Improving Anti-VEGF Drugs in the Vitreous by Eda Isil Altiok A

Improving Anti-VEGF Drugs in the Vitreous

Eda Isil Altiok

A dissertation submitted in partial satisfaction of the

Requirement for the degree of

Joint Doctor of Philosophy with UC San Francisco

Bioengineering

In the graduate division

Of the

University of California, Berkeley

Committee in charge:

Professor Kevin E. Healy, Chair Professor David Schaffer Professor Tejal Desai Professor Anthony Adams

Fall 2015

Improving Anti-VEGF Drugs in the Vitreous

Eda Isil Altiok

University of California, Berkeley

Abstract

Improving Anti-VEGF Drugs in the Vitreous

Eda Isil Altiok

Doctor of Philosophy in Bioengineering

University of California, Berkeley

Professor Kevin Healy, Chair

The work described in this dissertation present a novel technique utilizing multivalent hyaluronic acid bioconjugates with an anti-VEGF protein for improving the action of drugs in the vitreous. This technology, which has shown efficacy both in vitro and in vivo has the potential to enhance the bioactivity of drugs used for treating patients with diseases including diabetic retinopathy, wet AMD and other neovascular diseases of the retina.

Chapter 2 described our initial efforts in creating multivalent conjugates of the anti-VEGF protein, sFlt. Before beginning in vivo studies, we wanted to determine what parameters would maximize the bioactivity of mvsFlt. We investigated the use of several HyA molecular weights and valencies of sFlt molecules to HyA chains. The characterization and in vitro experiments were carried out with 6 mvsFlt conjugates of 300 kDa, 650 kDa and 1 MDa molecular weights with feed ratios of 10 sFlt per 1 HyA chain (termed low conjugation ratio (LCR)) and 30 sFlt per 1 HyA chain (termed high conjugation ratio (HCR)). SDS-PAGE and SEC-MALS enabled us to examine the conjugation efficiency following the reaction as well as to investigate the composition of the conjugates focusing specifically on the contribution of unbound sFlt that remained in solution. The in vitro experiments were crucial in determining whether the conjugation of sFlt to the HyA resulted in a decrease in the affinity of sFlt for VEGF. Using an ELISA and a cell-based survival assay, we determined that all the conjugates, irrespective of their molecular weight and valency, equally inhibited VEGF activity and were unaffected by conjugation. Using HyA crosslinked gels, we created an in vitro model of the vitreous to study how the increase in size impacted movement of the mvsFlt conjugates through the gel. The largest mvsFlt conjugates (650 kDa and 1 MDa) were significantly slower in their movement through the gel. The 650 kDa mvsFlt conjugate was then used in the in vivo studies described in Chapter 3. Furthermore, this conjugate was used to confirm the protective effect of HyA on sFlt degradation by a protease that specifically targets sFlt, matrix metalloproteinase-7. Taken together, the work in this chapter demonstrated the efficacy of conjugation, characterization, in vitro bioactivity, slowed

1 diffusion and protection from proteases. The progress demonstrated in this chapter enabled the studies presented in Chapter 3.

The work in Chapter 3 details the in vivo studies used to show mvsFlt efficacy in two models of in vivo angiogenesis and a half-life model in the rat vitreous. As shown in Chapter 2, all the mvsFlt conjugates performed equally at inhibiting VEGF-dependent processes. Thus, we chose the conjugate that displayed slowest diffusion in the crosslinked HyA vitreous model, the 650 kDa mvsFlt conjugate. The first question we tried to address was whether the mvsFlt conjugate remained bioactive in vivo. The corneal angiogenesis model enabled us to examine the effect of sFlt and mvsFlt treatment on the growth of blood vessels in a corneal injury model of angiogenesis. Due to the fact that VEGF is one of the main mediators of angiogenesis in vivo, we expected that the addition of sFlt and mvsFlt would inhibit the formation of new blood vessels. Our data showed that both the sFlt and mvsFlt were equally capable of inhibiting angiogenesis in this model, indicating that the conjugation did not decrease the ability of sFlt to inhibit angiogenesis in vivo. We next demonstrated that the conjugation of sFlt to HyA significantly enhanced the half-life of sFlt in the vitreous of the rat eye by an order of magnitude. Finally, we utilized an oxygen-induced retinopathy rat model to examine the indirect effect of increased half-life on the prolonged anti-angiogenic effect of mvsFlt on neovascularization in the retina. mvsFlt was more effective than sFlt at inhibiting retinal neovascularization, likely due to its increased half-life in the vitreous. Taken together, the mvsFlt conjugate showed superior activity to sFlt in vivo, results that could substantially improve upon currently available drugs for treating retinal disorders.

Chapter 4 gives an overall conclusion and details future directions the project can go in. There are some questions that remain unanswered and this chapter is a guideline to help fill in those gaps.

Dedicated to the memory of Tayfur Altiok

Table of Contents

Abstract ...... 1 List of figures ...... iv List of tables ...... v List of abbreviations ...... iv

Acknowledgements ...... ix

Chapter 1. Introduction

1.1 Diabetic retinopathy and its public health impact...... 1 1.2 Posterior segment biology...... 3 1.2.1 Retinal structure...... 3 1.2.2 Vitreous biology...... 5 1.2.3 Modes of drug clearance from the vitreous...... 6 1.3 Pathogenesis of diabetic retinopathy...... 6 1.3.1 Introduction to angiogenesis...... 6 1.3.2 The vascular endothelial growth factor family ...... 7 1.3.3 Mechanisms of DR progression...... 9 1.4 Ocular drug delivery and current strategies for DR management...... 10 1.4.1 Pharmacological interventions...... 10 1.4.2 Retinal drug delivery systems...... 12 1.4.2.1 Implants...... 12 1.4.2.2 Particulate systems...... 17 1.4.2.3 Hydrogels...... 20 1.4.2.4 Polymer conjugation...... 21 1.5 Dissertation approach and outline...... 22

Chapter 2. Multivalent hyaluronic acid bioconjugates improve sFlt activity in vitro

2.1 Introduction...... 33 2.2 Materials and methods...... 34 2.2.1 Expression of soluble Flt-1 receptor...... 34 2.2.2 mvsFlt conjugate synthesis...... 35 2.2.3 SEC-MALS Characterization of mvsFlt...... 35 2.2.4 SDS-PAGE Analysis of mvsFlt...... 35 2.2.5 DLS size characterization of mvsFlt...... 35 2.2.6 Binding competition ELISA...... 35 2.2.7 HUVEC survival assay...... 36 2.2.8 HUVEC tube formation assay...... 36 2.2.9 HUVEC migration assay...... 36 2.2.10 Retention of mvsFlt in crosslinked HyA gels ...... 36 2.2.11 FRAP diffusivity measurement...... 37 2.2.12 Enzymatic degradation with MMP-7...... 37

2.2.13 Statistical analysis...... 38 2.3 Results and discussion...... 38 2.4 Conclusion...... 48 2.5 Acknowledgements...... 48 2.6 References...... 48

Chapter 3. sFlt multivalent conjugates inhibit angiogenesis and improve half-life in vivo

3.1 Introduction...... 54 3.2 Materials and methods...... 55 3.2.1 Expression of soluble Flt-1 receptor...... 55 3.2.2 mvsFlt conjugate synthesis...... 55 3.2.3 Corneal angiogenesis assay...... 56 3.2.4 Determination of mvsFlt intravitreal half-life...... 56 3.2.5 OIR angiogenesis model ...... 57 3.2.6 Statistical analysis...... 57 3.3 Results...... 58 3.3.1 Multivalent sFlt synthesis and characterization...... 58 3.3.2 sFlt and mvsFlt equally inhibit corneal angiogenesis...... 58 3.3.3 mvsFlt has significantly longer half-life in the vitreous...... 58 3.3.4 mvsFlt is a more potent inhibitor of retinal angiogenesis...... 58 3.4 Discussion...... 59 3.5 Acknowledgements ...... 66 3.6 References...... 66

Chapter 4. Summary and future directions ...... 71

Appendix I: Determination of the pore size in crosslinked HyA gels presented in Chapter 2...... 76

Appendix II: Optimization of the rat vitreous as a model for studying the in vivo half-life of mvsFlt presented in Chapter 3 ...... 81

iii

List of figures

Figure 1.1: Prevalence of obesity and diagnosed diabetes among U.S. adults ...... 1

Figure 1.2: Retinal anatomy...... 3

Figure 1.3: Anatomy of retinal vasculature...... 4

Figure 1.4: Structural schematic of the vitreous...... 5

Figure 1.5: The VEGF family of proteins...... 7

Figure 1.6: Commonly used anti-VEGF drugs for treating diabetic retinopathy...... 10

Figure 1.7: Schematics of implant-based ocular drug delivery...... 15

Figure 1.8: Basics of particulate-based drug delivery vehicles...... 17

Figure 2.1: Multivalent sFlt synthesis and schematics...... 38

Figure 2. Characterizing mvsFlt conjugation efficiency and size...... 40

Figure 2.3. All mvsFlt bioconjugates maintain their ability to inhibit VEGF165 dependent activities in VEGF165 ELISA and VEGF165-dependent HUVEC survival assays...... 41

Figure 2.4. mvsFlt inhibits HUVEC tube formation...... 42

Figure 2.5. mvsFlt inhibits VEGF165-driven HUVEC migration...... 43

Figure 2.6. sFlt conjugation to HyA decreases mvsFlt mobility and diffusion in HyA gels.. 44

Figure 2.7. Conjugation to HyA reduces susceptibility to protease degradation by MMP-7. 46

Figure 3.1: Synthesis of mvsFlt schematic...... 59

Figure 3.2: sFlt and mvsFlt equally inhibit corneal angiogenesis...... 60

Figure 3.3: mvsFlt has longer residence time in the rat vitreous...... 61

Figure 3.4: mvsFlt inhibits retinal angiogenesis...... 62

Figure 3.5: Schematic demonstrating the proposed mechanism of mvsFlt action...... 63

List of Tables

Table 1.1: Summary of implants used for treating retinal diseases...... 13

Table 2.1: SEC-MALS analysis of all mvsFlt conjugates...... 39

Table 2.2: IC50 values from ELISA and HUVEC survival assays examining mvsFlt inhibition of VEGF165...... 41

Table 2.3: �! and mesh size calculations based on swelling and rheological data...... 44

List of abbreviations

ADH Adipic dihydrazide AGE Advanced glycation end products AMD Age-related macular degeneration ANOVA Analysis of variance BCA Bicinchoninic acid BRVO Branch retinal vein occlusion BSA Bovine serum albumin CMV Cytomegalovirus CNTF Ciliary neurotrophic factor CNV Choroidal neovascularization CRVO Central retinal vein occlusion Da Dalton DLS Dynamic light scattering DM Diabetes mellitus DME Diabetic macular edema DMSO Dimethyl sulfoxide DR Diabetic retinopathy ECT Encapsulated cell technology EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride EDGMA Ethylene glycol dimethacrylate ELISA Enzyme linked immunosorbent assay ELM External limiting membrane EMCH 3,3′-N-(ε-maleimidocaproic acid) hydrazide EVA Ethylene-vinyl acetate FBS Fetal bovine serum Fc Antibody fragment FDA United States Food and Drug Administration FGF Fibroblast growth factor FITC Fluorescein isothiocyanate FRAP Fluorescence recovery after photobleaching GCL Ganglion cell layer GTPase Guanosine triphosphate enzyme HCR High conjugation ratio mvsFlt HEMA Hydroxyethyl methacrylate HOBt 1-hydroxybenzotriazole hydrate HUVEC Human umbilical cord vein endothelial cells HyA Hyaluronic acid IgG Immunoglobulin G ILM Inner limiting membrane INL Inner nuclear layer IOP Intraocular pressure IPL Inner plexiform layer IPTG Isopropyl β-D-1-thiogalactopyranoside IS Cone inner segment IVT Intravitreal

vi kDa Kilodalton LCR Low conjugation ratio mvsFlt MDa Megadalton MES 0.1 M 2-(N-morpholino)ethanesulphonic acid buffer MMP Matrix metalloproteinase Mn Number average molecular weight MT Macular telangiectasia mvsFlt Multivalent sFlt Mw Weight average molecular weight MWCO Molecular weight cutoff NADPH Nicotinamide adenine dinucleotide phosphate NF-kB nuclear factor kappa-light-chain-enhancer NFL Nerve fiber layer NNLS Non-negative least squares test NPDR Nonproliferative diabetic retinopathy NRP1/2 Neuropilin 1/2 NTC-200 Retinal pigment epithelium cell line OIR Oxygen-induced retinopathy ONL Outer nuclear layer OPL Outer plexiform layer OS Cone outer segment PBS Phosphate buffered saline PDGF Platelet-derived growth factor PDGFR Platelet-derived growth factor receptor PDI Polydispersity index PDR Proliferative diabetic retinopathy PEDF Pigment epithelium-derived factor PEG Polyethylene glycol PG Proteoglycans PGA Poly(glycolic) acid PI-3K Phosphatidyl inositol-3 kinase PIGF Placenta growth factor PLA Poly(lactic) acid PLGA Poly(lactic-co-glycolide) PLP Pan-retinal laser photocoagulation PMMA Poly(methyl methacrylate) PVA Poly(vinyl alcohol) RAGE Advanced glycation end product receptor RIPA Radioimmunoprecipitation assay buffer RNA Ribonucleic acid RPE Retinal pigment epithelium RVO Retinal vein occlusion SD Standard deviation SEC-MALS Size exclusion chromatography with multiangle light scattering SF9 Spodoptera frugiperda-9 (insect cells) sFlt-1 Soluble fms-like tyrosine kinase receptor-1

vii t1/2 Half-life TA Triamcinolone acetonide TEOA Triethanolamine buffer TGFB Transforming growth factor beta UV Ultraviolet VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor µg microgram

viii

Acknowledgements

The success of this work is due largely in part to the amazing group of people I am lucky enough to call my friends and family. The five and a half years I spent doing my Ph.D. at Berkeley was filled with plenty of high points and low points as well and if it had not been for these people, the successes I saw would not have realized.

I would like to sincerely thank Prof. Kevin Healy for his constant help and guidance through the completion of my project. He encouraged me to break out of my comfort zone which enabled me to grow as a researcher and a scientist, and for that I am very grateful.

I was lucky enough to have found a great mentor in Prof. Christine Wildsoet early on in graduate school. I have very fond memories of being overly caffeinated on coffee from Strada and barging into her office with some of my very first scientific ideas involving drug delivery to the eye. She was always incredibly patient, calming, supportive and ready to give a hug. Her support throughout graduate school was invaluable and I will always cherish our bond.

I am also indebted to Anthony Adams, Dave Schaffer and Tejal Desai who were instrumental in the finalizing of my dissertation project. Tony, thank you for your support over the years. Although we did not meet as often as I would have liked, our meetings were always light spirited and enlightening. Thank you for helping me see the big picture in my work. Dave, thank you for your encouragement over the years. You guided me through expressing this impossible protein, to conjugating it and finally to finding it in the eye. Your input has been invaluable to the development of this project.

I also made incredible friendships while in the Vision Science program. The two years I spent in the Chen laboratory were made easier by daily trips to Yogurt Land and Café Strada with Tatiana Ecoiffier and Stella Kang. Tatiana, our trip to Phoenix together will always hold a special place in my heart vortex. She is one of my very best and dependable friends and I am so thankful for that friendship. I am also incredibly grateful for the friendship I have with Christy Sheehy who was my partner in rapping and video making early on in graduate school. That single, amazing YouTube video still has more views than any of my other videos combined.

I was also lucky enough to join an incredible ultimate frisbee team upon moving to the bay called Classy. Over the five and a half years of graduate school, this team has become like my family. Words cannot express how much I love this team and how much each individual means to me. We’ve celebrated birthdays, marriages, graduations, new jobs, moves, babies and been there for each other through incredible personal losses as well. Some special shout- outs go to Rebecca Busch, Dana Nelson, Dhwani Shah, Josh Mitts, Joe Bryan, Doug Hannah, Tien-tien Chan, Ben Raudabaugh, Allison Yochim, Sam Haynor, Katie Rouse, Henry Riekena and so many more.

My labmates in the Healy Laboratory have also been incredibly supportive and helpful over the years. Willie Mae Reese, your smile and laugh always gave me the strength to continue after each failed experiment. Shane Browne, you arrived just when I needed your guidance to pull this all together. Thanks for sitting through hours of injections with me with the only

ix job but to clean a syringe. Peter Loskill and Anurag Mathur- you helped me see the light and then some. Wesley Jackson was another great mentor early on. You helped me trouble shoot so many of the early experiments that would eventually work and go into the papers. You helped mold the project and our chats established the road I would follow for the next 3 years. A very special thanks goes to Jorge Santiago-Ortiz. In the early days we had to work together to get this tricky protein expressed. Without you, the experiments would not have happened! Thanks for sticking it through and being a great friend along the way.

I also had the pleasure of being a part of the treehouse crew, which included the wonderful Tenzing Joshi, Lowry Kirkby, Ryan Arant and Alex Polo. Countless bottles of wine, Halloween parties, dinner parties, slumber parties and coffee breaks later we’ve all successfully joined the Dr. club. Thanks for getting through this before me and giving me the inspiration and motivation to do it too. Sunsets from that house brought us together like no other could. Thanks for taking a chance on me that day in April 2012- I’m so grateful you that you did.

This dissertation was possible in large part because of the support I received from my parents. Baba, I distinctly remember being on the phone with you while working in Australia and telling you that I was not coming back. Thank you for putting your foot down. I wish I could have shared the successes I’ve had since then with you but I am sure you know. Anne, I know you have done your best these last few years when trying to figure out what to say to me when nothing was working. Your encouragement and support mean the world to me. Selen, my seastar, I am so proud of you. I am eternally grateful that I can call someone so amazing, so strong and such a beautiful person inside and out my sister. Thank you for all the blackmail-worthy photos, cute animal photos and bay area visits. I could not have done this without your support.

And finally, thank you to Matt/Ruby/askim. I am at a loss as to how to thank you for the last 5 years. I feel so incredibly lucky to have you in my life. You held my hand and pulled me through broken bones and the biggest loss of my life not to mention months and years of failed experiments. Words cannot express the gratitude I feel for you every day. Together, we made it through an incredible chapter of our lives and I am so excited to see what the other chapters hold for us as well.

Chapter 1: Introduction

Summary

Drug delivery to the posterior segment of the eye is a significant challenge to overcome for the pharmaceutical industry. Patients suffering from diseases including diabetic retinopathy, macular degeneration, posterior uveitis and choroidal neovascularization require drugs for diseases management and treatment, however due to the difficulty in delivering drugs through the superficial layers of the eye, many conventional approaches are ineffective. This chapter goes into detail on the anatomy and physiology of the retina and discusses a wide range of approaches taken to deliver drugs to the retina through the use of polymeric implants, particulate systems and hydrogels. An introduction to the multivalent anti-VEGF approach is also introduced and discussed in greater detail in chapters 2 and 3.

1.1 Diabetic retinopathy and its public health impact

Diabetes mellitus (DM) is a disease in which the body makes insufficient levels of insulin to process blood sugar levels. Although several types of this disease exist, the two major forms of DM are type 1 and type 2. Type 1 DM is the genetic form of the disease, which is characterized by the failure of the pancreas to produce enough insulin [1]. In this case, the body has an autoimmune reaction mediated by T cells that leads to a complete depletion of beta cells, which are responsible for producing insulin. Type 2 DM is tightly linked to obesity and begins with insulin resistance and can progress to complete lack of insulin production [2].

Figure 1.1: Prevalence of obesity and diagnosed diabetes among U.S. adults aged 18 and older. The prevalence of diabetes is increasing steadily with the upward trend in obesity. The top panel indicates the prevalence of obesity (BMI > 30 kg/m2) and the bottom panel indicates the prevalence of diabetes. Numbers are expected to double by 2030 posing an incredible strain on healthcare costs national and worldwide. (www.cdc.gov/diabetes)

Obesity is a major problem facing the United States (Figure 1.1) and countries worldwide. It is estimated that 65% of Americans are overweight of this population, 23% are obese [3]. Evidence shows a direct correlation between the rise in obesity and the increasing prevalence of type 2 diabetes. Of the patients with type 2 diabetes, 90% of them are significantly overweight or obese. As a consequence of this staggering increase in obesity, there has been a substantial increase in the prevalence of obesity-dependent diabetes in the last 20 years, a pattern that that is projected to skyrocket in the coming decades [4]. In the United States alone, it is estimated that there are more than 23.6 million people with diabetes, 95% of which are represented by the preventable form, type 2 and the other 5% is accounted for by the genetic form, type 1. This accounts for approximately 8% of the US population and it is estimated that another 57 million people are considered to be “pre- diabetic” as a result of abnormally high blood glucose levels [3]. It is estimated that at this rate, the prevalence of diabetes in the national population will exceed 30 million by 2030. These numbers are different based on which population is being examined. The prevalence of diabetes is significantly higher in studies examining African Americans, a population where 18.2% of the people have diabetes type 2 [5]. Although there is no current preventative measure for diabetes type 1, diabetes type 2 can be prevented and controlled due to its dependence on body weight and behavior. People at risk for diabetes can prevent its progression by reducing body weight, increasing physical exercise and following a healthy diet [6]. Sugary beverages and red meat have also been associated with an increased risk of diabetes as well as smoking habits. Despite the ease of diabetes prevention, the prevalence of this disease is on the rise. Due to this drastic increase in the prevalence of type 2 diabetes, there has also been a surge in the devastating symptoms associated with diabetes including stroke, peripheral vascular disease and diabetic retinopathy. Diabetic retinopathy is currently the leading cause of blindness among individuals aged 20-74 in the United States [7]. It is estimated that there are over 200 million people worldwide suffering from the consequences of diabetic retinopathy and this number is expected to double by 2030 with the increasing prevalence of diabetes mellitus [8]. Diabetic retinopathy is one of the most severe symptoms of diabetes and time is a major risk factor in its development. Over the course of their disease, all patients with diabetes type I will develop symptoms of diabetic retinopathy within two decades in comparison to 60% of patients with type 2 diabetes [7]. Diabetic retinopathy is a treatable symptom of diabetes, however, the disparity between drug availability and the increase in patients presenting with retinopathy symptoms points to severe barriers in disease management. Rates of non-adherence to diabetes regimens are extremely high when examining diet restrictions, insulin administration, blood 2 glucose testing and exercise. Non-adherence to diabetes management is one of the primary reasons why symptoms of diabetes continue to progress with time, with the additional problem of very limited access to medical care [9]. In developing countries, access to proper medical care is quite limited for the majority of the population [10]. For these reasons, treatable symptoms of diabetes like diabetic retinopathy can easily progress to debilitating vision loss without proper care. There are ample therapeutics on the market currently for treatment of diabetic retinopathy, however these drugs require monthly injections in order to maintain effective concentrations of drug within the vitreous. These injections are not only expensive (monthly injections of Lucentis cost $2000), but also uncomfortable for patients who often chose not to receive timely treatments, which can cause the disease to progress to debilitating levels. Thus, developing therapeutics that will lead to better patient quality of life is of utmost importance. The overarching goal of this thesis was to improve upon currently available drugs for treating diabetic retinopathy by increasing the overall residence time of drugs within the vitreous, thereby reducing the number of injections patients are required to get while still keeping the disease under control. Although the results from this project indicated that our approach could decrease the number of injections, this approach cannot be used without injection directly into the vitreous. The rationale behind that was to simply decrease the number of required injections in order to increase patient comfort and thus compliance. Other approaches that are discussed in this chapter are non-injection based but do require surgical placement of implants. Decreasing the injection frequency could lead to better patient compliance, reducing the overall healthcare costs this disease imposes on society and reducing prevalence of diabetic vision loss.

1.2 Posterior segment biology

1.2.1 Retinal structure

The human eye is divided into two main compartments: the posterior and anterior segments. The anterior segment consists of the front third of the eye and is comprised of the lens, ciliary body, suspensory ligaments, cornea and aqueous humor. The posterior segment is the part of the eye primarily affected by diabetic retinopathy and consists of the vitreous humor, retina, choroid and optic nerve.

Figure 1.2: Retinal anatomy. From the vitreous to the choroid, the retinal layers are the internal limiting membrane (ILM), nerve fiber layer (NFL), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL), external limiting membrane (ELM), rod and cone inner and outer segments (IS/OS), and the retinal pigment epithelium (RPE). (www.retinareference.com/anatomy)

The retina is composed of five major types of cellular elements including neurons, glial cells, microglia, blood vessels and pigment epithelium (Figure 1.2). The proper communication of all these cells is required for normal vision. Neurons and glial cells comprise more than 95% of all cells within the retina [11]. Neuronal cells are made up of photoreceptors, amacrine, bipolar, and horizontal cells and electrical input from these cell types converge via ganglion cells on the optic nerve for delivery of electrical signals to the brain through the ganglion cell neuronal axons. Glial cells, Müller cells and astrocyotes serve as a cell glue for supporting neurons and blood vessels by regulating extracellular ion potentials to facilitate action potentials, metabolize neurotransmitters like glutamate and transportation of glucose and amino acids for retinal metabolism from blood vessels to neurons [12].

Figure 1.3: Anatomy of retinal vasculature. The schematic shows the vasculature in the retina and choroid. Retinal arterioles and venules are proximal to the vitreous on the surface of the retina while the capillary plexi lie just beneath the surface and in the INL. Neovascularization in diabetic retinopathy results from angiogenesis in the most superficial blood vessels, the superficial capillary plexus [13].

