Formulation of Particulate-based Immunomodulatory Therapeutics for the

Treatment of Diseases

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Kevin J. Peine

Graduate Program in Molecular, Cellular and Developmental Biology

The Ohio State University

2014

Dissertation Committee:

Dr. Kristy M. Ainslie, Advisor

Dr. Caroline Whitacre

Dr. Amy Lovett-Racke

Dr. Eric M. Bachelder

Copyright by

Kevin J. Peine

2014

Abstract

Immunomodulatory therapies provide a promising route for the treatment of infectious or autoimmune diseases. Generally, treatments for infectious diseases target pathogens directly, which overtime, can lead to antimicrobial resistance. Amplifying the host immune response for clearance of pathogens could likely limit the emergence of resistance. Compounds such as the toll-like receptor (TLR) agonists polyinosinic:polycytidylic acid (poly I:C), cytosine bonded to guanine on a phosphodiester backbone (CpG), and resiquimod, or other products that amplify an immune response could be used for the clearance of however, systemic delivery is not ideal due to and toxicity concerns. Conversely, autoimmune disease therapies induce wide-spread immune suppression which can result in opportunistic diseases. specific immunomodulatory therapies could solve this problem by inhibiting the immune response in an antigen specific manner, therefore inhibiting disease, while the remainder of the immune system is functional. For both the delivery of TLR agonists and antigen specific suppression of the immune system, particulate vehicles can provide significant advantages such as increased solubility, reduced toxicity, decreased clearance from circulation and passive targeting of phagocytic cells, like antigen presenting cells (APCs). Targeting APCs is beneficial for the creation of immunomodulatory therapies because APCs are vital for the initiation of a ii variety of immune responses. This work shows liposomal encapsulation of resiquimod or a novel immunostimulatory compound, pentalinonsterol, decreases parasite burden in the liver, spleen, and bone marrow of mice infected with Leishmania donovani. Furthermore, both compounds enhance T cell proliferation and increase production of cytokines that indicate a shift towards a TH1 immune response. Another particulate carrier formulated from the acid sensitive polymer acetalated (Ac-DEX) encapsulated CpG and poly

I:C and induced a pro-inflammatory cytokine profile when cultured with RAW macrophages, indicating its potential use for therapies or subunit formulations.

Finally, Ac-DEX encapsulating a tolerogenic compound (ex. dexamethasone or rapamycin) and a disease-associated antigen were able to decrease symptoms in a multiple sclerosis animal model and inhibit onset of disease in a type 1 diabetes mouse model, in an antigen specific manner. These symptom decreases coincided with lower inflammatory cytokine production from splenocytes, after being re-stimulated with a disease-associated antigen. Here, particulate-based therapies are used to mediate both inflammatory responses towards and infectious pathogen and to induce antigen specific immune suppression for the treatment of autoimmune disorders.

iii

Dedication

This document is dedicated to my family.

iv

Acknowledgments

I thank my adviser Dr. Kristy Ainslie for her guidance, support, and patience throughout my graduate studies. Additionally, I thank her for always encouraging me to pursue my goals.

I thank Dr. Eric Bachelder for his constant scientific and moral support. I would especially like to thank him for expanding my knowledge of immunology and providing invaluable support for this work.

I am grateful to my committee members for enhancing my graduate education through scientific and career guidance.

I would like to thank my fellow lab-mates for providing collaboration, friendship, and support.

I would like to thank the lab of Dr. Amy Lovett-Racke for their extensive assistance with the multiple sclerosis project and the experimental autoimmune encephalomyelitis animal

v model. Specifically, I would like to thank Dr. Mireia Guerau-de-Arellano, Priscilla Lee,

Mary Severin, Dr. Haiyan Peng, Dr. Yuhong Yang, and Dr. Amy Lovett-Racke.

I would like to thank Dr. Tracy Papenfuss for histological and immunological assistance, as well as Zachary Vangundy for support with bone marrow-derived culture.

I acknowledge the lab of Dr. Abhay Satoskar including Dr. Gaurav Gupta, Dr. Steve Oghumu, and Sanjay Varikuti for their extensive support with the Leishmania animal model and subsequent mechanistic experiments.

I would like to thank Dr. A Douglas Kinghorn, Dr. Jim Fuchs, Dr. Li Pan, Dr. Dahlia

Abdelhamid, and Ben Naman for their roles in isolating, synthesizing, and detecting pentalinonsterol.

