAGEs from the gel (Figure A.1B). Poloxamer 10 wt% and SELP-415K 4 wt% did not result in the formation of gels under these conditions. All other combinations resulted in the formation of gels with no observed separation (Figure A.1). A.2.2 Confocal Microscopy Based upon in vitro screening, selected formulations were then chosen to evaluate in vivo for rectal accumulation. A utilized nuclear stain allowed for viewing of the cells and tissues. Observed fluorescently labeled SAGEs were present within all tissue samples at 6 hours (Figure A.2). These decreased over time and appeared to be enhanced by polymeric delivery. 148 Figure A.1: Macroscopic phase separation of polymer SAGE combinations. A) Qualitative macroscopic phase separations after 1 hour at 37 °C. B) Quantitative % of GM-0111 excluded from gels exhibiting gelation. (* indicates no observed macroscopic phase separation observed). 149 Figure A.2: Confocal microscopy of polymer SAGE combinations instilled in the rectums. APPENDIX B CHAPTER 4 SUPPLEMENTAL INFORMATION B.1 Methods B.1.1 Histological Scoring of Mice After 3-Days SELP-415K (11 wt.%) and SAGE (100 mg/mL) were instilled into the rectum of mice with volumes of 100 µL. The lower abdominal region BDF-1 mice were irradiated with 37 Gy. After three days mice were sacrificed, and rectal tissue was collected. Tissues were fixed in 4% neutral buffered formalin and submitted for processing at ARUP, where sections of 2 µM were produced and stained with H &E. Stained sections were then provided to a certified pathologist and scored in a blinded manner. This scoring occurred in a manner consistent with prior literature regarding RIP [1,2]. B.1.2 Histological Scoring of Mice at the Time of Sacrifice SELP-415K (11 wt.%) and SAGE (100 mg/mL) were instilled into the rectum of mice with volumes of 100 µL. The lower abdominal region of BDF-1 mice were irradiated with 37 Gy. After >20% weight loss, mice were sacrificed, and rectal tissue was collected. Tissues were fixed in 4% neutral buffered formalin and submitted for processing at ARUP, where sections of 2 µM were produced and stained with H &E. Stained sections were then provided to a certified pathologist and scored in a blinded manner. This scoring occurred in a manner consistent with prior literature regarding RIP 151 [1,2]. B.1.3 Casual Analysis of RNA Sequencing Results SELP-415K (11 wt.%) and SAGE (100 mg/mL) were instilled into the rectum of mice with volumes of 100 µL. The lower abdominal region of BDF-1 mice were irradiated with 37 Gy. After three days mice were sacrificed, and rectal tissue was collected. Tissues were flash frozen and submitted to GENEWIZ (Cambridge, MA) for sequencing. A mouse genome (GRCm38) and gene feature files were obtained from the Ensembl release 102[3]. A reference database was generated using STAR (2.7.6a) with optimized splice junctions of 150 base pair reads. Reads were trimmed using cutadapt 1.16[4] and aligned using STAR in two pass mode to the reference database. Reads were assigned to genes using featureCounts (1.6.3) [5]. Output files were summarized with MultiQC to assess for outliers [6]. Differentially expressed genes were identified using a false discovery rate of 5% with DESeq2 (1.30.1) [7] and pathways analyzed with Ingenuity Pathway Analysis [8]. Gene Ontology was assessed using EnrichR [9–11]. B.2 Results B.2.1 Histological Scoring of Mice After 3-Days After 3-days mouse tissue was evaluated histologically to determine early onset of RIP. The blinded scoring of this tissue provided several notable tissue manifestations. These included luminal migration of epithelial nuclei and inflammation (Figure B.1). All other evaluated tissue aspects were not presented after 3 days. 152 Figure B.1: Scoring of histological observations 3 days after treatment and irradiation. Scoring was performed in a blinded manner. 153 B.2.2 Histological Scoring of Mice at the Time of Sacrifice After animals lost >20% of body weight, we evaluated tissues histologically to assess terminal tissue toxicities. All other evaluated parameters scored higher than 3-day outcomes, indicating the pathological development of this model (Figure B.2). The SELP-SAGE combination resulted in decreased observance of toxicities in 8/10 evaluated parameters. B.2.3 Casual Analysis of RNA Sequencing Results The use of the SELP-415K/GM-0111 prophylactic in this RIP model resulted in 263 differentially expressed genes (adjusted p < 0.1) compared to mice receiving only PBS. Of these differentially expressed genes, 10 genes had a 2-fold change greater than 1, and 67 had a 2-fold change less than -1 (Table B.1). Pathway analysis of SELP415K/GM-0111 protection in mice revealed 91 significantly (-log(p) ≥ 1.3) enriched canonical pathways (Table B.2). The differentially expressed genes were further used to identify key upstream regulators of the protective benefits of GM-0111 delivery via SELP. A total of 697 potential upstream regulators were identified to be significant (pvalue of overlap < 0.05) (Table B.3). To determine significantly enriched biological processes (p < 0.05), significant differentially expressed genes were then analyzed using EnrichR. Analysis of the protective action of the SELP-415K/GM-0111 prophylactic revealed 185 significant biological processes (Table B.4). 154 Figure B.2: Scoring of histological observations 3 days after treatment and irradiation. Scoring was performed in a blinded manner. 155 Table B.1: Differentially expressed genes. Expr Intensity/RPKM/ FPKM/Counts Expr Log Ratio Expr pvalue ID Symbol ENSMUSG00000105526 Gm43490 2.893834383 -1.849555556 0.010674637 ENSMUSG00000000617 GRM6 14.18125913 -1.601333928 0.020761614 ENSMUSG00000116368 Cyp2d33-ps 8.927521351 -1.505725055 0.01109524 ENSMUSG00000108035 Gm44021 10.20503952 -1.484658704 0.010458882 ENSMUSG00000079186 Gzmc 4.194724809 -1.463422951 0.066550155 ENSMUSG00000053044 CD8B 8.549413265 -1.428885964 0.040367095 ENSMUSG00000094377 Gm24407 131.4995477 -1.373629139 0.040367095 ENSMUSG00000098197 BC051537 8.1563078 -1.370519595 0.055683231 ENSMUSG00000108955 Gm44775 8.752798106 -1.359193215 0.040367095 ENSMUSG00000113502 AC158115.1 7.203932877 -1.358104658 0.067690375 ENSMUSG00000118294 Gm9028 7.822760103 -1.355909243 0.051810203 ENSMUSG00000038357 CAMP 4.839044804 -1.355874893 0.067690375 ENSMUSG00000096243 Gm24265 136.9786009 -1.306690565 0.049847812 ENSMUSG00000076939 Iglv3 28.38205988 -1.278656751 0.025513338 ENSMUSG00000077578 Gm25631 5.343781371 -1.274731466 0.073627868 ENSMUSG00000043931 GIMAP7 55.76123971 -1.269749549 0.032359052 ENSMUSG00000049362 OR5K1 5.754197966 -1.268285707 0.086945121 ENSMUSG00000104925 Gm43061 8.443148208 -1.257727368 0.067690375 ENSMUSG00000100291 2310069B03Rik 132.9863752 -1.24060802 0.071426754 ENSMUSG00000101414 Gm29101 8.349708115 -1.23307551 0.095451128 ENSMUSG00000106024 A530083M17Rik 7.6776956 -1.226636112 0.085353947 ENSMUSG00000039691 TSPAN10 6.821407103 -1.217717622 0.070284455 ENSMUSG00000043168 4930426D05Rik 33.18319908 -1.214753969 0.05606828 ENSMUSG00000084796 Mir142hg 118.6112601 -1.212788682 0.055683231 ENSMUSG00000005465 IL27RA 59.35218864 -1.191381626 0.048159799 ENSMUSG00000076615 Ighg3 39.69825973 -1.185284471 0.082153564 ENSMUSG00000020279 IL9R 50.06939546 -1.184022375 0.067690375 ENSMUSG00000001588 ACAP1 509.1700566 -1.176160978 0.062680882 ENSMUSG00000106443 Gm42702 7.705600762 -1.165285809 0.095451128 ENSMUSG00000076490 Trbc1 46.15180227 -1.157664039 0.005280586 ENSMUSG00000094872 Igkv9-120 8.651581206 -1.138530905 0.082153564 ENSMUSG00000115647 AC107453.4 12.49803383 -1.137798897 0.095451128 ENSMUSG00000036634 MAG 12.70179497 -1.135850188 0.067690375 ENSMUSG00000108169 Gm43958 26.00506593 -1.130408273 0.069014393 ENSMUSG00000041247 LAMP3 9.136973405 -1.125799609 0.095451128 ENSMUSG00000024670 CD6 13.98693444 -1.123693012 0.071426754 156 Table B.1 (Continued) Expr Intensity/RPKM/ FPKM/Counts Expr Log Ratio Expr pvalue -1.119779075 0.073627868 ID Symbol ENSMUSG00000037944 CCR7 65.30254309 ENSMUSG00000029380 CXCL2 51.90851623 -1.11178404 0.048159799 ENSMUSG00000109162 2900027M19Rik 6.581175196 -1.107439897 0.09789155 ENSMUSG00000102326 Gm37788 19.49213788 -1.099142218 0.062838479 ENSMUSG00000031390 AVPR2 22.89151923 -1.098349526 0.062838479 ENSMUSG00000048031 FCRL5 15.05478002 -1.096563101 0.095451128 ENSMUSG00000110529 RP23-314A15.5 9.068196902 -1.095902918 0.067153848 ENSMUSG00000022491 Glycam1 220.1106645 -1.085780751 0.072462682 ENSMUSG00000096349 Gm22513 41.65792879 -1.08275261 0.095451128 ENSMUSG00000097352 C920009B18Rik 15.13444998 -1.081116908 0.055683231 ENSMUSG00000105547 Iglc3 194.3045946 -1.080503297 0.05606828 ENSMUSG00000095616 Gm26244 37.68899487 -1.076418017 0.066408139 ENSMUSG00000052142 RASAL3 253.7698874 -1.075094355 0.095451128 ENSMUSG00000040592 CD79B 489.6371075 -1.069127628 0.095451128 ENSMUSG00000031304 IL2RG 208.8330768 -1.033232958 0.050100911 ENSMUSG00000095630 Igkv6-23 17.47187335 -1.03181428 0.086945121 ENSMUSG00000102562 Gm37694 22.6734929 -1.027563608 0.095451128 ENSMUSG00000023078 CXCL13 841.8441792 -1.027455632 0.095002406 ENSMUSG00000046793 GPR61 18.36158617 -1.022355281 0.067690375 ENSMUSG00000115497 AC131033.1 19.06676655 -1.021729344 0.095451128 ENSMUSG00000050357 CARMIL2 178.8204195 -1.019084135 0.071052972 ENSMUSG00000020722 CACNG1 12.23540256 -1.018578001 0.089704864 ENSMUSG00000097146 Gm4211 8.210602458 -1.014786073 0.095451128 ENSMUSG00000090214 Gm15657 80.39225797 -1.014292296 0.010458882 ENSMUSG00000041202 PLA2G2D 126.89744 -1.011327887 0.066740989 ENSMUSG00000019874 FABP7 28.30408036 -1.010650242 0.073627868 ENSMUSG00000115381 AC102847.2 48.60061412 -1.001198023 0.066550155 ENSMUSG00000022416 CACNA1I 44.34679933 -0.998150204 0.095451128 ENSMUSG00000035697 ARHGAP45 827.6573774 -0.995974258 0.095002406 ENSMUSG00000050100 HMX2 28.46628715 -0.994194788 0.086945121 ENSMUSG00000087361 0610043K17Rik 15.06753247 -0.983251985 0.090005847 ENSMUSG00000076937 Iglc2 246.8710767 -0.983233798 0.098326685 ENSMUSG00000010142 TNFRSF13B 115.3283289 -0.981834283 0.095451128 ENSMUSG00000114842 AC173482.5 11.10616106 -0.980847326 0.095451128 ENSMUSG00000021880 RNASE6 83.68794097 -0.975868964 0.069014393 ENSMUSG00000040061 PLCB2 114.8981628 -0.972813939 0.040367095 ENSMUSG00000087480 Gm15910 41.72844112 -0.970951221 0.032359052 ENSMUSG00000008318 RELT 61.11592964 -0.966754795 0.069014393 ENSMUSG00000028492 SAXO1 39.10293736 -0.958459915 0.065374363 157 Table B.1 (Continued) ID Symbol Expr Intensity/RPKM/ FPKM/Counts ENSMUSG00000052336 CX3CR1 240.3869229 ENSMUSG00000108912 E230020D15Rik 41.20566665 -0.93290317 0.095451128 ENSMUSG00000004837 GRAP 190.0088761 -0.927028376 0.076603434 ENSMUSG00000104724 Gm43162 48.72494296 -0.917545254 0.095451128 ENSMUSG00000023274 CD4 80.43072959 -0.915258573 0.081406958 ENSMUSG00000035095 FAM167A 48.05279434 -0.913221646 0.064740549 ENSMUSG00000114246 AC102542.1 23.05771745 -0.901830428 0.090005847 ENSMUSG00000031506 PTPN7 93.63272863 -0.900156141 0.098326685 ENSMUSG00000038147 CD84 147.7578486 -0.899289848 0.079705654 ENSMUSG00000109555 Gm44891 110.912031 -0.897894456 0.090005847 ENSMUSG00000018654 IKZF1 180.2904673 -0.889824567 0.095451128 ENSMUSG00000031389 ARHGAP4 540.8776557 -0.886143436 0.063388333 ENSMUSG00000030149 Klrk1 18.86848025 -0.884196184 0.066476847 ENSMUSG00000057058 SKAP1 44.16080321 -0.883680761 0.095451128 ENSMUSG00000030830 ITGAL 152.852734 -0.880111127 0.095451128 ENSMUSG00000060586 HLA-DRB5 2990.049899 -0.878726725 0.084484482 ENSMUSG00000036526 CARD11 185.9621616 -0.872894044 0.073627868 ENSMUSG00000001741 IL16 170.8566549 -0.855412342 0.095451128 ENSMUSG00000020651 SLC26A4 23.98340566 -0.853380104 0.095002406 ENSMUSG00000022488 NCKAP1L 344.243561 -0.846980833 0.086945121 ENSMUSG00000034041 LYL1 177.3136711 -0.837346657 0.095451128 ENSMUSG00000020159 GABRP 40.31045084 -0.829802069 0.089704864 ENSMUSG00000036322 HLA-DRA 1341.191468 -0.823643131 0.088851423 ENSMUSG00000024696 LPXN 111.7823234 -0.822555074 0.09570569 ENSMUSG00000015396 CD83 268.5544525 -0.814469351 0.01109524 ENSMUSG00000034786 GPSM3 151.5378168 -0.811890049 0.087872091 ENSMUSG00000021123 RDH12 36.60991953 -0.80847651 0.095451128 ENSMUSG00000028581 LAPTM5 566.1126635 -0.807303717 0.074689023 ENSMUSG00000102037 BCL2A1 52.14794828 -0.806672386 0.095451128 ENSMUSG00000100215 Gm8292 44.66593936 -0.805248635 0.098326685 ENSMUSG00000003283 HCK 126.5998635 -0.802660135 0.025513338 ENSMUSG00000071714 CSF2RB 151.4771501 -0.785145201 0.073627868 ENSMUSG00000060550 HLA-A 301.5126496 -0.785016994 0.095451128 ENSMUSG00000040751 LAT2 74.60334856 -0.775779612 0.099482486 ENSMUSG00000090019 GIMAP1 217.0582468 -0.772786585 0.066858876 ENSMUSG00000107120 Gm43059 29.3353051 -0.767517912 0.086945121 ENSMUSG00000048895 CDK5R1 118.3476186 -0.743725744 0.08085727 ENSMUSG00000107336 Gm43461 37.91353774 -0.696630172 0.095451128 ENSMUSG00000096950 Gm9530 61.07962843 -0.694500021 0.071430759 Expr Log Ratio Expr pvalue -0.94698633 0.043168462 158 Table B.1 (Continued) ID Symbol Expr Intensity/RPKM/ FPKM/Counts ENSMUSG00000070000 FCHO1 232.4864869 -0.658103683 0.073627868 ENSMUSG00000004266 PTPN6 1296.183418 -0.638747268 0.062838479 ENSMUSG00000021025 NFKBIA 3019.106939 -0.59694261 0.095451128 ENSMUSG00000116940 Gm7450 150.5591621 -0.582458008 0.055683231 ENSMUSG00000105176 Gm43668 86.8160011 -0.579038143 0.095451128 ENSMUSG00000104649 Gm43712 53.21518866 -0.578332898 0.095451128 ENSMUSG00000024621 CSF1R 416.3264463 -0.573292625 0.025513338 ENSMUSG00000012126 UBXN11 119.3461099 -0.569152197 0.073537417 ENSMUSG00000036905 C1QB 406.0278427 -0.558822269 0.055683231 ENSMUSG00000097415 AU020206 989.1492923 -0.55067746 0.095451128 ENSMUSG00000051124 Gimap9 234.402101 -0.550207443 0.095451128 ENSMUSG00000047767 ATG16L2 461.6848349 -0.544796139 0.040367095 ENSMUSG00000022020 NAA16 526.3724142 -0.54407415 0.095451128 ENSMUSG00000083992 Gm11478 334.721166 -0.504324609 0.066476847 ENSMUSG00000036908 UNC93B1 1489.26057 -0.459920142 0.05606828 ENSMUSG00000013155 ENKD1 173.7732632 -0.408528024 0.099482486 ENSMUSG00000027220 SYT13 320.7532393 -0.407626348 0.095794033 ENSMUSG00000031107 RBMX2 223.1981213 -0.405256791 0.095451128 ENSMUSG00000033099 NOL12 748.6378822 -0.342660817 0.084484482 ENSMUSG00000056708 IER5 1254.997906 -0.339706855 0.072462682 ENSMUSG00000021431 SNRNP48 1883.465819 -0.322076977 0.079832661 ENSMUSG00000040128 PNRC1 2850.959062 -0.293804741 0.095451128 ENSMUSG00000035278 PLEKHJ1 1375.042859 -0.215648251 0.059243521 ENSMUSG00000071072 PTGES3 3263.097733 -0.15761023 0.096119371 ENSMUSG00000004789 DLST 4881.412033 0.167702726 0.079832661 ENSMUSG00000015095 FBXW5 1189.308093 0.178812008 0.095451128 ENSMUSG00000042626 SHC1 1827.768173 0.183213036 0.095451128 ENSMUSG00000026626 PPP2R5A 3820.146163 0.192559739 0.066408139 ENSMUSG00000002546 GOLGA2 2200.879136 0.21024036 0.069766511 ENSMUSG00000003233 DVL3 1464.371692 0.237934848 0.066408139 ENSMUSG00000039318 RAB3GAP2 1023.62716 0.250084185 0.098326685 ENSMUSG00000022770 DLG1 2092.711649 0.252773836 0.010458882 ENSMUSG00000028868 WASF2 2642.676374 0.259187882 0.089704864 ENSMUSG00000021748 PDHB 3055.740244 0.262324629 0.098326685 ENSMUSG00000030421 URI1 1243.895728 0.262837499 0.095451128 ENSMUSG00000001761 SMO 753.8425238 0.263239295 0.067690375 ENSMUSG00000024759 ATL3 2564.352476 0.273089359 0.095451128 ENSMUSG00000020456 OGDH 8031.939337 0.273143121 0.095451128 ENSMUSG00000045767 C5orf24 1524.204255 0.283475024 0.095451128 Expr Log Ratio Expr pvalue 159 Table B.1 (Continued) ID Symbol Expr Intensity/RPKM/ FPKM/Counts ENSMUSG00000043061 Tmem18 539.059315 0.287457362 0.095451128 ENSMUSG00000024887 ASAH2 399.878446 0.287780777 0.067690375 ENSMUSG00000020883 FBXL20 1291.438625 0.29307777 0.095451128 ENSMUSG00000022297 FZD6 423.6481812 0.296397266 0.066476847 ENSMUSG00000014956 PPP1CB 8918.715116 0.297371014 0.095451128 ENSMUSG00000031865 DCTN1 3085.270557 0.306070493 0.095002406 ENSMUSG00000025968 NDUFS1 2799.304007 0.311940187 0.066858876 ENSMUSG00000041842 FHDC1 585.3465484 0.313393391 0.032359052 ENSMUSG00000041935 C5orf51 1525.847432 0.319161534 0.066408139 ENSMUSG00000024143 RHOQ 1141.153809 0.329762361 0.067690375 ENSMUSG00000039461 TCTA 932.3383065 0.330356832 0.088310478 ENSMUSG00000031299 PDHA1 6208.744418 0.333587626 0.081389123 ENSMUSG00000039414 HEATR5B 497.7054632 0.339649664 0.066858876 ENSMUSG00000028082 SH3D19 4309.350108 0.342407685 0.089704864 ENSMUSG00000025241 FYCO1 937.8905462 0.343443534 0.010674637 ENSMUSG00000042350 AREL1 817.2223009 0.354184567 0.069014393 ENSMUSG00000035064 EEF2K 3599.349754 0.354883685 0.066550155 ENSMUSG00000032411 TFDP2 931.9887406 0.355256805 0.066476847 ENSMUSG00000041037 IRGQ 570.9186361 0.356688608 0.063148077 ENSMUSG00000039789 ZNF597 315.5755838 0.362718372 0.049847812 ENSMUSG00000034109 GOLIM4 1668.908162 0.36273811 0.049097668 ENSMUSG00000018931 NATD1 252.2191831 0.363258565 0.073627868 ENSMUSG00000062098 BTBD3 578.1570779 0.372906159 0.067690375 ENSMUSG00000025745 HADHA 5152.540207 0.392377498 0.095002406 ENSMUSG00000037949 ANO10 1295.680225 0.401271142 