The retina is also an extremely vascularized tissue with tight organization through several layers as demonstrated in Figure 1.3 [13]. The central retinal artery with four main branches and the choroidal blood vessels are two main blood supplies to the retina with the choroid contributing 65-85% of the blood flow. Microcirculation around the retina is carried out through extensive capillary beds, precapillary arterioles and postcapillary venules. Under healthy conditions, the retinal vasculature balances nutrient delivery to every cell layer with waste removal with retinal metabolism [13].

1.2.2 Vitreous biology

The vitreous is one of the main ocular components responsible for maintaining mechanical stability of the eye. Made up of approximately 97% water, the vitreous is a gel- like tissue that separates the retina from the anterior segment. In addition to water, the vitreous is a meshwork of structured glycosaminoglycans, collagens and proteoglycans as shown in Figure 1.4. Hyalocytes are the main cellular component of the vitreous found on the peripheral part of the vitreous body and are responsible for secreting hyaluronic acid and collagen [14]. Glycosaminoglycans are long chains of repeating carbohydrate disaccharide units. The prominent glycosaminoglycan in the vitreous is hyaluronic acid (HyA), which is composed of the repeating disaccharide unit glucuronic acid and N-acetylglucosamine [15,16]. The concentration of HyA in the adult human vitreous is estimated to be 100-400 µg/mL with the concentration varying depending on location within the vitreous. Higher concentrations of HyA are present in the posterior section of the vitreous and progressively decreases closer to the lens. The molecular weight of HyA in the adult human vitreous has been estimated to be between 2 and 4 million daltons with high polydispersity. Although chemical crosslinks are absent in the vitreous, the vitreous is held together by both intra chain and inter chain entanglements of HyA molecules other vitreous components. Recently, it has been proposed the HyA chains can form entanglements through hydrophilic and hydrophobic interactions to form extended network sheets and tubule structures [17].

Figure 1.4: Structural schematic of the vitreous. The vitreous has a high concentration of hyaluronic acid (HyA), various proteoglycans (PG) and several types of collagen. Proteoglycans form bridges across collagen fibrils to provide stability.

Collagen is another major component of the vitreous and is typically found at concentrations of 300 µg/mL in adult humans [18]. Similar to HyA, collagens are not evenly distributed in the vitreous. The highest concentration of collagen is found at the vitreous base and decreases towards the central and posterior vitreous. The collagen fibrils are typically composed of several different types of collagens including types II, V, XI and IX. Collagen type II is the prominent type in the vitreous and accounts for approximately 70- 5

80% of total collagen. Proteoglycans associate with collagen fibrils at binding sites along the collagen backbone and are separated by 60 nm to add structural stability to the vitreous gel. There is an association between the extent to which the vitreous is a gel and age. As people get older, the HyA and collagen networks in the vitreous break down [18]. By the age of 80 years old, the vitreous is on average 50% liquid compared to 20% at adolescence. This may have direct consequences on the diffusion and mobility of drugs within the vitreous due to the fact that molecular entities move slower through gel-like substances as opposed to liquids.

1.2.3 Modes of drug clearance from the vitreous

The current approach to treating retinal neovascularization is with intravitreal injection through the sclera. Once injected into the vitreous, drugs distribute through the vitreous in a charge and size dependent manner and are cleared from the vitreous through two main routes: anterior and posterior elimination [19]. Small molecules are able to diffuse through the vitreous rapidly and have very low half-lives whereas large molecules have retarded diffusion that results in longer half-lives [20]. Large, hydrophilic molecules are able to passively diffuse through the vitreous, around the lens into the anterior chamber and are cleared through the Schlemm’s canal or the uveoscleral outflow pathway. This pathway is slower than the posterior elimination pathway and is the main route of clearance for the majority of substances cleared from the tissue [19]. The posterior route implies loss across the retina through active receptor-mediated transcytosis across the retinal pigment epithelium and is reserved for small, lipophilic molecules. This mode of transport is often impermeable to even proteins such as albumin, but actively transports small lipophilic molecules like iodide, fluorescein and indotheacin. Upon elimination through the retina, these substances are eliminated through the extensive choroidal vasculature that underlies the retina [19]. Due to the large surface area of the retina, molecules capable of posterior clearance have significantly lower half-lives in comparison to those eliminated through the anterior route [21].

1.3 Pathogenesis of diabetic retinopathy

1.3.1 Introduction to angiogenesis

Angiogenesis is the process by which new blood vessels sprout from pre-existing ones. It is a process critical to human development, reproduction and wound healing but also is a key player in the progression of cancer to metastatic levels and vision loss in various types of retinopathies [22]. Situations resulting in the demand for oxygen and nutrients such as diabetic retinopathy, tumor growth or myocardial infarction result in angiogenesis through

6 activation of endothelial cells and supporting cells. Several signaling pathways are responsible for the tight regulation of angiogenesis in development and wound healing as well as its rampant activity in disease progression [23]. Numerous signaling families have been identified as major players in angiogenesis. Pro-angiogenic factors include members of the vascular endothelial growth factor (VEGF) family, angiopoietins, transforming growth factors (TGF), platelet-derived growth factor (PDGF), tumor necrosis factor-α, interleukins and members of the fibroblast growth factor (FGF) family [22]. In addition to protein-based control, cell-matrix and cell-cell interactions through integrins such as α vβ3 and α vβ5 as well as inputs from collagens and HyA has also been implicated in angiogenesis [24]. Negative regulators of angiogenesis have also been identified and include proteins such as thrombospondin, angiostatin, soluble fms-like toll like receptor-1 (sFlt-1), and pigment epithelium-derived factor (PEDF) [25]. These anti- angiogenesis factors are activated once tissue vascularity to supply adequate nutrients and oxygen has been reached. Although all the proteins contribute significantly to the process of angiogenesis, this review will cover the importance of only the vascular endothelial growth factor family.

1.3.2 The vascular endothelial growth factor family

The VEGF family is composed of several growth factors and receptors. Five major growth factors are expressed that are able to bind to 4 receptors, as demonstrated in Figure 1.5. In our studies, we were mainly concerned about inhibiting VEGF-A, as this is the VEGF that is responsible for inducing the strongest pro-angiogenic response. The human VEGF-A gene is made up of eight exons and seven introns, which, through alternative splicing, results in the expression of four different VEGF-A isoforms: VEGF-121, VEGF- 165, VEGF-189 and VEGF-206 (the isoform number corresponds to the number of amino acids that isoform contains) [26]. VEGF-165 is the predominant isoform of VEGF-A and is found as a 45 kDa heterodimeric glycoprotein with a heparin binding domain. VEGF-121 is freely soluble due to a loss of the heparin binding domain whereas isoforms -189 and -206 are completely membrane bound due to their basic nature and high affinity to heparin [27].

Figure 1.5: The VEGF family of proteins. The VEGF proteins, which include VEGF-A, -B, -C, -D and PIGF, are able to bind to 5 receptors: VEGFR-1, VEGFR-2, VEGFR-3, neuropilin-1 and neuropilin-2 (NRP1/2). The receptors are tyrosine kinases that are able to induce downstream signaling activation once the ligand binds. VEGFR-1, -2 and -3 contain seven Ig-like loops, a transmembrane domain and a cytoplasmic tyrosine kinase domain. Arrows indicate binding capabilities. Receptors are also able to form heterodimers between themselves, i.e. one VEGFR-1 receptor can bind with one VEGFR-2 receptor to form a VEGFR- 1/VEGFR-2 heterodimer upon binding of the ligand.

The importance of VEGF has been demonstrated in many studies using both in vitro and in vivo models. Using cell-based models, researchers determined early on that VEGF was required for endothelial cell survival by preventing apoptosis [28]. Disruption of even a single VEGF allele leads to premature death of neonatal mice, however does not affect the viability of adult mice [29,30]. Interestingly, loss of VEGF in established tumor vasculature does not result in endothelial cell death, a process that is hypothesized to be due to the presence of stabilizing pericytes surrounding endothelial cells [31]. VEGF is also a critical regulator of inflammatory processes through inducing vascular permeability [32]. High concentrations of VEGF in the vitreous not only results in extensive retinal angiogenesis but also increases fenestrations between endothelial cells, resulting in the formation of pooled blood [33]. The activity of VEGF is mediated primarily through two receptors within the VEGF family as demonstrated in Figure 1.5. VEGF receptor 1 (VEGFR1) and 2 (VEGFR2) are found on endothelial cells as well as bone marrow-derived cells [26]. Both receptors have 7 Ig-domains and are able to form homo- and heterodimers with one another upon VEGF binding to one receptor monomer. Although the receptors are structurally very similar, the two receptors have several significant differences. VEGFR1 (Flt1) has a high affinity for VEGF on the endothelial cell surface, however it has very weak tyrosine kinase activity, implying weak mitogenic capabilities. An alternatively spliced form of VEGFR1 (soluble Flt- 1, sFlt-1) is freely soluble and has strong anti-VEGF activity [26]. It has been hypothesized that the primary functions of VEGFR1 and sFlt-1 are to act as decoy receptors for VEGF to inhibit excess angiogenesis in tissues [34]. This hypothesis has been confirmed using Flt1-/- knockout mice, which showed that lack of the Flt1 gene results in excessive proliferation and disorganization of angioblasts and blood vessels [35]. VEGFR2 (Flk1), on the other hand, is an active receptor tyrosine kinase that is able to activate downstream signaling cascades resulting in endothelial cell proliferation, adhesion, migration and overall organization into new blood vessels. The importance of this protein is evidenced by Flk1-/- knockout models where mice without VEGFR2 protein fail to develop proper vasculature and die between day 8.5 and 9.5 in utero [36]. VEGF activation of VEGFR2 results in the phosphorylation of several downstream proteins including phospholipase C-γ, PI-3 kinase, Ras GTPase- activating protein and the Src family [23].

1.3.3 Mechanisms of DR Progression

The retina is one of few tissues that do not require insulin for transportation of glucose into retinal cells. In patients with diabetes, high glucose content in the blood drives more glucose to accumulate within retinal cells. The increase in intracellular glucose leads to an increase in reactive dicarbonyls that can react with amino groups on intracellular and extracellular proteins to form advanced glycation end products (AGE)[37]. AGEs damage cells through altering protein activity, affecting the binding of cell surface integrins to AGE- modified extracellular matrix components, and inducing NF-kB activation by binding to AGE receptors on endothelial cells and macrophages[37]. Pericytes specifically express high levels of AGE receptors (RAGEs), which make these cells more susceptible to changes in hyperglycemic by-products, most likely as a protective mechanism to shield the neuronal retina from damage [38]. RAGE activation induces nuclear factor kappa B (NF-kB) signaling which controls several genes involved in inflammation. High concentrations of RAGE ligands, i.e. AGE in diabetes establishes a positive feed back cycle leading to chronic inflammation in the retina and recruitment of leukocytes to the microvasculature resulting in occlusion. AGE receptor binding induces the formation of intracellular reactive oxygen species, which leads to pericyte apoptosis due to disruption of mitochondrial electron transfer chain or enzymatic activities such as NADPH oxidases or lipoxygenases [39,40]. To reverse the detrimental effects of hyperglycemia in the retina, endothelial cells attempt to mitigate the hypoxic situation through upregulation of pro-angiogenic growth factors such as VEGF [41]. In diabetic retinopathy, VEGF-driven retinal angiogenesis is initiated by hypoxic retinal neurons and due to pericyte loss and lack of inhibitory signaling, endothelial cells proliferate to form disorganized, leaky blood vessels and microaneurysms, one of the first visible hallmarks of disease initiation. Progression of DR from nonproliferative (NPDR, lack of angiogenesis) to proliferative DR (PDR) is characterized by angiogenesis of compromised vessels into the vitreous[41]. Several studies have examined the presence and distribution of the VEGF family in post-mortem patient samples, which have revealed significantly increased levels of VEGF throughout samples taken from diabetic eye tissues including the vitreous, aqueous humor and retina[42]. Animal models of NPDR have shown increases in VEGF and VEGFR2 expression, which lead to an increase in vascular permeability[43,44]. Furthermore, transitioning from NPDR to PDR results in a significant increase of intravitreal VEGF concentration which initiates the growth of blood vessels into the vitreous[45,46]. Symptoms of both types of diabetic retinopathy are primarily driven by physiological changes in the retinal microvasculature[47,48]. In non-proliferative diabetic retinopathy (NPDR), these changes primarily manifest through loss of vascular pericyte coverage, formation of sac-like microaneurysms, gradual thickening of the remaining endothelial cell vessels through cell proliferation, occlusion and death of capillaries. Furthermore, pericyte drop-off leads to the leakage of vascular contents into retina resulting in macular edema, and thus a significant reduction in visual acuity. Without sufficient treatment, NPDR gradually

9 develops into proliferative diabetic retinopathy (PDR), which is characterized by the detachment of retinal blood vessels from the retina and subsequent growth into the vitreous. This stage is driven by extensive endothelial cell proliferation and angiogenesis in the formation of vascular loops extending perpendicular to the retina infiltrating into the vitreal chamber, a process driven by heightened levels of intraocular VEGF[7,38,48]. Due to a lack of pericytes, these newly formed blood vessels have wide pores and release red blood cells into the vitreous, resulting in a substantial reduction in visual clarity. Anchoring of these blood vessels into the matrix of the vitreous can cause retinal detachment as a result of vascular contractions.

1.4 Ocular drug delivery and current strategies for DR management

1.4.1 Pharmacological interventions There are several anti-VEGF drugs on the market that are currently used for treating patients with diabetic retinopathy and other retinal neovascular diseases. Schematics depicting the most commonly used anti-VEGF drugs are shown in Figure 1.6.

Bevacizumab (Avastin, Genentech) Bevacizumab is a humanized recombinant full-length antibody at 148 kDa that is clinically identical to ranibizumab in its ability to inhibit all isoforms of VEGF in vivo [49]. Bevacizumab works by completely blocking the ability of all VEGF isoforms from binding to their receptors on the cell surface, resulting in blockage of all downstream pro-angiogenic activity. In pre-clinical studies with rhesus monkey, the terminal half-life of bevacizumab was 5.6 days [50]. In human trials, the half-life was between 2.5 and 7.3 days, with a mean of 4.9 days and depended heavily on state of ocular health [51]. Despite its large size, Heiduschka et al. used immunohistochemistry and radio active labeling to demonstrate that bevacizumab was able to penetrate through the ILM, ganglion cell layer, inner plexiform layer, photoreceptor, and outer segments after two weeks following intravitreal injection in cynomolgus monkeys [52]. Cost-wise, bevacizumab injections are much cheaper and cost $50 per injection, which is the reason many ophthalmologists use bevacizumab off label for treating patients with DR instead of ranibizumab.

Figure 1.6: Commonly used anti-VEGF drugs for treating diabetic retinopathy. Bevacizumab is a full- length recombinant humanized antibody that can bind all isoforms of VEGF. Ranibizumab is FDA approved for the treatment of eye diseases and is an IgG antibody fragment that binds all isoforms of VEGF. Pegaptanib is composed of a 28 kDa aptamer conjugated to two 20 kDa PEG chains. Aflibercept is composed of the 2nd Ig-like domain of VEGFR-1 (yellow), the 3rd Ig-like domain from VEGFR-2 conjugated to an Fc antibody fragment. (Purple indicates antibody heavy chain and its fragments in bevacizumab, ranibizumab and aflibercept. Green indicates light chain in bevacizumab and ranibizumab. Orange indicates optimized antigen binding site on ranibizumab. Blue chain indicates PEGylation (2x 20 kDa PEG chains). Blue region on aflibercept corresponds to the VEGFR-2 Ig-like domain 3 and the yellow region depicts the VEGFR-1 Ig-like domain 2)

Ranibizumab (Lucentis, Genentech) Ranibizumab is a 48 kDa IgG antibody fragment that is able to bind all isoforms of VEGF. By binding to the receptor binding site on all VEGF isoforms, ranibizumab is able to completely inhibit the interaction between VEGF and receptors responsible for initiating angiogenesis [53]. Ranibizumab is smaller than bevacizumab and was created with the intention to allow for drug penetration into the retina to target neovascular diseases that affect deeper retinal layers, a theory confirmed by Gaudrault et al. [54]. Studies in rabbits have demonstrated that an injection of 0.5 mg ranibizumab yields a half-life of 2.88 days [55], which matches studies in monkeys where the half-life is 2.9 days [50]. Humans receive 0.5 mg of ranibizumab, which yields an intravitreal half life of 7.2 days [56]. Lucentis is the currently approved FDA drug for treatment of diabetic retinopathy and costs $2,000 per injection, which is cost limiting for many patients.

Pegaptanib (Macugen, Bausch + Lomb) Pegaptanib is a 50 kDa PEGylated drug composed of a 28 kDa nucleic acid aptamer and 20 kDa PEG chain. It is highly specific to VEGF165 and has a high dissociation constant (50 pmol/L) [57]. The binding site for pegaptanib on VEGF165 is on its heparin-binding domain that is responsible for stabilizing and intensifying the interaction of VEGF165 and its receptor on the cell surface for activation of neovascularization. Although its high affinity for VEGF165 is advantageous, this specificity does not allow pegaptanib to bind any other isoforms of VEGF in contrast to the other anti-VEGF drugs [58]. An advantage to using an RNA-based molecule for drugs is that these molecules have very little immunogenicity and toxic side effects. Despite increasing the overall molecular weight of pegaptanib with a PEG chain, this drug still suffers from low half-lives within the vitreous. The half-life of 2 mg pegaptanib injected intravitreally in Rhesus monkeys is 89.9 hours (3.7 days) [59] whereas a 3 mg injection in humans has a half-life of 10 days (±4 days) [60].

Aflibercept (Eylea, Regeneron) Aflibercept is a highly potent 115 kDa anti-VEGF fusion protein developed using the “trap technology” in which the 2nd Ig domain from VEGFR1 and the 3rd Ig domain from VEGFR2 were fused to the Fc region of a human IgG antibody [61]. Similar to ranibizumab and bevacizumab, aflibercept is able to bind all VEGF isomers in addition to VEGF-B and 11 placental growth factor, which may make aflibercept more effective than its competitors [62]. Aflibercept was optimized for maximal VEGF binding and has 140 greater binding affinity for VEGF and its isomers in comparison to ranibizumab and bevacizumab [63]. Although there is a significant increase over the other anti-VEGF drugs in binding ability, the half-life of aflibercept is still very short (5 days in humans) and requires the same dosing regimen as the other drugs.

1.4.2 Retinal drug delivery systems

Drug delivery systems are in high demand for treating retinal diseases [64–66]. Commonly used modalities such as eye drops, subconjunctival injections and systemic drug delivery suffer from inability to make it through retinal and ocular barriers to the disease site. Topical eye drops are rapidly cleared from the tear film resulting in residence times of less than 5 minutes. Systemically delivered drugs are often impeded by the blood-retina barrier and penetration through the sclera is the main limiting barrier for subconjunctivally delivered drugs. Thus, the majority of drugs for retinal diseases are delivered through intravitreal injection. However, these drugs, including the anti-VEGF drugs discussed above, suffer from short half-lives in the vitreous and require frequent injections. For these reasons, there are been many attempts at using biomaterials and biomedical devices for increasing the half- life of drugs in the vitreous. This review will cover drug delivery systems for all retinal diseases, as there have been significant strides made in drug delivery for other disorders such as uveitis.

1.4.2.1 Implants

The goal of intravitreal implants is to deliver drugs over a period of time that can span months to several years, thus reducing the number of invasive procedures patients receive. In general, implants are surgically placed at the pars plana, posterior to the lens and anterior to the retina. This approach presents several benefits including: (1) by-passing the blood- retinal barrier; (2) delivery of drug at the site of action; (3) avoidance of the side effects associated with frequent intravitreal and systemic injections; and, (4) smaller quantity of drug used during treatment. Several types of implants have been developed and vary in degradability, duration of effect, size, delivery method and composition. Implants are generally defined as degradable or non-degradable where non-degradable implants often require surgical removal but are able to release drugs for much longer periods of time. A comparison of several implants is shown in Table 1.1 and schematics of implants discussed in this chapter are shown in Figure 1.7.

Table 1.1: Summary of implants used for treating retinal diseases.

Abbreviations: AMD: age-related macular degeneration, BRVO: branch retinal vein occlusion, CMV: cytomegalovirus, CNTF: ciliary neurotrophic factor, CNV: choroidal neovascularization, CRVO: central retinal vein occlusion, DME: diabetic macular edema, EVA: ethylene-vinyl acetate, IVT: intravitreal, MT: macular telangiectasia, NTC-200: genetically modified retinal pigment epithelium cell line, PDGFR: platelet-derived growth factor receptor, PLGA: poly(lactide-co-glycolide), PMMA: poly(methyl methacrylate), PVA: poly(vinyl alcohol), RVO: retinal vein occlusion, VEGFR-Fc: vascular endothelial growth factor receptor-Fc fusion protein.

Retisert The Retisert implant was developed by Bausch & Lomb Inc. (Rochester, NY) as a non- degradable, sustained-release system for the long-term treatment of posterior uveitis, DME and RVO. The implant contains 0.59 mg of the small molecule fluocinolone acetonide (molecular weight 452.5 Da) and releases 0.6 ug/day initially and decreases to 0.3-0.4 ug/day for up to 30 months [67]. Retisert is approximately the size of a grain of rice fixed to the pars plana with a suture and is composed of silicon and polyvinyl alcohol for encasing of the aqueous insoluble corticosteroid. A 3 year-clinical trial with 197 subjects showed that visual acuity was improved by 3 or more lines in 28% of implanted eyes in comparison to 15% of the control [68]. Treatment with Retisert also decreased the recurrence rate of uveitis from 54% to 7% in addition to improving visual acuity in 80% of patients. Despite positive outcomes in clinical trials, 30% of patients required glaucoma surgery by 24 months and 93- 100% of patients required cataract surgery [69]. Due to these significant side affects, Retisert becomes a reasonable alternative when patients do not respond to systemic or topical delivery of corticosteroids.

Iluvien (Medidur) Iluvien (Alimera Sciences, GA, USA) is another corticosteroid eluting implant used in treating uveitis, macular edema and retinal vein occlusion. The device is non-degradable and contains 180 ug of fluocinolone acetonide for delivery up to 3 years [70]. Contrary to

Retisert, which requires surgical implantation, Iluvien is inserted through a 25-gauge needle without the need for sutures. The implant is 3.5 mm in length and 0.37 mm in diameter and releases 0.23-0.45 ug of drug per day for 18-36 months. Clinical trials with this implant showed that 20% of patients with DME showed significant improvements in visual acuity tests by the end of 12 months [71]. One major drawback of this approach is that the implant is not retrievable, meaning that patients would have several Iluvien implants over time, which could cause visual defects. However, clinical outcomes with Iluvien have shown that only 0-5.9% of patients need glaucoma surgery and 15-29% require cataract after treatment, much lower than patients treated with Retisert [71].

I-vation (Surmodics) I-vation (Surmodics Inc.) is another non-biodegradable implant used for treating diabetic macular edema and uveitis through the sustained delivery of 0.93 mg triamcinolone acetonide (TA) [72]. The titanium implant is in the shape of a helical coil and is composed of the non-degradable polymers poly(methylmethacrylate) and ethylene-vinyl acetate, which can release drug for up to 2 years. Its unique helical shape enables for high surface area and maximal drug loading as well as sutureless placement through a 25-gauge hole in the sclera [66]. In clinical trials, 64% in the slow-releasing group and 72% in the fast-releasing group showed significant increases in visual acuity with 28.6% of patients in the fast-releasing group showing greater than 15-letter improvement after 6 months. Despite these positive results, further clinical trials have been suspended when data released showed better results with photocoagulation over TA treatment for DME [72].

Vitrasert Similar to Retisert, Iluvien and I-vation, Vitrasert (Bausch + Lomb) is an FDA-approved, non-degradable implant that has a drug reservoir of ganciclovir, another corticosteroid used for the treatment of cytomegalovirus CMV retinitis and posterior uveitis [73]. Two separate polymer coatings tightly control release from the drug reservoir. Polyvinyl alcohol is highly permeable to allow the release of drug and ethylene vinyl acetate is impermeable and limits the surface area through which the drug can be released. This system is also being tested for the release of other corticosteroids including dexamethasone, fluocinolone acetonide and cyclosporine. Similar to Retisert, Vitrasert is associated with high likelihood of cataract development and increased IOP [73]. Unlike the other implants available, insertion of Vitrasert requires a large 4-5 mm scleral incision at the pars plana in an operating room and after drug release is complete 5-8 months later, a second surgery is required for excision of the implant followed by replacement, if necessary.

Ozurdex Unlike Iluvien and Retisert, Ozurdex (Allergan, Inc.) is a biodegradable implant made of PLGA that releases 0.7 mg of dexamethasone over 6 months [74]. It has been approved for use in macular edema following branch or central retinal vein occlusion and is currently

14 under clinical trials for posterior and intermediate uveitis. Similar to Iluvien, the Ozurdex implant does not require a suture placement like Retisert. One main advantage to using Ozurdex is that the implant slowly degrades over the course of 6 months to lactic and glycolic acid, leaving no trace in the eye. Implantation is simple and facilitated through a specialized applicator with a 22-gauge needle. In clinical trials, 53% of patients showed improvement of at least 3 lines and only 0.5% and 4% needed glaucoma or cataract surgery, respectively [74,75].