I am forever appreciative to my family for always ensuring I had the chance to attain my goals.

vi

Vita

June 2006 ...... Chardon High School, Chardon OH

June 2010 ...... B.S. Biology, DePaul University

September 2010 to present ...... Graduate Research Associate, Molecular

Cellular and Developmental Biology

Graduate Program, The Ohio State

University

Publications

Peine KJ, Guerau-de-Arellano M, Lee P, Kanthamneni N, Severin M, Probst G.D, Peng H, Yang

Y, Vangundy Z, Papenfuss TL, Lovett-Racke A.E, Bachelder EM, Ainslie KM. “Treatment of

Experimental Autoimmune Encephalomyelitis by Co-Delivery of MOG35-55 and

Dexamethasone in Acetalated Dextran Microparticles.” Mol Pharm. 2014 Feb 4.

Peine KJ, Bachelder EM, Vangundy Z, Brackman DJ, Gallovic M, Papenfuss TL, Schully KL,

Pesce J, Keane-Meyers A, Ainslie KM. “Efficient Delivery of the TLR-Agonists Poly I:C and

CpG to Macrophages by Acetalated Dextran Micoparticles.” Mol. Pharm. 2013 Aug

5;10(8):2849-57.

vii

Peine KJ, Gupta G, Brackman DJ, Papenfuss TL, Ainslie KM, Satoskar AR, Bachelder EM.

“Liposomal resiquimod for the treatment of Leishmania donovani . J. Antimicrob.

Chemother. 2014 Jan;69(1):168-75.

Collier MA, Gallovic MD, Peine KJ, Gunn JS, Schlesinger LS, Ainslie KM. “Delivery of host cell-directed therapeutics for intracellular pathogen clearance.” Expert Review of Anti-infective

Therapy. November 2013, Vol. 11, No. 11 , Pages 1225-1235.

Borteh HM, Gallovic M, Sharma S, Peine KJ, Maio S, Brackman DJ, Gregg K, Xu Y, Guan J,

Bachelder EM, Ainslie KM. “Electrospun Acetalated Dextran Scaffolds for Temporal Release of

Therapeutics.” Langmuir. 2013 Jun 11.

Schully KL, Sharma S, Peine KJ, Pesce J, Elberson MA, Fonseca ME, Prouty AM, Bell MG,

Borteh H, Gallovic M, Bachelder EM, Keane-Meyers A, Ainslie KM. “Rapid Vaccination Using

Acetalated Dextran Microparticulate Subunit Vaccine Confers Protection Against Triplicate

Challenge by Bacillus Anthracis.” Pharm Res. 2013 Jan 25.

Duong AD, Sharma S, Peine KJ, Gupta G, Satoskar AR, Bachelder EM, Wyslouzil BE, Ainslie

KM. “Electrospray encapsulation of toll-like receptor agonist resiquimod in polymer microparticles for the treatment of visceral Leishmaniasis.” Mol Pharm. 2013 Mar 4;10(3):1045-

55.

Rajah TT, Peine KJ, Du N, Serret CA, Drews NR. “Physiological concentrations of genistein and

viii

17β-estradiol inhibit MDA-MB-231 breast cancer cell growth by increasing BAX/BCL-2 and reducing pERK1/2.” Anticancer Research 2012 Apr;32(4):1181-91.

Fields of Study

Major Field: Molecular, Cellular and Developmental Biology

ix

Table of Contents

Abstract ...... ii

Dedication ...... iv

Acknowledgments...... v

Vita ...... vii

Publications ...... vii

Fields of Study ...... ix

Table of Contents ...... x

Chapter 1: Introduction ...... 1

1.1 Drug Delivery ...... 1

1.1.1 Liposomes ...... 2

1.1.2 Polymer Particles ...... 3

1.2 Leishmaniasis ...... 5

1.2.1 Current Therapies for Leishmaniasis ...... 6

1.2.2 Parasite Resistance to Therapies ...... 7

1.2.3 Particulate Delivery Vehicles for the Treatment of Leishmaniasis ...... 9 x

1.3 Autoimmunity ...... 10

1.3.1 Immune Tolerance ...... 11

1.3.2 Inducing Tolerance with Particulate based Therapeutics ...... 12

1.4 Objective and Research Contribution ...... 14

Chapter 2: Efficient Delivery of the TLR-Agonists Poly I:C and CpG to Macrophages by