0.066858876 ENSMUSG00000019899 LAMA2 222.8014585 0.407706764 0.066408139 ENSMUSG00000032202 RAB27A 1029.512221 0.410580913 0.095002406 ENSMUSG00000095362 ZNF442 124.2494341 0.420329036 0.095451128 ENSMUSG00000041961 ZNRF3 315.5552768 0.420790217 0.086945121 ENSMUSG00000039976 TBC1D16 397.8002586 0.422194769 0.012715457 ENSMUSG00000025509 PNPLA2 3278.997463 0.443448926 0.095451128 ENSMUSG00000038167 PLEKHG6 1045.87458 0.446388435 0.073627868 ENSMUSG00000062908 ACADM 4202.200657 0.448114405 0.079705654 ENSMUSG00000068742 CRY2 919.9049069 0.449016234 0.067690375 ENSMUSG00000023806 RSPH3 221.5959254 0.461962338 0.066476847 ENSMUSG00000015659 SERAC1 188.6478065 0.467369964 0.067690375 ENSMUSG00000001983 TACO1 174.9181945 0.484369973 0.08643769 ENSMUSG00000033096 APMAP 1165.891617 0.484950634 0.067690375 ENSMUSG00000091498 Mpc1-ps 164.0517182 0.488491196 0.086945121 Expr Log Ratio Expr pvalue 160 Table B.1 (Continued) ID Symbol Expr Intensity/RPKM/ FPKM/Counts ENSMUSG00000035284 VPS13C 636.7993213 0.497000618 0.09570569 ENSMUSG00000033059 PYGB 5718.160783 0.497287017 0.032359052 ENSMUSG00000071656 LRRN4CL 127.1385939 0.51755914 0.086531787 ENSMUSG00000028542 SLC6A9 200.8316109 0.521629927 0.095451128 ENSMUSG00000046541 ZNF526 127.1842653 0.522069964 0.062838479 ENSMUSG00000066571 GARRE1 199.5524183 0.532666932 0.067690375 ENSMUSG00000055730 Ces2a 1492.288913 0.53609906 0.098326685 ENSMUSG00000064105 CNNM2 164.7551495 0.544795178 0.095451128 ENSMUSG00000037016 FREM2 362.6519036 0.555303386 0.095451128 ENSMUSG00000002996 HBP1 636.8509431 0.55580474 0.010458882 ENSMUSG00000021823 VCL 1991.941072 0.562405812 0.098326685 ENSMUSG00000025262 FAM120C 90.28025431 0.564929732 0.064740549 ENSMUSG00000030616 SYTL2 1625.705824 0.585417053 0.095451128 ENSMUSG00000022032 SCARA5 160.7569438 0.609791499 0.078878073 ENSMUSG00000005580 ADCY9 143.1194831 0.610067964 0.049847812 ENSMUSG00000027204 FBN1 1090.898924 0.611626398 0.086945121 ENSMUSG00000027375 MAL 609.8428432 0.613323309 0.095451128 ENSMUSG00000040584 ABCB1 10475.57444 0.613999073 0.086945121 ENSMUSG00000024327 SLC39A7 307.0944412 0.625511156 0.049097668 ENSMUSG00000022519 SRL 110.2707271 0.630929516 0.050069013 ENSMUSG00000045281 GPR20 200.1838239 0.63355842 0.067690375 ENSMUSG00000027637 RAB5IF 653.7971968 0.640272973 0.073627868 ENSMUSG00000082361 BTC 200.636709 0.645407901 0.081699043 ENSMUSG00000085830 Grin1os 59.42621064 0.6612661 0.09570569 ENSMUSG00000039238 ZNF750 280.9700596 0.668514318 0.067690375 ENSMUSG00000036377 Cracd 1955.144489 0.678710125 0.095451128 ENSMUSG00000041695 KCNJ2 98.93255963 0.680007434 0.095451128 ENSMUSG00000087543 Gm16576 65.03381063 0.687650498 0.095451128 ENSMUSG00000028246 FAXC 212.6858369 0.688601239 0.069014393 ENSMUSG00000003752 ITPKC 324.85551 0.701345104 0.095451128 ENSMUSG00000054200 FFAR4 238.5699495 0.71819574 0.099482486 ENSMUSG00000033855 STON1 278.4169838 0.732997371 0.025513338 ENSMUSG00000047497 ADAMTS12 48.84762818 0.781563691 0.089246817 ENSMUSG00000037106 FER1L6 1294.811863 0.789934133 0.055683231 ENSMUSG00000117278 Gm36684 26.55030286 0.799715781 0.095451128 ENSMUSG00000089704 GALNT2 35.14423938 0.808759692 0.086945121 ENSMUSG00000034107 ANO7 700.5476335 0.81879875 0.095794033 ENSMUSG00000085595 Gm16090 29.89381644 0.898582969 0.086945121 ENSMUSG00000047730 FCGBP 43225.49797 0.930168031 0.066476847 Expr Log Ratio Expr pvalue 161 Table B.1 (Continued) ID Symbol Expr Intensity/RPKM/ FPKM/Counts ENSMUSG00000032226 GCNT3 1456.163932 0.935932263 0.055683231 ENSMUSG00000097852 4933405D12Rik 12.34312283 0.939022689 0.095451128 ENSMUSG00000105456 Gm43745 148.2833567 0.963989734 0.068442377 ENSMUSG00000049350 ZG16 38482.30486 0.999022892 0.048159799 ENSMUSG00000025515 MUC2 29997.53455 1.003895682 0.067690375 ENSMUSG00000083907 Plk-ps1 11.81002948 1.004674755 0.09570569 ENSMUSG00000013523 BCAS1 2777.248845 1.05600499 0.049097668 ENSMUSG00000079105 C7 14.82009712 1.061197289 0.079443522 ENSMUSG00000079645 Gm17193 18.67976083 1.094454819 0.066476847 ENSMUSG00000107215 Gm43197 19.45427114 1.118049202 0.067690375 ENSMUSG00000052305 HBB 404.7193049 1.146734426 0.055683231 ENSMUSG00000028255 CLCA1 56226.35101 1.186839345 0.040367095 ENSMUSG00000112902 RP23-193A14.3 2.875855527 1.23751697 0.095451128 ENSMUSG00000069917 HBA1/HBA2 84.49308608 1.369393603 0.01109524 Expr Log Ratio Expr pvalue 162 Table B.2: Identified canonical pathways. -log(pvalue) 1.65 5.94 3.19 2.84 2.66 1.37 2.79 2.03 1.82 1.49 1.42 Ratio 0.0179 0.0879 0.0492 0.0361 0.0472 0.0202 0.0286 0.0336 0.036 0.0286 0.0272 T Cell Receptor Signaling ICOS-ICOSL Signaling in T Helper Cells p70S6K Signaling Erythropoietin Signaling Pathway Production of Nitric Oxide and Reactive Oxygen Species in Macrophages Integrin Signaling Glioblastoma Multiforme Signaling Hepatic Fibrosis Signaling Pathway WNT/Ca+ pathway GM-CSF Signaling STAT3 Pathway Actin Cytoskeleton Signaling White Adipose Tissue Browning Pathway ERK/MAPK Signaling Gustation Pathway 2.6 1.51 3.01 2.37 0.0212 0.0178 0.0455 0.0339 2.21 1.99 1.8 1.55 2.65 2.51 2.21 1.72 1.53 1.43 2.12 0.0314 0.0282 0.0292 0.0191 0.0625 0.0571 0.037 0.0245 0.0294 0.0234 0.03 Phagosome Formation GPCR-Mediated Nutrient Sensing in Enteroendocrine Cells D-myo-inositol-5-phosphate Metabolism Insulin Secretion Signaling Pathway Endothelin-1 Signaling Role of NFAT in Cardiac Hypertrophy Protein Kinase A Signaling Breast Cancer Regulation by Stathmin1 2.59 2.49 2.2 2.07 1.61 1.39 1.64 2.73 0.0203 0.0431 0.0312 0.0261 0.0262 0.0227 0.0199 0.022 Ingenuity Canonical Pathways PKCθ Signaling in T Lymphocytes Crosstalk between Dendritic Cells and Natural Killer Cells Th1 Pathway Synaptic Long Term Depression PD-1, PD-L1 cancer immunotherapy pathway Senescence Pathway Neuroinflammation Signaling Pathway Corticotropin Releasing Hormone Signaling Antioxidant Action of Vitamin C Insulin Receptor Signaling Endocannabinoid Neuronal Synapse Pathway 163 Table B.2 (Continued) -log(pvalue) 2.62 2.55 2.4 2.32 2.09 1.75 1.75 1.65 1.64 1.52 1.46 4.81 4.49 4.24 3.91 3.74 3.47 3.36 Ratio 0.0223 0.0446 0.0302 0.0331 0.0191 0.0284 0.0284 0.0166 0.0266 0.0248 0.0238 0.0753 0.116 0.0452 0.0465 0.0511 0.103 0.5 Glucocorticoid Receptor Signaling Acetyl-CoA Biosynthesis I (Pyruvate Dehydrogenase Complex) 3.29 0.0241 2.83 0.286 Communication between Innate and Adaptive Immune Cells FcγRIIB Signaling in B Lymphocytes Primary Immunodeficiency Signaling Telomerase Signaling Molecular Mechanisms of Cancer PCP (Planar Cell Polarity) Pathway TCA Cycle II (Eukaryotic) Neuregulin Signaling Sphingosine-1-phosphate Signaling Renin-Angiotensin Signaling Role of NANOG in Mammalian Embryonic Stem Cell Pluripotency Role of JAK1 and JAK3 in γc Cytokine Signaling Basal Cell Carcinoma Signaling GABA Receptor Signaling 2.56 2.21 1.93 1.87 1.83 1.83 1.75 1.74 1.73 1.7 0.0183 0.0471 0.0545 0.0374 0.0202 0.05 0.0833 0.0342 0.0339 0.0333 1.7 1.67 1.62 1.58 0.0333 0.0435 0.0417 0.0305 Ingenuity Canonical Pathways Cardiac Hypertrophy Signaling (Enhanced) CDK5 Signaling Superpathway of Inositol Phosphate Compounds Dopamine-DARPP32 Feedback in cAMP Signaling B Cell Receptor Signaling D-myo-inositol (1,4,5,6)-Tetrakisphosphate Biosynthesis D-myo-inositol (3,4,5,6)-tetrakisphosphate Biosynthesis Systemic Lupus Erythematosus In B Cell Signaling Pathway 3-phosphoinositide Degradation 3-phosphoinositide Biosynthesis Thrombin Signaling IL-4 Signaling B Cell Development PI3K/AKT Signaling Th1 and Th2 Activation Pathway Th2 Pathway Antigen Presentation Pathway 2-ketoglutarate Dehydrogenase Complex 164 Table B.2 (Continued) Ingenuity Canonical Pathways Role of MAPK Signaling in Inhibiting the Pathogenesis of Influenza Dopamine Receptor Signaling Role of WNT/GSK-3β Signaling in the Pathogenesis of Influenza IL-3 Signaling Chemokine Signaling G-Protein Coupled Receptor Signaling Role of JAK2 in Hormone-like Cytokine Signaling Fatty Acid β-oxidation I CD28 Signaling in T Helper Cells PEDF Signaling IL-9 Signaling HIPPO signaling Dilated Cardiomyopathy Signaling Pathway Agranulocyte Adhesion and Diapedesis MIF-mediated Glucocorticoid Regulation Regulation Of The Epithelial Mesenchymal Transition In Development Pathway Cellular Effects of Sildenafil (Viagra) Complement System Regulation of Cellular Mechanics by Calpain Protease Docosahexaenoic Acid (DHA) Signaling Melanocyte Development and Pigmentation Signaling April Mediated Signaling -log(pvalue) Ratio 1.56 1.55 0.0395 0.039 1.53 1.52 1.5 1.5 1.47 1.47 1.46 1.45 1.44 1.44 1.43 1.43 1.42 0.0385 0.038 0.0375 0.0217 0.0588 0.0588 0.0174 0.0357 0.0571 0.0353 0.0274 0.0234 0.0556 1.41 1.4 1.4 1.39 1.38 1.33 1.3 0.0345 0.0268 0.0541 0.0337 0.0526 0.0319 0.0476 165 Table B.3: Identified upstream regulators. Upstream Regulator Expr Log Ratio Predicted Activation Activation z-score State p-value of overlap TNF -0.711 Inhibited -3.784 0.00334 IFNG AHR E. coli B5 lipopolysaccharide Inhibited -0.072 Inhibited -3.694 -3.268 0.000314 0.000668 Inhibited -3.234 0.0000219 Lipopolysaccharide Inhibited -3.193 0.000000174 IL6 E. coli B4 lipopolysaccharide Inhibited -3.064 0.000261 Inhibited -2.918 0.00022 Dexamethasone CSF2 IL10RA TLR7 Activated Inhibited -0.769 Activated -0.783 Inhibited 2.918 -2.905 2.813 -2.764 0.0017 0.00291 0.00667 0.00014 Tretinoin CD28 Salmonella minnesota R595 lipopolysaccharides TNFSF11 Inhibited -0.556 Inhibited -2.677 -2.621 0.117 0.0385 Inhibited -0.306 Inhibited -2.581 -2.572 0.0000991 0.00069 CD40LG CLPP ZBTB10 Resiquimod IL1 OSM Inhibited -0.041 Inhibited 0.262 Inhibited Inhibited Inhibited -1.009 Inhibited -2.461 -2.449 -2.449 -2.416 -2.395 -2.39 0.000228 0.0000521 0.00288 0.000253 0.0932 0.00442 166 Table B.3 (Continued) Upstream Regulator Expr Log Ratio Predicted Activation Activation State z-score p-value of overlap SIRT1 0.091 Activated 2.324 0.00000766 INSR IL18 CDKN2A mono-(2ethylhexyl)phthalate PSMB11 0.148 Activated 0.246 Inhibited -0.219 Inhibited 2.316 -2.236 -2.236 0.0000352 0.0282 0.132 Activated Activated 2.236 2.236 0.00335 0.00468 IL2 USP22 GATA2 BCL3 Tlr IL1A Fenofibrate Nr1h TLR9 FN1 TP73 Inhibited 0.169 Inhibited -0.138 Inhibited -0.294 Activated Inhibited -0.446 Inhibited Activated Activated -0.317 Inhibited 0.469 Inhibited -0.117 Activated -2.23 -2.219 -2.219 2.213 -2.2 -2.19 2.186 2.18 -2.171 -2.169 2.151 3.43E-08 0.0000367 0.00034 0.000232 0.00378 0.0713 0.0311 0.0127 0.0658 0.0397 0.0291 IL33 Ige -0.179 Inhibited Inhibited -2.093 -2.043 0.00000411 0.00296 Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited Inhibited -2.024 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 -2 0.00353 0.000598 0.00365 0.00521 0.00647 0.0203 0.0205 0.0227 0.0657 0.106 0.126 0.518 poly rI:rC-RNA Pembrolizumab CBX5 PLX5622 ESRRG IL21 MAP4K4 Cardiotoxin Raloxifene CXCL12 AKT1 GABA 0.154 0.228 -0.161 -0.181 0.202 167 Table B.3 (Continued) Upstream Regulator KDM1A NFKBIB Arachidonic acid DIO3 TAZ CIITA Go 6976 Fingolimod TNFSF15 CDK9 Bay 11-7082 APP IL27 Filgrastim Interferon alpha Enterotoxin B TNFRSF1A TLR3 Expr Log Ratio Predicted Activation Activation State z-score 2 2 2 2 -1.997 -1.985 1.982 1.982 -1.98 -1.98 1.98 -1.979 -1.974 1.97 -1.969 -1.969 -1.96 -1.96 0.00000512 0.0013 0.0139 0.0668 0.004 0.0005 0.00289 0.00595 0.000339 0.00139 0.00675 0.335 0.0419 0.0418 0.00253 0.00545 0.0571 0.202 E. coli lipopolysaccharide -1.954 0.00413 Peptidoglycan IFNAR1 TCR SPI1 PRKCD IL5 TICAM1 TRAF3IP2 Metformin IKBKG TLR4 IL3 Glucocorticoid -1.954 -1.951 -1.945 -1.944 -1.942 -1.941 -1.934 -1.93 -1.925 -1.915 -1.909 -1.89 1.869 0.0168 0.0124 0.0019 0.00521 0.00154 0.252 0.0402 0.00393 0.0432 0.0345 0.28 0.0831 0.00157 -1.859 -1.833 0.00196 0.0116 NFkB (complex) CHUK 0.089 Activated -0.013 Activated Activated -0.347 Activated -0.054 -0.566 p-value of overlap -0.094 -0.115 0.206 0.046 0.316 -0.073 -0.786 0.119 0.298 0.112 0.283 -0.005 -0.04 168 Table B.3 (Continued) Upstream Regulator Expr Log Ratio Predicted Activation Activation State z-score p-value of overlap PRDM1 -0.118 1.811 0.00000352 IL4 FAS PPARGC1A IL17A BNIP3L RNF31 CPT1B TREM1 Camptothecin CD40 Fluticasone propionate Pam3-Cys-Ser-Lys4 IRF8 MYD88 -0.188 -0.082 0.145 -1.782 -1.777 1.766 -1.752 -1.732 1.732 -1.633 -1.633 -1.633 -1.55 1.544 -1.527 -1.519 -1.504 0.000000938 0.000534 0.0988 0.00472 0.00193 0.00413 0.000122 0.00195 0.0443 0.0145 0.0000235 0.00000396 0.00899 0.00125 IL1B EIF4E P38 MAPK RELA CpG oligonucleotide EGR1 Ciprofibrate NCOA2 3M-002 PML Cholesterol NR1H2 ITK RB1 Methotrexate -0.795 0.012 -1.49 -1.48 -1.476 -1.463 -1.457 -1.452 1.451 -1.432 -1.432 -1.432 -1.432 1.414 -1.408 1.408 1.406 0.00283 0.000106 0.00634 0.026 0.0221 0.0282 0.0402 0.00306 0.00484 0.0155 0.0183 0.000302 0.000105 0.0161 0.00584 -0.262 -0.062 0.535 -0.732 -0.719 -0.244 0.011 -0.147 0.013 0.18 -0.111 0.041 -0.675 0.166 PTEN 0.28 1.405 0.00164 STAT6 0.102 -1.348 0.000888 169 Table B.3 (Continued) Upstream Regulator IFN alpha/beta 5-O-mycolyl-beta-araf-(1>2)-5-O-mycolyl-alphaaraf-(1->1')-glycerol CD44 INSIG1 SYVN1 IL15 GATA1 Expr Log Ratio -0.246 0.528 -0.172 0.052 Predicted Activation Activation State z-score -1.342 p-value of overlap 0.000788 -1.342 -1.342 1.342 1.342 -1.318 1.309 0.000928 0.0384 0.00302 0.00874 0.000855 0.00125 CD3 Salmonella enterica serotype abortus equi lipopolysaccharide -1.29 0.000556 -1.231 0.017 Immunoglobulin IgG Calcitriol SATB1 STAT3 Pyrrolidine dithiocarbamate -1.229 -1.222 -1.205 -1.195 -1.179 1.172 0.000000371 0.00705 0.0114 0.000708 0.00637 0.00314 1.155 1.154 0.0166 0.0005 -0.288 -0.034 Levodopa Isoprenaline AGT MAP3K14 -0.304 -0.303 -1.152 -1.091 0.0148 0.0057 MTOR Niacinamide MYB 0.198 -1.071 1.067 1.067 0.0000973 0.00792 0.0212 1.003 -1.002 -1 0.00161 0.00152 0.0126 -1 0.0126 Beta-estradiol IL22 Erlotinib N-nitro-L-arginine methyl ester 0.316 170 Table B.3 (Continued) Upstream Regulator TAL1 RUNX3 Epicatechin Tacrolimus ADORA2A Z-LLL-CHO Hyaluronic acid Thapsigargin Expr Predicted Log Activation Activation Ratio State z-score 0.222 1 -0.664 1 1 1 -0.387 1 0.949 -0.943 -0.916 p-value of overlap 0.00386 0.0059 0.0103 0.0231 0.0345 0.0357 0.000068 0.000126 D-glucose IFIH1 0.149 -0.916 -0.914 0.012 0.00077 OGA 0.084 -0.898 0.0000987 0.879 -0.849 0.842 -0.832 -0.816 -0.816 -0.816 -0.816 0.816 0.816 -0.808 -0.798 -0.771 3.25E-08 0.00801 0.0000402 0.0253 0.000184 0.000206 0.00993 0.0121 0.000694 0.00651 0.015 0.0344 0.0492 Genistein Eicosapentenoic acid CXCL8 Alpha catenin -0.764 0.762 -0.747 0.73 0.0000687 0.0122 0.00675 0.000713 Tetradecanoylphorbol acetate Fcer1 MAP2K1 CSF3 -0.725 -0.692 -0.689 -0.647 0.00000767 0.000232 0.0298 0.000185 IL10 KITLG CAMP C5 BMP10 daidzein ARNT2 SIM1 Cyclosporin A ADIPOQ IKBKB JUN IFNB1 0.302 -1.356 -0.353 -0.228 0.145 -0.032 -0.252 0.455 -0.661 171 Table B.3 (Continued) Expr Predicted Log Activation Activation Ratio State z-score -0.597 -0.636 -0.627 0.625 0.571 -0.257 -0.557 -0.557 -0.16 0.555 0.532 0.119 -0.522 p-value of overlap 0.0212 0.00521 0.0371 0.0343 0.00208 0.0068 0.0025 0.0211 0.00384 0.522 0.00107 -0.496 -0.447 -0.447 -0.447 0.447 0.0297 0.000978 0.0118 0.031 0.0182 Butyric acid -0.44 0.00024 Forskolin NOTCH1 IL13 TRAF3 5-azacytidine Sirolimus Prostaglandin E2 -0.42 -0.415 0.402 0.394 0.348 -0.343 -0.3 0.0155 0.00994 0.0171 0.0016 0.0199 0.0183 0.00554 -0.285 0.281 -0.276 -0.271 0.265 0.243 -0.24 0.239 0.00702 0.000017 0.0194 0.00208 0.0473 0.00137 0.0158 0.00121 Upstream Regulator NFKBIA MAP3K7 Triamcinolone acetonide PD98059 NFKB2 Paclitaxel NFATC2 Resveratrol NR4A1 Trichostatin A TGFB1 Histamine TCF3 Tnf (family) S100A9 TP53 IKBKE Calcimycin NFAT5 IGF1R KLF2 VIP S100A8 -0.482 -0.157 -0.319 0.075 -0.013 -0.042 0.149 0.009 -0.249 -0.313 -0.32 -0.596 172 Table B.3 (Continued) Upstream Regulator Phytohemagglutinin 17-alpha-ethinylestradiol ELF4 Ionomycin CTCF RUNX1 RIPK2 Alitretinoin NFKB1 HSPA5 ATP-gamma-S 1,4-bis[2-(3,5dichloropyridyloxy)]benzene SP600125 YBX1 CCR2 Cisplatin OGT TREM2 PPIF 2-amino-5-phosphonovaleric acid HDAC1 semaxinib DYSF MAPT Ferritin Interferon beta-1a BIRC5 Ap1 gamma CAMK2D TNFSF13B NOD2 mir-150 LY6E RECQL4 Expr Log Ratio Predicted Activation Activation State z-score 0.219 -0.218 -0.218 -0.179 -0.152 0.152 -0.147 0.13 0.124 -0.117 -0.106 p-value of overlap 0.00574 0.0000775 0.000305 0.0287 0.0069 0.0273 0.00356 0.0134 0.00472 0.00105 0.0000274 0.088 -0.407 0.106 -0.101 -0.037 -0.034 0.0211 0.00718 0.00302 0.000496 0.223 -0.326 0.43 -0.032 0 0 0 0.0248 0.000353 0.00139 0.00922 0.032 0 0 0.0377 0.0412 0.000000358 0.00000212 0.332 -0.032 -0.22 -0.049 -0.069 0.011 -0.219 -0.438 0.017 -0.046 -0.512 -0.125 0.033 -0.046 0.000029 0.0000541 0.000116 0.000133 0.000229 0.000345 0.000356 0.000524 0.000547 0.000684 0.000756 173 Table B.3 (Continued) Upstream Regulator RFXANK LTBR Lipoarabinomannan NR1H3 Lestaurtinib BCL2L11 DAB1 CLIC4 Muc4 Clec2d (includes others) TRADD MALP-2s B2M IRAK3 POU3F3 Rfx5 ULBP1 TYROBP CLEC7A PTX3 PSMB9 RP 73401 BQ 123 OSCAR Rhesus theta-defensin 1 TLR2/3/4/9 Sobetirome CTTN Perilla alcohol TBP RYR1 SELP RC3H1 FLT3LG NCSTN NLRP12 Vinblastine Expr Predicted Log Activation Activation Ratio State z-score 0.069 0.03 0.101 -0.2 0.081 0.161 0.755 -0.694 0.112 -0.265 -0.004 -0.556 0.186 0.335 -0.542 -0.564 -0.3 -0.24 0.273 0.018 -0.147 -0.016 0.154 -0.562 0.185 -0.275 p-value of overlap 0.000756 0.000902 0.00104 0.00107 0.00113 0.00134 0.00157 0.00157 0.00157 0.00157 0.00187 0.00187 0.00207 0.00207 0.00208 0.00208 0.00208 0.00217 0.00228 0.00251 0.00266 0.00266 0.00266 0.00328 0.00331 0.00331 0.00331 0.00331 0.00331 0.00356 0.00356 0.00356 0.00374 0.00374 0.00386 0.00386 0.00386 174 Table B.3 (Continued) Upstream Regulator ZBTB7B 1-hydroxy-2-methyl-2butenyl 4-diphosphate Traj18 CARD14 CAMK2G STAT5A mir-130 CD4 ATG16L1 Diaminopimelic acid RARB TLR7/8 Zymosan TNIP1 BPIFA1 HLA-A B4GALNT1 PLA2G2E PCGEM1 S-nitrosoglutathione MHC II CD2 SELPLG MVP Metoprolol DMP1 Maslinic acid HBB TIFA AIMP1 RFX5 Vadimezan NPC1 MYOC Expr Predicted Log Activation Activation Ratio State z-score 0.199 -0.166 0.023 -0.227 -0.915 0.059 0.213 0.24 -0.785 -0.554 0.53 -0.938 -0.607 0.106 -0.267 1.147 -0.37 0.009 0.144 0.319 p-value of overlap 0.00393 0.00402 0.00402 0.00402 0.00402 0.00409 0.00418 0.00418 0.00479 0.00479 0.00486 0.00521 0.00521 0.00558 0.00563 0.00563 0.00563 0.00563 0.00598 0.00598 0.00638 0.00638 0.00638 0.00653 0.00653 0.00681 0.00703 0.00725 0.00749 0.00749 0.00749 0.00749 0.00762 0.00762 175 Table B.3 (Continued) Upstream Regulator SENP3 Pam3-Cys TLR8 IL1RL2 POU3F2 LY96 CXCL2 VIPR1 BCL10 Cyclopiazonic acid Eicosa-11Z, 14Z-dienoic acid Patched Ryr Coactivator-Dtx-NotchRbpsuh Miocamycin Loxoprofen ZNF496 FOXN2 SNORD21 HDLBP B3GNT6 USP9Y MACROD1 PPP3CC DNAJC15 IL34 PGS1 SMG6 cis-vaccenic acid PRPF4 TRAK2 mir-583 EPSTI1 Raet1d/Raet1e LINC00278 Expr Predicted Log Activation Activation Ratio State z-score -0.005 0.495 0.194 -0.001 -0.06 -1.112 0.3 0.086 p-value of overlap 0.0077 0.0077 0.00817 0.00851 0.00851 0.00851 0.00851 0.00851 0.00851 0.00851 0.00879 0.00879 0.00879 -0.071 0.215 0.309 -0.24 0.05 -0.266 0.216 -0.095 -0.161 0.041 -0.073 0.22 0.105 0.39 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 176 Table B.3 (Continued) Upstream Regulator DPF2 GUCA2B EPS15 TONSL CTNNA1 AM114 GRM7 HSP-990 Clomoxir Mitoguazone Triton X-100 Anthraquinone Gemtuzumab ozogamicin NEF inhibitor B9 Oxfenicine ZFP36 CHD1 PSMB8 CD70 Hsp70 ITGB2 FOLR1 NRAS EBF1 Pomalidomide Inosine Carrageenan Bee venom LDL ABCB1 C2CD5 PHLPP1 S1PR2 PAEP PLIN1 KLF13 Expr Predicted Log Activation Activation Ratio State z-score 0.125 -0.005 0.17 0.06 0.163 -0.318 -0.114 0.197 -0.255 -0.745 -0.112 -0.071 -0.587 0.614 0.151 0.222 0.037 0.298 0.108 p-value of overlap 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00879 0.00922 0.00959 0.00959 0.00959 0.00969 0.0102 0.0103 0.0104 0.0106 0.0107 0.0108 0.0108 0.011 0.0111 0.0114 0.0119 0.0119 0.0119 0.0119 0.0119 0.0119 177 Table B.3 (Continued) Upstream Regulator PS-1145 RNA polymerase II MOG ICAM1 STAP2 NOD1 MSR1 HRG Fumonisin B1 S-(2,3bispalmitoyloxypropyl)cysteine-GDPKHPKSF CAT CEBPE mir-142 CD5 Lymphotoxin DHX9 RNASE1 SUV39H1 CD14 EBI3 Caffeic acid phenethyl ester TNFSF9 PTGER2 QKI LTB TFPI2 RARRES2 SUPT20H Evodiamine AZprime5576 L-leucine Miglitol CASK FAM168A ARL16 Expr Log Ratio -0.488 0.092 -0.065 -0.593 -0.06 -0.507 -0.485 -0.25 0.189 -0.321 -0.101 0.105 -0.708 -0.283 -0.046 -1.048 -0.233 -0.268 0.281 0.134 -0.014 Predicted Activation Activation State z-score p-value of overlap 0.0119 0.0124 0.0126 0.0126 0.0132 0.0132 0.0132 0.0132 0.0132 0.0132 0.0132 0.0132 0.0132 0.0138 0.0145 0.0145 0.0145 0.0145 0.0145 0.0145 0.0158 0.0158 0.0163 0.0166 0.0172 0.0172 0.0172 0.0172 0.0172 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 178 Table B.3 (Continued) Upstream Regulator Raet1a BCL6 peptide inhibitor Raet1b AP1M1 MMP10 HLTF CELA1 RAET1E ACP3 H60a DCTN4 Poly-L-glutamic acidpeptoid 1 conjugate QM56 NAGLU ATP6V0C ADGRV1 SLC25A12 12-hydroxyeicosapentaenoic acid HCST GBP2 HBG1 CLEC2D Iberdomide E-c-HDMAPP Palytoxin Satavaptan Acetoxyacetylaminofluorene Miltefosine Sulfonylurea Talniflumate Phenyl butyrate AZD4573 Brimapitide 3,20-pregnanedione MRTFB Ciap RNF138 Expr Log Ratio Predicted Activation Activation State z-score 0.021 p-value of overlap 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.149 0.188 -0.102 0.078 0.0175 0.0175 0.0175 0.0175 0.0175 -0.101 -0.472 0.093 0.461 0.408 -0.115 0.145 0.308 0 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0175 0.0182 0.0187 0.0187 179 Table B.3 (Continued) Upstream Regulator TF CXCR2 Poly dA-dT L-buthionine (SR)sulfoximine KIT BCL6 Plumbagin Calpain NfkB-RelA NODAL SLPI BTRC MTA2 TRPV4 EIF4EBP2 RHO Leukotriene B4 Resolvin E1 AHI1 IL36G Cuprizone FLI1 MMP12 KCNE3 Trovafloxacin ECSIT ELOVL5 ACE SHC1 MMP2 MUC1 Map3k7 KRAS L-ornithine Chloride HS-243 Expr Predicted Log Activation Activation Ratio State z-score -0.495 0.067 -0.136 0.812 0.158 0.056 -0.211 0.312 -0.338 -0.235 -0.598 -0.514 0.242 0.022 -0.029 0.149 0.183 0.195 0.18 0.142 p-value of overlap 0.0187 0.0187 0.0187 0.0187 0.0188 0.0194 0.0202 0.0202 0.0202 0.0202 0.0202 0.0202 0.0202 0.0202 0.0202 0.0213 0.0218 0.0218 0.0218 0.0218 0.0218 0.0221 0.0221 0.0221 0.0221 0.0234 0.025 0.025 0.0256 0.0256 0.0256 0.0256 0.0259 0.0262 0.0262 0.0262 180 Table B.3 (Continued) Upstream Regulator Stat5b dimer Clobetasol DEFA1 (includes others) FREM1 ADRM1 LRRC8E S1PR4 MTCH2 OXGR1 FERMT3 KLHL21 TRPC5 GNLY RPL13A DHPS YARS1 MADD CARD8 ZNRD1ASP IFI30 TBC1D10A CACNA2D1 ZFX RECQL ERC1 Cacnb1 CYP2C9 HERC5 HILPDA Prinaberel Fomepizole p-chloroamphetamine Oenothein B Senexin B KDM8 TARDBP mir-27 Expr Log Ratio -0.357 0.036 0.27 -0.826 0.196 0.303 -0.621 0.091 0.052 -0.018 0.176 0.093 -0.141 -0.071 -0.064 0.095 0.218 0.139 0.777 0.027 0.191 0.104 Predicted Activation Activation State z-score p-value of overlap 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0262 0.0266 0.0266 0.0266 181 Table B.3 (Continued) Upstream Regulator 2-deoxyglucose ROCK1 Hemozoin Magnesium Rituximab BAK1 BGN SFRP4 VAV3 CD69 GNAI2 MIF tetracycline CARD9 MALT1 IDR-1002 KRT17 CNR2 VAV2 Fluvoxamine Suramin Ursolic acid PRNP RELB ID3 TIRAP SFTPA1 KDM6B Cdk TAB1 Sodium tungstate TAS4464 TCF12 Rsk Chitinase Aluminum hydroxide Smectite Expr Log Ratio -0.374 0.285 0.356 0.22 0.092 -0.945 -0.094 -0.164 -0.344 -0.184 -0.195 -0.709 -0.232 -0.192 -0.096 -0.298 -0.065 0.211 0.14 0.049 Predicted Activation Activation State z-score p-value of overlap 0.0266 0.0267 0.0267 0.0267 0.0285 0.0285 0.0285 0.0285 0.0285 0.0285 0.0285 0.0294 0.0295 0.0303 0.0303 0.0303 0.0303 0.0303 0.0303 0.0303 0.0303 0.0305 0.0315 0.0315 0.0316 0.0321 0.0325 0.034 0.034 0.034 0.034 0.034 0.0345 0.0347 0.0347 0.0347 0.0347 182 Table B.3 (Continued) Upstream Regulator CACTIN ZXDC IKBIP CIRBP TRIL NXF1 FZD1 UFD1 miR-27a-5p (miRNAs w/seed GGGCUUA) miR-330-5p (and other miRNAs w/seed CUCUGGG) miR-324-5p (miRNAs w/seed GCAUCCC) PALS1 CBFA2T2 SNX17 K-604 IK IGL TAF12 NPLOC4 DCC Macf1 GGT1 PTP4A2 WARS1 ERO1A Gzmb Cyp2a12/Cyp2a22 Phenylacetic acid Apicularen A Ascomycin Polymethyl methacrylate Phenanthridine Salsalate Expr Predicted Log Activation Activation Ratio State z-score -0.093 0.185 -0.115 -0.057 -0.045 -0.187 0.3 0.023 p-value of overlap 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 -0.021 0.13 0.022 0.025 -0.121 0.144 -0.118 -0.042 -0.1 0.213 0.149 -0.332 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 0.0347 183 Table B.3 (Continued) Upstream Regulator 1,1-diethyl-2-hydroxy-2nitrosohydrazine AC083837.1 Succinylacetone Fenclonine D,L-propargylglycine Firre TGFA C3 Etanercept Secretase gamma bryostatin 1 ELAVL1 GCG CpG ODN 1826 ITGAM Deoxycorticosterone acetate/potassium chloride/sodium chloride ACE2 PD173074 FOXL2 CYBB CORT HDAC5 IFNL1 POU2AF1 LYN IL7R RNASE2 IKZF2 GNB2 GTF2B ozone KLF6 NSC 172285 p38 Sapk Teniposide Expr Log Ratio 0.169 0.271 -0.354 -0.016 -0.367 0.308 -0.623 -0.299 0.058 -0.979 -0.218 -0.462 -0.498 -0.032 0.074 0.078 -0.105 Predicted Activation Activation State z-score p-value of overlap 0.0347 0.0347 0.0347 0.0347 0.0347 0.0357 0.0357 0.0357 0.0359 0.0359 0.0359 0.0364 0.0369 0.0369 0.0379 0.0379 0.0379 0.038 0.0391 0.0399 0.0399 0.0403 0.0403 0.0403 0.0403 0.0415 0.0419 0.0419 0.0419 0.0419 0.0419 0.0427 0.0432 0.0432 0.0432 184 Table B.3 (Continued) Upstream Regulator Asialo GM1 ganglioside Integrinα Edratide CpG ODN 1555 IRF3-IRF7 MARCHF3 Propolis YIPF6 MCOLN2 RPE65 GPR68 TAF9 Nc2 Mucin RAP1B DAPK1 UBB PGRMC1 TLN1 TIAL1 NBR1 SEPTIN9 RPS3 CCL22 GJB1 SLAMF6 ADAM8 AQP3 NUBPL ORC2 PELI2 SSBP1 Muc1 PILRA RAB4A Fluticasone H3B-8800 Expr Log Ratio -0.054 0.364 0.302 -0.22 0.057 -0.008 -0.061 -0.052 0.084 -0.034 0.192 0.141 0.079 -1.028 0.053 -0.839 -0.468 -0.054 0.335 -0.031 0.304 -0.105 0.576 -0.705 0.143 Predicted Activation Activation State z-score p-value of overlap 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 0.0432 185 Table B.3 (Continued) Upstream Regulator Ranitidine Fenoldopam Roflumilast Cathelicidin-WA N(1)-guanyl-1,7diaminoheptane Trans-aminocyclopentane1,3-dicarboxylic acid Halofuginol Lactose Lovastatin CBFB LCN2 NCOA3 TCL1A MBTD1 CERK NEUROG2 TLR5 IGHM Ethylene glycol tetraacetic acid BRD4 SERPINE2 GNB1 CIDEC MAP3K8 EIF4EBP1 Parthenolide ID2 Expr Log Ratio Predicted Activation Activation State z-score p-value of overlap 0.0432 0.0432 0.0432 0.0432 0.0432 0.078 -0.447 0.004 -0.095 -0.147 0.388 -0.958 -0.436 0.052 0.336 -0.423 0.063 -0.361 0.0432 0.0432 0.0432 0.0437 0.0439 0.0439 0.0439 0.0439 0.044 0.044 0.044 0.044 0.044 0.044 0.0446 0.0461 0.0461 0.0461 0.0473 0.0482 0.0482 0.0483 Table B.4: Identified gene ontology terms. p-value 3.77E-05 4.42E-05 7.85E-05 0.000122 0.000255 0.000299 0.000458 0.00047 0.00059 0.00059 0.000627 0.000714 0.000811 0.001305 0.001348 0.001691 0.001691 0.001789 0.002047 0.002195 0.002514 0.002514 0.002514 0.002514 0.002514 q-value 0.033893 0.033893 0.04016 0.046709 0.076418 0.076418 0.087543 0.087543 0.087543 0.087543 0.087543 0.091387 0.095789 0.13797 0.13797 0.148431 0.148431 0.148431 0.148431 0.148431 0.148431 0.148431 0.148431 0.148431 0.148431 186 Term Cellular response to organic substance (GO:0071310) Positive regulation of cell-cell adhesion mediated by integrin (GO:0033634) Positive regulation of cell adhesion mediated by integrin (GO:0033630) Cortical actin cytoskeleton organization (GO:0030866) Regulation of cell-cell adhesion mediated by integrin (GO:0033632) Positive regulation of protein polymerization (GO:0032273) Positive regulation of actin filament polymerization (GO:0030838) Positive regulation of T cell activation (GO:0050870) Cellular response to interleukin-15 (GO:0071350) Interleukin-15-mediated signaling pathway (GO:0035723) Cytokine-mediated signaling pathway (GO:0019221) Cellular response to cytokine stimulus (GO:0071345) Antigen receptor-mediated signaling pathway (GO:0050851) Positive regulation of supramolecular fiber organization (GO:1902905) Regulation of metal ion transport (GO:0010959) Regulation of barbed-end actin filament capping (GO:2000812) Succinyl-CoA metabolic process (GO:0006104) Positive regulation of T cell proliferation (GO:0042102) Cortical cytoskeleton organization (GO:0030865) Lipopolysaccharide-mediated signaling pathway (GO:0031663) Peptide antigen assembly with MHC protein complex (GO:0002501) Cellular response to cold (GO:0070417) Regulation of endoplasmic reticulum tubular network organization (GO:1903371) Cell junction disassembly (GO:0150146) Negative regulation of myeloid leukocyte mediated immunity (GO:0002887) Table B.4 (Continued) p-value 0.002791 0.00314 0.003325 0.003489 0.003489 0.003489 0.003489 0.003833 0.004608 0.004612 0.004612 q-value 0.158684 0.1623 0.1623 0.1623 0.1623 0.1623 0.1623 0.173027 0.191329 0.191329 0.191329 0.004852 0.005218 0.005313 0.005595 0.005878 0.005878 0.005878 0.005878 0.005878 0.00619 0.007095 0.007205 0.007284 0.195989 0.196149 0.196149 0.196149 0.196149 0.196149 0.196149 0.196149 0.196149 0.202172 0.215015 0.215015 0.215015 187 Term Regulation of actin filament polymerization (GO:0030833) Positive regulation of immune response (GO:0050778) Regulation of T cell proliferation (GO:0042129) Regulation of natural killer cell chemotaxis (GO:2000501) Oxygen transport (GO:0015671) Positive regulation of cAMP-mediated signaling (GO:0043950) Golgi disassembly (GO:0090166) Response to cytokine (GO:0034097) Positive regulation of cytoskeleton organization (GO:0051495) Positive regulation of CD4-positive, alpha-beta T cell differentiation (GO:0043372) Immunological synapse formation (GO:0001771) Positive regulation of plasma membrane bounded cell projection assembly (GO:0120034) Cellular response to lipopolysaccharide (GO:0071222) Negative regulation of apoptotic process (GO:0043066) Regulation of humoral immune response (GO:0002920) Negative regulation of leukocyte degranulation (GO:0043301) Cellular response to interleukin-9 (GO:0071355) Positive regulation of CD8-positive, alpha-beta T cell activation (GO:2001187) Interleukin-9-mediated signaling pathway (GO:0038113) Synapse pruning (GO:0098883) Regulation of inflammatory response (GO:0050727) Positive regulation of phosphate metabolic process (GO:0045937) Regulation of calcium ion transport (GO:0051924) Acetyl-CoA biosynthetic process (GO:0006085) Table B.4 (Continued) p-value 0.007284 0.008502 0.008825 0.008825 0.008825 0.008825 0.008825 0.009262 0.009825 0.010061 0.010061 q-value 0.215015 0.233564 0.233564 0.233564 0.233564 0.233564 0.233564 0.233564 0.233564 0.233564 0.233564 0.010149 0.010499 0.010499 0.010499 0.010499 0.010499 0.010499 0.011614 0.011843 0.012301 0.012301 0.012301 0.01249 0.233564 0.233564 0.233564 0.233564 0.233564 0.233564 0.233564 0.254675 0.255155 0.255155 0.255155 0.255155 0.255624 188 Term Regulation of podosome assembly (GO:0071801) ERBB2 signaling pathway (GO:0038128) Positive regulation of integrin activation (GO:0033625) Golgi inheritance (GO:0048313) Interleukin-2-mediated signaling pathway (GO:0038110) Actin filament network formation (GO:0051639) Cellular response to interleukin-2 (GO:0071352) Positive regulation of organelle organization (GO:0010638) Regulation of immune response (GO:0050776) B cell receptor signaling pathway (GO:0050853) Positive regulation of interleukin-12 production (GO:0032735) Antimicrobial humoral immune response mediated by antimicrobial peptide (GO:0061844) Lysine catabolic process (GO:0006554) Regulation of CD4-positive, alpha-beta T cell differentiation (GO:0043370) Negative regulation of leukocyte apoptotic process (GO:2000107) Positive regulation of actin filament depolymerization (GO:0030836) Gas transport (GO:0015669) Lysine metabolic process (GO:0006553) Inflammatory response (GO:0006954) Antigen processing and presentation of exogenous peptide antigen (GO:0002478) Purine ribonucleoside triphosphate metabolic process (GO:0009205) Positive regulation of CD4-positive, alpha-beta T cell activation (GO:2000516) Golgi ribbon formation (GO:0090161) Interferon-gamma-mediated signaling pathway (GO:0060333) Table B.4 (Continued) p-value 0.013779 0.013779 0.01403 0.014227 0.014227 0.014227 0.014227 0.014227 0.014227 0.015866 0.016274 0.016274 0.016274 0.016274 0.016274 0.017362 0.018439 0.018439 0.018439 0.018611 0.018929 0.018943 0.018943 0.01903 0.019687 q-value 0.256921 0.256921 0.256921 0.256921 0.256921 0.256921 0.256921 0.256921 0.256921 0.274514 0.274514 0.274514 0.274514 0.274514 0.274514 0.289682 0.292106 0.292106 0.292106 0.292106 0.292106 0.292106 0.292106 0.292106 0.29921 189 Term Neutrophil chemotaxis (GO:0030593) Defense response to Gram-positive bacterium (GO:0050830) Regulation of ERK1 and ERK2 cascade (GO:0070372) Positive regulation of T cell chemotaxis (GO:0010820) Positive regulation of macrophage chemotaxis (GO:0010759) Positive regulation of glycoprotein biosynthetic process (GO:0010560) Cell junction maintenance (GO:0034331) Antigen processing and presentation of endogenous peptide antigen (GO:0002483) 2-oxoglutarate metabolic process (GO:0006103) Granulocyte chemotaxis (GO:0071621) Regulation of integrin activation (GO:0033623) Regulation of lymphocyte activation (GO:0051249) Regulation of T cell chemotaxis (GO:0010819) GTP metabolic process (GO:0046039) Macrophage differentiation (GO:0030225) Positive regulation of lymphocyte proliferation (GO:0050671) Epiboly involved in wound healing (GO:0090505) Positive regulation of alpha-beta T cell proliferation (GO:0046641) Dendritic cell chemotaxis (GO:0002407) T cell receptor signaling pathway (GO:0050852) Cytosolic transport (GO:0016482) Neutrophil migration (GO:1990266) Positive regulation of phosphatidylinositol 3-kinase signaling (GO:0014068) Positive regulation of leukocyte mediated cytotoxicity (GO:0001912) MAPK cascade (GO:0000165) Table B.4 (Continued) p-value 0.020718 0.020718 0.021466 0.022265 0.022265 0.022745 0.022745 0.023107 0.023107 0.023107 0.023107 0.023107 0.024064 0.024064 0.024064 0.025164 0.025425 0.025604 0.025604 0.025604 0.025604 0.026875 0.028205 0.028205 0.028205 q-value 0.305784 0.305784 0.311134 0.311134 0.311134 0.311134 0.311134 0.311134 0.311134 0.311134 0.311134 0.311134 0.315717 0.315717 0.315717 0.319528 0.319528 0.319528 0.319528 0.319528 0.319528 0.332689 0.335616 0.335616 0.335616 