On Demand Therapeutics On Demand Therapeutics developed ODTx, a unique multi-reservoir implant that is driven by laser-activation of individual drug-containing reservoirs [76,77]. Similar to Iluvien, ODTx is a small rod that can be easily injected and does not require scleral suturing. The drug reservoirs can store small molecule or protein-based drugs until release is initiated by targeting of the device with a standard ophthalmic laser in a routine office-based procedure. In testing with protein release, the device was able to maintain protein stability for up to 1- year post-device injection. The devices are meant to give the ophthalmologist more control over drug dosing, unlike the other implantable devices, which have no mechanism for dose control. Once the device is depleted of drug, ODTx is unretrieveable, much like Iluvien. Furthermore, targeting a small implant and accurately choosing loaded drug reservoirs may be difficult in practice.

Figure 1.7: Schematics of implant-based ocular drug delivery. These implants have been developed to treat a range of retinal diseases including uveitis, DME, and RVO. See text for details of each device.

Encapsulated cell technology Encapsulated cell technology (ECT) is a platform developed by Neurotech Pharmaceuticals that harnesses the ability of a modified retinal pigment epithelial cell line to secrete therapeutic levels of protein that can impede the progression of chronic disorders in the central nervous system and the eye [78,79]. There are currently 3 implants being developed, 2 of which are in phase 2 of clinical trials for treatment of wet AMD and macular telangiectasia. The main advantage of this approach is that it presents an efficient and continuous way of delivering protein-based therapies unlike the other implants and does so over for at least 2 years. The basis of the technology is dependent on protein secreted from a proprietary cell line derived from human retinal pigment epithelial cells. Retinal pigment epithelial (RPE) cells were screened from a large library of different cell types and ultimately chosen due to their ability to thrive under low oxygen and nutrient conditions for many months and being amenable to genetic manipulation [78]. The RPE cells are genetically modified to suppress proliferative genes and are programmed to overexpress proteins including a VEGF receptor (NT-503 ECT), platelet-derived growth factor (PDGF, NT-506 ECT) and ciliary neurotrophic factor (CNTF, NT-501 ECT). The protein secreting cells are encapsulated within a semi-permeable membrane that prevents immune cell and antibody infiltration from the vitreous and surrounding tissues but allows for the secretion of protein from inside the implant. In comparison to the other implants, this implant is larger and measure 1 cm in length [78] and 1 mm in width and is anchored to the sclera with a titanium loop, requiring surgical implantation similar to many of the other implants. Extensive clinical trials have been carried out to test the first- generation implant, Renexus (NT-501) for treatment of retinitis pigmentosa, which secretes CNTF. After 6 months, implants retrieved from 10 patients in a phase I clinical trial contained viable cells with minimal cell loss [80]. In a 12-month phase II trial, patients with the high-dose NT-501 implant showed a dose-dependent increase in retinal thickness that lasted through 12 months which was associated with visual function stabilization and stable visual acuity. A trend in greater vision loss was observed for the low-dose/sham treated eyes. Their findings suggest that CNTF delivery can help prevent vision loss in patients with wet AMD [81]. This technology has been further developed and now spans implants, which are capable of having multiple cell reservoirs. The NT-503 implant secretes a VEGF receptor fusion protein, which has a 20-fold increase in binding to VEGF compared to ranibizumab (Lucentis). This implant secretes the anti-VEGF protein for more than 2 years with currently on-going clinical trials to test its efficacy in patients with wet AMD [82]. The NT-506 implant secretes a combination of the VEGFR-Fc fusion protein and PDGF as a response to clinical results that indicated improved outcomes with such combination therapies. Optimization studies at Neurotech Pharmaceuticals have been able to increase release rates of protein by 30-fold through increasing the number of cell-chambers, cell counts, membrane permeability and overall device size.

1.4.2.2 Particulate systems

Another approach to controlling intravitreal drug delivery is through various encapsulation technologies. The use of nanoparticles and liposomes has been investigated in depth over the last two decades and is schematically shown in Figure 1.8. Generally biodegradable polymers such as gelatin, albumin, polyesters, polyanhydrides, poly-lactic acid (PLA),poly-glycolic acid (PGA), and PLGA copolymers are preferred because they slowly degrade at the site of injection over time. Of those polymers, PLA, PGA and PLGA used most frequently because they are materials used in other FDA approved devices for use in the clinic. Particle systems have been successful at delivering various types of drugs over long periods of time ranging from weeks to even several months. Although the technology is promising, clinical success has been minimal.

Figure 1.8: Basics of particulate-based drug delivery vehicles. Nanoparticles and liposomes are formed by organization of repeating units consisting of a hydrophilic head group and a hydrophobic tail group. Both particles can be used to delivery hydrophilic and hydrophobic drugs. Nanoparticles can be used to delivery hydrophilic and hydrophobic drugs in on the surface of the vesicle or in the hydrophobic core, respectively. Liposomes can also deliver both types of drugs and can store hydrophilic drugs in its aqueous core or on the liposomal surface. Hydrophobic drugs can be stored within the lipid bilayer of liposomes.

The major advantages to using particle-based systems are similar to other approaches and include drug stabilization, reduction in toxicity of certain drugs, reduction in burst drug exposure and localized delivery. The main drawback to this approach is vitreous clouding, which is well documented and is a result of particle aggregation. The permanence is variable and depends on the degradability of the polymers and amount of particle injected. Furthermore, long-term storage of particle systems results in the formation of heterogeneous aggregates due to particle instability.

Nanoparticles Nanoparticles are less than 1um in diameter, made of biodegradable polymers such as PLA, PLGA, natural biopolymers such as chitosan, gelatin, alginate, albumin, and lipids. Drugs can be stored either within the nanoparticle if they are hydrophobic/lipophilic or tethered to the outside of the nanoparticle if they are hydrophilic (Figure 1.8). Due to their small size, nanoparticles are able to distribute throughout the posterior segment, penetrating retinal layers and accumulating in the retinal pigment epithelium cells unlike microspheres. Nanoparticles with these pharmacokinetic properties could be very useful in treating diseases such as choroidal neovascularization and due to their ability to penetrate to the posterior layers of the retina where these diseases are situated. Albumin nanoparticles have shown promise in bioavailability and duration of release. In vivo rat studies showed that after a single injection, anticytomegaloviral drug containing albumin nanoparticles were detected in the vitreous and ciliary body for at least two weeks after a single intravitreal injection [83]. Nanoparticles have the added benefit of being small enough to penetrate layers of the retina, as demonstrated by Kim et al. [84]. The authors used albumin nanoparticles between 110 and 170 nm in size to examine the effect of overall nanoparticle charge on ocular distribution following intravitreal injection. Anionic albumin nanoparticles easily penetrated the vitreous and localized with in retinal cell layers however the cationic nanoparticles were bound to the negatively charged proteoglycans like collagen and hyaluronic that caused nanoparticle sequestration in the vitreous [84]. Similarly, Bourges et al. [85] showed significant transretinal movement of fluorophore containing PLA nanoparticles with preferential localization in RPE cells after 18-24 hours and remained detectable in the RPE layer 4 months after a single injection of PLA nanoparticles [85]. Santos et al. [86] investigated the efficacy of delivering anti-TGFB-2 oligonucleotide- polyethylenimine complexes, which formed nanoparticles in solution. After 24 hours, these complexes were uniformally distributed throughout the retina and at 72 hours had specifically accumulated in their cell of interest, the retinal Müller glial cells [86]. In addition to penetrating retinal layers following intravitreal injections, nanoparticles have also been shown to cross the blood-retinal barrier following intravenous injection as shown by Kim et al. [87]. Nanoparticles that were 20nm in size could easily pass through the blood-retinal barrier and were evenly distributed in all layers of the retina. In contrast, 100 nm particles were not detected in any retinal cell layer [87].

Liposomes Liposomes were first introduced in 1965 as a novel drug delivery carrier [88]. These vesicles can vary widely in size ranging from 10 nm to 1 um or greater. Liposomes have an aqueous core surrounded by phospholipid bilayers made of natural or synthetic substances. Incorporation of cholesterol adds rigidity to the liposomes. The drug loading capacity of liposomes depends heavily on its size as well as the type of molecule being delivered. An advantage to using liposomes is their ability to encapsulate both lipophilic and hydrophilic molecules within the phospholipid bilayer and aqueous core, respectively (Figure 1.8). Similar to the other particle-based drug delivery systems, liposomes can improve drug pharmacokinetics and reduce toxicity associated with higher drug doses and help avoid burst drug delivery. The use of liposomes in ocular drug delivery systems has been extensively explored due to the versatility of the system. Positively charged liposomes have been tested for their ability to increase the half life of drugs in the vitreous due to the charge association between the positively charged liposomal surface and negatively charged biopolymers found naturally in the vitreous like collagen and hyaluronic acid. There are several liposome-based drug delivery systems that are clinically approved including Ambisome, DaunoXome, and Novasome however only Visudyne is approved for retinal applications, specifically for the treatment of age-related macular degeneration. Verteporfin (Visudyne, Novartis) is currently the only FDA approved liposomal technology for clinical use in ocular applications [89,90]. The drug is injected intravenously and is able to locate to the retina where it can be activated by applying a non-thermal red laser. Activated verteporfin causes localized damage to the neovascular endothelium and results in occlusion of the targeted vessels in treatment of patients with choroidal neovascularization and age-related macular degeneration. Visudyne is still considered a suboptimal therapy and other liposomal technologies are being developed to improve upon it. Rostaporfin (Photrex, Miravant Medical Technologies) is a newer photosensitizing agent that aims to treat age-related macular degeneration with pending FDA approval. Rostaporfin improves significantly on the verteporfin technology by decreasing the number of required treatments [91]. The pharmacokinetics of liposomes in the vitreous were initially investigated by Barza et al. Larger vesicles were cleared faster than smaller ones and intraocular inflammation significantly increased vesicle clearance rate for both sizes. Furthermore, the addition of cholesterol to the liposome stabilized the vesicle and slowed drug release into the vitreous [92]. Paasonen et al. investigated the incorporation of gold-nanoparticles for user- mediated control of drug release. The addition of nanoparticles to the inner and outer layers of the liposomes enabled the vesicles to have a heat-responsive element for controlling the release of calcein [93]. Upon UV irradiation, the gold nanoparticles absorbed the heat, which caused the liposome to undergo a phase change and release its calcein. Without the incorporation of the gold nanoparticles, the liposomes had no response to UV light. Bevacizumab has also been incorporated into liposomes made of phosphatidylcholine and cholesterol to increase its intravitreal half-life. The authors found that incorporation into

19 liposomes increased the intravitreal concentration of bevacizumab by 1 and 5 times at days 28 and 42, respectively. Despite some successes, the advancement of liposomes to clinical trials has been limited due to their short shelf life, limited drug cargo capacity, complexity of preparation and stability, and problems in sterilization.

1.4.2.3 Hydrogels

Verisome Verisome is a versatile hydrogel technology made of benzyl benzoate that can be used to deliver a broad range of pharmaceutical agents such as small molecules, peptides, proteins and antibodies and can take the form of a liquid, gel or solid [94]. The technology is being tested for several applications including retinal drug delivery. Verisome is injected through a 30-gauge needle in the form of a drug-containing hydrogel that coalesces into a spherule and slowly degrades to release the active agent. Similar to the Ozurdex biodegradable implant, Verisome drug release can be indirectly monitored by examining the size of the spherule in the patient’s vitreous. Phase I clinical trials demonstrated safety and showed drug efficacy in patients with CME secondary to RVO. Despite promising early results, this delivery system quickly degrades and would require more injections than the comparable biodegradable device, Ozurdex [94]. Verisome has also been used to deliver ranibizumab, which is currently undergoing a phase II clinical trial. At 120 days post injection, 3 out of 6 patients needed reinjection and at 180 days, only 1 of 5 needed a repeat injection, indicating that this technology may helped to reduce the number of required drug injections. Kang-Derwent et al. demonstrated the efficacy of using thermoresponsive hydrogels for slow release of bevacizumab and ranibizumab in vitro as another potential drug delivery system [95]. Thermoresponsive hydrogels were synthesized with poly(N- isopropylacrylamide) (PNIPAAm) crosslinked with poly(ethylene glycol) diacrylate (PEG- DA). Gels showed an expected burst release in the first 48 hours and then steady drug release for 3 weeks. The crosslinking density could be adjusted to control the rate of release. Although promising, this work was not followed up with in vivo studies to demonstrate efficacy. Hydrogel-based delivery of ocular therapeutics has also been explored using iontophoresis [96]. This technique is suitable for the charge-dependent transport of small molecules across tissue membranes. Eljarrat-Binstock et al. showed that gentamicin, methylprednisolone and carboplatin (drugs that are used to treatment of infection, uveitis and retinoblastoma, respectively) could be effectively delivered by encapsulating drugs in hydrogels of hydroxyethyl methacrylate (HEMA) crosslinked with ethylene glycol dimethacrylate (EDGMA). Sponges were surgically placed at the pars plana of rabbits and cathodal iontophoretic administration was performed by using a current intensity of 1.0 mA

20 for 5 minutes. Significantly higher concentrations of drug were found in the retina, vitreous and aqueous humor after iontophoresis compared with control groups [97–99].

Cortiject Cortiject is an emulsion-based drug delivery system for the sustained release of the pro-drug dexamethasone for treatment of DME [77]. This approach utilizes an oily carrier and a phospholipid as a surfactant for the encapsulation of the drug. Once released, the dexamethasone pro-drug is de-esterified by esterases in the retina to become activated. A single intravitreal injection is anticipated to release drug for 6-9 months. Phase I/II clinical trials are currently underway in Europe.

1.4.2.4 Polymer conjugation

Polymer conjugation of drugs for ocular applications is in developmental stages with few approved for clinical use. The most famous example is the PEGylated drug, Macugen (pegaptanib sodium, EyeTech Pharmaceuticals/Pfizer), which is FDA approved for treating age-related macular degeneration. Macugen is an RNA aptamer that is conjugated to two chains of 20 kDa PEG and specifically targets VEGF-165 [57]. PEGylation of the aptamer significantly improved its half-life in the vitreous [100], however the half-life of Macugen is comparable to the other protein-based therapeutics targeting VEGF such as Lucentis, Avastin and Eylea. We recently demonstrated that conjugation of the anti-VEGF protein, sFlt-1, to hyaluronic acid (HyA) showed significant improvements over its unconjugated form in preliminary studies. We demonstrated using several different molecular weights and valencies of sFlt to HyA that the conjugation did not reduce the affinity of sFlt for VEGF. We also created an in vitro model of the vitreous using crosslinked HyA gels and showed that conjugation to HyA significantly slowed the diffusion of sFlt out of the gels, a good indication that the same effect would be seen in vivo [101]. Our in vivo studies utilized 2 in vivo models of angiogenesis and a rat vitreous model to determine whether conjugation affected the ability of the conjugate to inhibit angiogenesis in vivo and whether conjugation would lead to increased half-life in the vitreous of rats. Our results showed that the conjugate was equally able to inhibit corneal angiogenesis in comparison to the unconjugated sFlt but was more effective at inhibiting retinal angiogenesis in a rat oxygen-induced retinopathy model, most likely due to the significantly increased drug half-life. Finally, the sFlt conjugate showed significantly improved half-life in the rat vitreous. Our results are promising when compared to the other conjugation technology, PEGylation, because we demonstrate that bioactivity is maintained following the conjugation whereas PEGylation results in significantly reduced bioactivity [102]. This is likely due the large PEG chain sterically inhibiting the active site of the protein from binding to its ligands. In contrast, the technology we use allows for the conjugation of several proteins per chain of HyA, enabling

21 for the presentation of multiple bioactive proteins, which likely compensates for steric effects.

1.5 Dissertation approach and outline

The work in this dissertation aims to improve upon anti-VEGF drugs that are currently used in the clinic by increasing the half-life of drugs in the vitreous of the eye.

A hyaluronic acid-based multivalent drug conjugate system has been investigated as a treatment for patients with diabetic retinopathy. We hypothesized that multivalent drug conjugates with hyaluronic acid would significantly increase the half-life of drugs in the vitreous of the eye, which would lead to a prolonged anti-angiogenic effect of the drug in vivo. This work could represent a significant step forward in improving therapeutics used for treating retinal diseases due to the versatility of this technology.

This dissertation is organized into 3 sections describing two distinct aims (Chapters 2 and 3). A concluding chapter (Chapter 4) summarizes the dissertation’s main findings and discusses potential future directions for the project.

v Chapter 2: Multivalent hyaluronic acid bioconjugates improve sFlt activity in vitro. The synthesis and characterization of a range of multivalent sFlt (mvsFlt) bioconjugates are described. In depth in vitro assays were carried out to investigate the effect of conjugation on the affinity of sFlt for VEGF. An in vitro vitreous model synthesized from crosslinked HyA is described for the analysis of conjugate diffusion. Lastly, the effect of conjugation on protease degradation is presented.

v Chapter 3: sFlt multivalent conjugates inhibit angiogenesis and improve half- life in vivo. Optimization studies from Chapter 2 were used to select one conjugate to study in vivo. The mvsFlt was initially tested for its ability to inhibit angiogenesis in a corneal angiogenesis model in mice. The effect of conjugation on intravitreal half- life was examined in using the vitreous in rats. Finally, the ability of the mvsFlt conjugate to inhibit angiogenesis over a longer period of time due to increased half- life was examined using an oxygen-induced retinopathy model in rats.

v Chapter 4: Summary of results. Summarizes dissertation’s main findings and explores future steps for the use of multivalent technology in ocular drug delivery.

1.6 References

[1] J. a Bluestone, K. Herold, G. Eisenbarth, Genetics, pathogenesis and clinical interventions in type 1 diabetes., Nature. 464 (2010) 1293–1300. doi:10.1038/nature08933.

[2] M. Stumvoll, B.J. Goldstein, T.W. van Haeften, Type 2 diabetes: principles of pathogenesis and therapy., Lancet. 365 (2010) 1333–46. doi:10.1016/S0140- 6736(05)61032-X.

[3] C.L. Ogden, M.D. Carroll, L.R. Curtin, M. a McDowell, C.J. Tabak, K.M. Flegal, Prevalence of overweight and obesity in the United States, 1999-2004., JAMA. 295 (2006) 1549–1555. doi:10.1001/jama.295.13.1549.

[4] S. Smyth, A. Heron, Diabetes and obesity: the twin epidemics., Nat. Med. 12 (2006) 75–80. doi:10.1038/nm0106-75.

[5] D.P. Schuster, K. Osei, Diabetes in African Americans, Clin. Diabetes. (2006) 535– 549. doi:10.1016/B978-1-4160-0273-4.50044-8.

[6] T.J. Orchard, M. Temprosa, R. Goldberg, S. Haffner, R. Ratner, S. Marcovina, et al., The Effect of Metformin and Intensive Lifestyle Intervention on the Metabolic Syndrome: The Diabetes Prevention Program Randomized Trial, Ann. Intern. Med. 142 (2005) 611–619. doi:15838067.

[7] R. Fong, D.S., Aiello, L., Gardner, T.W., King, G.L., Blankenship, G., Cavallerano, J.D., Ferris, F.L., Klein, Retinopathy in Diabetes, Diabetes Care. 27 (2004) S84–87. doi:10.2337/diacare.27.2007.S84.

[8] D.A. Antonetti, R. Klein, T.W. Gardner, Diabetic retinopathy., N. Engl. J. Med. 366 (2012) 1227–39. doi:10.1056/NEJMra1005073.

[9] E.R. Schoenfeld, J.M. Greene, S.Y. Wu, M.C. Leske, Patterns of adherence to diabetes vision care guidelines, Ophthalmology. 108 (2001) 563–571. doi:10.1016/S0161-6420(00)00600-X.

[10] D.B. Mukamel, G.H. Bresnick, Q. Wang, C.F. Dickey, Barriers to compliance with screening guidelines for diabetic retinopathy, Ophthalmic Epidemiol. 6 (1999) 61–72. doi:10.1076/opep.6.1.61.1563.

[11] R.H. Masland, The fundamental plan of the retina., Nat. Neurosci. 4 (2001) 877–886. doi:10.1038/nn0901-877.

[12] J. Stone, Z. Dreher, Relationship between astrocytes, ganglion cells and vasculature of the retina., J. Comp. Neurol. 255 (1987) 35–49. doi:10.1002/cne.902550104.

[13] E.A. Newman, Functional hyperemia and mechanisms of neurovascular coupling in the retinal vasculature., J. Cereb. Blood Flow Metab. 33 (2013) 1685–95. doi:10.1038/jcbfm.2013.145.

[14] P. Bishop, The biochemical structure of mammalian vitreous, Eye. 10 (1996) 664– 670. doi:10.1038/eye.1996.159.

[15] C.S. Nickerson, J. Park, J. a Kornfield, H. Karageozian, Rheological properties of the vitreous and the role of hyaluronic acid., J. Biomech. 41 (2008) 1840–6. doi:10.1016/j.jbiomech.2008.04.015.

[16] T.C. Laurent, Studies on Hyaluronic Acid in the Vitreous Body, J. Biol. Chem. 216 (1955) 263–271.

[17] T.N. Wight, S. Evanko, F. Kolodgie, A. Farb, R. Virmani, Chemistry and Biology of Hyaluronan, 2004. doi:10.1016/B978-008044382-9/50045-5.

[18] M.M. Le Goff, P.N. Bishop, Adult vitreous structure and postnatal changes, Eye. 22 (2008) 1214–1222. doi:10.1038/eye.2008.21.

[19] S. Maurice, D.M., Mishima, Ocular Pharmacokinetics, Springer, Verlag Berlin Heidelberg, 1984. doi:10.1007/978-3-642-69222-2_2.

[20] L. Pitkänen, M. Ruponen, J. Nieminen, A. Urtti, Vitreous is a barrier in nonviral gene transfer by cationic lipids and polymers., Pharm. Res. 20 (2003) 576–83.

[21] A. Urtti, Challenges and obstacles of ocular pharmacokinetics and drug delivery., Adv. Drug Deliv. Rev. 58 (2006) 1131–5. doi:10.1016/j.addr.2006.07.027.

[22] P. Carmeliet, R.K. Jain, Molecular mechanisms and clinical applications of angiogenesis., Nature. 473 (2011) 298–307. doi:10.1038/nature10144.

[23] N. Ferrara, H.-P. Gerber, J. LeCouter, The biology of VEGF and its receptors., Nat. 24

Med. 9 (2003) 669–76. doi:10.1038/nm0603-669.

[24] C.J. Avraamides, B. Garmy-Susini, J. a Varner, Integrins in angiogenesis and lymphangiogenesis., Nat. Rev. Cancer. 8 (2008) 604–17. doi:10.1038/nrc2353.

[25] R.P. Million, Therapeutic area crossroads: anti-angiogenesis, Nat. Rev. Drug Discov. 7 (2008) 115–116. doi:10.1038/nrd2507.

[26] G. Neufeld, T. Cohen, S. Gengrinovitch, Z. Poltorak, Vascular endothelial growth factor (VEGF) and its receptors., FASEB J. 13 (1999) 9–22.

[27] A. Hoeben, B. Landuyt, M.S. Highley, H. Wildiers, A.T. Van Oosterom, E. a De Bruijn, Vascular endothelial growth factor and angiogenesis., Pharmacol. Rev. 56 (2004) 549–580. doi:10.1124/pr.56.4.3.549.

[28] N. Ferrara, T. Davis-Smyth, The biology of vascular endothelial growth factor., Endocr. Rev. 18 (1997) 4–25. doi:10.1210/edrv.18.1.0287.

[29] J. Plouët, J. Schilling, D. Gospodarowicz, Isolation and characterization of a newly identified endothelial cell mitogen produced by AtT-20 cells., EMBO J. 8 (1989) 3801–3806.

[30] D.W. Leung, G. Cachianes, W.J. Kuang, D. V Goeddel, N. Ferrara, Vascular endothelial growth factor is a secreted angiogenic mitogen., Science. 246 (1989) 1306– 1309. doi:10.1126/science.2479986.

[31] G. Bergers, S. Song, N. Meyer-Morse, E. Bergsland, D. Hanahan, Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors., J. Clin. Invest. 111 (2003) 1287–95. doi:10.1172/JCI17929.

[32] D.O. Bates, Vascular endothelial growth factors and vascular permeability, Cardiovasc. Res. 87 (2010) 262–271. doi:10.1093/cvr/cvq105.

[33] M.J. Tolentino, J.W. Miller, E.S. Gragoudas, F. a Jakobiec, E. Flynn, K. Chatzistefanou, et al., Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate., Ophthalmology. 103 (1996) 1820–1828. doi:10.1097/00006982-199805000-00031.

[34] R.L. Kendall, K.A. Thomas, Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor, Proc Natl Acad Sci U S A. 90 25

(1993) 10705–10709.

[35] G.H. Fong, L. Zhang, D.M. Bryce, J. Peng, Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice., Development. 126 (1999) 3015–3025.

[36] F. Shalaby, J. Rossant, T.P. Yamaguchi, M. Gertsenstein, X.F. Wu, M.L. Breitman, et al., Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice., Nature. 376 (1995) 62–66. doi:10.1038/376062a0.

[37] M. Brownlee, Biochemistry and Molecular Cell Biology of Diabetic Complications, Nature. 414 (2001) 813–820.

[38] H.-P. Hammes, Pericytes and the pathogenesis of diabetic retinopathy., Horm. Metab. Res. 37 Suppl 1 (2005) 39–43. doi:10.1055/s-2005-861361.

[39] G.R. Barile, S.I. Pachydaki, S.R. Tari, S.E. Lee, C.M. Donmoyer, W. Ma, et al., The RAGE axis in early diabetic retinopathy., Invest. Ophthalmol. Vis. Sci. 46 (2005) 2916–24. doi:10.1167/iovs.04-1409.