190 Term Ruffle organization (GO:0031529) Positive regulation of regulated secretory pathway (GO:1903307) Negative regulation of T cell activation (GO:0050868) Protein homooligomerization (GO:0051260) Cellular response to interferon-gamma (GO:0071346) Erythrocyte differentiation (GO:0030218) Positive regulation of organelle assembly (GO:1902117) Dendritic cell migration (GO:0036336) Phosphatidylglycerol acyl-chain remodeling (GO:0036148) Epithelial structure maintenance (GO:0010669) Positive regulation of lymphocyte chemotaxis (GO:0140131) Maintenance of gastrointestinal epithelium (GO:0030277) Epidermal growth factor receptor signaling pathway (GO:0007173) Positive regulation of cell-cell adhesion (GO:0022409) Regulation of T cell activation (GO:0050863) Lymphocyte differentiation (GO:0030098) Regulation of interleukin-2 production (GO:0032663) Aspartate family amino acid catabolic process (GO:0009068) Positive regulation of actin cytoskeleton reorganization (GO:2000251) Positive regulation of macrophage migration (GO:1905523) Positive regulation of neutrophil chemotaxis (GO:0090023) Positive regulation of ERK1 and ERK2 cascade (GO:0070374) Negative regulation of lipid storage (GO:0010888) Negative regulation of osteoclast differentiation (GO:0045671) Regulation of granulocyte chemotaxis (GO:0071622) Table B.4 (Continued) p-value 0.028205 0.029621 0.029749 0.029953 0.030542 0.030907 0.030907 0.030907 0.032833 0.032833 0.033621 0.033707 0.033707 0.033707 0.033707 0.033707 0.034074 0.034435 0.034435 0.034435 0.035413 0.036601 0.036601 0.036601 0.037759 q-value 0.335616 0.348316 0.348316 0.348316 0.348838 0.348838 0.348838 0.348838 0.357147 0.357147 0.357147 0.357147 0.357147 0.357147 0.357147 0.357147 0.357147 0.357147 0.357147 0.357147 0.364826 0.369626 0.369626 0.369626 0.376362 191 Term Mesoderm development (GO:0007498) Defense response to bacterium (GO:0042742) Regulation of interleukin-12 production (GO:0032655) Positive regulation of intracellular signal transduction (GO:1902533) Negative regulation of programmed cell death (GO:0043069) Negative regulation of protein localization to cell periphery (GO:1904376) Negative regulation of protein localization to plasma membrane (GO:1903077) Hydrogen peroxide catabolic process (GO:0042744) Regulation of immune effector process (GO:0002697) Positive regulation of phagocytosis (GO:0050766) T cell activation (GO:0042110) Positive regulation of neutrophil migration (GO:1902624) Positive regulation of granulocyte chemotaxis (GO:0071624) Regulation of macrophage chemotaxis (GO:0010758) Cell projection assembly (GO:0030031) Negative regulation of B cell activation (GO:0050869) Negative regulation of cytokine production (GO:0001818) Cytoplasmic microtubule organization (GO:0031122) Positive regulation of leukocyte chemotaxis (GO:0002690) Inorganic anion transport (GO:0015698) Regulation of inflammatory response to antigenic stimulus (GO:0002861) Negative regulation of phosphoprotein phosphatase activity (GO:0032515) Regulation of mononuclear cell migration (GO:0071675) Triglyceride catabolic process (GO:0019433) Peptidyl-tyrosine modification (GO:0018212) Table B.4 (Continued) p-value 0.038315 0.039588 0.039588 0.039588 q-value 0.379439 0.384605 0.384605 0.384605 0.040926 0.040926 0.041241 0.042664 0.042664 0.042664 0.042664 0.042664 0.042726 0.04304 0.04304 0.043546 0.044878 0.045637 0.045825 0.045825 0.045825 0.045825 0.045825 0.045825 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 192 Term Enzyme linked receptor protein signaling pathway (GO:0007167) Positive regulation of lamellipodium assembly (GO:0010592) Wound healing, spreading of cells (GO:0044319) Protein kinase B signaling (GO:0043491) Antigen processing and presentation of exogenous peptide antigen via MHC class II (GO:0019886) Positive regulation of defense response (GO:0031349) Regulation of phagocytosis (GO:0050764) Positive regulation of T cell migration (GO:2000406) Regulation of neutrophil chemotaxis (GO:0090022) Phosphatidylglycerol metabolic process (GO:0046471) Negative regulation of antigen receptor-mediated signaling pathway (GO:0050858) Regulation of cAMP-mediated signaling (GO:0043949) Transmembrane receptor protein tyrosine kinase signaling pathway (GO:0007169) Response to reactive oxygen species (GO:0000302) Positive regulation of tyrosine phosphorylation of STAT protein (GO:0042531) Antigen processing and presentation of peptide antigen via MHC class II (GO:0002495) Cellular response to chemokine (GO:1990869) Positive regulation of cytosolic calcium ion concentration (GO:0007204) Positive regulation of interleukin-2 production (GO:0032743) Mononuclear cell differentiation (GO:1903131) Positive regulation of T cell mediated cytotoxicity (GO:0001916) Membrane depolarization during action potential (GO:0086010) Positive regulation of microtubule polymerization or depolymerization (GO:0031112) Positive regulation of filopodium assembly (GO:0051491) Table B.4 (Continued) Term Negative regulation of interleukin-1 beta production (GO:0032691) Positive regulation of ion transmembrane transporter activity (GO:0032414) O-glycan processing (GO:0016266) Entrainment of circadian clock by photoperiod (GO:0043153) Regulation of epidermal growth factor-activated receptor activity (GO:0007176) Positive regulation of cellular component movement (GO:0051272) p-value 0.045825 0.045825 0.046755 0.04907 0.04907 0.04907 q-value 0.385142 0.385142 0.385142 0.385142 0.385142 0.385142 193 194 B.3 References (1) Hovdenak, N.; Fajardo, L. F.; Hauer-Jensen, M. Acute Radiation Proctitis: A Sequential Clinicopathologic Study during Pelvic Radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 2000, 48 (4), 1111–1117. https://doi.org/10.1016/S03603016(00)00744-6. (2) Jullien, N.; Blirando, K.; Milliat, F.; Sabourin, J.-C.; Benderitter, M.; François, A. Up-Regulation of Endothelin Type A Receptor in Human and Rat Radiation Proctitis: Preclinical Therapeutic Approach w ith Endothelin Receptor Blockade. Int. J. Radiat. Oncol. 2009, 74 (2), 528–538. https://doi.org/10.1016/j.ijrobp.2008.12.086. (3) Dobin, A.; Davis, C. A.; Schlesinger, F.; Drenkow, J.; Zaleski, C.; Jha, S.; Batut, P.; Chaisson, M.; Gingeras, T. R. STAR: Ultrafast Universal RNA-Seq Aligner. Bioinformatics 2013, 29 (1), 15. https://doi.org/10.1093/BIOINFORMATICS/BTS635. (4) Martin, M. Cutadapt Removes Adapter Sequences from High-Throughput Sequencing Reads. EMBnet.journal 2011, 17 (1), 10–12. https://doi.org/10.14806/EJ.17.1.200. (5) Liao, Y.; Smyth, G. K.; Shi, W. featureCounts: An Efficient General Purpose Program for Assigning Sequence Reads to Genomic Features. Bioinformatics 2014, 30 (7), 923–930. https://doi.org/10.1093/BIOINFORMATICS/BTT656. (6) Ewels, P.; Magnusson, M.; Lundin, S.; Käller, M. MultiQC: Summarize Analysis Results for Multiple Tools and Samples in a Single Report. Bioinformatics 2016, 32 (19), 3047. https://doi.org/10.1093/BIOINFORMATICS/BTW354. (7) Love, M. I.; Huber, W.; Anders, S. Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2. Genome Biol. 2014, 15 (12), 1–21. https://doi.org/10.1186/S13059-014-0550-8. (8) Kramer, A.; Green, J.; Pollard, Jr., J.; Tugendreich, S. Causal Analysis Approaches in Ingenuity Pathway Analysis. Bioinformatics 2014, 30 (4), 523–530. https://doi.org/10.1093/BIOINFORMATICS/BTT703. (9) Xie, Z.; Bailey, A.; Kuleshov, M. V.; Clarke, D. J. B.; Evangelista, J. E.; Jenkins, S. L.; Lachmann, A.; Wojciechowicz, M. L.; Kropiwnicki, E.; Jagodnik, K. M.; et al. Gene Set Knowledge Discovery with Enrichr. Curr. Protoc. 2021, 1 (3), e90. https://doi.org/10.1002/CPZ1.90. (10) Kuleshov, M. V.; Jones, M. R.; Rouillard, A. D.; Fernandez, N. F.; Duan, Q.; Wang, Z.; Koplev, S.; Jenkins, S. L.; Jagodnik, K. M.; Lachmann, A.; et al. Enrichr: A Comprehensive Gene Set Enrichment Analysis Web Server 2016 Update. Nucleic Acids Res. 2016, 44 (W1), W90–W97. https://doi.org/10.1093/NAR/GKW377. 195 (11) Chen, E. Y.; Tan, C. M.; Kou, Y.; Duan, Q.; Wang, Z.; Meirelles, G. V.; Clark, N. R.; Ma’ayan, A. Enrichr: Interactive and Collaborative HTML5 Gene List Enrichment Analysis Tool. BMC Bioinformatics 2013, 14. https://doi.org/10.1186/1471-2105-14-128. APPENDIX C MATRIX-MEDIATED VIRAL GENE DELIVERY: A REVIEW C.1 Abstract Polymeric matrices inherently protect viral vectors from pre-existing immune conditions, limit dissemination to off target sites, and can sustain vector release. Advancing methodologies in development of particulate-based vehicles have led to improved encapsulation of viral vectors. Polymeric delivery systems have contributed to increasing cellular transduction, responsive release mechanisms, cellular infiltration, and cellular signaling. Synthetic polymers are easily customizable, and are capable of balancing matrix retention with cellular infiltration. Natural polymers contain inherent biorecognizable motifs adding therapeutic efficacy to the incorporated viral vector. Recombinant polymers use highly conserved motifs to carefully engineer matrices, allowing for precise design including elements of vector retention and responsive release mechanisms. Composite polymer systems provide opportunities to create matrices with unique properties. Carefully designed matrices can control spatiotemporal release patterns that synergize with approaches in regenerative medicine and antitumor therapies. Reprinted with permission. Steinhauff, D.; Ghandehari, H. Matrix Mediated Viral Gene Delivery: A Review. Bioconjugate Chem. 2019, 30 (2), 384–399. https://doi.org/10.1021/acs.bioconjchem.8b00853. Copyright 2019 American Chemical Society. 197 C.2 Introduction Delivery of biological therapeutics can result in altered cellular function and improved pathophysiology. Protein delivery often results in low and short temporal responses insufficient for effective treatment. Gene delivery can provide sustained therapeutic responses to correct existing abnormalities or provide new cellular functions in vivo through viral or non-viral methods. The total number of clinical trials for gene therapeutics is nearing 2600 worldwide, with 132 trials occurring in 2017 alone [1]. Applications include, but are not limited to, treatment of cancers, infectious, cardiovascular and neurological diseases with more expected to soon achieve clinical approval [1–5]. While non-viral approaches are relatively nonimmunogenic, they suffer from poor transfection efficiencies and short transgene expressions. Viral vectors exhibit high transduction efficiencies and potential for long-term transgene expression, but are plagued with patient immunity, dissemination to non-target sites, and toxicities. Viral delivery can be local or systemic and depending on the vector, can persist for the duration of vector presence or can be inserted into host genome leading to permanent effects. Preclinical studies have failed to predict the extent of innate, humoral, and T-cell immune responses towards viral vectors [6,7] leading to challenges in clinical translation. Intravenous administration can result in transient liver toxicity, off target effects, and inflammatory responses. Despite these challenges, viral vectors have still made it to the market with recent approvals for treatment of head and neck cancer, lipoprotein lipase deficiency, and melanoma [8–11]. Polymeric matrix-mediated (MM) delivery of viral vectors has been investigated since the 1990s for controlled release, improved localization at target sites, immune shielding, and reduction of other toxicities [12–17]. These benefits have led to the formulations entering clinical trials, showing safety and 198 efficacy for treatment of non-healing diabetic foot ulcers and vaccination against prostate cancer [18–21]. Applications of regenerative medicine and antitumor therapies, can further benefit from carefully designed MM delivery. Regenerative medicine can benefit from MM delivery through increased transduction of target cells and benefits of inherent properties exhibited by matrices. Antitumor therapies utilizing matrices can ensure an immune balance towards an antitumor response rather than an antiviral response [22]. Temporal release patterns are critical parameters affecting therapeutic outcome and can be tuned using polymeric matrices. This review will focus on the additional benefits provided by polymeric matrices for viral gene delivery and how synergy between matrices and gene therapies can achieve enhanced outcomes. C.3 Viral Vectors Various viral vectors have been investigated in clinical and preclinical stages, providing a range of transgene expression profiles, genetic payload, tropism, inflammatory potential, and physiochemical properties (Table C.1). Physiochemical properties of vectors such as size, shape and surface properties influence their interactions with polymeric matrices. In the choice of viral vector to be encapsulated in a biomaterial matrix, factors such as vector geometry (spherical, icosahedral, long vs. short axial ratios), size, enveloped vs. non-enveloped, major and minor capsid proteins, along C.4 Matrix-Mediated Delivery of Viral Gene Vectors In the modern sense, the use of biomaterials for delivery of therapeutic agents began in the 1960s evolving from macroscopic drug delivery systems towards Table C.1: Various viral vectors used in gene therapy and respective physiochemical properties with the desired duration of transgene expression need to be considered. Virus Tropism Retargeting Dividing Cells Transgene expression Long Payload Enveloped Geometry Dimensions 8 kb Yes Spherical/Ovoid23 Dividing/Nondividing Cells Dividing/Nondividing Cells Dividing/Nondividing Cells Nondividing cells Short 8 kb No Icosahedral Long 5 kb27 No Icosahedral Diameter: 80120 nm24 Diameter: 70100 nm25 Diameter: 20 nm Short Yes Icosahedral Long 30-40 kb 8 kb Yes Spherical/Ovoid 23 Vaccinia virus Dividing Cells Short 25 kb Yes Ellipsoidal/barrel/brick29 Baculovirus Dividing/Nondividing Cells Long/Short no known limit Yes Rod-shaped35–37 Retrovectors Adenovectors Adeno-associated vectors Herpes Simplex Vectors Lentivectors Diameter: 110200 nm Diameter: 166 nm28 Surface Charge Major Capsid Proteins Gag, Env Negative Hexon, penton, fibre26 Negative Negative Glycoprotein, Large protein, Phosphoprotein, Matrix Protein, Nucleoprotein 320-380 x 260340 x240-290 nm30–34 30-60 nm x 250300 nm35–37 199 200 nanoscopic designs capable of delivering fragile cargo such as proteins and genetic vectors [38]. Spatiotemporal release of therapeutics can be achieved using synthetic, natural, and recombinant polymers. Synthetic polymers offer a wide range of properties and can be tuned for functionality and degradability, depending on the synthetic methodology and the resulting monomer sequence, molecular weight and polydispersity. Naturally derived polymer properties will depend on extraction method and source. These polymers must be carefully processed to remove immunogenic components and may contain inherent recognition motifs for cells or enzymes. Recombinant polymers are designed genetically and are created utilizing cellular machinery. This method allows precise control over polymer structure, motif incorporation, and molecular weight [39– 42]. These three polymer types are reviewed elsewhere [43–45]. This review will encompass polymers that have been specifically used in matrixmediated viral gene delivery through the formation of gel and particulate structures. Gel systems, in this context are considered to be hydrogels and scaffolds and are largely used for regenerative medicine and localized delivery. Hydrogels are three-dimensional polymeric matrices, which hydrate upon submersion in aqueous solvents swelling to water contents of 70-99% [46]. This hydrated state provides delicate packaging of fragile cargo such as viral vectors. Scaffolds support cellular differentiation and proliferation. Some matrices can be hydrogels and scaffolds depending on hydration and ability to regenerate tissues. The particulate systems covered in this review consist of microspheres and microgels, which provide improved viral stability in vivo and sustained release. They largely rely on the physical entanglement of viral vectors within the matrix and are advantageous for systemic treatments. Several polymer structures discussed in this review can be found in Figure C.1. 201 Figure C.1: Several polymer systems discussed in this review. Fibrin schematic adapted from McDowall 2006 [196]. 202 C.5 Particulate Systems Particulate systems can be administered locally or systemically presenting an advantageous option to treat widespread disease such as metastatic cancers. Microencapsulation can result in sustained release of viral vectors, increasing stability, and shielding from neutralizing antibodies [12,15,47–50]. Past methodologies for particulate synthesis relied upon harsh conditions for encapsulation, and subsequent loss of virus activity. Recently new methods have been developed making microencapsulation of viral vectors more attractive. A list of particulate systems discussed in this article can be found in Table C.2. Poly(lactic-co-glycolic acid) (PLGA) copolymers (Figure C.1) are widely used biocompatible systems with a wide variety of molecular weights and copolymer ratios available commercially. These polymers degrade upon hydrolysis. Methods to synthesize PLGA microspheres include the use of organic solvents and their degradation products provide an acidic environment which may deactivate viral vectors resulting in needs for gentler encapsulation methods [51]. Microencapsulation of PEGylated adenoviruses (ADs) modified with poly(ethylene glycol) (PEG) (Figure C.1) resulted in increased physical stability compared to naked vectors in a double emulsion technique. PEGylated ADs exhibited a decreased burst release in comparison to ADs, before following a similar release profile due to PEG-PLGA entanglement resulting in sustained release of 10 days in vitro [48]. PLGA microparticles encapsulating ADs exhibited increased infectivity when synthesized using total recirculation one-machine system apparatus (TROMS) compared to traditional emulsion preparation techniques, ameliorating the need for aggressive homogenization that may deactivate viral vectors. Intramuscular administration of PLGA formulations prepared by TROMS in Table C.2: Particulate matrix systems used to encapsulate viral vectors. Parent Material Poly(lactic-co-glycolic acid) Variations in Structure Fabrication Method Structure Shape Particle Size (µm) PEGylated AD Double Emulsion Microspheres Spherical Double Emulsion Microspheres Spherical TROMS Microspheres Double Emulsion Modified double emulsion Microspheres Spherical 1.965-3.320 AD-TIMP Microspheres Spherical 1.250- 3.320 Microfluidics Microgel Spherical 0.126 (1%) 0.127 (2%) 2.9 (2%) 4.8 (1%) Microfluidics Microgel Spherical Microfluidics On-chip polymer blending On-chip polymer blending On-chip polymer blending On-chip polymer blending Microgel Spherical 0.152 (1%) 0.158 (2%) 0.104 (1%) 0.0985 (2%) 9.9 (2%) 18 (1%) 2.1 (2% 3.4 (1%) Microgel Spherical Microgel Poly-DL-lactide Poly-DL-lactide-poly (ethylene glycol) Alginate (Low MW) Alginate (AF350/AF555) Calcium carbonate and glucono delta-lactone (CaCO3-GDL) (wt %) Ethylenediaminetetraacetic acid chelated calcium (CaEDTA-AcOH) (wt%) Calcium chloride (CaCl2) (wt%) 1% Microgel AF350 1.5% Microgel (50/50 blend) 2% Microgel AF555 2% Microgel nondegradable 2% Microgel (50/50 degradable/nondegradable blend) 2% Microgel degradable On-chip polymer blending On-chip polymer blending Mesh Size (nm) Cargo Application Refs 9.3 AD-GFP HeLa Cells [48] 9.43 AD-GFP ADβgalactosidase [48] AD-TIMP HeLa Cells Intramuscular Administration Hepatocellular Carcinoma Hepatocellular Carcinoma LV-GFP HEK-293T [55] Discontinued due to inactivity [55] LV-GFP HEK-293T [55] 35 LV-VEGF CAM Assay [56] Spherical 24 LV-VEGF CAM Assay [56] Microgel Spherical 22 LV-VEGF CAM Assay [56] Microgel Spherical 82 LV-VEGF CAM Assay [56] Microgel Spherical 33 LV-VEGF CAM Assay [56] Microgel Spherical 22 LV-VEGF CAM Assay [56] 8.4-10.2 [50] [52] [54] 203 204 immunocompetent mice showed sustained expression of β-galactosidase for at least 7 weeks. This sustained expression was attributed to immune shielding of encapsulated vectors and increased stability within microparticles [50]. Encapsulation of ADs encoding for tissue inhibitors of metalloproteinase into poly-DL-lactide-poly(ethylene glycol) microspheres led to encapsulation efficiencies of 60%, posing as plausible candidates for treatment of hepatocellular carcinoma due to large blood filtration of the liver and ability to minimize tumor cell migration and invasion [52,53]. The transduction efficiency of HepG2 cells was enhanced by 90%, however this may be due to the replication-competent nature of the vectors used [54]. These microencapsulation methodologies provide gentler conditions for viral vectors resulting in a greater infectivity compared to previous techniques. TROMS reduces the use of high forces that deactivate vectors. The inclusion of PEG domains in multiple synthetic techniques, whether on the viral capsids or the polymeric vehicle, aids in stabilization of the vector capsids leading to improved activity of vectors. Further developments in microencapsulation technologies have resulted from droplet microfluidic technologies with the ability to form micrometer sized hydrogels with discreet volumes [57]. These have been used for lentivirus (LV) encapsulation within alginate microgels (Table C.2) [55]. Alginate is extracted from brown algae and consists of L-guluronate (G) and D-mannonate (M) to form block copolymers [58]. The G-blocks can be crosslinked with divalent cations to form intermolecular crosslinking, with crosslinking mechanics relying on molecular weight, G-block length, and M/G ratio [59]. Alginates do not support cellular infiltration and can be tuned for precise degradation by mismatched cross-linking junction size, alterations in molecular weight 205 (MW), and crosslinking with hydrolytically susceptible crosslinkers [60–64]. These polymers have been extensively investigated in tissue engineering and drug delivery [58]. The use of calcium-ethylenediaminetetraacetic acid (EDTA) as a crosslinker resulted in drastically decreased LV activity due to a drop in pH upon calcium-EDTA disassociation and subsequent LV vector instability [55,65]. Microgels crosslinked with calcium chloride and calcium carbonate/glucono delta-lactone did not result in reduced LV activity and achieved 60% cumulative release by day 10, despite the drastically smaller mesh sizes compared to LV size (Tables C.1, C.2). This is possibly attributed to an increased matrix surface area and an unideal mesh network consisting of closed polymer loops, dangling ends, and slipping chain entanglements [55,66]. On-chip polymer blending, a novel microfluidic method to create composite microgels, was employed to optimize material properties and therapeutic potential of encapsulated LV encoding vascular endothelial growth factor (VEGF). Blending provided variation in mechanical properties, degradability and controllable release kinetics sustained over 10 days. The LV-VEGF microgels produced a marked increase in proangiogenic response compared to controls in a chick chorioallantoic membrane (CAM) assay, resulting from controlled delivery of vectors and limited dissemination to somatic tissues often observed by naked vectors [56,67]. This methodological synthesis provides capability to create composite microgel systems in a controlled manner with tunable functionality. Microfluidic encapsulation results in an alternative methodology for successful incorporation of vectors, with the ability to discretely control volumes and polymer composition for carefully tuned properties. The use of particulate systems for encapsulation of viral vectors has long lagged 206 behind the use of larger structures. While these systems were the first developed, the methodologies of encapsulation resulted in poor yields and/or poor activity of vectors. The recent developments have resulted in methods that can maintain vector activity and providing a level of control that has been long obtainable with macroscopic systems. C.6 Gel Structures Larger gel structures have long been studied for the encapsulation and controlled release of viral vectors. These systems are largely composed of polymers consisting of synthetic, natural, and recombinant polymers. These structures have been used for local delivery of vectors for regenerative medicine and localized antitumor therapies. Each polymer system has distinct advantages depending on chain structure and properties. Matrices confer additional benefits including but not limited to, localized release, sustained release, cellular infiltration, and mechanical support. In many instances, matrices will produce their own therapeutic response that is similar or better than matrices with viral vectors. In the clinic formulated collagen gels with AD encoding platelet derived growth factor beta (PDGFβ) performed similar to collagen gels alone for treatment of non-healing diabetic foot ulcers [19]. C.6.1 Synthetic Polymers Some of the first polymers used for matrix-mediated delivery are amphiphilic Pluronic (poloxamer) and Tetronic (poloxamine) copolymers composed of PEG and poly(propylene oxide) (PPO) blocks (PEG-PPO-PEG) (Table C.3). In response to increasing temperatures PPO blocks dehydrate and polymers form micelles at low Table C.3: Polymeric matrices used in matrix-mediated viral delivery. Polymer System 3D Structures Modifications Modification Effects Applications References Cellular infiltration; Increased vector retention; Programming pH responsivity Vectors Encapsulated LV, AD, rAAV, bacteriophage Poly(ethylene glycol) Hydrogel Poly(ethylene glycol1-(3aminopropyl)imidazol e-DL-aspartic acid) Pluronic Gel Macropore generation; Affinity peptides; Chitosan/Heparin nanoparticles; Poly histidine Polyethyleneimine (PEI800) Angiogenesis; Immune shielding; pH tuning [81– 83,88,89,16 0] Programming for pH responsivity AAV Tumor environment [161] Gel α-cyclodextran blend; Alginate composite; Combinatorial delivery; All-in-one vaccine Mechanical properties; Phenotype maintenance; Increased nucleus localization AD, LV, rAAV [13,14,69,7 2,73,76,80,1 62,163] Gel α-cyclodextran blend rAAV Poly(lactic-co-glycolic acid) Microsphere; Scaffold Fibrin Hydrogel; Scaffold PEGylated vector; PolyL-lysine; Hydroxyapatite; Chitosan; Hyaluronan Polyurethane discs; Hydroxyapatite Mechanical properties; Phenotype maintenance Increased encapsulation efficiency; Vector Retention; Increased Transduction Cartilage regeneration; Central nervous system; Vascular injury; Cardiovascular disease Cartilage regeneration Bone regeneration, infections Tetronic Alginate Hydrogel; Microgel; Microspheres Hydrogel; Fiber; Nanogel Mechanical properties LV, rAAV, AD MMP/Inflammation responsive AD, GLV1h68, oAD Silk-Elastinlike Protein Polymers Polymer blending; Crosslinking agent; Poloxamer composite MMP-degradable sites AD, rAAV, AAV, bacteriophage AAV, LV, AD Wound healing; Glue; Mucosal surfaces Angiogenesis; Skeletal muscle regeneration Head and neck cancer; [162] [15,48,50,8 5,90,164– 166] [102,103,10 8,109,111– 113,167,168 ] [47,55,56,9 8–100,169] 207 [17,69,170, 142,143,145 ,147– 149,151,157 ] Table C.3 (Continued) Polymer System 3D Structures Modifications Modification Effects Poly-Lactide Scaffold; Microspheres Ice Porogens One-step fabrication; Mechanical properties Poly-DL-lactidepoly(ethylene glycol) Polymer System Microspheres 3D Structures Modifications Modification Effects Gelatin Stereolithographic printing Matrix shaping Polyester urethane urea Polyester ether urethane urea Collagen Hydrogel; Scaffold Solid fibers; Core shell fibers Solid fibers; Core shell fibers Sponge; Gel Chitosan/Collagen Scaffold Elastinlike polypeptides/Poly (ϵcaprolactone) Poly (ϵ-caprolactone) Fibers Core Shell Fibers Vectors Encapsulated AAV, AD Applications References Bone induction, Hepatocellular carcinoma Hepatocellular carcinoma Applications [52,171] [126,172] AAV Oncolytic therapy, Bone induction Heart infarction AAV Heart infarction [95] LVs, AD Spinal cord injury; Non-healing diabetic foot ulcers; Neurons; Prostate cancer vaccine; Bone regeneration Dental bone regeneration; Periodontal tissue engineering Tissue engineering [18,19,178, 20,21,102,1 73–177] Regenerative medicine [97] rAAV Combinatorial delivery All in one tumor vaccine Vectors Encapsulated oAD, LV AD Composite Blending Mechanical Properties AAV AD [54] References [95] [128–131] [179] 208 209 concentrations and gels of intertangled micelles at higher concentrations (Figure C.2) [68]. These structures have short residence times in vivo, clearing due to the highly viscous flow exhibited by these polymer types, leading to release of viral vectors. We have observed the dissolution of intratumorally injected Poloxamer 407 within one week in vivo [69]. Tetronics, analogs of Pluronics, contain four pluronic chains branching from a charged EDTA core. The amphiphilic nature of these polymers results in increased transduction efficiencies and their wide use in matrix-mediated delivery of viral vectors (Figure C.2) [13,14,70–75]. Recently Pluronics have been shown to facilitate delivery of LVs to the central nervous system without toxicities or loss of infectivity [74,76]. LVs encoding Lingo-1 short hairpin RNA have been incorporated into these gel systems to promote the functional recovery of spinal cord injuries [76]. Lingo-1 is a negative regulator of axonal sprouting, myelination, and silencing may facilitate favorable remodeling upon injury [77–79]. Formulation within gels required fewer vectors to achieve a therapeutic effect compared to naked vectors in vivo. Increased biomarkers for neurogenesis were observed in gel formulations along with increased sprouting of axons compared to non-gel formulations [76]. Formulation of Pluronic synperonics with LVs resulted in increased transduction compared to polybrene assisted transduction [75]. Inclusion of small compounds within Pluronic gels has further increased transduction efficiencies, possibly due to assistance with nuclear localization of genetic cargo [80]. The enhanced transduction exhibited by these materials has led to the development of improved composite gel structures which will be discussed later. PEG is widely used in biomedical applications and is regularly used to create hydrogels. PEG hydrogels are easily customizable and can serve as a model for studying 210 Figure C.2: Poloxamers form micelles at low concentrations and gels of intertangled micelles at higher concentrations. Interactions with cell membranes can cause changes in microviscosities and membrane fluidization resulting in higher rates of transduction. 211 factors that influence matrix-mediated delivery of viral vectors. Vector retention has been investigated through incorporation of affinity peptides or nanoparticles [81–83]. Phage display identified affinity interactions with the pseudotyped vesicular stomatitis virus glycoprotein G (VSV-G) LVs, resulting in a 20-fold increase in expression [83,84]. Phages with histidine at positions 4-7 showed preference to LVs, with the addition of lysine in other prominent phages [83]. The viral retention exhibited by these phages suggest the presence of hydrogen bonding and/or electrostatic interactions with the LVs. Matrix retention has also been achieved using heparin-chitosan nanoparticles [82]. Hydrogels containing negatively charge heparin/chitosan nanoparticles were able to retain an increased number of LVs compared to non-functionalized hydrogels, supported by other matrices using heparin and chitosan modifications [85]. This interaction with heparin-chitosan and phage displayed peptides is likely dependent on the charge density presented to the viral capsid [82]. Viral retention may be dependent on hydrogen bonding as histidine, heparin, and chitosan provide opportunity for dipole-dipole interactions. Despite the increased retention using heparin-chitosan nanoparticles, delivery of LVVEGF from PEG hydrogels did not result in observable blood vessels after 8 weeks, possibly due to limited areas for cell ingrowth and interactions with vectors presented in dense matrices [82]. The larger mesh size of macroporous PEG hydrogels (PEGmp) led to less physical entrapment of LVs, greater opportunities for transduction, cell attachment, migration, and proliferation [86,87]. In vivo PEGmp hydrogels containing LV-VEGF exhibited angiogenesis after just 4 weeks with infiltration of endothelial cells through the interconnected macropores generated using gelatin porogens. PEGmps with LVs were able to support an increased number of endothelial cells compared to other 212 formulations. Nanoporous PEG hydrogels entrapped viral vectors with similar expression levels to PEGmp, but with shorter expression periods resulting from less cellular infiltration and higher viral entrapment [88]. Generally, vector retention is dependent on matrix density. Decreases in matrix density can result in cell infiltration and desirable remodeling. It may also lead to rapid vector release and insufficient temporal transgene expression for tissue regeneration. Therefore, matrices achieving appropriate vector retention and properties for tissue regeneration are crucial for effective therapeutic outcomes. PEG and PLGA matrices have been explored for the delivery of bacteriophages. PEG-maleimide hydrogels crosslinked with collagen mimetic peptides released a mixture of bacteriophages within 24 hours with responsive release achieved in the presence of collagenase. In a mouse radial segment defect, phage treatments resulted in significantly lower amount of recoverable bacteria compared to controls [89]. PLGA matrices manufactured via melt-processing were used to encapsulate Bacteriophage QB nanoparticles for vaccination. Matrices loaded with 10 wt % QB generated similar levels of anti-QB compared to a 3 injection immunization schedule in vivo, illustrating the slow release potential of this system and its ability to attenuate needs for repeated dosing [90]. The use of phages in therapeutics provides another mechanism to treat infectious disease and may prove useful especially due to rising antibiotic resistance. Electrospinning utilizes high voltage gradients to shear polymer solution into nano-scale fibers capable of providing topographical cues for cells [91–93]. Coaxial spinning of fibers can overcome limitations of single stream electrospinning protecting bioactive agents from harsh conditions compared to conventional single stream spinning 213 [94]. Electrospinning of polyester urethane urea (PEUU) and polyester ether urethane urea (PEEUU) into solid and core-sheath fibers influenced release of adeno-associated viruses (AAVs) with sustained release of 2 months exhibited by core-sheath matrices. Solid fibers exhibited minimal release, likely due to inactivity resulting from harsh synthesis conditions. The incorporation of PEG into the PEUU backbone led to variations in release with higher PEG contents leading to more rapid vector diffusion through swollen scaffolds. PEUU core-sheath scaffolds exhibited cellular infiltration with pores of 40 μm compared to little infiltration in solid fibers with mean pore sizes of 3 μm. In vivo PEEUU core-sheath patches with and without AAV encoding green fluorescent protein (GFP) provided functional benefit in reducing the effects of ischemic cardiomyopathy compared to naked AAVs illustrating the benefit provided by the PEEUU matrix [95]. Incorporation of low MW PEG in coaxial spinning of poly(αcaprolactone) results in porogen like affects [96]. Increasing concentrations of PEG resulted in increasing rates of AD release with transgene expression of HEK 293 in vitro [97]. These methods provide tunable and bendable matrices containing topographical cues for cells with sustained release of vectors. This customization allows tuning of matrix properties and resulting changes in therapeutic outcome. Synthetic polymer systems can be synthesized for customized matrix properties. Properties can be balanced to enhance temporal vector retention and cellular responses in vivo. The ease of programming makes these systems attractive for studying fundamental matrix properties. However, synthetic systems lack cues inherently found in other polymeric materials that can result in favorable remodeling and cellular proliferation. 