[40] U. Denis, M. Lecomte, C. Paget, D. Ruggiero, N. Wiernsperger, M. Lagarde, Advanced glycation end-products induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction., Free Radic. Biol. Med. 33 (2002) 236–47.

[41] J.W. Miller, J. Le Couter, E.C. Strauss, N. Ferrara, Vascular endothelial growth factor a in intraocular vascular disease., Ophthalmology. 120 (2013) 106–14. doi:10.1016/j.ophtha.2012.07.038.

[42] G. Lutty, S. Mcleod, C. Merges, A. Diggs, J. Plouet, Localization of Vascular Endothelial Growth Factor in Human Retina and Choroid, Arch. Ophthalmol. 114 (1996) 971–977.

[43] T. Murata, T. Ishibashi, A. Khalil, Y. Hata, H. Yoshikawa, H. Inomata, Vascular Endothelial Growth Factor Plays a Role in Hyperpermeability of Diabetic Retinal Vessels, Ophthalmic Res. 27 (1995) 48–52.

[44] J.W. Miller, a P. Adamis, D.T. Shima, P. a D’Amore, R.S. Moulton, M.S. O’Reilly, et al., Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model., Am. J. Pathol. 145

(1994) 574–84.

[45] D. Ray, M. Mishra, S. Ralph, I. Read, R. Davies, P. Brenchley, Association of the VEGF gene with proliferative diabetic retinopathy but not proteinuria in diabetes., Diabetes. 53 (2004) 861–4.

[46] L. Aiello, R. Avery, P. Arrigg, B. Keyt, N. Ferrara, G. King, Vascular Endothelial Growth Factor in Ocular Fluid of Patients with Diabetic Retinopathy and Other Retinal Disorders, N. Engl. J. Med. (1994).

[47] D. Cogan, D. Toussaint, T. Kuwabara, Retinal Vascular Patterns. IV Diabetic Retinopathy, Arch. Ophthalmol. (1963).

[48] R.L. Engerman, Perspectives in Diabetes Pathogenesis of Diabetic Retinopathy, Diabetes. 38 (1989) 1203–1206.

[49] A. Grothey, E. Galanis, Targeting angiogenesis: progress with anti-VEGF treatment with large molecules., Nat. Rev. Clin. Oncol. 6 (2009) 507–518. doi:10.1038/nrclinonc.2009.110.

[50] J. Mordenti, R. a Cuthbertson, N. Ferrara, K. Thomsen, L. Berleau, V. Licko, et al., Comparisons of the intraocular tissue distribution, pharmacokinetics, and safety of 125I-labeled full-length and Fab antibodies in rhesus monkeys following intravitreal administration., Toxicol. Pathol. 27 (1999) 536–544. doi:10.1177/019262339902700507.

[51] E. Moisseiev, M. Waisbourd, E. Ben-Artsi, E. Levinger, A. Barak, T. Daniels, et al., Pharmacokinetics of bevacizumab after topical and intravitreal administration in human eyes, Graefe’s Arch. Clin. Exp. Ophthalmol. 252 (2014) 331–337. doi:10.1007/s00417-013-2495-0.

[52] P. Heiduschka, H. Fietz, S. Hofmeister, S. Schultheiss, A.F. Mack, S. Peters, et al., Penetration of bevacizumab through the retina after intravitreal injection in the monkey, Investig. Ophthalmol. Vis. Sci. 48 (2007) 2814–2823. doi:10.1167/iovs.06- 1171.

[53] P.J. Rosenfeld, D.M. Brown, J.S. Heier, D.S. Boyer, P.K. Kaiser, C.Y. Chung, et al., Ranibizumab for neovascular age-related macular degeneration, N. Engl. J. Med. 355 (2006) 1419–1431. doi:10.1056/NEJMoa054481.

[54] J. Gaudreault, D. Fei, J. Rusit, P. Suboc, V. Shiu, Preclinical pharmacokinetics of Ranibizumab (rhuFabV2) after a single intravitreal administration., Invest. Ophthalmol. Vis. Sci. 46 (2005) 726–33. doi:10.1167/iovs.04-0601.

[55] S.J. Bakri, M.R. Snyder, J.M. Reid, J.S. Pulido, M.K. Ezzat, R.J. Singh, Pharmacokinetics of intravitreal ranibizumab (Lucentis)., Ophthalmology. 114 (2007) 2179–82. doi:10.1016/j.ophtha.2007.09.012.

[56] T.U. Krohne, Z. Liu, F.G. Holz, C.H. Meyer, Intraocular pharmacokinetics of ranibizumab following a single intravitreal injection in humans, Am. J. Ophthalmol. 154 (2012) 682–686. doi:10.1016/j.ajo.2012.03.047.

[57] E.W.M. Ng, D.T. Shima, P. Calias, E.T. Cunningham, D.R. Guyer, A.P. Adamis, Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease., Nat. Rev. Drug Discov. 5 (2006) 123–32. doi:10.1038/nrd1955.

[58] J. Ruckman, L.S. Green, J. Beeson, S. Waugh, W.L. Gillette, D.D. Henninger, et al., 2’-Fluoropyrimidine RNA-based Aptamers to the 165-Amino Acid Form of Vascular Endothelial Growth Factor (VEGF165), J. Biol. Chem. 273 (1998) 20556–20567. doi:10.1074/jbc.273.32.20556.

[59] D.W. Drolet, J. Nelson, C.E. Tucker, P.M. Zack, K. Nixon, R. Bolin, et al., Pharmacokinetics and safety of an anti-vascular endothelial growth factor aptamer (NX1838) following injection into the vitreous humor of rhesus monkeys, Pharm. Res. 17 (2000) 1503–1510. doi:10.1023/A:1007657109012.

[60] S. a. Vinores, Pegaptanib in the treatment of wet, age-related macular degeneration, Int. J. Nanomedicine. 1 (2006) 263–268.

[61] J. Holash, S. Davis, N. Papadopoulos, S.D. Croll, L. Ho, M. Russell, et al., VEGF- Trap: a VEGF blocker with potent antitumor effects., Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 11393–8. doi:10.1073/pnas.172398299.

[62] M.W. Stewart, S. Grippon, P. Kirkpatrick, Aflibercept, Nat. Rev. Drug Discov. 11 (2012) 269–270. doi:10.1038/nrd3700.

[63] M.W. Stewart, P.J. Rosenfeld, Predicted biological activity of intravitreal VEGF Trap., Br. J. Ophthalmol. 92 (2008) 667–668. doi:10.1136/bjo.2007.134874.

[64] T.R. Thrimawithana, S. Young, C.R. Bunt, C. Green, R.G. Alany, Drug delivery to the

posterior segment of the eye., Drug Discov. Today. 16 (2011) 270–7. doi:10.1016/j.drudis.2010.12.004.

[65] K.G. Janoria, S. Gunda, S.H.S. Boddu, A.K. Mitra, Novel approaches to retinal drug delivery., Expert Opin. Drug Deliv. 4 (2007) 371–388. doi:10.1517/17425247.4.4.371.

[66] S.S. Lee, M.R. Robinson, Novel drug delivery systems for retinal diseases. A review., Ophthalmic Res. 41 (2009) 124–35. doi:10.1159/000209665.

[67] G.J. Jaffe, D. Martin, D. Callanan, P.A. Pearson, B. Levy, T. Comstock, Fluocinolone Acetonide Implant (Retisert) for Noninfectious Posterior Uveitis. Thirty-Four-Week Results of a Multicenter Randomized Clinical Study, Ophthalmology. 113 (2006) 1020–1027. doi:10.1016/j.ophtha.2006.02.021.

[68] P.A. Pearson, T.L. Comstock, M. Ip, D. Callanan, L.S. Morse, P. Ashton, et al., Fluocinolone acetonide intravitreal implant for diabetic macular edema: A 3-year multicenter, randomized, controlled clinical trial, Ophthalmology. 118 (2011) 1580– 1587. doi:10.1016/j.ophtha.2011.02.048.

[69] J.H. Kempen, M.M. Altaweel, J.T. Holbrook, D.A. Jabs, T.A. Louis, E.A. Sugar, et al., Randomized comparison of systemic anti-inflammatory therapy versus fluocinolone acetonide implant for intermediate, posterior, and panuveitis: the multicenter uveitis steroid treatment trial., Ophthalmology. 118 (2011) 1916–1926. doi:10.1016/j.ophtha.2011.07.027.

[70] F.E. Kane, J. Burdan, A. Cutino, K.E. Green, Iluvien: a new sustained delivery technology for posterior eye disease, Expert Opin. Drug Deliv. 5 (2008) 1039–1046. doi:10.1517/17425247.5.9.1039.

[71] P. a Campochiaro, G. Hafiz, S.M. Shah, S. Bloom, D.M. Brown, M. Busquets, et al., Sustained ocular delivery of fluocinolone acetonide by an intravitreal insert., Ophthalmology. 117 (2010) 1393–9.e3. doi:10.1016/j.ophtha.2009.11.024.

[72] N. Haghjou, M. Soheilian, M.J. Abdekhodaie, Sustained release intraocular drug delivery devices for treatment of uveitis, J. Ophthalmic Vis. Res. 6 (2011) 317–319.

[73] J.L. Bourges, C. Bloquel, a. Thomas, F. Froussart, a. Bochot, F. Azan, et al., Intraocular implants for extended drug delivery: Therapeutic applications, Adv. Drug Deliv. Rev. 58 (2006) 1182–1202. doi:10.1016/j.addr.2006.07.026.

[74] J.A. Haller, F. Bandello, R. Belfort, M.S. Blumenkranz, M. Gillies, J. Heier, et al., Randomized, sham-controlled trial of dexamethasone intravitreal implant in patients with macular edema due to retinal vein occlusion., Ophthalmology. 117 (2010) 1134– 1146.e3. doi:10.1016/j.ophtha.2010.03.032.

[75] D.S. Boyer, Y.H. Yoon, R. Belfort, F. Bandello, R.K. Maturi, A.J. Augustin, et al., Three-Year, Randomized, Sham-Controlled Trial of Dexamethasone Intravitreal Implant in Patients with Diabetic Macular Edema, Ophthalmology. (2014). doi:10.1016/j.ophtha.2014.04.024.

[76] Laser-Activated , On-Demand Drug Delivery Technology Tested In Vivo, Retin. Today. (2010).

[77] B.N. Kuno, S. Fujii, Ocular Drug Delivery Systems for the Posterior Segment : A Review, Retin. Today. (2012).

[78] W. Tao, Application of encapsulated cell technology for retinal degenerative diseases., Expert Opin. Biol. Ther. 6 (2006) 717–726. doi:10.1517/14712598.6.7.717.

[79] W. Tao, R. Wen, M.B. Goddard, S.D. Sherman, P.J. O’Rourke, P.F. Stabila, et al., Encapsulated cell-based delivery of CNTF reduces photoreceptor degeneration in animal models of retinitis pigmentosa, Investig. Ophthalmol. Vis. Sci. 43 (2002) 3292–3298.

[80] P.A. Sieving, R.C. Caruso, W. Tao, H.R. Coleman, D.J.S. Thompson, K.R. Fullmer, et al., Ciliary neurotrophic factor (CNTF) for human retinal degeneration: phase I trial of CNTF delivered by encapsulated cell intraocular implants., Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 3896–901. doi:10.1073/pnas.0600236103.

[81] K. Zhang, J.J. Hopkins, J.S. Heier, D.G. Birch, L.S. Halperin, T. a Albini, et al., Ciliary neurotrophic factor delivered by encapsulated cell intraocular implants for treatment of geographic atrophy in age-related macular degeneration., Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 6241–6245. doi:10.1073/pnas.1018987108.

[82] V. Ling, Encapsulated cell technology: a new therapy for biologics delivery, in: Present. Annu. Meet. Antib. Soc., San Diego, n.d. doi:10.1371/journal.pone.0010202.

[83] J.M. Irache, M. Merodio, a Arnedo, M. a Camapanero, M. Mirshahi, S. Espuelas, Albumin nanoparticles for the intravitreal delivery of anticytomegaloviral drugs., Mini Rev. Med. Chem. 5 (2005) 293–305.

[84] H. Kim, S.B. Robinson, K.G. Csaky, Investigating the movement of intravitreal human serum albumin nanoparticles in the vitreous and retina, Pharm. Res. 26 (2009) 329–337. doi:10.1007/s11095-008-9745-6.

[85] J.-L.L. Bourges, S.E. Gautier, F. Delie, R. a. Bejjani, J.-C.C. Jeanny, R. Gurny, et al., Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles, Invest. Ophthalmol. Vis. Sci. 44 (2003) 3562–3569. doi:10.1167/iovs.02-1068.

[86] A.L. Gomes dos Santos, A. Bochot, N. Tsapis, F. Artzner, R.A. Bejjani, B. Thillaye- Goldenberg, et al., Oligonucleotide-polyethylenimine complexes targeting retinal cells: structural analysis and application to anti-TGFbeta-2 therapy, Pharm. Res. 23 (2006) 770–781. doi:10.1007/s11095-006-9748-0.

[87] J.H. Kim, J.H. Kim, K.-W. Kim, M.H. Kim, Y.S. Yu, Intravenously administered gold nanoparticles pass through the blood-retinal barrier depending on the particle size, and induce no retinal toxicity., Nanotechnology. 20 (2009) 505101. doi:10.1088/0957- 4484/20/50/505101.

[88] A.D. Bangham, M.M. Standish, J.C. Watkins, Diffusion of univalent ions across the lamellae of swollen phospholipids, J. Mol. Biol. 13 (1965) 238–252. doi:10.1016/S0022-2836(65)80093-6.

[89] I.A. Barbazetto, B.S. Takahashi, Verteporfin photodynamic therapy in the age of antiangiogenic therapy, Expert Rev. Ophthalmol. 3 (2008) 365–383. doi:http://dx.doi.org/10.1586/17469899.3.4.365.

[90] B.J. Rosenblatt, G.K. Shah, K. Blinder, Photodynamic therapy with verteporfin for peripapillary choroidal neovascularization., Retina. 25 (2005) 33–7. doi:10.1097/00006982-200501000-00004.

[91] Anonymous, Rostaporfin: PhotoPoint SnET2, Purlytin, Sn(IV) etiopurpurin, SnET2, tin ethyl etiopurpurin, Drugs R D. 5 (2004) 58–61.

[92] M. Barza, M. Stuart, F. Szoka, Effect of size and lipid composition on the pharmacokinetics of intravitreal liposomes, Investig. Ophthalmol. Vis. Sci. 28 (1987) 893–900.

[93] L. Paasonen, T. Laaksonen, C. Johans, M. Yliperttula, K. Kontturi, A. Urtti, Gold nanoparticles enable selective light-induced contents release from liposomes., J. Control. Release. 122 (2007) 86–93. doi:10.1016/j.jconrel.2007.06.009. 31

[94] A.E. Fung, A Novel Sustained-Release Intravitreal Drug Delivery System for Retinal Vascular Disease, Retin. Today. (2010) 51–53.

[95] J.J. Kang Derwent, W.F. Mieler, Thermoresponsive hydrogels as a new ocular drug delivery platform to the posterior segment of the eye., Trans. Am. Ophthalmol. Soc. 106 (2008) 206–213; discussion 213–214.

[96] E. Eljarrat-Binstock, A.J. Domb, Iontophoresis: A non-invasive ocular drug delivery, J. Control. Release. 110 (2006) 479–489. doi:10.1016/j.jconrel.2005.09.049.

[97] E. Eljarrat-Binstock, A.J. Domb, F. Orucov, A. Dagan, J. Frucht-Pery, J. Pe’er, In Vitro and In Vivo Evaluation of Carboplatin Delivery to the Eye Using Hydrogel- Iontophoresis, Curr. Eye Res. 33 (2008) 269–275. doi:10.1080/02713680701871140.

[98] E. Eljarrat-Binstock, F. Orucov, J. Frucht-Pery, J. Pe’er, A.J. Domb, Methylprednisolone Delivery to the Back of the Eye using Hydrogel Iontophoresis, J. Ocul. Pharmacol. Ther. 24 (2008) 344–350. doi:10.1089/jop.2007.0097.

[99] E. Eljarrat-Binstock, A.J. Domb, F. Orucov, J. Frucht-Pery, J. Pe’er, Methotrexate delivery to the eye using transscleral hydrogel iontophoresis., Curr. Eye Res. 32 (2007) 639–46. doi:10.1080/02713680701528674.

[100] U. Chakravarthy, P. Adamis, E.T. Cunningham, M. Goldbaum, D.R. Guyer, B. Katz, et al., Year 2 efficacy results of 2 randomized controlled clinical trials of pegaptanib for neovascular age-related macular degeneration., Ophthalmology. 113 (2006) 1508.e1–25. doi:10.1016/j.ophtha.2006.02.064.

[101] E.I. Altiok, J.L. Santiago-Ortiz, A. Zbinden, A.K. Jha, D. Bhatnagar, W.M. Jackson, et al., Multivalent Hyaluronic Acid Bioconjugates Improve sFlt Activity In Vitro, (under Rev. (2015).

[102] S.C. Fishburn, The pharmacology of PEGylation: Balancing PD with PK to generate novel therapeutics, J. Pharm. Sci. 97 (2010) 4167–4183. doi:10.1002/jps.

Chapter 2: Multivalent hyaluronic acid bioconjugates improve sFlt activity in vitro

2.1 Introduction

Diabetic retinopathy (DR) is one of the most detrimental consequences of diabetes, which imposes enormous healthcare costs in the U.S. and worldwide, and affects over 25 million American adults. Within two decades of their initial diagnosis, all patients with diabetes type I and 60% of patients with diabetes type II will exhibit symptoms of DR [1]. The main mediator of retinal neovascularization in diabetic retinopathy is vascular endothelial growth factor (VEGF), which is upregulated by hypoxic neurons in the retina [2–4]. Elevated intravitreal levels of VEGF results in the aberrant growth of blood vessels with loose cell-cell junctions leading to areas of vision loss caused by pooled blood and edema in and around the macula [5]. The reference-standard treatment for DR is pan-retinal laser photocoagulation (PLP), which forestalls retinal neovascularization by destroying ischemic neurons and reducing the number of VEGF-producing cells in the retina [6]. While this therapy can reduce the risk of disease progression and severe vision loss, the best outcomes for improved visual acuity occur when PLP is used in combination with anti-angiogenic therapies [7], as intravitreal VEGF remains elevated even after treatment by PLP [8]. The most commonly used anti-VEGF therapies for DR are Lucentis (ranibizumab, Genentech, a 48 kDa humanized antibody fragment) [9], Avastin (bevacizumab, Genentech, a 150 kDa humanized antibody) [10], and Eylea (aflibercept, Regeneron, a 110 kDa VEGF- receptor fusion protein) [11]. Due to limited drug retention in the vitreous as a result of their small molecular size, a single administration of these drugs provides only a limited therapeutic benefit for a finite period of time. In order to maintain an effective drug dose in the vitreous to inhibit VEGF-mediated retinal neovascularization, patients require monthly injections. Consequently, low patient compliance to these treatment protocols continues to be the most significant factor limiting the ability of anti-VEGF treatments to maintain improvements in visual acuity [12,13]. Therefore, novel approaches are required to enhance the half-life of anti- VEGF therapies within the vitreous, which would in turn reduce the frequency of required intravitreal drug injections and improve patient quality of life. In this work, we describe a novel multivalent drug bioconjugate composed of the anti- angiogenic VEGF decoy receptor sFlt-1, and hyaluronic acid (HyA), a naturally occurring biopolymer present in high concentrations throughout the body and in particular within the vitreous of the eye [14]. The overall goal of this study was to create high-molecular weight multivalent bioconjugates of sFlt (mvsFlt) that were capable of binding and inhibiting

VEGF165 activity in vitro, improving protein stability, and increasing mvsFlt residence time over 33 unconjugated sFlt. For the latter, we modeled the vitreous in vitro using a cross-linked hyaluronic acid hydrogel and observed that mvsFlt bioconjugates diffused slower and had reduced mobility in comparison to unconjugated sFlt. We anticipate that the future clinical use of multivalent conjugates of sFlt may significantly increase the intravitreal drug residence time and inhibit retinal angiogenesis over a longer period of time.

2.2 Materials and Methods

2.2.1 Expression of Soluble Flt-1 Receptor The sFlt sequence for the first 3 Ig-like extracellular domains of sFlt-1 [15] was cloned into the pFastBac1 plasmid (Life Technologies) and then transformed into DH10Bac E.coli, which were plated on triple antibiotic plates containing kanamycin (50 µg/mL), gentamicin (7 µg/mL, Sigma Aldrich), tetracycline (10 µg/mL, Sigma Aldrich), IPTG (40 µg/mL, Sigma Aldrich) and Bluo-gal (100 µg/mL, Thermo Fisher Scientific). The sFlt gene-containing bacmid was isolated from DH10Bac E.coli (Life Technologies) and transfected into SF9 insect cells for virus production (provided by the Tissue Culture Facility, UC Berkeley). Virus was then used to infect High Five insect cells (provided by the Tissue Culture Facility, UC Berkeley) to induce sFlt protein expression. After 3 days, protein was purified from the supernatant using Ni-NTA agarose beads (Qiagen Laboratories). Recombinant sFlt was eluted from the Ni-NTA beads using an imidazole gradient and then concentrated and buffer exchanged with 10% glycerol/PBS using Amicon Ultra-15mL Centrifugal devices (EMD Millipore). The protein solution was sterile filtered and the concentration was determined using a BCA assay (Thermo Fisher Scientific).

2.2.2 mvsFlt Conjugate Synthesis Conjugation of sFlt to HyA was carried out according to the schematic in Figure 2.1A, as previously described [16–18]. To make thiol-reactive HyA intermediates, 3,3′-N-(ε- maleimidocaproic acid) hydrazide (EMCH, Thermo Fisher Scientific, 1.2 mg/mL), 1- hydroxybenzotriazole hydrate (HOBt, Sigma Aldrich, 0.3 mg/mL) and 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC, Thermo Fisher Scientific, 10 mg/mL) were added to a 3 mg/mL solution of HyA (Lifecore Biotechnology) of various molecular weights in 0.1 M 2-(N-morpholino)ethanesulphonic acid (MES) (Sigma Aldrich) buffer (pH 6.5) and allowed to react at 4 °C for 4 hours. The solution was then dialyzed into pH 7.0 PBS containing 10% glycerol. Recombinant sFlt was treated with 2-iminothiolane at 10 molar excess to create thiol groups for conjugation to the maleimide group on EMCH. sFlt was then added to activated HyA-EMCH at 10:1 and 30:1 molar ratios of sFlt to HyA to create the mvsFlt bioconjugates and allowed to react at 4 °C overnight. The mvsFlt reactions were dialyzed exhaustively using 100 kDa molecular weight cut-off (MWCO) Float-A-Lyzer G2 (Spectrum Labs) dialysis tubes in pH 7.0 PBS to remove unreacted sFlt. The BCA assay was used to measure the protein concentrations of the mvsFlt bioconjugates. We defined 34 conjugates with 10:1 sFlt to HyA molar feed ratios as ‘low conjugation ratio’ (LCR) mvsFlt and 30:1 molar feed ratios ‘high conjugation ratio’ (HCR) mvsFlt.

2.2.3 SEC-MALS Characterization of mvsFlt Protein conjugation was characterized using size exclusion chromatography with multiangle light scattering (SEC-MALS) as previous described [19]. Briefly, the SEC-MALS setup consisted of an Agilent HPLC 1100 with a DAWN-HELEOS II multiangle laser light scattering detector and Optilab relative refractive interferometer (Wyatt Technology, Santa Barbara, CA). Refractive index change was measured differentially with 690nm laser and UV absorbance was measured with the diode array detector at 280 nm. Shodex OH pak SB-804 columns were used for separation (Phenomenex Inc.). Prior to analysis, the mvsFlt conjugates were sterile filtered through a 0.45 µm filter and 200 µL was injected at HyA concentration between 0.2-0.5 mg/mL. The dn/dc values were determined using SEC-MALS to be 0.1447 and 0.185 for HyA-EMCH and sFlt, respectively. UV extinction coefficients used for HyA- EMCH and sFlt were also determined on SEC-MALS and were 0.022 and 0.894, respectively. Data analysis was carried out using the Astra software (Wyatt Technologies).

2.2.4 SDS-PAGE Analysis of mvsFlt Samples were prepared with 5X SDS dye loading buffer, 2-mercaptoethanol and boiled for 5 minutes at 95°C. Precast Mini-Protean TGX 4-20% gradient gels (Bio-Rad Laboratories) were run for 90 minutes at 110 volts. Gels were then stained with Bio-Safe Coomassie Stain (Bio- Rad Laboratories) for 2 hours and then imaged using a BioRad Molecular Imager ChemiDoc XRS+. The intensities of protein in the stacking and gradient gel were analyzed using ImageJ to determine the amount of protein that was conjugated versus free unconjugated protein that did not dialyze out of solution following the conjugation reaction.

2.2.5 DLS Size Characterization of mvsFlt A Brookhaven Goniometer & Laser Light Scattering system (BI-200SM, Brookhaven Instruments Corporation) was used to determine the hydrodynamic diameter of mvsFlt bioconjugates. Each sample was filtered at 0.45 µm and loaded into a 150 µL cuvette (BI-SVC, Brookhaven). Data acquisition was performed at 90 degrees with a 637nm laser for 2 minutes. Data analysis was carried out with BIC Dynamic light Scattering software (Brookhaven) using the BI-9000AT signal processor. The intensity average particle size was obtained using a non-negative least squares (NNLS) analysis method.