214 C.6.2 Natural Polymers In some of the first MM delivery of vectors, alginate matrices were directly shown to limit systemic dissemination in mice upon intratumoral injections compared to free vectors, attributed to sealing of damaged blood vessels upon administration [98]. LVs delivered from alginate hydrogels resulted in noticeable luciferase expression in murine hindlimb muscles for 11 weeks compared to naked vectors upon administration into hind limbs. Naked vectors induced a maximal transgene expression after 14 days followed by a gradual decrease in the following two weeks. Peak expression of alginate loaded hydrogels was observed after 21 days and sustained for the following two months [99]. The sustained expression likely results from the shielding of immune response and vector stabilization. The in vivo transgene expression follows patterns exhibited by the hydrogel degradation rates in vitro, which was tuned using binary MW polymers and partial oxidation of chains [99]. Tuning alginate degradation allows customization of temporal release patterns of vectors. Furthermore, encapsulation of oncolytic ADs (oADs) in alginate hydrogels has been shown to sustain bioactivity and vector release for an extended period. Naked vectors lost 93% ability to express GFP over 1 week compared to minimal losses by alginate formulations. In vivo oADs-alginate formulations had 1.9- (U343) and 2.4-fold (C33A) increased antitumor activity with broader tumor area expression and higher densities of expression compared to naked oADs, likely achieved by preserved bioactivity, sustained release, and viral propagation [100]. Additionally, matrix-mediated immune shielding likely led to a higher degree of antitumor immunity and less innate/adaptive cell priming towards antiviral responses. Fibrin is a naturally occurring network involved in coagulation with intrinsic 215 wound healing properties [101]. Maintenance of hemostasis and hemocompatibility along with recognition motifs for endothelial cells makes it a popular choice for regenerative medicine and encapsulation of viral vectors [102,103]. Fibrin matrices are composed of protofibers making up larger fibers in a hexagonal close packed crystal like structure resulting in viscous, viscoelastic matrices with water volumes of 70-90% of fiber volume (Figure C.1) [104–107]. The use of fibrin scaffolds has illustrated therapeutic effects, with and without ADs in murine increased wound closure models [108]. Release rates of ADs and LVs show strong dependence on concentration of scaffolds with sustained release greater than one week [103,108,109]. This dependence of fibrin concentration on release and transduction of both LVs and ADs likely arises from entrapment of vectors within the matrix, either entangled within protofibers, fibers, or between fibers. Fibrin scaffolds polymerized on polyurethane discs showed maximal transduction at higher fibrin concentrations in vivo. The retention of vectors within the matrix increased target cellular interactions with entrapped vectors improving wound repair cell transduction. Maximal transduction within the matrix occurred at day 10 [110]. In vivo, fibrin matrices were able to retain ADs at the wound site up to 7 days, with minimal transgene expression at day 14, transducing infiltrating fibroblasts, endothelial cells, and inflammatory cells [108]. Fibrinolysis occurs with the activation of plasmin from plasminogen. This conversion can occur by soluble or bound cell membrane enzymes expressed by wound cells (Figure C.3). As fibrin matrices degrade entrapped vectors are released and increase cell-vector interactions. Due to advantageous rheological properties and inherent wound healing abilities fibrin glue encapsulating viral vectors have been investigated for multiple applications [111–113]. Fibrin glues provided external support 216 Figure C.3: Enzymatic matrix degradation can increase target cell transduction. The expression of enzymes or conversion of enzymes by target cells can lead to their infiltration into matrices while releasing entrapped viral vectors. Wound cells activate plasmin by membrane bound proteins, such as urokinase plasminogen activator receptor or tissue plasminogen activator resulting in the conversion of proenzymes to active forms in proximity of matrices. 217 for vein grafts inhibiting intimal hyperplasia. Glues have been shown to prevent overdistension and preserve distensibility in the high pressure range of the human saphenous vein [114]. Sustained gene expression in perivenous applications has been achieved out to 14 days in vivo solving issues with vector dissemination and providing opportunities to ameliorate intimal hyperplasia [112,115]. Further studies have illustrated the ability of fibrin glues to deliver AAVs to esophageal epithelium and promising in vitro results for cartilage engineering [111,113]. The inherent cellular recognition motifs and mechanical properties exhibited by fibrin gels makes them an attractive matrix for increasing target cellular transduction, applications requiring matrix elasticity, and wound healing. Tuning of concentration and degradation can lead to variations in release patterns and increased transduction of target cells which is best evaluated in vivo. Collagen is a triple helix polypeptide chain (G-P-X) with left hand helices twisted together producing a right handed coil, with quaternary structure stabilized by Van der Waals, intermolecular hydrogen bonding, and covalent bonding (Figure C.1) [116–118]. Cellular interactions with collagen provide essential signals for migration, differentiation, survival, proliferation, and anchorage [119]. Collagen naturally provides support for skin, tendons, cartilage, blood vessels, and ligaments making it a viable candidate for tissue engineering applications [120,121]. Gelatin is a denatured form of collagen with amorphous regions of interconnected coiled-coil chains and spatially-ordered microcrystallites [122]. The biocompatibility and osteoconductivity of gelatin has led to its use in bone regeneration [123–125]. Intramuscular injections of ADs encapsulated in collagen gels resulted in bone formation after 4 weeks. This long period before formation was attributed to cellular migration and infection, which may have taken longer periods 218 of time than that of release [102]. Stereolithographic fabrication, a rapid 3D printing technology, of a nanoporous gelatin matrix was used to encapsulate human mesenchymal stem cells (hMSCs) and LV encoding bone morphogenetic protein 2 (BMP2). This approach aimed to reduce unregulated bone formation by entrapping vectors and cells resulting in observable BMP2 production within 5 days and sustained production for 3 months in vitro. In vivo bone formation was slow with minimal vascularization impairing the long-term potential of this system. The inclusion of angiogenic factors may be an additional strategy to enhance regeneration [126]. This rapid 3D printing technology may lead to the formation of anatomically shaped matrices with mechanical properties similar to native tissues and limitation of unregulated bone formation. Collagen blends with chitosan create hardened scaffolds that have been extensively used for bone regeneration in dental engineering [127–131]. These scaffolds exhibit pore structures that appear to provide good environments for growth of human periodontal ligament cells (HPLCs). Scaffolds releasing AD encoding bone morphogenetic protein 7 (BMP7) exhibited highest activity of alkaline phosphatase and expression of osteopontin and bone sialoprotein when cultured with HPLCs. Implantation into defects led to highest bone formation in AD-BMP7 scaffolds at 4 and 8 weeks [131]. When AD-PDGFβ were evaluated in these scaffolds, there was enhanced proliferation of HPLCs [130]. Combination of AD-BMP2 and VEGF proteins within these scaffolds resulted in a rapid release of protein and sustained transgene expression. This formulation performed superior to scaffolds containing both AD-BMP2 and AD-VEGF in canine defects around the implants [128]. The rapid release of VEGF and long-term expression of BMP2 follows regenerative patterns indicated by the occurrence of angiogenesis 219 followed by osteogenesis in bone fracture models [132–134]. The combination of chitosan and collagen produces matrices with mechanical properties favorable for bone regeneration. Silk fibroin is a naturally derived polymer with high mechanical strength and has been frequently used in tissue engineering for bone regeneration [135–138]. Scaffolds of this polymer have been used to incorporate AD-BMP7 and evaluated in calvarial defects [139,140]. Scaffolds were capable of transfecting bone marrow derived stem cells for 14 days with production of BMP7 up to 21 days in vitro. The highest production of BMP7 occurred on day 7, and is in accordance with optimal timing of BMP7 administration for regeneration of bone within defects [139,141]. In SCID mice, scaffolds containing ADBMP7 enhanced bone formation compared to negative controls confirmed by histological staining of markers for new bone formation. The additional incorporation of bone marrow derived stem cells did not enhance in vivo efficacy suggesting AD release to surrounding environment [139]. Inflammatory responses to scaffolds containing vectors were evaluated in BALB/C mice. After 1-week post-implantation there was a 2.5-fold increase of interleukin-2 compared to controls, indicating T cell receptor stimulation. TNF-α expression peaked after 1 week in vector matrices, an indication of T cell activation. Both interleukin-2 and TNF-α levels returned to normal after 4 and 2 weeks respectively. Interleukin-6 levels did not have noticeable change throughout the study [140]. These matrices are promising due to their high mechanical strength, natural breakdown, and clearance, but suffer from immunogenicity in vivo. The motifs in natural polymers can provide additional therapeutic benefits to MM viral delivery systems. Motifs for cell infiltration and enzymatic breakdown can enhance 220 target cellular transduction. Furthermore, motifs from polymers such as silk fibroin can be utilized to engineer matrices with enhanced mechanical properties. These motifs can be incorporated in carefully designed recombinant polymer systems for exact programming of polymer properties. C.6.3 Recombinant Polymers In our laboratory we have extensively studied silk-elastinlike recombinant protein polymers (SELPs) and their ability to control release of genetic materials [17,69,150– 152,142–149]. SELPs are composed of alternating motifs of silk (GAGAGS) and elastin (GVGVP) [153]. Using recombinant control, we have synthesized several variations of SELPs (Figure C.4A). The elastin units result in a thermoresponsive polymer capable of forming hydrogels upon an increase in temperature and the resulting crosslinking by hydrogen bonding of silk motifs to form beta sheets (Figure C.4B, C.4C) [150,154]. The primary protein structure and hydrogel concentration results in varying properties (Figure C.4D). Data from our lab indicates a dependence of AD release on polymer structure and hydrogel concentration. Polymers with longer elastin blocks or lower silk: elastin ratios result in increased and more complete release, due to differences in pore size and swelling ratios between the polymers (Figure C.4D). SELP matrices ameliorate immune response of viral vectors, decrease hepatotoxicity, and increase safety of gene-directed enzyme-prodrug therapy (GDEPT) compared to naked ADs. Localized transduction is also greater via SELP delivery, resulting in a 55-fold increase in tumor/liver transduction ratio. These findings have been summarized in a previous review [155]. SELP hydrogels are robust and capable of residing in vivo over 12 weeks, allowing release through a 221 Figure C.4: Silk-elastinlike protein polymers for gene delivery.: A). Primary sequences of SELPs. MMP responsive sites are inserted once into SELP-815K monomer at indicated positions to produce three different responsive polymers. B). Illustration of SELP showing the crosslinking via beta sheet formation and pores created by elastin motifs. C). Scanning electron microscopy image of SELP-815K 12 wt. % hydrogel. D). Physical characteristics of SELPs with dependence on primary polymer structure. Adapted from Gustafson 2010 [155]. E). Sequences of SELPs with MMP degradable site [158]. F). Physical characteristics of MMP responsive SELPs [158]. 