2.2.6 Binding Competition ELISA

The mvsFlt conjugates were analyzed using a VEGF165 Quantikine Sandwich ELISA (R&D

Systems) to examine the effect of HyA conjugation on sFlt inhibition of VEGF165. The assay was carried out according to the manufacturer’s instructions. Briefly, VEGF165 was added to

PBS with varying concentrations of sFlt or mvsFlt. Free VEGF165 that bound to the capture

35 antibodies on the plate surface were detected using a horseradish peroxidase conjugated detection antibody and quantified using a spectrophotometer at 450 nm.

2.2.7 HUVEC Survival Assay Human umbilical cord vein endothelial cells (HUVECs) were purchased from ATCC and cultured in EBM-2 media (Lonza) in a humidified incubator at 37°C and 5% CO2. In order to examine the effect of HyA conjugation on the ability of sFlt to bind and inhibit VEGF165 activity in vitro, a survival assay was carried out with HUVECs grown in the presence of

VEGF165 and mvsFlt conjugates. HUVECs were added to 96 well plates coated with 0.2% gelatin at 10,000 cell/well in M199 media. Cells were grown in 2% FBS and 20 ng/mL

VEGF165 (R&D Systems) in the presence of sFlt or mvsFlt. 72 hours after plating, the media was aspirated and the cells were washed with PBS prior to freezing for analysis with CyQuant (Life Technologies). Total cell number per well was determined by reading fluorescence at 480 nm excitation and 520 nm emission using a fluorometer (Molecular Devices).

2.2.8 HUVEC Tube Formation Assay HUVEC tube formation assays were carried out in 96-well plates coated with 80 µL of Matrigel (Corning, NY) and incubated at 37°C for 1 hour to allow gelation to occur. HUVECs were trypsinized and resuspended in M199 with 2% FBS and 20 ng/mL VEGF165 and treated with mvsFlt LCR conjugates. Wells were imaged 18 hours after plating and tube formation was quantified using ImageJ software.

2.2.9 HUVEC Migration Assay Wells of a 12-well plate were coated with 0.2% gelatin. HUVECs were added at 150,000 cells/well in EBM-2 and allowed to attach and spread overnight. Using a 1 mL pipette tip, crosses were scratched into the confluent layer of HUVECs. The wells were then washed with excess PBS to remove cell debris and media and replaced with media containing VEGF165 and mvsFlt. Scratches were imaged at 0 and 24 hours post scratch and the area without cells was quantified using ImageJ and T-scratch software (CSE Laboratory software, ETH Zurich). The percent open wound area was calculated by comparing the open scratch area at 24 hours to the open scratch area at 0 hours.

2.2.10 Retention of mvsFlt in Crosslinked HyA Gels To model the chemical and network structure of the vitreous, acrylated HyA (AcHyA) hydrogels were synthesized as described previously [20,21]. Briefly, adipic acid dihydrazide (ADH, Sigma Aldrich) was added in 30 molar excess to HyA in deionized water (DI). EDC (3 mmol) and HOBt (3 mmol) were dissolved in DMSO/water and added to the HyA solution. The solution was allowed to react for 24 hours and then dialyzed exhaustively against DI water. HyA-ADH was precipitated in 100% ethanol and reacted with acryloxysuccinimide to generate acrylate groups on the HyA. The resulting AcHyA was exhaustively dialyzed and

36 lyophilized for storage. The presence of grafted acrylate groups on HyA chains was confirmed using H1-NMR. To make 1% AcHyA hydrogels, 8 mg of AcHyA was dissolved in 800 µL of triethanolamine-buffer (TEOA; 0.3 M, pH 8). Either 5 µg of Alexafluor 488 5-SDP ester (Life Technologies) tagged sFlt, 650 KDa LCR mvsFlt or bovine serum albumin (BSA) were added in 50 µL volumes to the AcHyA solution prior to crosslinking. Thiolated 5 kDa-PEG crosslinker (Laysan Bio, Inc.) was dissolved in 100 µL of TEOA buffer and added to dissolved AcHyA. Cell culture inserts with 4 µm sized pores (Millipore Corporation, Billerica, MN) were added to wells of a 24-well plate and 70 µL of gel containing sFlt, mvsFlt or BSA was added to each insert. The gels were allowed to crosslink at 37°C for 1 hour before adding 150 µL of PBS to the wells and submerging the hydrogels. To determine release kinetics, the well supernatant was collected and fully replaced at 0, 1, 2, 3, 7, 10 and 14 days. Samples were read with a fluorometer to detect the fluorescently tagged sFlt, mvsFlt and BSA in the supernatant.

2.2.11 Fluorescence Recovery After Photobleaching (FRAP) Diffusivity Measurement FRAP measurements were performed on 1% AcHyA hydrogels containing FITC labeled sFlt and 650 kDa LCR mvsFlt. Total fluorescence intensity of the hydrogels was acquired using a Zeiss LSM710 laser-scanning microscope (Carl Zeiss, Jena, Germany) with a 20X magnification objective and an argon laser set at 488 nm with 50% power. Photobleaching was done by exposing a 100x100-µm spot in the field of view to high intensity laser light. The area was monitored by 15 pre-bleach scanned images at low laser intensity (2%), then bleached to 75% of the starting fluorescence intensity at 100% laser power. A total of about 300 image scans of less than 1 second were collected for each sample. The mobile fraction of fluorescent sFlt and mvsFlt molecules within the hydrogel was determined by comparing the fluorescence in the bleached region after full recovery (�!) with the fluorescence before bleaching (��) and just after bleaching (�!). The mobile fraction R was defined according to equation (1):

(�! − �!) � = (1) (�� − �!)

2.2.12 Enzymatic Degradation with MMP-7 Matrix metalloproteinase-7 (MMP-7) has been previously shown to specifically degrade sFlt [22]. To determine whether HyA conjugation shielded mvsFlt from degradation, we treated sFlt and mvsFlt with varying amounts of MMP-7 (EMD Millipore). sFlt and mvsFlt were incubated with matrix metalloproteinase-7 (MMP-7) for 12 hours at 37C while shaking at high (1:1 molar ratio MMP-7:sFlt), medium (1:2), and low (1:4) molar ratios of MMP-7 to sFlt. The enzyme-treated sFlt and mvsFlt were then loaded into precast Mini-Protean TGX 4-20% gradient gels (Bio-Rad Laboratories) and run at 110 volts for 90 minutes. Gels were then stained using a silver staining kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The degree of enzymatic degradation was assessed by quantifying the total amount of protein remaining in the gel following treatment with MMP-7. Each well was 37 normalized to their respective no-MMP-7 well and background intensity was subtracted according to the blank well between the groups on the gel.

2.2.13 Statistical Analysis All quantitative experiments were performed in triplicate. Values are expressed as means ± standard deviations (SD). One-way ANOVA with Tukey post-hoc analysis was used to compare treatment groups in the quantitative measurements where appropriate and p<0.05 was used to assess statistical significance.

2.3 Results and Discussion

The overall goal of this study was to synthesize protein-polymer bioconjugates to increase the residence time of anti-VEGF drugs in the vitreous for use in treating patients with DR. In contrast to drugs currently used for the treatment of DR that suffer from short half- lives, we have developed large multivalent protein bioconjugates with unperturbed affinity for

VEGF165 and good enzymatic stability that show delayed diffusion and mobility in an in vitro model of the vitreous.

Figure 2.1: Multivalent sFlt synthesis and schematics. A) mvsFlt bioconjugates were synthesized using a 3- step reaction in which HyA was reacted with EDC and EMCH to create a cysteine reactive HyA-EMCH intermediate. sFlt was then treated with 2-iminothiolane and then reacted with the HyA-EMCH intermediate for the synthesis of the final product. B) Schematic of protein conjugation to HyA and subsequent binding to

VEGF165. The ratio a:b represents the valency of sFlt molecules (a) covalently bound to a single chain of HyA (b). C) Schematic of low conjugation ratio (LCR) mvsFlt conjugates which are synthesized by reacting 10 molecules of sFlt with 1 HyA chain. This reaction has 61% conjugation efficiency as determined by SEC-MALS (see Table 2.1). D) Schematic of high conjugation ratio (HCR) mvsFlt conjugates which are synthesized by reacting 30 molecules of sFlt with 1 HyA chain (same molecular weight of HyA as in (C)). This reaction has 52% conjugation efficiency as determined by SEC-MALS (see Table 2.1).

The synthesis of mvsFlt conjugates was carried out according to the schematics shown in Figure 2.1. We created mvsFlt at low (LCR, Figure 2.1C) and high (HCR, Figure 2.1D) conjugation ratios in order to determine whether a certain valency would provide an enhanced effect on VEGF binding. We were able to successfully conjugate sFlt to HyA at several different molecular weights of HyA and valencies, which were significantly larger than the sFlt in its unconjugated form (Figure 2.2). The molecular weights of the protein and polymer components of the conjugates were characterized using SEC-MALS as shown in Table 2.1 and Figure 2.2A. Conjugates with 10:1 feed ratios (termed low conjugation ratio, LCR) of sFlt to HyA averaged 61.2±12.5% conjugation efficiency whereas conjugates with 30:1 sFlt to HyA feed ratios (termed high conjugation ratio, HCR) had conjugation efficiencies averaging 51.8±4.1%. SDS-PAGE of unbound sFlt exhibited a protein band at the predicted 50 kDa. Conversely, mvsFlt bioconjugates only migrated into the stacking portion of the gel, indicating inhibited mobility as a result of covalent attachment to the much larger multivalent conjugate (Figure 2.2B). Gel analysis using ImageJ indicated that on average 76.4±6.7% of sFlt in the mvsFlt bioconjugates was covalently bound whereas the rest of the detected sFlt was presumably non-specifically interacting with the hyaluronic acid chain in solution and thus could not be removed by dialysis (Figure 2.2C).

Table 2.1: SEC-MALS analysis of all mvsFlt conjugates. 300 kDa 300 kDa 650 kDa 650 kDa 1 MDa 1 MDa LCR* HCR+ LCR* HCR+ LCR* HCR+ HyA Mna 2.7E5 2.1E5 5.9E5 6.4E5 9.7E5 1.0E6 HyA Mwb 3.1E5 3.4E5 7.1E5 7.1E5 1.2E6 1.1E6 HyA PDIc 1.1 1.6 1.2 1.1 1.3 1.1 sFlt : HyAd 7.9 15.4 5.3 17.1 5.2 14.1 aNumber average molecular weight given in g/mol. bWeight average molecular weight given in g/mol cPolydispersity index given as Mw/Mn dFinal stoichiometric ratio of sFlt to HyA calculated by dividing total attached protein Mw by sFlt MW (50 kDa) *LCR is low conjugation ratio conjugates (10:1 sFlt per HyA chain feed ratio) 39

+HCR is high conjugation ratio conjugates (30:1 sFlt per HyA chain feed ratio)

All mvsFlt conjugates were also characterized by DLS to determine conjugate hydrodynamic diameter in solution, as drug size is a critical factor that determines its mobility through biological hydrogels such as the vitreous. Unconjugated sFlt had a diameter of 22.6nm±3.1nm whereas mvsFlt conjugates made with HyA of molecular weights of 300 kDa, 650 kDa, and 1 MDa had diameters of 123.9±23.1 nm, 236.3±38.7 nm, and 223±13.9 nm, respectively (Figure 2.2D). The size of the mvsFlt conjugates was dependent on the HyA molecular weight for the 300 and 650 kDa conjugates; however, there was no significant difference between the 650 kDa and 1 MDa conjugates (Figure 2.2D). Interestingly, HCR conjugates of 300 and 650 kDa molecular weight had lower diameters than their respective LCR conjugates, which could be due to increased positive charge from sFlt on the negatively charged HyA backbone with increasing sFlt attachment causing the conjugate to fold in tighter around itself.

Figure 2.2 Characterizing mvsFlt conjugation efficiency and size. A) SEC-MALS chromatogram depicting cumulative weight fraction versus molar mass of 650 kDa LCR and HCR bioconjugates. Dotted line, dashed line and solid line represent total molar mass of all covalently attached sFlt proteins, HyA molar mass and total bioconjugate molar mass (all given as g/mol), respectively. B) 4-20% SDS-page gradient gel of sFlt and mvsFlt. Protein bands in the stacking gel indicate successful protein conjugation to HyA. Protein bands within gel represent the proportion of protein that was not covalently bound, but remained in solution after dialysis. C) Quantified protein band intensities of SDS-PAGE gel. Percent bound sFlt was determined by dividing the intensity of protein in the stacking gel by total protein intensity within the respective well. Free sFlt was determined by dividing the intensity of protein within the separating gel by the total protein intensity within the respective well. D) Dynamic light scattering analysis of conjugates. sFlt was significantly smaller than all mvsFlt bioconjugates (***p<0.001). In the case of 300 and 650 kDa mvsFlt bioconjugates, the LCR mvsFlt was significantly larger than its respective HCR conjugate (**p<0.01). Values are given as ±SD.

Figure 2.3. All mvsFlt bioconjugates maintain their ability to inhibit VEGF165 dependent activities in VEGF165 ELISA and VEGF165-dependent HUVEC survival assays. A) Dose-dependent inhibition of VEGF165 binding to a capture antibody by mvsFlt bioconjugates. Inhibition was independent of whether the sFlt was bound to HyA or free in solution. There were no significant differences between any of the groups (Table 2.2). B) Dose-dependent inhibition of HUVEC survival with mvsFlt at different molecular weights and protein valencies in the presence of VEGF165. Inhibition was independent of whether the sFlt was bound to HyA or free in solution (Table 2.2). Values are given as means ±SD.

Table 2.2: IC50 values from ELISA and HUVEC survival assays examining mvsFlt inhibition of VEGF165 ELISA (ng/mL) HUVEC Survival (ng/mL) sFlt unconjugated 3.8 ± 2.4 39.3 ± 4.4 300 kDa LCR 4.5 ± 1.5 46.9 ± 9.9 300 kDa HCR 4.7 ± 1.7 44.4 ± 2.6 650 kDa LCR 3.9 ± 2.6 41.7 ± 6.7 650 kDa HCR 2.0 ± 0.1 43.2 ± 13.6 1 MDa LCR 2.2 ± 0.1 44.9 ± 10.7 1 MDa HCR 3.3 ± 1.6 45.4 ± 2.2

We employed several different assays to determine whether conjugation of sFlt to HyA affected sFlt affinity VEGF165 and alter VEGF165-dependent cell function. Using a VEGF165 specific ELISA we found a dose-dependent response that indicated conjugation of sFlt to

HyA did not alter the ability of mvsFlt to bind VEGF165 (Figure 2.3A). ELISA results

41 indicated that the IC50 value of sFlt was 3.8±2.4 ng/mL and the IC50 value of the various mvsFlt conjugates averaged to 3.4±1.1 ng/mL (Table 2.2).

Figure 2.4. mvsFlt inhibits HUVEC tube formation. A) Representative images of inhibited HUVEC tube formation on Matrigel (BD Biosciences) when treated with 1 µg/mL of all LCR mvsFlt bioconjugates. Cells were seeded at 20,000 cells per well of a 96-well plate on 100 µL of matrigel and imaged at 18 hours. Scale bar = 500 µm. B-E) Quantification of total number of tubes per well (B), average tube length (C), total number of nodes (branching points, D), and total tube length per well (E) (***p<0.001).

In survival assays with HUVECs, mvsFlt demonstrated a dose-dependent decrease in survival, and the effect was independent of conjugation to HyA similar to the ELISA results (Figure 2.3B, Table 2.2). These results indicate that covalent conjugation of sFlt to HyA does not reduce the ability of sFlt to bind VEGF. This is extremely promising due to the fact that other conjugation technologies such as PEGylation, have previously reported that conjugation significantly reduces the bioactivity of the protein [23,24]. For subsequent studies, we decided only to study the LCR conjugates of different molecular weights, since all conjugates performed equally well when examining in vitro

VEGF165 inhibitory activity in ELISA and survival assays. In vitro tube formation and migration assays enabled us to examine the effect of mvsFlt conjugates in vitro on two additional processes involved in angiogenesis in vivo, organization into tubes and cell migration. Similar to the survival data, sFlt and mvsFlt had similar inhibition profiles of VEGF165 in these two assays where addition of sFlt and mvsFlt equally inhibited organization into tubes (Figure 2.4) and closure of wounds (Figure 2.5). Taken together, the ELISA and in vitro angiogenesis assays indicated that conjugation of sFlt to HyA did not affect the ability of sFlt to bind

VEGF165 and that all mvsFlt conjugates maintained their ability to inhibit endothelial cell functions mediated by VEGF165 signaling.

Figure 2.5. mvsFlt inhibits VEGF165-driven HUVEC migration. Representative images of inhibition of HUVEC migration with LCR mvsFlt bioconjugates of varying molecular weights. HUVECs were allowed to grow to confluence in 12-well plates prior to making a scratch and were treated with 20 ng/mL VEGF165 in the presence of 200 ng/mL mvsFlt. Cells were stained with CellTracker Green (Life Technologies) prior to seeding. Scale bar = 20 µm. B) Quantified HUVEC migration following treatment with LCR mvsFlt showing percent open wound area calculated by comparing open wound area at 24 hours to open wound area at time 0 (***p<0.001).

We used crosslinked HyA gels [20,21] to examine how diffusion conjugation of sFlt to HyA affects diffusion. This hydrogel was chosen as a model system for studying the vitreous in vitro based primarily on compositional similarity to the vitreous with respect to high HyA content. The stiffness of the HyA hydrogel (Figure S1A) was higher than published reports examining bovine and porcine vitreous stiffness [25], suggesting the predictions of drug clearance from the vitreous using this model may not be sufficient to estimate actual in vivo rates. However, we anticipate that the diffusion of sFlt and mvsFlt through these gels will be instructive to predict the benefits of conjugation to HyA given the compositional similarities of the model with the vitreous. We used several different models as described in the supplemental methods to characterize the mesh size of HyA hydrogels (Table 2.3). Preliminary experiments analyzing size dependent diffusion using 40 kDa and 2 MDa fluorescently tagged dextrans (Life Technologies) were used to confirm that this hydrogel system would be appropriate for examining molecular weight and size dependent diffusion (Figure S1B). Using this system, we analyzed the release kinetics of sFlt and all LCR mvsFlt bioconjugates from the HyA gels. We expected that the main effect of mvsFlt bioconjugates would be size-dependent decreases in mobility, and thus we chose to analyze only conjugates of varying sizes while holding valency constant. After 14 days, only 30.8±1.9% of sFlt remained in the gel in comparison to 38.3%±2.2%, 63.8±0.5% and 62.8% ±0.4% of 300 kDa, 650 kDa, and 1 MDa LCR conjugates, respectively (Figure 2.6A). In comparison, 100% of BSA released after 24 hours, a difference more likely due to significantly different isoelectric points (5.4 for BSA [26] and 9.5 for sFlt [27]) and very low protein affinity for HyA rather than protein size since BSA and sFlt have similar molecular weights.

Table 2.3: �� and mesh size calculations based on swelling and rheological data. Model + �� (g/mol)* � (nm) Peppas/Merrill [28] – Affine (Q)a 299,030 377 Erman [29] – Phantom (Q) a 388,940 430 b Mooney [30] – Affine (�′) 247,750 343 b Erman [29] – Phantom (�′) 123,880 242

*�� - molecular weight between crosslinks + � - mesh size of 1% HyA gel calculated according to [31] a �� calculated from mass swelling data b �� calculated from rheology data

Figure 2.6. sFlt conjugation to HyA decreases mvsFlt mobility and diffusion in HyA gels. A) Alexafluor 488-tagged LCR mvsFlt bioconjugates 650 kDa and 1 MDa encapsulated in 1% HyA hydrogels diffused out significantly slower than unconjugated sFlt and 300 kDa mvsFlt after day 1 (*p<0.05) and persisted until the last time point, day 14 (***p<0.001). B) Representative confocal images corresponding to FRAP experiment of FITC tagged 650 kDa LCR mvsFlt. Finitial depicts mvsFlt in the gel prior to bleaching.; F0 is the fluorescence measurement immediately after 75% photobleaching; F∞ corresponds to the maximal recovery of fluorescence at the end of the experiment. C) Normalized fluorescence recovery [f(t)] of FITC labeled sFlt and 650 kDa LCR mvsFlt after photobleaching.

To assess whether diffusion through the gel was Fickian, we fit the curves in Figure 2.6A using equation (2) as described by Ritger et al. [32]: � � = �� (2) �! The diffusional exponent, n, is indicative of whether diffusion is Fickian and if n is equal to 0.5, the transport is Fickian. The n value for BSA, sFlt and mvsFlt release was determined to be 0.3-0.4 (Fig. S2), indicating that the diffusion through the gel is not Fickian. BSA was depleted from the gel through rapid burst release due to the extremely small size of the protein

44 and very low affinity to the HyA hydrogel due to charge repulsion, leading to non-Fickian diffusion. The sFlt and mvsFlt conjugates release slower in comparison due in part to ionic affinity with the HyA hydrogel and size. Although the sFlt and BSA are similar in size (50 kDa and 66 kDa, respectively), the sFlt has a much stronger ionic interaction with the matrix, which slows its diffusion from the gel, also resulting in non-Fickian diffusion due to this strong affinity. The 300 kDa mvsFlt conjugate is small enough in size ( < 150 nm, see Figure 2.2D) relative to the estimated hydrogel � (Table 2.3) to release as rapidly as sFlt. The two largest conjugates were close to the mesh size in diameter ( > 225 nm) and were significantly impeded in their release due to size, resulting in gel release that followed the reptation mechanism of diffusion [33]. Based on data from the sFlt release studies, we chose to study only the 650 kDa LCR bioconjugate using FRAP, since this conjugate displayed the highest difference in gel retention in comparison to unconjugated sFlt. The mobile fraction of sFlt in the gel was 73.8±4.4% whereas the mobile fraction of the mvsFlt within the gel was 48.3±3.0%, indicating that a significant portion of the mvsFlt bioconjugate was large enough to become immobile within the gel due to the similarity in diameter between the mvsFlt and the mesh size of the HyA gel. Experimental data in Figure 2.6C was fit according to Soumpasis [34] to obtain characteristic diffusion times. Interestingly, the characteristic diffusion time for mvsFlt (94.8±19.5 s) was significantly faster than sFlt (176±18.1 s). We believe that this difference is due to ionic shielding of the positively charged sFlt by the negatively charged hyaluronic acid within the multivalent conjugate, which reduces the overall affinity of the conjugate for the gel allowing for faster diffusion. In contrast, the unconjugated sFlt remains highly positively charged resulting in stronger ionic affinity within the HyA gel that slow its characteristic diffusion time. The mvsFlt in the hydrogel also recovered fluorescence to a significantly lower degree, 85.3±0.8%, in comparison to sFlt, which recovered to 91.6%±2.4%. Although the mvsFlt conjugate displayed faster diffusion, a much smaller percentage of the mvsFlt bioconjugate is actually mobile and the size significantly limited the total fluorescence recovery, results that are also supported by the gel release data in Figure 2.6A. It becomes clear that even though mvsFlt can diffuse faster as shown by FRAP in Figure 2.6B,C a much smaller portion of this sample is able to move due to size and thus much less of it is released over time as evidenced by the gel release data in Figure 2.6A. Taken together, we anticipate that the effect of size will be the strongest determinant of mvsFlt residence time in vivo. We examined how conjugation of sFlt to HyA affected protease degradation of the protein using MMP-7, a protease that has been shown to specifically degrade sFlt [22]. We found that conjugation of sFlt to HyA shielded the degradation of sFlt at all molar ratios of MMP-7 to sFlt (Figure 2.7A). At high concentrations of protease (1:1 molar ratio of MMP-7 to sFlt), only 6.8±6.6% of sFlt remained detectable in comparison to 34.8±1.8% of mvsFlt (Figure 2.7B). Decreasing the ratio of MMP-7 to sFlt from 1:1 to 1:4 still resulted in significant degradation of sFlt and degradation shielding of the conjugated form, where detectable sFlt increased to 34±2.7% in comparison to detectable mvsFlt at 74.8±4.9% (Figure 2.7B). We believe that the shielding effect of HyA will be crucial for maintaining

45 mvsFlt stability and bioavailability in vivo, aiding in the prolonged anti-angiogenic effect of mvsFlt bioconjugates.

Figure 2.7. Conjugation to HyA reduces susceptibility to protease degradation by MMP-7. A) 4-20% SDS-page gradient gel of sFlt and mvsFlt (650 kDa LCR) following 12 hour treatment with MMP-7 at high, medium and low molar ratios of MMP-7 to sFlt, which correspond to 1:1, 1:2 and 1:4 molar ratios of MMP- 7 to sFlt. Band intensity was normalized to the no MMP-7 treatment within each group and background was subtracted from each sample using the blank well. B) Quantification of 4-20% gradient gel of sFlt and mvsFlt treated with MMP-7 at high, medium, and low molar ratios of sFlt to MMP-7 for 12 hours (*p<0.05; **p<0.01).