222 stable matrix system. Other polymers such as Poloxamer 407 undergo dissolution mediated release resulting in shorter release periods [69,156]. The ability of SELPs to sustain release over 22 days results from carefully tuned physiochemical properties. Incorporation of lysine in SELP constructs (815K, 415K, 47K) may increase polymerAD electrostatic interactions with Hexon, a major AD capsid protein (Table C.1), and therefore vector retention [148,157]. Prolonged matrix residence times can lead to the formation of a fibrous capsules and has prompted our lab to develop matrixmetalloprotease (MMP) responsive SELPs with varying locations of enzymatic degradable sites and properties (Figure C.4E,F) [69,144,158]. MMPs are proteolytic enzymes that are naturally upregulated in sites of inflammation and solid cancers [159]. By incorporating an MMP responsive sequence (GPQGIFGQ) into different locations of the SELP-815K backbone we can control degradation rates in inflammatory environments (Figure C.4E,F) [158]. This degradable system was evaluated in vivo using a GDEPT approach for head and neck cancer, showing increased degradation compared to SELP-815K, and increased mice survival from 29% to 100% over 50 days compared to control groups [142]. The recombinant programing of degradation sequences within these polymers has given them a responsive release mechanism which results in specific payload unloading at inflamed sites and the ability to be naturally cleared without the formation of a fibrous capsule. Others have also illustrated the benefits of localized oADs MM delivery via SELPs. Encapsulation of oADs in SELPs results in enhanced bioactivity, with reports up to 1000-fold greater infectivity than naked ADs after incubation for one week at 37 ⁰C, explained by reported preservation of bioactivity exhibited by SELPs [17,143,170]. This 223 preservation and sustained delivery of oncolytic adenoviral vectors encoding short hairpin RNA from SELP-47K matrices may be responsible for a 1.5-fold increase in antitumor efficacy, wider areas of tumor apoptosis, and higher tumor transduction compared to free vectors [143]. Replication competent oncolytic vaccinia viruses (GLV1h68) have also been encapsulated within SELP-47K matrices for treatment of thyroid carcinoma. Topical intraoperative administration of SELP-GLV-168h resulted in decreased tumor volume compared to naked vector due to increased and stable interactions with target cells rather than transient interactions exhibited by PBS formulations [170]. When mechanically disrupted, SELP gels broke into particles and were capable of the highest levels of luciferase expression and tumor responses, attributed to increased matrix surface area [170]. The use of SELPs in antitumor therapies has been well established. SELP matrices have shown promise in tissue engineering applications but have not yet been utilized for regenerative approaches in combination with viral gene therapy [180,181]. The specific incorporation of motifs designed for cellular infiltration and migration could further enhance these regenerative approaches in conjunction with viral vectors. C.6.4 Composite Polymer Systems Composite polymer systems are composed of multiple polymer types to create matrices with increased mechanical or therapeutic properties. The benefits of pluronics and natural polymers have been combined into single matrices for cartilage regeneration. Pluronics/Tetronics have been used to develop supramolecular polypsuedorotaxane (PPDs) gels through inclusion of α-cyclodextrin (αCD) [182]. These thoroughly studied 224 structures form gels through the formation of inclusion domains and crystalline regions [183]. The tunable thermal and mechanical properties exhibited by PPDs reinforce initial Pluronic/Tetronic properties, resulting in materials with enhanced storage and elastic moduli [162,184]. These supramolecular structures were evaluated for release of recombinant adeno-associated virus (rAAV) from an interpenetrating gel in combination with glycosaminoglycans abundant in the cartilage extracellular matrix, hyaluronic acid (HA) and chondroitin sulfate (CS) [162,185]. HA has previously been reported to exhibit high levels of biocompatibility, and the presence of HA within PPDs increased cytocompatibility [162,185]. The incorporation of αCD in Pluronic- and Tetronic-CS formulations resulted in increased release rates, while the opposite was true of Pluronicand Tetronic-HA gels. The increased release rates may be due to crosslink disruption by the charged, interpenetrating CS. The interactions between CS and αCDs would result in formation of fewer PPD gels and possible formation of Pluronic micelles acting as porogens. Tetronic gels showed the most sustained release, resulting in more dense matrices from electrostatic interactions between EDTA core (+) and the rAAVs (-) [162,186]. The increased matrix density is evident from higher viscosities and leads to higher diffusion resistance. The inclusion of αCD and interpretation exhibited by CS/HA resulted in altered mechanical properties and spatiotemporal release of rAAVs [162]. The presence of HA increased activity of rAAVS possibly due to interactions with CD44 in mesenchymal stem cells [187]. The matrix-mediated signals of HA and CS still need to be evaluated within these systems, but independent studies have shown the ability of HA and CS to modulate chondrogenesis of stem cells [188,189]. Other cyclodextrin-based constructs have been developed using adamantane functionalized PEG polymers, to 225 release ADs exhibiting enhanced transgene expression of GFP compared to naked vectors and collagen matrices [190]. Interpenetrating networks have further been explored by creating Pluronics and alginate matrices (PF-Alg) to transduce human mesenchymal stem cells with rAAVs towards chondrogenesis. PF-Alg contains modified rheological properties of individual polymers exhibiting a biphasic system resulting from chain-chain interactions through hydrogen bonding in addition to divalent alginate crosslinking [191,192]. The biphasic nature of the PF-Alg hydrogels contains more porous structures, yet a slower release rate of rAAV, suggesting increased vector-polymer interactions. Hydrogel formation at higher temperatures created more porous structures due to entanglement of micellar PF127 acting as a porogen, releasing rAAVs more quickly. These PF-Alg systems resulted in high and stable transduction efficiencies at least 21 days. The inclusion of Pluronics in hydrogels increased gene transfer efficiencies and transgene expression levels [169]. This blended construct may have resulted in multiple polymer-virus interactions, enhancing matrix retention of rAAVs. Disruption of native alginate structures by Pluronics may have resulted in modified divalent electrostatic interactions, in which Ca2+ complexes with alginate and the negatively charged rAAVs. Polymer interpenetration interrupted native alginate/Pluronic architecture creating less semicrystalline domains and pore interconnectivity. PF-Alg resulted in less undesirable, hypertrophic differentiation in human mesenchymal stem cells compared to AlgPH155 alone [169]. Recombinant elastinlike protein polymers have been combined with polycaprolactone to produce electrospun scaffolds containing AAVs. Variations in polymer blends resulted in scaffolds with varying mechanical properties, cell compatibilities, and vector 226 transduction depending on polymer blend ratios [179]. The development of polymeric matrices has led to synergism between biomaterial and gene delivery approaches to treat disease. Synthetic polymers can be designed and tuned due to vast methodologies in matrix synthesis and customization. Natural polymers are capable of mimicking native tissue properties while providing cellular signals and degradation in vivo. Recombinant polymers can utilize natural motifs for programmable mechanical properties and ability to interact with tissues. MM delivery systems can be tuned to achieve appropriate temporal release of viral vectors in addition to providing necessary matrix cues for cellular migration and proliferation. C.7 Remaining Challenges and Future Directions Current MM systems for the delivery of viruses have shown promise in preclinical studies, for regenerative medicine and oncolytic therapies, requiring smaller doses than naked vectors with less immune neutralization. In clinical studies both an empty collagen matrix and matrix encapsulating AD-PDGFB showed safety and efficacy to treat non-healing diabetic foot ulcers [18,19]. Biomaterial matrices alone can have therapeutic effects depending on inherent material properties and composite systems such as interpenetrating networks have potential to increase integration with native tissues. Matrices provide opportunities to engineer regenerative microenvironments, all-in-one cancer vaccines, and localized antitumor therapies all easily customizable through the inclusion of cells, matrix components, small molecular weight compounds, and more [80,126,163]. Unique materials and formulation strategies may lead to the improvement of 227 current polymeric MM systems. The combination of cationic gold nanoparticles and anionic tobacco mosaic virus particles generate highly organized super lattices consisting of a 2D square lattice geometry through electrostatics. This level of organization may assist with polymer engineering by defining exact colloidal requirements for material self-assembly [193]. Kostiainen et al. have illustrated self-assembly using dendron-virus complexes and crystalline arrays [194,195]. Elucidated matrix requirements may be exactly programmed using recombinant design and production. Further developments of MM delivery need to address specific requirements of tissue microenvironment. For example, bone regeneration requires vascularization prior to osteogenesis and matrices of high mechanical strength. 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The addition of a charged foldable region provides these protein subunits with varied thermoresponsive properties from their parent ELPs. These subunits have responsive secondary structures, dependent on pH, indicating the capability to form coiled-coils with a complementary peptide. A Rituximab conjugate was generated herein, containing this complementary peptide. Upon mixing of the ELP and Rituximab subunits, the resulting protein complexes targeted CD20 receptors on Raji B cells, resulting in at least 2-fold increase in mean fluorescent intensities. These ELP subunits folded in vitro with complimentary the generated Rituximab conjugate, providing the basis for the design of a therapeutic stimuli-responsive biomacromolecule for targeting CD20 receptors. 248 D.2 Introduction Recombinant protein production utilizes naturally occurring sequences to generate biologically relevant structures, where motifs from nature can be combined to generate proteins with unique properties. Elastinlike polypeptides (ELPs) are a family of peptide motifs derived from tropoelastin, a 72 kDa protein with abundant domains of proline and glycine rich domains [1]. The first ELPs were developed from these proline rich domains consisting of VPGXG repeats, where X (the guest residue) represents any amino acid except proline (single letter amino acid residue abbreviations are used). Upon heating to a specific temperature, ELPs undergo a collapse with increased intra- and intermolecular interactions and decreased interactions with solvent. This temperature is known as the lower critical solution temperature (LCST) and depends on several factors including ELP chain length, concentration, solution ionic strength, pH, and mean chain hydrophobicity [2]. Below this LCST, ELPs exist as hydrated random coils. Upon heating above the LCST, ELP chains will undergo expulsion of water from their surface, collapse hydrophobically leading to an increased structural change of β-strands [3]. Fusion of ELPs to bioactive therapeutics further influences resulting protein LCSTs [4,5]. These fusion proteins can maintain bioactivity and have been used for numerous biomedical approaches [6,7]. However, the fusion of ELPs to larger proteins may be problematic due to limitations in recombinant technology, specifically the molecular sizes that can be produced successfully [8]. Combining the properties of large, therapeutically active proteins and ELPs can result in novel macromolecular delivery systems with well-defined structures. Coiled-coils are highly conserved motifs consisting of two or more α-helices that 249 spontaneously wind together to form a superhelical structure. The core of this structure is formed from the regular meshing of individual helices consisting of a heptad repeat unit abcdefg containing hydrophobic residues at positions ‘a’ and ‘d’ with polar residues making up the remaining portions of the chain [9]. Charged residues at positions ‘e’ and ‘g’ engage in electrostatic interactions between chains and can either stabilize or destabilize the coiled-coil stability between specific helices [10]. Two complimentary peptides, CCE (E VSALEKE VSALEKK NSALEKE VSALEKE VSALEK) and CCK (K VSALKEK VSALKEE VSANKEK VSALKEK VSALKE), have been shown to form heterodimeric, antiparallel coiled-coils in physiologically relevant media [11]. These charged CCK and CCE peptides form alpha helices or homodimers in basic or acidic pH due to charge neutralization. However, at neutral pH they can only form heterodimers due to self-charge repulsion [11] (Figure D.1A). This highly specific peptide pair when conjugated to copolymers results in clustering of CD20 receptors on malignant B cells [12–14]. The established biorecognition of these peptides make them a promising complimentary pair for rational design of novel fusion proteins. The combination of an ELP with coiled-coil peptide (CCE) can produce a foldable, thermoresponsive protein subunit (Figure D.1B). These subunits could then be used to fold with CCK tagged biotherapeutics, providing thermoresponsive properties to the resulting protein complex. In this work, we have designed two thermoresponsive protein subunits, with variations in the length of ELP chain. Subunit secondary structures and thermoresponsive properties were characterized. We hypothesized these subunits could then be used to fold with a clinically used antibody, Rituximab, which has been tagged with CCK peptides. The resulting complexes were then assessed for their ability 250 Figure D.1: Protein subunits and approach. A) Diagram of CCE-CCK coiled-coil formation. B) Schematic of CCE-ELP protein subunit consisting of either 48 or 192 ELP(V) repeats. C) Schematic or protein complex targeting cell receptors. 251 to target the cell surface receptor, CD20 (Figure D.1C). D.3 Results D.3.1. Generation and Confirmation of CCE-ELPs CCE-ELP genes were constructed via PCR amplification of the CCE gene, vector restriction digestion, and ligation. The PCR amplification of the CCE gene was confirmed by electrophoresis, yielding a single band between 100 and 200 bp (144 expected). This product was then ligated to the N-terminus of ELP genes of 48 (CCEV48) and 192 (CCE-V192) repeats (Figure D.2A and D.2B). These genes were confirmed with Sanger Sequencing of both termini and restriction digest (Figure D.2C). Expected genes of the CCE-V48 and CCE-V192 were 863 and 3023 base pairs respectively. These corresponded with banding observed during agarose gel electrophoresis. Protein production from these genes resulted in quantifiable yields via ITC purification. SDS-PAGE analysis of CCE-V48 and CCE-V192 yielded varying results (Figure D.2D). The CCE-V48 band appears at an increased molecular weight than expected. Further investigation of CCE-V48 protein molecular weight with matrix assisted laser desorption ionization-time of flight revealed a molecular weight of 23,749 Da similar to the expected 23,880 Da. The accuracy of molecular weight determination on SDS-PAGE depends on specific protein properties and regions with a high amount of acidic residues, as found in CCE, may repel the negatively charged SDS molecules, thus altering electrophoretic mobility [15]. CCE-V192 banding revealed a protein between 75 and 100 kDa. This is in alignment with an expected molecular weight of 82,846.04 Da. Proteins were analyzed for amino acid contents. Amino acid analysis showed conservation of both the ELP and CCE motifs. CCE was confirmed by probing the ratio 252 Figure D.2: Confirmation of ELP-CCE protein subunits. A) Schematic of protein subuntis generated. B) Primary sequence of subunits. C) Electrophoresis of digested plasmids illustrating the gene of interest size. D) SDS-PAGE of purified protieins. E) Amino acid analysis to confirm conserved ELP and CCE motifs. 253 of K, E, and L, to A. The ELP portion of the protein subunit was confirmed by comparing V and P to G (Figure D.2E). The confirmation of genetic sequence and length, along with protein characteristics of molecular mass and amino acid composition, indicates successful generation of both the CCE-V48 and CCE-V192. D.3.2 Characterization of Protein Subunits Circular dichroism and turbidimetry were then used to gain insight into the secondary structures and turbidimetry properties of the CCE-ELP subunits. CCE-V48 was assessed for secondary structures at varying pH. At neutral pH values CCE-V48 exhibited a broad negative peak near 210 nm indicating a random coil structure (Figure D.3A). The less pronounced negative peak near 220 nm suggests a β-turn structure, which is commonly exhibited by ELPs. Analysis of the CCE-V48 at pH 2 and pH 5 revealed an increased alpha helical tendency with minima near 208 and 222 nm. This alpha helical structure arises due to neutralized charges on the CCE peptide at low pH and indicates CCE-ELP subunit folding under dynamic conditions [11]. The thermoresponsive properties of the macromolecules were assessed by turbidimetry. At 10 µM the V48 has a sharp LCST of 40.83 ± 0.29 °C while the CCEV48 did not contain a discernable LCST below 80 °C (Figure D.3B). By increasing the concentration of CCE-V48 to 25 and 100 µM an LCST was observed at 67.00 ± 0.87 and 63.00 ± 2.17 °C respectively, however these transitions were of much less magnitude than that of the parent V48. This initial investigation prompted the development of the CCE-V192, as an increased ELP chain length lowers LCSTs [16]. For the higher molecular weight V192 ELP (10 µM) an LCST of 27.67 ± 0.29 °C was observed, lower 254 Figure D.3: Characterization of ELP-CCE subunits. A) Secondary structure of CCE-V48 at varying pH values. B) Turbidimetry of V48 and CCE-V48 at 10 µM in PBS. C) Turbidimetry of V192 and CCE-V192 at 10 µM in PBS. D) Qualitative solubility of transtioned V192 and CCE-V192 at 10 µM at 37 °C. E) Quantified solubility of V192 and CCE-V192 using absorbance turbidimetry. 255 than that of a 10 µM CCE-V192 (30.00 ± 0.00 °C) (Figure D.3C). The LCST of CCEV192 was dependent on the concentration with 2.5 µM and 1 µM solutions resulting in LCST values of 31.83 ± 0.29 and 32.00 ± 0.00 °C respectively. In both instances of the parent ELP protein (V48 and V192), LCSTs were recorded by a sharp increase in absorbance. At 43°C and 31°C, 10 µM solutions of V48 and V192 had >90% of total maximum absorbance. At temperatures slightly higher, both protein solutions started to exhibit drops in absorbance (Figure D.3B,3D.C). This sharp LCST is not observed in the CCE-V48 and CCE-V192 in which >90% absorbance was achieved at temperatures of 75.5°C and 59.5°C, respectively. However, the CCE-V48 only had a minimal change in absorbance throughout the temperature ramp (0.041 ± 0.001). The observed differences suggest that ELPs undergo a rapid LCST event and subsequent precipitation, indicated by the drop off of absorbance. On the other hand, CCE-V192 exhibits a much-prolonged change in molecular properties and does not precipitate similarly to ELPs. This was observed qualitatively with 10 µM solutions of the V192 and CCE-V192 (Figure D.3D). At 37°C and t = 0 solutions were clear and after 15 minutes both underwent transition, appearing turbid. After 12 hours the plain V192 was no longer turbid and the CCE-V192 remained turbid. When quantified using absorbance it is clear that the solubility of transitioned CCE-V192 at 37 °C remained for 12 hours, while the V192 crashed out of solution (Figure D.3E). Furthermore, it is evident that CCE-V192 may exhibit continued restructuring upon increase in temperatures as absorbance remains constant at 37 °C, but rises with temperature (Figure D.3C, D.3E). The addition of a CCE tag results in increased solubility of the resulting fusion protein. This could be a product of increased solvent-protein interactions, 256 intermolecular charge repulsion, or both. D.3.3 Generation of Rituximab-CCK Subunits Multivalent Rituximab-CCK conjugates were generated to produce a complimentary subunit for CCE-ELPs (Figure D.4A). This multivalent approach was used to increase interactions between the targeting component (Rit-CCK) and the CCEELPs. Following conjugation and purification of Rit-CCK, the conjugates were assessed by SDS-PAGE (Figure D.4B). Successful conjugation can be noted when Rituximab is compared to the Rit-CCK lane. No small molecular weight impurities and several valences of conjugation were observed. However, the nature of SDS-PAGE makes it difficult to assess the number of CCK per Rituximab. To overcome this, samples were further evaluated for amino acid analysis. Peptides associated with CCK (K, V, S, A, L, E) were compared to an amino acid not present within the peptide, M. By comparing these ratios between a control Rituximab and Rit-CCK we were able to estimate an average of 2.88 ± 0.16 CCK molecules per Rituximab (Figure D.4C). This average and deviation were based on the analysis of the individual amino acids (K, V, S, A, L, E) and is considered to be an estimation. Together these data indicate the successful generation of a multi-valent Rit-CCK with 2-3 CCK peptides per molecule. D.3.4 Complex Biorecognition at the Cell Surface Upon successful confirmation of each protein subunit (CCE-V48, CCE-V192, and Rit-CCK), complexes were then assessed to target CD20 receptors on Raji B Cells. Fluorescently labeled CCE components were mixed with Rit-CCK overnight at 4 °C. These complexes were then evaluated for CD20 recognition via flow cytometry. These 257 Figure D.4: Generation of Rit-CCK subunit. A) Schematic of synthesis. B) Analysis of Rituximab and Rit-CCK subunit via SDS-PAGE. C) Amino acid analysis estimating number of CCK per mol Rituximab. 258 custom protein complexes were generated at two separate concentrations of Rit-CCK (0.3 and 1.2 µM). All complexes resulted in increased mean fluorescent intensity indicating successful binding to the cell surface CD20 receptors (Figure D.5A). Complexes containing CCE-V48 resulted in significantly increased mean fluorescent intensities when they were generated at 0.3 µM Rit CCK concentration (9805.7 ± 4145.4 RFUs) compared to the control subunit CCE-V48 (1453.3 ± 669.8 RFUs) and complexes generated with 1.2 µM Rit-CCK (2995.3 ± 753.3 RFUs) (p < 0.05) (Figure D.5B). Similar trends were observed with complexes formed with the CCE-V192, in which complexes with 0.3 µM Rit-CCK had significantly increased mean fluorescent intestines (8156.0 ± 1648.1 RFUs) than the control CCE-V192 subunit (3695.7 ± 1260.5 RFUs) (p < 0.05). In both instances the observed decrease in fluorescence in 1.2 µM Rit-CCK solutions indicates saturation of the cell surface receptors with the Rit-CCK targeting moiety and subsequent washing away of fluorescence of unbound complexes prior to analysis. The increase in all CCE-ELP/Rit-CCK solutions indicates the formation of protein complexes, as the CCE-ELP subunits lack receptor targeting abilities. Those formed with 0.3 µM Rit-CCK/CCE-V48 and CCE-V192 resulted in a 6.7- and 2.2-fold increase in fluorescent signal compared to controls, respectively. The difference between these macromolecules may be due to the difference in the chain lengths of ELPs used, with the V192 chain likely providing more steric hinderance of the CCE recognition site. These complexes efficiently targeted CD20 receptors on Raji B cells and can be used for further design of therapeutic strategies. 259 Figure D.5: Analysis of protein complex binding to CD20. A) Flow cytometry histograms of ELP-CCE (red), ELP-CCE/Rit-CCK (0.3 µM) (blue), and ELP-CCE/Rit-CCK (1.2 µM) (orange). B) Mean intensitys of respective protein subunits (control) and complexes. 260 D.4 Discussion In this work a novel protein subunit, consisting of an ELP and coiled-coil peptide was developed. This proof of concept study provides the rational for further subunit design and protein complex targeting of cell surface receptors and therapeutic development. This includes a temperature- or concentration-based approach to clustering of CD20 receptors. Clustering of this nature has been shown to induce cell death as a strategy for non-Hodgkin’s Lymphoma [14]. Several other ELP fusion proteins capable of coiled-coil formation have also been explored [17–19]. However, most of these applications focus on the use of these systems to form multi-valent nanoparticles, which may not be representative of in vivo conditions. While protein complexes in this work can also form nanoparticles, as evidenced via turbidimetry, the ability to target cell receptors in a soluble state provides additional opportunities for therapeutic development and design. Due to their canonical thermoresponsive properties, ELPs have been explored in numerous therapeutic applications. Conjugation of ELPs to small molecular weight drugs can maintain therapeutic activity and can be programmed to contain responsive release mechanisms of drug cargo [20,21]. The genetic programming and recombinant production of ELPs has allowed for the development of ELP fusion proteins [22]. These fusion proteins can combine supramolecular protein assembly and targeting capabilities. The fusion of single chain antibody domains and ELPs have produced nanoparticle-like structures which were able to produce biological responses via interactions of CD20 and PD-1. However, both of these approaches utilized a multivalent nanostructure [23,24]. A soluble protein or protein complex could provide in situ stimulus responsive behavior, 261 enabling therapeutics to have dynamic activation states based upon location, temperature, and concentrations. Naturally occurring proteins contain intrinsically disordered regions which play vital roles in protein interactions, signaling pathways, and cellular processes. Spatiotemporal dysregulation of these regions is commonly associated with diseased states. Intrinsically disordered proteins can also produce phase separation of regulatory proteins in a concentration-dependent manner [25]. Producing a therapeutic of this nature could greatly improve rational mimicry of natural phenomena. In a sense, ELPs contain some characteristics of intrinsically disordered proteins with low sequence complexity, lacking defined secondary structures, and varied states dependent on the environment [26]. The characteristics of ELPs and their LCST present a unique therapeutic opportunity. For example, the fusion of ELPs to EGFRs have resulted in an intracellular approach to cluster cell receptors. The clustering of EGFR-ELP fusion proteins is temperature and ELP sequence- dependent, resulting in receptor phosphorylation and activation of ERK1/2 pathways [27]. This illustrates the capability of ELPs to mediate receptor clustering through an LCST-based mechanism. However, the intracellular nature of this approach limits translatability. Extracellular approaches to receptor clustering have largely focused on multivalent structures [23,24]. Future development of extracellular approaches to mediate receptor clustering through an LCST mechanism can result in enhanced clustering of target receptors. This investigation provides a basis for well-defined customized macromolecular therapeutic. ELPs are a well-characterized family of biopolymers, especially with regards to their LCST, allowing for easy customization of properties. Further rational design of 262 ELP subunits can focus on the folding of CCE with its CCK complement. This can be customized, and if needed, improved through the use of flexible linkers, increased CCE valence, and assessment of CCE location. Future studies will focus on the influence of CCE/CCK folding on ELP LCST properties, and how protein complexes with multiple ELP subunits respond to heat and concentration. The capability of this protein complex to undergo collapse at the cell surface, and subsequent receptor clustering, will also be evaluated. D.5 Conclusion In this work, thermoresponsive ELP subunits were developed and characterized. It’s altered thermoresponsive properties resulted from the charged residues found within the fused CCE peptide. This subunit is capable of folding with its counterpart a Rituximab conjugate containing a CCK peptide, the compliment to CCE. These protein complexes can target CD20 receptors on Raji B cells. This study provides the basis for further development of a variety of ELP-based subunits for therapeutic evaluation. D.6 Materials and Methods D.6.1 Materials Restriction enzymes, recombinant shrimp alkaline phosphatase, and T4 ligase were purchased from New England Biolab (Ipswich, MA. Plasmid vectors encoding the ELP genes were purchased from Addgene (Watertown, MA [28]. CCE gene was synthesized and provided by Integrated DNA Technologies (Coralville, IA. CCK (K VSALKEK VSALKEE VSANKEK VSALKEK VSALKE GGYC was synthesized and 263 provided by Biomatik (Wilmington, DE). Rituximab (Genentech) was obtained from Huntsman Cancer Institute, Salt Lake City, Utah. Sulfo-GMBS (N-γ-maleimidobutyryloxysulfosuccinimide ester was purchased from ThermoFisher (Waltham, MA). Raji B cells and cell media components were obtained from American Type Culture Collection (Manassas, VA). D.6.2 Gene Construction The plasmid vector, pET25b(+) encoding a 48mer (pET25b(+)-V48) and a 96mer (pET-25b(+)-V96) ELP (VPGVG) were purchased from Addgene. A 192mer ELP was then constructed by recursive directional ligation [29]. Briefly, the pET25b(+)-V96 was linearized with BseRi (10 U) and dephosphorylated with rSAP. In parallel the 96mer ELP gene was isolated through BseRi and AcuI (10 U) digestion. The purified 96mer gene was then ligated to the linearized BseRi gene and circularized using T4 ligase, resulting in a pET25b(+)-V192 plasmid. The gene encoding the CCE was generated by Integrated DNA Technologies. This gene was designed with flanking, complimentary BseRI restriction sites. These sites facilitated the ligation of CCE to the N-terminus of the ELP-48mer and -192mer. Briefly, the pET25b(+)-V48 and pEt25b(+)-V192 were digested with 10 U of BseRI and dephosphorylated with rSAP. The CCE gene was amplified with Phusion polymerase and digested with 10 U of BseRi. T4 ligase was used to insert digested CCE genes into the dephosphorylated pET-V4. Ligations were then transformed into NEB Stable Cells. Genes were confirmed with 1% gel electrophoresis and Sanger Sequencing (University of Utah). 264 D.6.3 Protein Production Confirmed plasmids were transformed into BL21 production cells. Frozen glycerol stocks were used to initiate cultures in 5 mL Terrific Broth with 100 μg/mL of ampicillin before passage into a 100 mL culture. Further passage to 500 mL cultures and growth to an OD600 of 2 – 2.5 was achieved before induction with Isopropyl β-D-1thiogalactopyranoside (IPTG) to a final concentration of 0.9 mM. Expression was allowed to proceed for 6-8 hours after which the cultures were placed on ice for 15 minutes, pelleted, and resuspended to 2 mL/g in PBS with Halt Protease Inhibitor Cocktail. Biomass was then stored at -20 °C until purification. Cells were lysed using freeze thaw cycling for no less than 5 cycles. Cell lysates were pelleted. CCE-ELP constructs were purified using inverse transition cycling (ITC) [22]. The lysate was spun at 4°C for 20 min at 15,000 RCF and the supernatant was collected. ITC was then initiated. Saturated NaCl was added to induce ELP phase separation. After observing phase separation, the protein was centrifuged (15,000 RCF) at room temperature for 20 minutes. The pellet was solubilized in cold PBS and centrifuged (15,000 RCF) at 4 0C for 20 minutes to remove further contaminants. The supernatant was then taken back through the cycle for a total of 5 cycles. Protein solutions were then run through a Pierce Endotoxin removal column, dialyzed, and frozen. Proteins were stored at -200C until use. D.6.4 Protein Confirmation Proteins were confirmed by molecular weight and amino acid analysis. Amino acid analysis was performed by AAA Service Laboratory, Inc. (Damascus, OR). A total 265 of 20-40 µg of protein was run on an SDS-PAGE to determine purity and molecular weight. Where needed, MALDI-TOF was performed by the University of Utah Proteomics core to confirm molecular weight. D.6.5 Subunit Secondary Structures ELP fusion protein solutions (0.25 mg/mL) were analyzed for CD spectra using the Jasco J-1500 CD Spectrometer (Jasco Products Company, Oklahoma City, OK). Solutions were prepared in either buffered solutions (pH 2 ± 0.05, pH 5 ± 0.05, pH 7 ± 0.05) as previously described [11]. The solutions were placed in quartz cuvettes (Hellma Analytics, Plainview, NY) with a 1 mm path length. Spectra was measured from 190 to 260 nm at a 0.1 nm resolution with a 10 nm/min scanning speed. CD spectra were obtained at 37 °C. All groups were run in triplicate. D.6.6 Absorbance Turbidimetry The phase transitions of the ELP and CCE-ELP fusion proteins were analyzed using absorbance measurements at 350 nm with a J-1500 CD Spectrometer (Jasco Instruments, Easton, MD). Protein solutions were prepared in PBS and loaded into10 mm pathlength quartz cuvettes (Jasco Instruments, Easton, MD). The temperature increased at a rate of 0.5 °C min-1 from 5 to 80 °C. At every 0.5 °C step the samples equilibrated for 10 s before a measurement was taken. Raw data was normalized by subtracting the minimum absorbance observed. LCSTs were determined by taking the maximum of the first derivative. Groups with deviations greater than 5 °C were considered to have no discernable LCST below 80 °C. To assess V192 and CCE-V192 solubility a similar 266 protocol was followed, however the temperature was kept constant at 37°C. D.6.7 Rituximab-CCK Generation Buffer exchange of Rituximab solutions was performed to result in a PBS (pH 7.2 ± 0.1) buffered solution. The bifunctional linker (Sulfo-GMBS (N-γ-maleimidobutyryloxysulfosuccinimide ester) was then added in x5 molar excess, mixed, and allowed to incubate with Rituximab for one hour before filtration removal. This activated amines on Rituximab. The CCK peptide was then added in x3 molar excess to activated amines and the reaction proceeded at 4 °C overnight before filtration to remove the reactants. D.6.8 Protein Complex Biorecognition on Raji B Cells The amines of CCE-V48 and CCE-V192 were fluorescently labeled with Cy5.5NHS. Labeling occurred overnight at 4 °C in PBS (pH 8.4 ± 0.1). Unconjugated dye was removed using PD-10 desalting columns to produce Cy5.5-CCE-V48 and Cy5.5-CCEV192. Rit-CCK (0.3 or 1.2 µM) and Cy5.5-CCE-ELP (0.2 µM) subunits were then mixed and incubated at 4 °C overnight. These complexes were mixed with a 1% BSA solution and used to target CD20 on Raji B cells (1e05) at 4 °C. After 30 minutes cells were washed with PBS and evaluated with a CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA). Data was then analyzed using FloJo to determine the mean fluorescence of single alive cells. 267 D.6.9 Statistical Analysis Statistical significance was determined with a student t-test. In cases of identical triplicate values, a one sample t-test was run. All statistical analyses and graphs were prepared utilizing GraphPad (San Diego, CA). 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