Intravitreally injected drugs are cleared from the eye via two main mechanisms: 1) the anterior elimination pathway powered by counter directional bulk aqueous flow and 2) the posterior elimination pathway via bulk flow due to osmotic pressure gradients of the posterior segment [35,36]. The posterior pathway is much faster than the anterior route and is reserved for small lipophilic drugs that are able to rapidly traverse the retinal pigment epithelium using transcellular routes. In contrast, larger drugs are only able to escape through the anterior pathway, a process that is highly dependent on drug size and molecular weight [37]. For these reasons, we predict that the mvsFlt bioconjugates would be cleared through the anterior route; however, we anticipate that the 650 kDa and 1 MDa bioconjugates would take significantly longer to clear in comparison to the 300 kDa and unconjugated sFlt based on gel release data (Fig. 2.6A). Better systems for improved drug delivery to the posterior segment of the eye have been in high demand with the increasing prevalence of retinal diseases including diabetic retinopathy [35,38–40]. Intravitreal injection (IVT) has become the current gold standard for delivering drugs to the retina in order to circumvent blood retinal barriers, which keep most drugs out of the eye when delivered topically, systemically and transsclerally [41]. However, repeated intravitreal injections are required to reach and maintain effective drug doses, which can damage the lens, cause retinal detachment, hemorrhage and result in poor patient compliance [42]. For these reasons, improvements to IVT technologies are needed for treating posterior segment diseases. The use of drug-containing liposomes has emerged as a popular drug delivery system; however, liposomes suffer from several drawbacks including low

46 solubility, high production cost, and leakage/fusion of encapsulated drugs and molecules [40,43,44]. Ocular implants represent another branch of drug delivery systems used for treating ocular diseases for providing drug release for several months to years, but can affect the patients’ line of sight and non-biodegradable implants often require surgical implantation and sometimes invasive device removal [38,39,45]. PEGylation of drugs also presents an interesting approach to increasing drug stability and residence time; however, clinical trials with PEGylated drugs like Pegaptanib (Macugen) have shown that this approach does not significantly increase drug half-life in the vitreous [23,46,47]. Furthermore PEGylated drugs typically have lower effectiveness/affinity as a result of the PEG grafting [23,24]. In contrast to these approaches, we used hyaluronic acid, a naturally occurring biopolymer found in very high concentrations within the vitreous [14,48]. Although hyaluronic acid has previously been shown to induce angiogenesis in certain systems [49], we do not anticipate that the HyA in our mvsFlt will induce the growth of new blood vessels due to the fact that HyA is naturally found in high concentrations ranging between 100 and 400 µg/g in the vitreous [50] and is naturally turned over without the induction of angiogenesis in the retina. Furthermore, previous research has shown that vitreal hyaluronic acid has anti- angiogenic properties [51], an effect that the HyA in our conjugates may supplement. In this work we have introduced a new technology using multivalent forms of an anti- VEGF protein, sFlt, that improves its activity with respect to diffusion time and mobility through hydrogels as well as shields the protein from protease degradation, promising results that may indicate potential for improvement upon clinically used anti-VEGF drugs. Specifically, conjugation to HyA decreased the release kinetics of mvsFlt from crosslinked HyA hydrogels, a system meant to model drug release from the vitreous of the eye. Although attachment of several sFlt molecules per HyA chain did not improve affinity for VEGF165, we showed that conjugates maintained their ability to bind VEGF165 following chemical conjugation. These findings illustrate the effectiveness of mvsFlt at inhibiting VEGF165 activity, improving sFlt protein stability, and decreasing conjugate mobility in hydrogels, all characteristics that we anticipate will be essential in improving anti-VEGF drug treatments for patients with diabetic retinopathy. Furthermore, our approach provides tunability and modularity by allowing us to control bioconjugate size through altering HyA molecular weight and protein attachment independently, a technology that is not limited in its use for diabetic retinopathy but can be applied in the development of drugs for other diseases as well.

2.4 Conclusion Developing drugs with suitable half-lives is a universal challenge and multivalent conjugates have immense promise for maintaining drug stability as well as prolonging residence time in a range of tissues. The multivalent conjugate technology presented here is facile and utilizes hyaluronic acid, a biopolymer ubiquitous in the human body including the vitreous and allows

47 for tunability in the number of grafted proteins and overall conjugate size. The technology is also easily adaptable for other therapeutic applications in the eye and other tissues. Therefore, we anticipate that multivalent protein bioconjugates will be not only be useful for improving drug delivery to patients with diabetic retinopathy, but also for clinical outcomes and patient quality of life for a wide range of diseases.

2.5 Acknowledgements This work was supported in part by the National Science Foundation Graduate Research Fellowship, National Heart Lung and Blood Institute of the National Institutes of Health R01HL096525 (K.E.H.), and the Jan Fandrianto Chair Fund (K.E.H.). We would like to thank Jonathan Winger and Xiao Zhu for guidance with the insect cell protein expression system and providing reagents. We would like to acknowledge Ann Fischer for help express the sFlt protein in the Tissue Culture Facility at UC Berkeley and Dawn Spelke and Anusuya Ramasubramanian for help optimizing protein purification from insect cells. We would also like to thank Felicia Svedlund for helping with SEC-MALS analysis of our protein-polymer conjugates.

2.6 References [1] R. Fong, D.S., Aiello, L., Gardner, T.W., King, G.L., Blankenship, G., Cavallerano, J.D., Ferris, F.L., Klein, Retinopathy in Diabetes, Diabetes Care. 27 (2004) S84–87. doi:10.2337/diacare.27.2007.S84.

[2] M. Boulton, D. Foreman, G. Williams, D. McLeod, VEGF localisation in diabetic retinopathy., Br. J. Ophthalmol. 82 (1998) 561–8. doi:10.1136/bjo.82.5.561.

[3] J.W. Miller, J. Le Couter, E.C. Strauss, N. Ferrara, Vascular endothelial growth factor a in intraocular vascular disease., Ophthalmology. 120 (2013) 106–14. doi:10.1016/j.ophtha.2012.07.038.

[4] A. Witmer, Vascular endothelial growth factors and angiogenesis in eye disease, Prog. Retin. Eye Res. 22 (2003) 1–29. doi:10.1016/S1350-9462(02)00043-5.

[5] D. a Antonetti, R. Klein, T.W. Gardner, Diabetic retinopathy., N. Engl. J. Med. 366 (2012) 1227–39. doi:10.1056/NEJMra1005073.

[6] J.G.F. Dowler, Laser management of diabetic retinopathy., J. R. Soc. Med. 96 (2003) 277–279. doi:10.1258/jrsm.96.6.277.

[7] K. a M. Solaiman, M.M. Diab, S. a Dabour, Repeated intravitreal bevacizumab injection with and without macular grid photocoagulation for treatment of diffuse diabetic macular edema., Retina. 33 (2013) 1623–9. doi:10.1097/IAE.0b013e318285c99d.

[8] P.L. Lip, F. Belgore, A.D. Blann, M.W. Hope-Ross, J.M. Gibson, G.Y.H. Lip, Plasma VEGF and soluble VEGF receptor FLT-1 in proliferative retinopathy: Relationship to endothelial dysfunction and laser treatment, Investig. Ophthalmol. Vis. Sci. 41 (2000) 2115–2119.

[9] Q.D. Nguyen, D.M. Brown, D.M. Marcus, D.S. Boyer, S. Patel, L. Feiner, et al., Ranibizumab for diabetic macular edema: Results from 2 phase iii randomized trials: RISE and RIDE, Ophthalmology. 119 (2012) 789–801. doi:10.1016/j.ophtha.2011.12.039.

[10] D.R.C.R. Network, A Phase II Randomized Clinical Trial of Intravitreal Bevacizumab for Diabetic Macular Edema, Ophthalmology. 114 (2007) 1860–1867. doi:10.1016/j.ophtha.2007.05.062.

[11] D. V. Do, Q.D. Nguyen, D. Boyer, U. Schmidt-Erfurth, D.M. Brown, R. Vitti, et al., One-year outcomes of the da VINCI study of VEGF trap-eye in eyes with diabetic macular edema, Ophthalmology. 119 (2012) 1658–1665. doi:10.1016/j.ophtha.2012.02.010.

[12] S. Pagliarini, S. Beatty, B. Lipkova, E. Perez-Salvador Garcia, S. Reynders, M. Gekkieva, et al., A 2-year, phase IV, multicentre, observational study of ranibizumab 0.5 mg in patients with neovascular age-related macular degeneration in routine clinical practice: The EPICOHORT study, J. Ophthalmol. 2014 (2014). doi:10.1155/2014/857148.

[13] S.Y. Cohen, G. Mimoun, H. Oubraham, A. Zourdani, C. Malbrel, S. Queré, et al., Changes in visual acuity in patients with wet age-related macular degeneration treated with intravitreal ranibizumab in daily clinical practice: the LUMIERE study, RETINA. 33 (2013) 474–481. doi:10.1097/IAE.0b013e31827b6324.

[14] J.E. Scott, The chemical morphology of the vitreous., Eye (Lond). 6 ( Pt 6) (1992) 553– 5. doi:10.1038/eye.1992.120.

[15] L. Ma, X. Wang, Z. Zhang, X. Zhou, a Chen, L. Yao, Identification of the ligand- binding domain of human vascular-endothelial-growth-factor receptor Flt-1., 49

Biotechnol. Appl. Biochem. 34 (2001) 199–204. doi:10.1042/BA20010043.

[16] S.T. Wall, K. Saha, R.S. Ashton, K.R. Kam, D. V Schaffer, K.E. Healy, Multivalency of Sonic hedgehog conjugated to linear polymer chains modulates protein potency., Bioconjug. Chem. 19 (2008) 806–12. doi:10.1021/bc700265k.

[17] A. Conway, T. Vazin, D.P. Spelke, N. a Rode, K.E. Healy, R.S. Kane, et al., Multivalent ligands control stem cell behaviour in vitro and in vivo., Nat. Nanotechnol. (2013) 1–8. doi:10.1038/nnano.2013.205.

[18] B.W. Han, H. Layman, N.A. Rode, A. Conway, D. V Schaffer, N. Boudreau, et al., Multivalent conjugates of sonic hedgehog accelerate diabetic wound healing., Tissue Eng. Part A. 21 (2015) 2366–2378. doi:10.1089/ten.TEA.2014.0281.

[19] J.F. Pollock, R.S. Ashton, N. a Rode, D. V Schaffer, K.E. Healy, Molecular characterization of multivalent bioconjugates by size-exclusion chromatography with multiangle laser light scattering., Bioconjug. Chem. 23 (2012) 1794–801. doi:10.1021/bc3000595.

[20] A.K. Jha, A. Mathur, F.L. Svedlund, J. Ye, Y. Yeghiazarians, K.E. Healy, Molecular Weight and Concentration of Heparin in Hyaluronic Acid-based Matrices Modulates Growth Factor Retention Kinetics and Stem Cell Fate, J. Control. Release. 209 (2015) 308–316. doi:10.1016/j.jconrel.2015.04.034.

[21] A. Jha, K. Tharp, J. Ye, J. Santiago-Ortiz, W. Jackson, A. Stahl, et al., Growth factor sequestering and presenting hydrogels promote survival and engraftment of transplanted stem cells, Biomaterials. 47 (2015) 1–12. doi:10.1016/j.biomaterials.2014.12.043.

[22] T.-K. Ito, G. Ishii, S. Saito, K. Yano, A. Hoshino, T. Suzuki, et al., Degradation of soluble VEGF receptor-1 by MMP-7 allows VEGF access to endothelial cells., Blood. 113 (2009) 2363–9. doi:10.1182/blood-2008-08-172742.

[23] F.M. Veronese, Peptide and protein PEGylation, Biomaterials. 22 (2001) 405–417. doi:10.1016/S0142-9612(00)00193-9.

[24] S.C. Fishburn, The pharmacology of PEGylation: Balancing PD with PK to generate

novel therapeutics, J. Pharm. Sci. 97 (2010) 4167–4183. doi:10.1002/jps.

[25] C.S. Nickerson, J. Park, J. a Kornfield, H. Karageozian, Rheological properties of the vitreous and the role of hyaluronic acid., J. Biomech. 41 (2008) 1840–6. doi:10.1016/j.jbiomech.2008.04.015.

[26] J. Lu, T. Su, R. Thomas, Structural Conformation of Bovine Serum Albumin Layers at the Air-Water Interface Studied by Neutron Reflection., J. Colloid Interface Sci. 213 (1999) 426–437. doi:10.1006/jcis.1999.6157.

[27] R. Thadhani, T. Kisner, H. Hagmann, V. Bossung, S. Noack, W. Schaarschmidt, et al., Pilot Study of Extracorporeal Removal of Soluble Fms-Like Tyrosine Kinase 1 in Preeclampsia, Circulation. 124 (2011) 940–950. doi:10.1161/CIRCULATIONAHA.111.034793.

[28] N. a. Peppas, E.W. Merrill, Crosslinked poly(vinyl alcohol) hydrogels as swollen elastic networks., J. Appl. Polym. Sci. 21 (1977) 1763–1770. doi:10.1002/app.1977.070210704.

[29] J.E. Mark, B. Erman, Rubberlike Elasticity: A Molecular Primer, 1st ed., John Wiley & Sons, Toronto, 1988.

[30] K.Y. Lee, J. a. Rowley, P. Eiselt, E.M. Moy, K.H. Bouhadir, D.J. Mooney, Controlling mechanical and swelling properties of alginate hydrogels independently by cross-linker type and cross-linking density, Macromolecules. 33 (2000) 4291–4294. doi:10.1021/ma9921347.

[31] J. Baier Leach, K. a. Bivens, C.W. Patrick Jr., C.E. Schmidt, Photocrosslinked hyaluronic acid hydrogels: Natural, biodegradable tissue engineering scaffolds, Biotechnol. Bioeng. 82 (2003) 578–589. doi:10.1002/bit.10605.

[32] P.L. Ritger, N. a. Peppas, A simple equation for description of solute release I. Fickian and non-Fickian release from non-swellable devices in the form of slabs, spheres, cylinders or discs, J. Control. Release. 5 (1987) 23–36. doi:10.1016/0168- 3659(87)90035-6.

[33] P.G. De Gennes, Conjectures on the transport of a melt through a gel, Macromolecules. 19 (1986) 1245–1249. doi:10.1021/ma00158a051.

[34] D.M. Soumpasis, Theoretical analysis of fluorescence photobleaching recovery experiments, Biophys. J. 41 (1983) 95–97. doi:10.1016/S0006-3495(83)84410-5.

[35] R. Gaudana, H.K. Ananthula, A. Parenky, A.K. Mitra, Ocular drug delivery., AAPS J. 12 (2010) 348–60. doi:10.1208/s12248-010-9183-3.

[36] S.S. Lee, M.R. Robinson, Novel drug delivery systems for retinal diseases. A review., Ophthalmic Res. 41 (2009) 124–35. doi:10.1159/000209665.

[37] H.F. Edelhauser, C.L. Rowe-Rendleman, M.R. Robinson, D.G. Dawson, G.J. Chader, H.E. Grossniklaus, et al., Ophthalmic drug delivery systems for the treatment of retinal diseases: basic research to clinical applications., Invest. Ophthalmol. Vis. Sci. 51 (2010) 5403–5420. doi:10.1167/iovs.10-5392.

[38] E.M. Del Amo, A. Urtti, Current and future ophthalmic drug delivery systems. A shift to the posterior segment., Drug Discov. Today. 13 (2008) 135–43. doi:10.1016/j.drudis.2007.11.002.

[39] T.R. Thrimawithana, S. Young, C.R. Bunt, C. Green, R.G. Alany, Drug delivery to the posterior segment of the eye., Drug Discov. Today. 16 (2011) 270–7. doi:10.1016/j.drudis.2010.12.004.

[40] S.K. Sahoo, F. Dilnawaz, S. Krishnakumar, Nanotechnology in ocular drug delivery., Drug Discov. Today. 13 (2008) 144–51. doi:10.1016/j.drudis.2007.10.021.

[41] D. Maurice, Review: practical issues in intravitreal drug delivery., J. Ocul. Pharmacol. Ther. 17 (2001) 393–401. doi:10.1089/108076801753162807.

[42] R.D. Jager, L.P. Aiello, S.C. Patel, E.T. Cunningham, Risks of intravitreous injection: a comprehensive review., Retina. 24 (2004) 676–698. doi:10.1097/00006982-200410000- 00002.

[43] E.J. Oh, J.S. Choi, H. Kim, C.K. Joo, S.K. Hahn, Anti-Flt1 peptide - Hyaluronate conjugate for the treatment of retinal neovascularization and diabetic retinopathy, Biomaterials. 32 (2011) 3115–3123. doi:10.1016/j.biomaterials.2011.01.003.

[44] A. Bochot, E. Fattal, Liposomes for intravitreal drug delivery: a state of the art., J. Control. Release. 161 (2012) 628–34. doi:10.1016/j.jconrel.2012.01.019.

[45] T. Yasukawa, Y. Ogura, Y. Tabata, H. Kimura, P. Wiedemann, Y. Honda, Drug delivery systems for vitreoretinal diseases., Prog. Retin. Eye Res. 23 (2004) 253–81. doi:10.1016/j.preteyeres.2004.02.003.

[46] F.M. Veronese, G. Pasut, PEGylation, successful approach to drug delivery, Drug Discov. Today. 10 (2005) 1451–1458.

[47] E.W.M. Ng, D.T. Shima, P. Calias, E.T. Cunningham, D.R. Guyer, A.P. Adamis, Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease., Nat. Rev. Drug Discov. 5 (2006) 123–32. doi:10.1038/nrd1955.

[48] P. Bishop, The biochemical structure of mammalian vitreous, Eye. 10 (1996) 664–670. doi:10.1038/eye.1996.159.

[49] D. Park, Y. Kim, H. Kim, K. Kim, Y.-S. Lee, J. Choe, et al., Hyaluronic acid promotes angiogenesis by inducing RHAMM-TGFβ receptor interaction via CD44-PKCδ, Mol. Cells. 33 (2012) 563–574. doi:10.1007/s10059-012-2294-1.

[50] U. Laurent, P. Törnquist, K. Granath, K. Lilja-Englind, D. Ytterberg, Molecular weight and concentration of hyaluronan in vitreous humour from diabetic patients, Acta Ophthalmol. 68 (1990) 109–112. doi:10.1111/j.1755-3768.1990.tb01972.x.

[51] G.A. Lutty, D.C. Thompson, J.Y. Gallup, R.J. Mello, A. Patz, A. Fenselau, Vitreous : An Inhibitor of Retinal Extract-Induced Neovascularization, Investig. Ophthalmol. Vis. Sci. 24 (1983) 52–56.

Chapter 3: sFlt multivalent conjugates inhibit angiogenesis and improve half-life in vivo

3.1 Introduction

Diabetes affects 285 million adults worldwide, which accounts for 6.4% of the world’s population. This number is expected to increase to 439 million people by 2030 [1]. In the United States alone, 25.8 million people have diabetes and it is estimated that another 57 million people are considered pre-diabetic as a result of abnormally high blood glucose levels resulting from poor diet. Diabetic retinopathy is one of the most debilitating consequences of diabetes and is currently the leading cause of blindness among individuals aged 20-64 [2–4]. In the development of diabetic retinopathy, hypoxic neurons upregulate growth factors that drive angiogenesis, a process primarily mediated by vascular endothelial growth factor (VEGF) [5–7]. Newly formed blood vessels have loose cell-cell junctions, causing macular edema and pooling of blood, which leads to areas of vision loss. Anti-VEGF therapeutics such as Lucentis (ranibizumab, Genentech, 48 kDa humanized antibody fragment), Avastin (bevacizumab, Genentech, a 150 kDa humanized antibody) and Eylea (aflibercept, Regeneron, a 110 kDa VEGF-receptor fusion protein) are the clinically used drugs for treating diabetic retinopathy in the clinic. However, these therapeutics suffer from short drug half-lives as a result of small molecular size and single injections of these drugs provide limited therapeutic effect over a finite period of time [8]. Consequently, low levels of patient compliance significantly limit the effect of these drugs to maintain visual acuity [9,10]. Therefore, the development of new drug delivery approaches is necessary to facilitate increased drug residence time within the vitreous. Such approaches will help to reduce the frequency at which patients are required to receive injections thereby improving quality of life as well as long term visual potential. We have developed a novel multivalent drug technology platform that allows for the long-term delivery of anti-VEGF drugs into the vitreous. Using a naturally occurring biopolymer, hyaluronic acid (HyA) we synthesized drug multivalent conjugates by chemically conjugating sFlt (fms-like tyrosine kinase-1 [11]), an anti-VEGF protein, to the biopolymer chain thereby significantly increasing the overall drug size. We have shown previously that these anti-VEGF multivalent conjugates, mvsFlt, maintain their ability to bind VEGF and inhibit endothelial cell proliferation, migration and tube formation in vitro. Furthermore, conjugation of sFlt to HyA significantly enhanced retention within in vitro models of the vitreous and protected sFlt from protease degradation by matrix metalloproteinase-7, an enzyme that specifically degrades sFlt (under review, [12]). The aim of this study was therefore to evaluate the in vivo activity of the mvsFlt conjugate. We used a corneal angiogenesis assay as an initial screen for examining the effect of HyA conjugation on sFlt bioactivity and then

investigated the half-life of mvsFlt in the rat vitreous in order to determine how HyA conjugation affected residence time of sFlt. Finally, we employed a rat oxygen-induced retinopathy (OIR) model to examine both bioactivity and the effect of increased mvsFlt half- life on retinal angiogenesis. This rat OIR model is one of the best and well-characterized models of diabetic retinopathy due to the similarity of symptoms that result in comparison to those seen in humans. The results from this study suggest our conjugation technology may have far reaching impacts on the development of pharmaceuticals with longer half-lives in the vitreous not only for treating diabetic retinopathy but also for retinal neovascular diseases such as wet AMD.

3.2 Materials and Methods

3.2.1. Expression of Soluble Flt-1 Receptor The sFlt sequence for the first 3 Ig-like extracellular domains of sFlt-1 [13] was cloned into the pFastBac1 plasmid (Life Technologies) and then transformed into DH10Bac E.coli, which were plated on triple antibiotic plates containing kanamycin (50 µg/mL), gentamicin (7 µg/mL, Sigma Aldrich), tetracycline (10 µg/mL, Sigma Aldrich), IPTG (40 µg/mL, Sigma Aldrich) and Bluo-gal (100 µg/mL, Thermo Fisher Scientific). The sFlt gene-containing bacmid was isolated from DH10Bac E.coli (Life Technologies) and transfected into SF9 insect cells for virus production (provided by the Tissue Culture Facility, UC Berkeley). Virus was then used to infect High Five insect cells (provided by the Tissue Culture Facility, UC Berkeley) to induce sFlt protein expression. After 3 days, protein was purified from the supernatant using Ni-NTA agarose beads (Qiagen Laboratories). Recombinant sFlt was eluted from the Ni-NTA beads using an imidazole gradient and then concentrated and buffer exchanged with 10% glycerol/PBS using Amicon Ultra-15mL Centrifugal devices (EMD Millipore). The protein solution was sterile filtered and the concentration was determined by BCA assay (Thermo Fisher Scientific).

3.2.2 mvsFlt Conjugate Synthesis Conjugation of sFlt to HyA was carried out according to the schematic in Figure 3.1, and as described previously ([12,14–16]. To make thiol-reactive HyA intermediates, 3,3′-N-(ε- maleimidocaproic acid) hydrazide (EMCH, Pierce, 1.2 mg/mL), 1-hydroxybenzotriazole hydrate (HOBt, Sigma, 0.3mg/mL) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, Pierce, 10 mg/mL) were added to a 3 mg/ml solution of 650 kDa HyA (Lifecore Biotechnology) in 0.1 M 2-(N-morpholino) ethanesulphonic acid (MES) (Sigma) buffer (pH 6.5) and allowed to react at 4 °C for 4 h. The solution was then dialyzed into pH 7.0 PBS containing 10% glycerol. Recombinant sFlt was treated with 2-iminothiolane at 10 molar excess to create thiol groups for conjugation to the maleimide group on EMCH. Activated HyA-EMCH was then added to sFlt at a 1:10 molar ratio (HyA to sFlt) and allowed to react at 4 °C overnight to synthesize the final mvsFlt conjugate. The mvsFlt conjugate was

dialyzed with 100 kDa molecular weight cut-off (MWCO) Float-A-Lyzer G2 (Spectrum Labs) in pH 7.0 PBS exhaustively to remove unreacted sFlt. The concentration of mvsFlt was measured using a BCA assay.

3.2.3 Corneal Angiogenesis Assay All experiments were performed with wild-type 7 to 12-week old male and female littermate FVB/n mice. Mice were maintained under pathogen-free conditions in the UCSF barrier facility and conducted in accordance with procedures approved by the UCSF Institutional Animal Care and Use Committee. Mice were anesthetized by isofluorane inhalation (Abbott Laboratories, Abbott Park, IL), 10mg/kg carprofren (Sigma, St. Louis, MO), and by topical application of 0.5% Proparacaine (Bausch & Lomb, Rochester, NY) placed on the cornea. An alkaline burn was created by applying filter paper 2.5mm in diameter soaked in 0.1N NaOH (Sigma, St. Louis, MO) for 30 seconds to the central cornea followed by rinsing with 250 µL of phosphate-buffered saline (PBS). After the chemical burn treatment, topical 0.5% proparacaine was added to the cornea for anesthesia. Mice were administered 5 µL subconjunctival injections with sFlt (150 µg/ml), mvsFlt (150 µg/ml), or PBS at day 1 and day 3 after burn. Ten days after treatment, eyes were enucleated and the corneas were dissected and fixed in 4% paraformaldehyde overnight at 4°C. Corneas were blocked with 3% BSA and stained with DAPI, rabbit anti-mouse CD31 primary antibody (Santa Cruz Biotechnology, Dallas, TX) and goat anti-rabbit Alexa Fluor 488 secondary antibody (Life Technologies, Carlsbad, CA) for quantification of blood vessels. Corneas were cut into quadrants and flat- mounted onto glass slides using Fluoromount Mounting Medium (Sigma, St. Louis, MO). Imaging was carried out with an automated slide scanner, Zeiss Axioscan Z1 (Zeiss Instruments). Corneal blood vessel coverage was quantified using NIH ImageJ software by comparing the total cornea area to the vascularized area.

3.2.4 Determination of mvsFlt Intravitreal Half-Life All half-life experiments were performed on 8-week old Brown Norway rats obtained from Charles River Laboratories and treated in accordance with protocols approved by the Animal Care and Use Committee at UC Berkeley. Rats were anesthetized using a mixture of ketamine and xylazine (50 mg and 10 mg/kg body weight, respectively) for the surgical procedure. Eyes were injected intravitreally 1 mm behind the limbus with 5 µl of PBS, sFlt or mvsFlt at 1 mg/mL using a 30-gauge Hamilton syringe and monitored daily for signs of inflammation. This concentration was selected to maximize fluorescence in the vitreous and remain in the detection limit of the fluorometer after 48 hours. Rats were sacrificed with CO2 asphyxiation in groups at 0, 4, 12, 24 and 48 hours post injection and eyes were immediately enucleated and placed on dry ice. Frozen vitreous was then extracted from the eye and immersed in 100 µL of RIPA buffer. After shaking on ice for 2 hours, each vitreous sample was homogenized with a Tissue Tearor (Bio Spec Products, Inc.) and the fluorescence measured using a fluorometer (Molecular Devices). Quantification was carried out by normalizing the fluorescence of

vitreous samples to the 0 hour vitreous fluorescence readings within their respective group. The half-lives of sFlt and mvsFlt were calculated according to equation (3.1): !�� = �!� (1) where �� is the concentration at time �, �! is the initial concentration and � is the elimination constant given by equation (3.2): log (2) = (2) � �!/! where �!/! is the half-life. The values used for calculating �!/! were based on data from the 48- hour time point.

3.2.5 OIR Angiogenesis Model Pregnant Brown Norway rats were obtained from Charles River Laboratories. All the animal experiments were performed in compliance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. Newborn pups were assigned to PBS, sFlt or mvsFlt treatment groups. Light was cycled on a 12 hour on, 12 hour off schedule and room temperature was maintained at approximately 21C. Rat pups were exposed to hyperoxia (75%

O2) from postnatal day 7 (P7) to P12. The oxygen-treated rats were housed in an incubator connected to an Oxycler Model A4 (Redfield, NY) with oxygen and nitrogen, allowing for adjustment of oxygen concentration to 75%±2%. The rats were placed in the oxygen chamber with enough food and water to sustain them for 5 days. The chamber was not opened during hyperoxia exposure from P7 to P12. On P12, the animals were returned to room air and administered intravitreal injections with 2 µL of PBS, sFlt or mvsFlt at 150 µg/mL. Rats at P17 were anesthetized and perfused with high-molecular weight FITC-dextran (2 x 106; Sigma- Aldrich, St. Louis MO) as described by Smith et al. (PMID 7507904). Retinas were dissected and flat-mounted and the vasculature was imaged using a fluorescence microscope (CKX41; Olympus, Center Valley, PA, USA). Vascular coverage at P17 was quantified using NIH ImageJ by comparing the total retinal area to the area of vascularization.

3.2.6 Statistical Analysis Values are expressed as means ± standard deviations (SD). Statistical analysis was performed with two-tailed t-tests to compare mean values. One-way (with Tukey post-hoc analysis) and Two-way ANOVA (with Bonferroni posttest) were also used to compare treatment groups in the quantitative measurements where appropriate (Prism, GraphPad Software). A P-value of less than 0.05 was considered to be statistically significant.

3.3 Results

3.3.1 Multivalent sFlt synthesis and Characterization The mvsFlt conjugate used in these studies was synthesized according to the schematic shown in Figure 3.1. The molecular weights of the protein and polymer components were characterized using SEC-MALS and confirmed with SDS-PAGE [12]. The mvsFlt conjugate used in the studies described here was conjugated with an average of 5.3 sFlt molecules per chain of 650 kDa HyA as determined by SEC-MALS [12]. SDS-PAGE results indicated that approximately 80% of the sFlt in the mvsFlt solution was covalently conjugated and the remainder was nonspecifically interacting with the HyA, likely through ionic interactions between the oppositely charged species. We also used DLS to characterize the hydrodynamic diameter of the sFlt and mvsFlt. The size of the unconjugated sFlt was 22.6±3.1 nm in diameter whereas the mvsFlt was 236.3±38.7nm in diameter [12].

3.3.2 sFlt and mvsFlt equally inhibit corneal angiogenesis The chemical injury-based corneal angiogenesis model was used as an initial screen to determine whether conjugation of sFlt to HyA reduced the affinity of sFlt for VEGF in vivo. Ten days-post corneal injury, all mice treated with sFlt and mvsFlt displayed similar inhibitory profiles of corneal angiogenesis (Figure 3.2). Corneas treated with PBS had 28.8±11.5% blood vessel coverage in contrast to corneas treated with sFlt and mvsFlt, which had 12.8±3.8% and 15.8±7.1% vascular coverage, respectively.

3.3.3 mvsFlt has significantly?? longer? half-life than sTlt in the vitreous We initially confirmed that the vitreous of Brown Norway rats could be used to determine intravitreal residence time of different sized molecules using fluorescently tagged dextrans of varying sizes (Appendix II). Differences in residence time between sFlt and mvsFlt were immediately apparent beginning at 4 hours where only 18.2±7.3% of sFlt remained compared to 105.8±9.8% of mvsFlt (Figure 3.3). By 12 hours, only 2.6±1.9% of sFlt remained detectable compared to 62.9±14.1% of mvsFlt. By 2 days post injection, sFlt was almost undetectable (1.2±0.5%) whereas 66.2±28.6% of mvsFlt remained in the vitreous. The half- life of sFlt in the vitreous was calculated to be 3.3 hours compared to 35 hours for mvsFlt.

3.3.4 mvsFlt is a more potent inhibitor of retinal angiogenesis We next used an OIR model of retinal angiogenesis assay to examine the effect of HyA conjugation of sFlt inhibition of retinal angiogenesis. This short-term model allowed us to also indirectly examine the effect of mvsFlt half-life on prolonged angiogenesis inhibition. Neovascular coverage was calculated by comparing the area of vascular coverage to total retinal area. After 5 days of treatment, retinal vascular coverage of PBS-injected eyes was 84.3±3.8% whereas retinas treated with intravitreal injections of sFlt were 85.4±6.1%. Both were statistically significant from intravitreal injections of mvsFlt, 72.9±3.4% (Figure 3.4).

3.4. Discussion The overall goal of this study was to determine whether increasing sFlt molecular size through conjugation to HyA would increase drug half-life in the vitreous and to investigate the ability of of the multivalent sFlt bioconjugate to inhibit in vivo angiogenesis. In contrast to other drugs that are currently used for treating diabetic retinopathy that suffer from short half- lives, we have developed large macromolecules of multivalent protein bioconjugates that maintain their ability in inhibiting VEGF-driven angiogenesis in vivo and show significantly longer residence time in the vitreous.

Figure 3.1: Synthesis of mvsFlt schematic. mvsFlt bioconjugates were synthesized using a 3-step reaction in which HyA was reacted with EDC and EMCH to create a thiol reactive HyA-EMCH intermediate. sFlt was then treated with 2-iminothiolane and reacted with the HyA-EMCH intermediate for the synthesis of the final mvsFlt product.

Previously, we described a chemical scheme for creating multivalent protein bioconjugates of HyA and the anti-VEGF molecule, sFlt as shown in Figure 3.1 [12,14–16].. We were able to make several mvsFlt bioconjugates of varying molecular weights and protein valencies, all of which were equally able to inhibit VEGF in several in vitro assays of angiogenesis. We also demonstrated that conjugation of sFlt to HyA shielded the protein from protease degradation with a matrix metalloproteinase that is specific to sFlt, MMP-7 [17]. We created an in vitro model of the vitreous using crosslinked HyA hydrogels to examine the effect of HyA conjugation of sFlt diffusion. We found that low molecular weight mvsFlt (300 kDa), which had hydrodynamic diameter of 123.9 nm (±23.1 nm), had very similar diffusion profiles to sFlt whereas higher molecular weight mvsFlt (650 kDa and 1 MDa mvsFlt) diffused significantly slower [12]. We chose to investigate the 650 kDa bioconjugate for its activity in vivo.

Figure 3.2: sFlt and mvsFlt equally inhibit corneal angiogenesis. A) Schematic depicting methods utilized for carrying out corneal burn model. Mice were treated twice with 5µl of PBS, sFlt or mvsFlt at day 1 and 3 following the chemical burn. B) Representative images of eyes treated with PBS, sFlt and mvsFlt. CD31 positive (green) staining of corneal blood vessels. B) Quantification of corneal angiogenesis at day 10 following treatment. One-way ANOVA gives p value **<0.01 (n.s.- not significant; *p<0.05; **p<0.01). Scale bars correspond to 20 µm.

Figure 3.3: mvsFlt has longer residence time in the rat vitreous. A) Schematic depicting methods used to determine the half-life of fluorescently tagged sFlt in the rat vitreous. The vitreous was injected with 5µl of Alexa Fluor 488-tagged sFlt or mvsFlt. After 0, 4, 12, 24, and 48 hours, the rats were sacrificed and their eyes were enucleated and frozen for analysis. The vitreous was then removed, immersed in RIPA buffer and homogenized for subsequent fluorescence measurements. B) Conjugation to HyA significantly improves residence time of sFlt in the vitreous after 48 hours in comparison to sFlt. Results are expressed as mean ±SD (*p<0.05, **p<0.01, ***p<0.0001). * indicates a difference between the mvsFlt and sFlt at the given time point. Two-way ANOVA gives p-value ***<0.001.

In this study, we used several different in vivo animal models for addressing three questions: 1) does the conjugation of sFlt to HyA affect the ability of sFlt to inhibit VEGF- driven angiogenesis; 2) does increasing the overall molecular size of sFlt lead to prolonged intravitreal half-life; and 3) does increasing intravitreal half-life prolong the inhibitory profile of sFlt. To answer our first question, we used a corneal angiogenesis assay as a facile method to visualize the effect of sFlt and mvsFlt on inhibition of angiogenesis. This assay is an excellent model for studying in vivo angiogenesis due to ease of vessel visualization on the physiologically avascular and transparent cornea, which is normally devoid of blood vessels and rapidly vascularizes upon stimulation with a chemical burn or injury [18,19]. Results from the corneal angiogenesis assay indicated that subconjunctivally administered sFlt and mvsFlt equally inhibited VEGF-driven angiogenesis as shown in Figure 3.2. We did not anticipate a difference in angiogenesis inhibition between the conjugated and unconjugated protein due to

the nature of clearance mechanisms in the cornea and the subconjunctival space [20]. Following injection of drugs into the subconjunctival space, drugs are rapidly cleared into systemic blood circulation via an extensive capillary network that is able to absorb large hydrophilic substances such as sFlt and mvsFlt due to the presence and abundance of large pores in the vascular epithelium [21,22]. We anticipated that the large pores in the vascular epithelium would clear both sFlt and mvsFlt at similar rates despite their significantly different sizes, which our results validate.

Figure 3.4: mvsFlt inhibits retinal angiogenesis. A) Schematic showing methods used for carrying out the OIR model. Newborn rat pups were housed in normoxic conditions (21% oxygen, room air) from post-natal day (P) 0-7 to allow for normal retinal vasculature development and then transferred to hyperoxic conditions from P7-P12, which induces vessel pruning. At P13, the pups are transferred back into normoxic conditions and treated with 2 µl of PBS, sFlt or mvsFlt and sacrificed at P17. Representative images of retinas treated with PBS, sFlt and mvsFlt. Green staining indicates CD31+ cells. Scale bar corresponds to 250 µm. Dashed boxes magnify that portion of tissue (scale bar corresponds to 100 µm). B) Quantified retinal vascularization after 5 days of treatment. Percent retinal vascularization was calculated by comparing the area of vascularization to the total retinal area in the image. One-way ANOVA gives p-value***<0.001 (n.s.-not significant; **p<0.01).

To address our second question, we wanted to examine the direct effect of HyA conjugation on the half-life of mvsFlt in the rat vitreous. We validated this model in the rat vitreous using fluorescently tagged dextrans. We found that greater than 94% of the 40 kDa and 2 MDa dextrans were cleared within 48 hours (Figure S1). Based on this data, we chose to examine the following 5 time points: 0, 4, 12, 24 and 48 hours. Significant differences in

residence time were observed between sFlt and mvsFlt beginning at 4 hours and sustained until the end of the study at 48 hours as demonstrated in Figure 3.3. The half-lives of sFlt and mvsFlt were calculated according to equations (1) and (2) and were determined to be 3.3 and 35 hours, respectively. Conjugation to HyA therefore increased the half-life of sFlt by 10-fold, an effect seldom seen in the literature, outside of implant technologies. In comparison, liposomal delivery of bevacizumab increased the drug half-life by 5 times [23] but this approach presents several challenges including complexity of synthesis, aggregation of liposomes and blurring of vision. PEGylation is another approach to improve the half-life of drugs. PEGylation of the aptamer in pegaptanib improved its half-life by 2.5 fold from 4.2 days [24] to 10±4 days [25]. The OIR model [26,27] enabled us to examine retinal neovascularization with the added advantage of also examining the effect of conjugation on prolonged inhibition due to increased residence time mvsFlt conjugate in the vitreous, which helped us address our third and final question. In contrast to the cornea where conjugation does not show a significant advantage, the vitreous is a tissue where clearance mechanisms are highly dependent on molecular size [28,29]. Due to its increased hydrodynamic diameter, we anticipated that the mvsFlt conjugate would have a longer half-life in the vitreous and result in a prolonged duration of angiogenesis inhibition. The mvsFlt conjugate showed superior inhibition of retinal angiogenesis in this model in comparison to sFlt, which did not inhibit angiogenesis in comparison to the PBS control (Figure 3.4).

Figure 3.5: Schematic demonstrating the proposed mechanism of mvsFlt action. A) sFlt (red, unconjugated) and mvsFlt (red conjugated blue chain of HyA) are injected into a diabetic retina where there is a high concentration of VEGF (green circles). B) After a given time, t, the majority of the sFlt has been cleared from the vitreous and VEGF is thus able to induce blood vessel growth. mvsFlt has a longer residence time in the vitreous and is thus able to bind and inhibit VEGF over much longer periods of time, leading to prolonged inhibition of retinal angiogenesis.

We believe that this stark difference in inhibition of retinal angiogenesis is primarily due to clearance of the sFlt from the vitreous more rapidly in comparison to mvsFlt, resulting in ineffective angiogenesis inhibition. At the time of injection, both sFlt and mvsFlt have similar concentrations in the vitreous (Figure 3.5A). Over time, sFlt is small enough to be cleared from the vitreous leaving very low concentrations of drug (Figure 3.5B, top). This allows for an increase in the intravitreal VEGF concentration, which induces angiogenesis. mvsFlt has longer intravitreal residence time and is thus able to act as a sponge for VEGF over time (Figure 3.5B, bottom), inhibiting angiogenesis and maintaining the basal level of vascularization in the retina. The time over which this occurs is highly dependent on the animal model being used. Lucentis, which is similar in size to sFlt and is also a protein therapeutic, has an intravitreal half-life of 2.9 days in rabbits [30] and 2.6 days in rhesus monkeys [31]. The disparity in the half-lives of our compounds and those of therapeutics tested in rabbit and monkey models is most likely due to anatomical differences in ocular globe size and differences in the rate of vitreous turnover between species. We anticipate that the unconjugated sFlt would perform similarly to Lucentis in rabbit and monkey models whereas the mvsFlt would likely show significant improvement in half-life over currently used therapeutics based on our preliminary studies shown in Figure 3.3. When comparing the injections in rats to injections in humans, we are injecting proportionally the same volume of drug into the vitreous. We believe that the results we see in the rats will be similar to what we will observe in humans because of the proportional size in injection volume. Although we only injected 5 uL into the rat eye (which has a total volume of ~100 uL), we would inject 50 uL into the human eye. Assuming that the clearance mechanisms of the rat and human eye are similar, the increase in half-life should be observed in the human eye as well. The current approach to treating retinal neovascularization including diabetic retinopathy is carried out through intravitreal injection through the sclera. Once injected into the vitreous, drugs distribute through the vitreous in a charge and size dependent manner and are cleared from the vitreous through two main routes: anterior and posterior elimination. Small molecules are able to diffuse through the vitreous rapidly and have very low half-lives whereas large molecules have retarded diffusion that results in longer half-lives. Large, hydrophilic molecules are able to passively diffuse through the vitreous, around the lens into the anterior chamber and are cleared through the Schlemm’s canal or the uveoscleral outflow pathway. Due to the large surface area of the retina, molecules capable of posterior clearance have significantly lower half-lives in comparison to those eliminated through the anterior route [20,32]. The development of drugs with longer residence times for patients with diabetic retinopathy has been in high demand with the increasing prevalence of diabetes in populations worldwide. Currently used drugs have short half-lives in the vitreous due to small molecular size and thus require patients to receive frequent intravitreal injections, leading to low patient compliance and worsening of the disease. Clinically approved drugs Avastin, Lucentis, and Eylea have half-lives of 7.4 [33], 6.7 [30,31], and 5 days [34,35], respectively and require patients to receive injections every 4-6 weeks to maintain effective levels of intravitreal drug

[8]. Several different approaches have been taken to increase drug residence time in the vitreous for treating diseases of the posterior segment, each with its own advantages and drawbacks. Intravitreal implants have received much of the attention in intravitreal drug delivery over the last two decades, primarily used in corticosteroid delivery for posterior uveitis, diabetic macular edema (DME), and retinal vein occlusion. Implantable devices are able to deliver a constant supply of drug over several months to several years. Several types of implants have been developed such as Iluvien [36], Ozurdex [37], and Retisert [38] and vary in degradability, duration of effect, size, delivery method and composition. Despite being able to deliver drug over long periods of time, implants often require surgical implantation and invasive removal of the implant after the drug is depleted. Particulate systems such as liposomes and nanoparticles have also emerged as another drug delivery option for treating retinal diseases. Although drug delivery with particulate systems provides stability for the entrapped drug and is more straightforward requiring minimally invasive intravitreal injections, this approach is accompanied by several disadvantages including the formation of precipitate that blurs vision, increased production cost and heterogeneity in formulation due to aggregation of particles [39,40]. PEGylation has also been employed to increase intravitreal half-life of drugs and is the core technology behind Macugen, a PEGylated anti-VEGF aptamer (Bausch + Lomb). Although similar in theory to our approach, PEGylation uses a synthetic polymer for conjugation to a single biological molecule, which often results in significantly decreased drug bioactivity [41,42]. Furthermore, PEGylation on Macugen did not result in a drug with significantly longer half-live compared to clinically available drugs, most likely due to the small size of polyethylene glycol chain that is conjugated to the aptamer [43]. In contrast to these approaches, we employed a multivalent conjugate approach with HyA, a naturally occurring biopolymer found in high concentrations within the vitreous, and conjugated several molecules of sFlt, a potent anti-VEGF protein, to it. HyA was chosen not only based on its natural ubiquity within the eye and body but also due to its exceptionally low turnover rate within the vitreous, 30 days [44]. We have shown that conjugation of sFlt to HyA does not negatively impact the ability of sFlt to bind and inhibit VEGF in vitro [12] or reduce its anti-angiogenic potency in vivo (Figure 3.2) affects that accompany other conjugation technologies such as protein PEGylation. Furthermore, we demonstrated that conjugation to HyA significantly improved the residence time of sFlt in the vitreous of the eye (Figure 3.3), which resulted in prolonged inhibition of retinal angiogenesis (Figure 3.4). Given these promising results, we anticipate that the future clinical use of multivalent conjugates of sFlt may significantly increase the intravitreal drug residence time and inhibit retinal angiogenesis over a longer period of time. Due to the ease of synthesis and tunability in this approach, we believe that our conjugation technology could be instrumental in improving the half-life of drugs in the human eye not only for treating diabetic retinopathy but other diseases as well.

3.5 Acknowledgements This work was supported in part by the National Science Foundation Graduate Research Fellowship, National Heart Lung and Blood Institute of the National Institutes of Health R01HL096525 (K.E.H.), and the Jan Fandrianto Chair Fund (K.E.H.). We would like to thank Jonathan Winger and Xiao Zhu for guidance with the insect cell protein expression system and providing reagents. We would like to acknowledge Ann Fischer for help express the sFlt protein in the Tissue Culture Facility at UC Berkeley and Dawn Spelke and Anusuya Ramasubramanian for help optimizing protein purification from insect cells. We are also grateful for the help from Leah Byrne and John Flannery at the Helen Wills Neuroscience Institute at UC Berkeley for aiding us in the development of the rat intravitreal residence time model and for allowing us to use their facilities.

3.6 References [1] J.E. Shaw, R. a. Sicree, P.Z. Zimmet, Global estimates of the prevalence of diabetes for 2010 and 2030, Diabetes Res. Clin. Pract. 87 (2010) 4–14. doi:10.1016/j.diabres.2009.10.007.

[2] D. a Antonetti, R. Klein, T.W. Gardner, Diabetic retinopathy., N. Engl. J. Med. 366 (2012) 1227–39. doi:10.1056/NEJMra1005073.

[3] R.L. Engerman, Perspectives in Diabetes Pathogenesis of Diabetic Retinopathy, Diabetes. 38 (1989) 1203–1206.

[4] R. Fong, D.S., Aiello, L., Gardner, T.W., King, G.L., Blankenship, G., Cavallerano, J.D., Ferris, F.L., Klein, Retinopathy in Diabetes, Diabetes Care. 27 (2004) S84–87. doi:10.2337/diacare.27.2007.S84.

[5] M. Boulton, D. Foreman, G. Williams, D. McLeod, VEGF localisation in diabetic retinopathy., Br. J. Ophthalmol. 82 (1998) 561–8. doi:10.1136/bjo.82.5.561.

[6] J.W. Miller, J. Le Couter, E.C. Strauss, N. Ferrara, Vascular endothelial growth factor a in intraocular vascular disease., Ophthalmology. 120 (2013) 106–14. doi:10.1016/j.ophtha.2012.07.038.

[7] A. Witmer, Vascular endothelial growth factors and angiogenesis in eye disease, Prog. Retin. Eye Res. 22 (2003) 1–29. doi:10.1016/S1350-9462(02)00043-5.

[8] M.W. Stewart, P.J. Rosenfeld, F.M. Penha, F. Wang, Z. Yehoshua, E. Bueno-Lopez, et

al., Pharmacokinetic rationale for dosing every 2 weeks versus 4 weeks with intravitreal ranibizumab, bevacizumab, and aflibercept (vascular endothelial growth factor Trap- eye), Retina. (2012) 1. doi:10.1097/IAE.0b013e31822c290f.

[9] S. Pagliarini, S. Beatty, B. Lipkova, E. Perez-Salvador Garcia, S. Reynders, M. Gekkieva, et al., A 2-year, phase IV, multicentre, observational study of ranibizumab 0.5 mg in patients with neovascular age-related macular degeneration in routine clinical practice: The EPICOHORT study, J. Ophthalmol. 2014 (2014). doi:10.1155/2014/857148.

[10] S.Y. Cohen, G. Mimoun, H. Oubraham, A. Zourdani, C. Malbrel, S. Queré, et al., Changes in visual acuity in patients with wet age-related macular degeneration treated with intravitreal ranibizumab in daily clinical practice: the LUMIERE study, RETINA. 33 (2013) 474–481. doi:10.1097/IAE.0b013e31827b6324.

[11] K. Tanaka, S. Yamaguchi, A. Sawano, M. Shibuya, Characterization of the extracellular domain in vascular endothelial growth factor receptor-1 (Flt-1 tyrosine kinase), Jpn.J.Cancer Res. 88 (1997) 867–876.

[12] E.I. Altiok, J.L. Santiago-Ortiz, A. Zbinden, A.K. Jha, D. Bhatnagar, W.M. Jackson, et al., Multivalent Hyaluronic Acid Bioconjugates Improve sFlt Activity In Vitro, (under Rev. (2015).

[13] L. Ma, X. Wang, Z. Zhang, X. Zhou, a Chen, L. Yao, Identification of the ligand- binding domain of human vascular-endothelial-growth-factor receptor Flt-1., Biotechnol. Appl. Biochem. 34 (2001) 199–204. doi:10.1042/BA20010043.

[14] B.W. Han, H. Layman, N.A. Rode, A. Conway, D. V Schaffer, N. Boudreau, et al., Multivalent conjugates of sonic hedgehog accelerate diabetic wound healing., Tissue Eng. Part A. 21 (2015) 2366–2378. doi:10.1089/ten.TEA.2014.0281.

[15] A. Conway, T. Vazin, D.P. Spelke, N. a Rode, K.E. Healy, R.S. Kane, et al., Multivalent ligands control stem cell behaviour in vitro and in vivo., Nat. Nanotechnol. (2013) 1–8. doi:10.1038/nnano.2013.205.

[17] T.-K. Ito, G. Ishii, S. Saito, K. Yano, A. Hoshino, T. Suzuki, et al., Degradation of soluble VEGF receptor-1 by MMP-7 allows VEGF access to endothelial cells., Blood. 113 (2009) 2363–9. doi:10.1182/blood-2008-08-172742.

[18] M.F. Chan, J. Li, A. Bertrand, A.-J. Casbon, J.H. Lin, I. Maltseva, et al., Protective effects of matrix metalloproteinase-12 following corneal injury., J. Cell Sci. 126 (2013) 3948–60. doi:10.1242/jcs.128033.

[19] C.A. Staton, M.W.R. Reed, N.J. Brown, A critical analysis of current in vitro and in vivo angiogenesis assays, Int. J. Exp. Pathol. 90 (2009) 195–221. doi:10.1111/j.1365- 2613.2008.00633.x.

[20] R. Gaudana, H.K. Ananthula, A. Parenky, A.K. Mitra, Ocular drug delivery., AAPS J. 12 (2010) 348–60. doi:10.1208/s12248-010-9183-3.

[21] M. Hornof, E. Toropainen, A. Urtti, Cell culture models of the ocular barriers., Eur. J. Pharm. Biopharm. 60 (2005) 207–25. doi:10.1016/j.ejpb.2005.01.009.

[22] A. Urtti, L. Salminen, Minimizing Systemic Absorption of Topically Administered Ophtalmic Drugs, Surv. Ophtalmol. 37 (1993) 435–456. doi:10.1016/0039- 6257(93)90141-S.

[23] M. Abrishami, S. Zarei-Ghanavati, D. Soroush, M. Rouhbakhsh, M.R. Jaafari, B. Malaekeh-Nikouei, Preparation, characterization, and in vivo evaluation of nanoliposomes-encapsulated bevacizumab (avastin) for intravitreal administration., Retina. 29 (2009) 699–703. doi:10.1097/IAE.0b013e3181a2f42a.

[24] D.W. Drolet, J. Nelson, C.E. Tucker, P.M. Zack, K. Nixon, R. Bolin, et al., Pharmacokinetics and safety of an anti-vascular endothelial growth factor aptamer (NX1838) following injection into the vitreous humor of rhesus monkeys, Pharm. Res. 17 (2000) 1503–1510. doi:10.1023/A:1007657109012.

[25] B.P. Nicholson, A.P. Schachat, A review of clinical trials of anti-VEGF agents for diabetic retinopathy., Graefes Arch. Clin. Exp. Ophthalmol. 248 (2010) 915–30. doi:10.1007/s00417-010-1315-z.

[26] B. Ricci, Oxygen-induced retinopathy in the rat model, in: Doc. Ophthalmol., 1990: pp.

171–177. doi:10.1007/BF02482606.

[27] J.D. Akula, T.L. Favazza, J.A. Mocko, I.Y. Benador, A.L. Asturias, M.S. Kleinman, et al., The anatomy of the rat eye with oxygen-induced retinopathy, Doc. Ophthalmol. 120 (2010) 41–50. doi:10.1007/s10633-009-9198-1.

[28] L.E. Tan, W. Orilla, P.M. Hughes, S. Tsai, J. a Burke, C.G. Wilson, Effects of vitreous liquefaction on the intravitreal distribution of sodium fluorescein, fluorescein dextran, and fluorescent microparticles., Invest. Ophthalmol. Vis. Sci. 52 (2011) 1111–8. doi:10.1167/iovs.10-5813.

[29] D. Maurice, Review: practical issues in intravitreal drug delivery., J. Ocul. Pharmacol. Ther. 17 (2001) 393–401. doi:10.1089/108076801753162807.

[30] S.J. Bakri, M.R. Snyder, J.M. Reid, J.S. Pulido, M.K. Ezzat, R.J. Singh, Pharmacokinetics of intravitreal ranibizumab (Lucentis)., Ophthalmology. 114 (2007) 2179–82. doi:10.1016/j.ophtha.2007.09.012.

[31] J. Gaudreault, D. Fei, J. Rusit, P. Suboc, V. Shiu, Preclinical pharmacokinetics of Ranibizumab (rhuFabV2) after a single intravitreal administration., Invest. Ophthalmol. Vis. Sci. 46 (2005) 726–33. doi:10.1167/iovs.04-0601.

[32] S.S. Lee, M.R. Robinson, Novel drug delivery systems for retinal diseases. A review., Ophthalmic Res. 41 (2009) 124–35. doi:10.1159/000209665.

[33] S.J. Bakri, M.R. Snyder, J.M. Reid, J.S. Pulido, R.J. Singh, Pharmacokinetics of intravitreal bevacizumab (Avastin)., Ophthalmology. 114 (2007) 855–9. doi:10.1016/j.ophtha.2007.01.017.

[34] A. Moradi, Y.J. Sepah, M.A. Sadiq, H. Nasir, S. Kherani, R. Sophie, et al., Vascular endothelial growth factor trap-eye (Aflibercept) for the management of diabetic macular edema., World J. Diabetes. 4 (2013) 303–309. doi:10.4239/wjd.v4.i6.303.

[35] M.W. Stewart, What are the half-lives of ranibizumab and aflibercept (VEGF Trap-eye) in human eyes? Calculations with a mathematical model, Eye Reports. 1 (2011) 5–7. doi:10.4081/eye.2011.e5.

[36] F.E. Kane, J. Burdan, A. Cutino, K.E. Green, Iluvien: a new sustained delivery technology for posterior eye disease, Expert Opin. Drug Deliv. 5 (2008) 1039–1046. doi:10.1517/17425247.5.9.1039.

[37] D.S. Boyer, Y.H. Yoon, R. Belfort, F. Bandello, R.K. Maturi, A.J. Augustin, et al., Three-Year, Randomized, Sham-Controlled Trial of Dexamethasone Intravitreal Implant in Patients with Diabetic Macular Edema, Ophthalmology. (2014). doi:10.1016/j.ophtha.2014.04.024.

[38] D.A. Mohammad, B. V Sweet, S.G. Elner, Retisert: is the new advance in treatment of uveitis a good one?, Ann. Pharmacother. 41 (2007) 449–54. doi:10.1345/aph.1H540.

[39] A. Bochot, E. Fattal, Liposomes for intravitreal drug delivery: a state of the art., J. Control. Release. 161 (2012) 628–34. doi:10.1016/j.jconrel.2012.01.019.

[40] S.K. Sahoo, F. Dilnawaz, S. Krishnakumar, Nanotechnology in ocular drug delivery., Drug Discov. Today. 13 (2008) 144–51. doi:10.1016/j.drudis.2007.10.021.

[41] F.M. Veronese, Peptide and protein PEGylation, Biomaterials. 22 (2001) 405–417. doi:10.1016/S0142-9612(00)00193-9.

[42] S.C. Fishburn, The pharmacology of PEGylation: Balancing PD with PK to generate novel therapeutics, J. Pharm. Sci. 97 (2010) 4167–4183. doi:10.1002/jps.

[43] E.W.M. Ng, D.T. Shima, P. Calias, E.T. Cunningham, D.R. Guyer, A.P. Adamis, Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease., Nat. Rev. Drug Discov. 5 (2006) 123–32. doi:10.1038/nrd1955.

[44] J. Necas, L. Bartosikova, P. Brauner, J. Kolar, Hyaluronic acid ( hyaluronan ): a review, Vet. Med. 2008 (2008) 397–411.

Chapter 4: Summary and Future Directions

4.2 Future Directions

The multivalent technology presented here has potential to improve upon many clinically available drugs, not only limited to diseases of the retina. Several questions remain unanswered and are presented below:

1) How long does the mvsFlt remain in the rat vitreous? Our study was optimized based on results from the dextran study presented in Appendix II. The dextrans were ~95% cleared by the end of 48 hours, however, the hydrodynamic diameter of the 2 MDa and mvsFlt are significantly different. Because of this difference in size, much more of the mvsFlt remains at 48 hours and our study did not address any time points after 2 days. It would be beneficial to know exactly how long it took for the mvsFlt conjugate to be cleared from the rat vitreous before moving on to bigger animal models. This could be simply addressed by repeating the experiment and examining the residence time of fluorescently tagged mvsFlt out to 2 weeks. Larger animal models generally examine the residence time of drugs out to 40 days but because the rat vitreous is smaller while maintaining similar rates of vitreous turnover, 2 weeks should be sufficient. After determining the half-life of sFlt in the rat vitreous, larger animal models that are more similar to the human eye will have to be used to carry out half- life experiments. Although the half-life of the mvsFlt in the rat eye may be on the order of a few days, this number will significantly increase when moving to larger animal models because of the size of the vitreous. For instance, in the monkey eye Lucentis has a half-life of 5 days whereas in the rat eye it is 3 hours. This difference demonstrates the stark difference in vitreal turnover between small and large animal models. Thus, we assume that an increase in half-life in the rat will lead to an exponential increase in half-life in larger animal models, thus significantly improving upon current techniques.

2) What is the half-life of sFlt and mvsFlt in bigger animal models? In general, the half-life of drugs in the eye are determined using large animal models because of the similarity eye shape and structure to the human. Preliminary half-life studies examining Avastin and Lucentis were carried out in rabbits and monkeys [1–3] and were on the order of 5-7 days, in comparison to the rat half-life values which were between 3 and 36 hours. We anticipate that sFlt would perform similarly to Lucentis based on protein composition and size in rabbit and/or monkey models, whereas mvsFlt may have a significantly longer half-life. The half-life of HyA in the vitreous is ~30 days [4], thus, it is possible that the mvsFlt conjugate may have a half-life similar to native HyA. If 71

that is the case, another question would then become- how effective is the mvsFlt if its half-life is so long? How many of the sFlt molecules are free after so many days to still bind VEGF (which leads us to our next question)?

3) What percent of the sFlt on mvsFlt remains free for VEGF binding? A possible concern about such high half-lives is whether any sFlt molecules remain that are not already VEGF bound. mvsFlt could remain in the retina for weeks but would be ineffective if all sFlt molecules were occupied by VEGF. This could be carried out using the OIR model and an ELISA to examine the intravitreal concentration of VEGF. Groups treated with mvsFlt should have significantly less detectable VEGF unless concentrations of mvsFlt are low. The dose of mvsFlt can be optimized to always be several times higher than the VEGF, thus creating a molar excess of drug to VEGF.

4) What is the effect of mvsFlt on retinopathy long-term? The studies described in Chapter 3 describe the OIR model as a way to look at inhibition of retinal angiogenesis on the short term- 5 days. We have shown that the addition of mvsFlt is more effective than sFlt at inhibiting retinal angiogenesis, however, pathological retinopathy happens on a much longer scale. Another retinopathy model that can be used is the Vldlr-/- knockout mouse model, which develop retinopathy on a much longer timescale- between 30-60 days [5]. This rat model can be used to determine when a second round of dosing may be necessary.

5) How do we bring this up to production scale? Despite being a very effective VEGF antagonist, sFlt is difficult to manufacture. It cannot be made in E.coli because it forms exclusion bodies that cause the bacteria to burst. We successfully made sFlt in insect cells (High Five) however our yields were very low and averaged 1 milligram per liter of cells. Furthermore, the protein structure has several disulfide bonds between and inside the Ig domains, which do not form intracellularly- secretion is necessary for these post-translational modifications. For these reasons, it may be necessary to choose another anti-VEGF protein that is easier to make in E.coli or simply use Avastin and Lucentis because these are commercially available. We anticipate that similar increases in half-life and prolonged anti-angiogenic effectiveness will still be observed with the use of another protein or drug. Although we used sFlt in these studies, we treated sFlt as a model protein to show the effectiveness of the technique. Other proteins such as Avastin and Lucentis are equally as effective and the technique should work for these as well. An added advantage to Avastin and Lucentis is that they are easily made in large-scale productions, which would be difficult to implement with the sFlt protein.

6) Can this technology be applied to other retinal diseases? As detailed in Chapter 1, there are many retinal diseases that require long-term delivery of drug such as posterior

uveitis (PU), diabetic macular edema (DME), central retinal vein occlusion (CRVO), choroidal neovascularization (CNV). Most of these diseases are currently treated with corticosteroids that can be released from polymeric devices such as Retisert, Iluvien, Vitrasert and Ozurdex. Multivalent technology can also be used to deliver corticosteroids and these conjugates would benefit from the increased half-life. Although the half-life of the conjugates would be significantly shorter than the implantable devices, surgical placement and removal would not be necessary.

7) Can this technology be applied to non-retinal diseases? Drug delivery is a concern that affects the treatment of many diseases. It is seldom that a disease that can be solved with a single drug administration. The majority of diseases need multiple administrations of drug to either manage or cure symptoms. For this reason, many diseases could potentially be improved by applying the multivalent technology. Another advantage of this technology is that the presentation of multiple drugs to the surface of a cell can actually induce receptor clustering and thus strengthening of the intracellular signal. Depending on the molecular targets and whether the target is in the extracellular matrix or cell-bound, the drug can be optimized as such. Other applications could include wound healing, an application being explored with treatment by multivalent fibroblast growth factor (mvbFGF).

8) What are possible side effects to this approach? Although both HyA and sFlt are found naturally in the human body, side effects of the mvsFlt are possible. HyA has been previously shown to induce angiogenesis at very low molecular weights [6–8]. Very low molecular weight (oligomers below 45 disaccharide units) has been shown to induce angiogenesis [9]. We do not anticipate this effect in the retina because the molecular weight of the HyA we are injecting is similar to that found natively in the vitreous. The degree of vascularization should be closely monitored to make sure such an effect does not occur. Furthermore, concerns have been raised that taking away VEGF that is required for endothelial cell survival [10] may damage normal retinal vasculature. Again, we believe this will not be a concern because the concentration of mvsFlt we inject is similar to that of clinically used Avastin and Lucentis. However, these drugs are cleared faster and thus may not bind as much VEGF in comparison to mvsFlt which will be cleared slower and absorb more VEGF. Thus, functional studies may need to be carried out to make sure vasculature stays healthy overtime following injections of mvsFlt.

As the prevalence of diabetes continues to skyrocket globally, the prevalence of its debilitating symptoms like diabetic retinopathy continue to become more prevalent. There exists a strong need for more effective anti-VEGF treatments for treating diabetic retinopathy and our approach has proven to be successful in its preliminary studies. If the concerns above are

73 considered and answered, we anticipate that our approach could be beneficial for improving quality of life for patients with diabetic retinopathy around the world.

4.3 References 1. Bakri SJ, Snyder MR, Reid JM, Pulido JS, Ezzat MK, Singh RJ. Pharmacokinetics of intravitreal ranibizumab (Lucentis). Ophthalmology. 2007 Dec;114(12):2179–82.

2. Bakri SJ, Snyder MR, Reid JM, Pulido JS, Singh RJ. Pharmacokinetics of intravitreal bevacizumab (Avastin). Ophthalmology. 2007 May;114(5):855–9.

3. Drolet DW, Nelson J, Tucker CE, Zack PM, Nixon K, Bolin R, et al. Pharmacokinetics and safety of an anti-vascular endothelial growth factor aptamer (NX1838) following injection into the vitreous humor of rhesus monkeys. Pharm Res. 2000;17(12):1503–10.

4. Necas J, Bartosikova L, Brauner P, Kolar J. Hyaluronic acid (hyaluronan): a review. Vet Med. 2008;2008(8):397–411.

5. Hu W, Jiang A, Liang J, Meng H, Chang B, Gao H, et al. Expression of VLDLR in the retina and evolution of subretinal neovascularization in the knockout mouse model’s retinal angiomatous proliferation. Investig Ophthalmol Vis Sci. 2008;49(1):407–15.

6. Park D, Kim Y, Kim H, Kim K, Lee Y-S, Choe J, et al. Hyaluronic acid promotes angiogenesis by inducing RHAMM-TGFβ receptor interaction via CD44-PKCδ. Mol Cells. 2012;33(6):563–74.

7. Slevin M, Krupinski J, Gaffney J, Matou S, West D, Delisser H, et al. Hyaluronan- mediated angiogenesis in vascular disease: uncovering RHAMM and CD44 receptor signaling pathways. Matrix Biol. 2007 Jan;26(1):58–68.

8. Slevin M, West D, Kumar P, Rooney P, Kumar S. Hyaluronan, angiogenesis and malignant disease. Int J Cancer. 2004 May 1;109(5):793–4; author reply 795–6.

9. West DC, Hampson IN, Arnold F, Kumar S. Angiogenesis induced by degradation products of hyaluronic acid. Science (80- ). 1985;228:1324–6.

10. Gerber HP, McMurtrey a, Kowalski J, Yan M, Keyt B a, Dixit V, et al. Vascular endothelial growth factor regulates endothelial cell survival through the 74

phosphatidylinositol 3’-kinase/Akt signal transduction pathway. Requirement for Flk- 1/KDR activation. J Biol Chem. 1998;273:30336–43.

Appendix I: Determination of crosslinked HyA gel pore size presented in Chapter 2

A1.1 Introduction

We created an in vitro model of the vitreous using crosslinked HyA gels for examining the diffusion of sFlt and the mvsFlt bioconjugates through size-limiting networks. We show in Figure 2.6A that the low molecular weight conjugate, 300 kDa LCR and sFlt are released from the gel much quicker than the 650 kDa and 1 MDa mvsFlt conjugates. In order to determine whether this difference in movement was due in large part to size, we decided to further characterize the pore size of the 1% HyA gel. We hypothesized that if the pore size of the gel was close to the hydrodynamic diameter of the conjugates (shown in Figure 2.2B and C) that would explain why the larger conjugates moved slower.

A1.2 Methods

A2.1 Rheology of 1% gels Viscoelastic properties of the HyA hydrogel were determined with an oscillatory rheometer (MCR 302 Modular Compact Rheometer; Anton Paar) with a parallel plate geometry (25 mm diameter) under 10% constant strain and frequency ranging from 0.1 Hz to 10 Hz as previously described [21].

A2.2 Swelling analysis of gels The swelling ratio of gels was determined as described previously [21]. Briefly, gels were dried by passing them through graded ethanol solutions, and the solvent was evaporated under vacuum. After the dry weight was recorded, the gel discs were immersed in PBS at 37oC overnight and the wet weight was measured.

A1.3 Calculations of Q and Mc

A1.3.1 Calculation of the volumetric swelling ratio (Q) The mass swelling ratio (�!) was deﬁned as the ratio of the ﬁnal wet weight to the initial dry weight. The mass swelling ratio, �! was used to calculate the volumetric swelling ratio, � according to equation (1):

�! � = 1 + �! − 1 (1) �! where �! is the density of dry polymer and �! is the density of solvent.

A1.3.2 Calculation of molecular weight between crosslinks We used both swelling and rheological measurements to independently characterize the molecular weight between crosslinks (�!) and mesh size (�) of the HyA gels used in the mvsFlt drug release studies.

A1.3.2.1 Calculation of Mc from swelling measurements For affine polymer networks from swelling measurements, we utilized the Peppas-Merrill [28] approximation for �! as shown in equation (2):

� ! ln 1 − �!,! + �!,! + �!�!,! 1 2 �! = − ! (2) �! �! �!,! ! 1 �!,! �!,! − �!,! 2 �!,! where � is the specific volume of bulk polymer in the amorphous state, �! is the molar volume of solvent, �!,! is the polymer volume fraction after crosslinking but prior to equilibration with more solvent (i.e., the relaxed state), �!,! is the final swollen equilibrium polymer volume fraction and χ1 is the Flory polymer-solvent interaction parameter). We used the theoretical value for �!,! as � ∗ �, where � is the concentration of the polymer at the time of formation in � 3 � = !,! � g/cm . We employed the identity for volumetric swelling, �!,! to calculate !,!.

For comparison, we used equation 3 as described by Erman [29] for calculating the �! for phantom networks from swelling data:

2 !/! !/! �(1 − )� � � � ! !,! !,! �! = ! (3) ln 1 − �!,! + ��!,! + �!,! where φ is the functionality (i.e., the number of sites from which chains can grow), assumed to be 4 in our case. Other parameters are the same as those defined for Equation 2. Parameters used to calculate �!for all equations are given below in Supplemental Table 2. Data is shown in Table 3.

A1.3.2.2 Calculation of Mc from rheological measurements We also used rheological data to calculate the �! with two models. We used equation (4) [30] for determining �! of affine polymer networks:

�� = (4) ! �′ where R is the gas constant, T is temperature at the time of measurement, c is concentration in g/cm3 as defined above, and G’ is the shear storage modulus of the hydrogel.

We used equation (5) as described by Erman [29] for calculating �! for phantom networks from rheological data:

2 �� = 1 − (5) ! � �!

Parameters used to calculate �! for all equations are given below in Supplemental Table 2. Data are presented in Table 3.

A1.4 Calculation of hydrogel mesh size

Following the calculation of �! using swelling and rheological data for affine and phantom networks, we carried out the calculation of the mesh size according to equation (6) defined by Leach et. Al [31]:

!/! !/! � = 0.1748�! � (6)

Values of �! used to calculate � are presented in Table 3.

Table A1: Summary of Variables and Values. The following parameters and values were used in the calculation of �! and �.

Variable Value Definition �! 50 Mass swelling ratio from [21] Q 61.2 Swelling ratio 3 �! 1.229 g/cm Density of dry polymer 3 �! 1 g/cm Density of solvent �! 600,000 Da Number average molecular weight � 0.814 Specific volume of bulk polymer 3 �! 18 cm /mol Molar volume of solvent 0.0081 Volume fraction after crosslinking before equilibration � !,! with solvent -4 �!,! 1.33*10 Final swollen equilibrium polymer volume fraction

�! 0.473 Flory polymer solvent interaction parameter 8314000 � (cm3*Pa)/(K*mol Gas constant ) � 298 K Temperature � 0.01 g/cm3 Polymer concentration �′ 90 Pa Shear modulus from Fig. S1A � 4 Polymer functionality parameter

78 Appendix II: Optimization of the rat vitreous as a model for studying the in vivo half-life of mvsFlt

A2.1 Introduction

The half-life of drugs in the vitreous is typically carried out in large animals such as rabbits or rhesus monkeys. Due to our lack of such resources, it was necessary to optimize a smaller animal model for determining the effect of HyA conjugation of the half-life of sFlt. This appendix describes the methods taken to determine the timeline over which the experiments would take place.

A2.2 Materials and methods

All half-life experiments were performed on 8-week old Brown Norway rats obtained from Charles River Laboratories and treated in accordance with protocols approved by the Animal Care and Use Committee at UC Berkeley. Rats were anesthetized using a mixture of ketamine and xylazine (50 mg and 10 mg/kg body weight, respectively) for the surgical procedure. Eyes were injected intravitreally 1 mm behind the limbus with 5 µl of 1 mg/mL 40 kDa or 2 MDa fluorescently tagged dextran particles (Life Technologies) using a 30-gauge Hamilton syringe and monitored daily for signs of inflammation. This concentration was selected to maximize fluorescence in the vitreous and remain in the detection limit of the fluorometer after 48 hours. Rats were sacrificed with CO2 asphyxiation in groups at 0, 4, 12, 24 and 48 hours post injection and eyes were immediately enucleated and placed on dry ice. Frozen vitreous was then extracted from the eye and immersed in 100 µL of RIPA buffer. After shaking on ice for 2 hours, each vitreous sample was homogenized with a Tissue Tearor (Bio Spec Products, Inc.) and the fluorescence measured using a fluorometer (Molecular Devices). Quantification was carried out by normalizing the fluorescence of vitreous samples to the 0 hour vitreous fluorescence readings within their respective group.

A2.3 Results and discussion

Differences in residence time between the 40 kDa and 2 MDa dextran were apparent beginning at 12 hours where only 3.9±4.4% of the 40 kDa dextran remained compared to 43.4±14.6% of 2 MDa (Figure A2.1). By 24 hours, only 1.2±0.8% of the 40 kDa dextran remained detectable compared to 10.5±11.6% of the 2 MDa. By 48 hours days post-injection, both the dextrans were almost undetectable. Only 1.1±0.3% of the 40 kDa and 5.4±1.9% of

79 the 2 MDa remained detectable in the vitreous. The half-lives of the 40 kDa and 2 MDa dextrans was calculated using data taken at 4, 12, 24 and 48 hours and is shown in Table A2.1.

Table A2.1 Half-lives of 40 kDa and 2 MDa fluorescent dextrans in the vitreous calculated based on data taken at various time points.

��/� of 40 kDa ��/� of 2 MDa 4 Hr 0.97 0.87 12 Hr 1.12 4.33 24 Hr 1.65 3.20 48 Hr 3.17 4.96

Figure A2.1 Higher molecular weight dextran displays longer residence time in vivo. A) Validation experiment demonstrating the effect of size on the retention of fluorescently tagged dextrans. The 2 MDa dextran (solid line) has significantly improved residence time over the 40 kDa (dashed line) over 48 hours. * indicates a difference between the 40 kDa and 2 MDa dextran at the given time point. Two-way ANOVA gives p-value* <0.05 (** corresponds to a P-value less than 0.01).

As shown in Figure A2.1, the largest difference in amount of dextran detected was observed at 12 hours post-injection. By 48 hours, both dextrans were almost completely cleared from the vitreous. Based on this data, we decided to study these time points only for the half-life determination study of mvsFlt. As we later discovered, the half-life of mvsFlt is significantly different than the larger dextran and this is likely due to the significant difference in hydrodynamic radius between the 2 MDa dextran and mvsFlt. Although it has a higher molecular weight, the 2 MDa dextran is significantly smaller in hydrodynamic radius and thus diffuses out of the vitreous faster. The sFlt and the 40 kDa dextran were more similar in size and thus showed similar profiles of vitreal clearance.