Acetalated Dextran Microparticles ...... 16

2.1 Introduction ...... 16

2.2 Materials and Methods ...... 20

2.2.1. Materials ...... 20

2.2.2. Cell Lines ...... 20

2.2.3. Synthesis and Analysis of Coverage of Ac-DEX ...... 20

2.2.4. Preparation of Double Emulsion Particles Encapsulating Poly I:C or CpG with

either Ac-DEX or PLGA ...... 21

2.2.5. Extraction and Quantification of Encapsulated Poly I:C and CpG ...... 21

2.2.6. Scanning Electron Microscopy (SEM) ...... 21

2.2.7. Dynamic Light Scatter ...... 22

2.2.8 Release Profile of Poly I:C Encapsulated in Ac-DEX ...... 22

2.2.9. Nitrite and Cyotkine Analysis of Macrophages Stimulated by Poly I:C or CpG

...... 22

xi

2.3 Results ...... 23

2.3.1. Poly I:C Particle Formulation and Analysis ...... 23

2.3.2 Free and Encapsulated poly I:C Stimulation ...... 25

2.3.3. CpG Particle Formulation and Analysis ...... 26

2.3.4. Free and Encapsulated CpG Show Increased Macrophage Viability ...... 29

2.3.5 Free and Encapsulated CpG Stimulation of Immune Response in Macrophages

...... 31

2.4 Discussion ...... 32

2.5 Conclusions ...... 36

Chapter 3: Liposomal Resiquimod for the Treatment of Leishmania donovani Infection 38

3.1 Introduction ...... 38

3.2 Materials and Methods ...... 40

3.2.1. Materials ...... 40

3.2.2. Animals ...... 42

3.2.3. Preparation of Empty or Resiquimod Loaded Liposomes ...... 42

3.2.4. Determining Size and Encapsulated Resiquimod in Liposomes ...... 43

3.2.5. Infection and Treatment Protocol ...... 44

3.2.6. Histology and LDU (L. donovani units) in Treated Mice ...... 44

3.2.7. T cell Proliferation and Cytokine ELISA ...... 45

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3.2.8. Measurement of Liver Aminotransferase Enzymes ...... 45

3.2.9. Cell Viability ...... 46

3.3 Results ...... 47

3.3.1. Liposomal Preparation ...... 47

3.3.2 Treatment of L. donovani Infection ...... 47

3.3.3. T cell Proliferation and Cytokines ...... 48

3.3.4. In vitro Viability, Histology and Liver Enzyme Activity ...... 52

3.4 Discussion ...... 54

3.5 Conclusions ...... 58

Chapter 4: Liposomal Synthetic Pentalinonsterol for the Treatment of Leishmania donovani Infection ...... 59

4.1 Introduction ...... 59

4.2 Materials and Methods ...... 61

4.2.1 Materials ...... 61

4.2.2 Preparation of Empty or Synthetic Pentalinonsterol Loaded Liposomes ...... 62

4.2.3 Determining Size and Encapsulated Synthetic Pentalinonsterol in Liposomes 62

4.2.4 Animals and Parasites ...... 63

4.2.5 Promastigote Assay ...... 63

4.2.6 Amastigote Assay ...... 64

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4.2.7 Cytotoxicity Assay in Non-Infected Macrophages ...... 64

4.2.8 In Vivo Infection and Treatment Protocol ...... 64

4.2.9 Parasite Burden Calculation ...... 65

4.2.10 Histopathology...... 65

4.2.11 T cell Proliferation and Cytokine ELISA ...... 66

4.2.12 Quantification of Transcript Levels by RT-PCR ...... 66

4.2.13 Measurement of Liver Aminotransferase Enzymes ...... 66

4.2.14 Statistical Analysis ...... 67

4.3 Results ...... 67

4.3.1 Antileishmanial Activity of Synthetic Pentalinonsterol against Leishmania

donovani Promastigotes and Intracellular Amastigotes ...... 67

4.3.2 Liposomal Synthetic Pentalinonsterol Treatment clears Leishmania donovani

from Infected Mice...... 71

4.3.3 Induction of Proliferative and Proinflammatory Cytokine Responses in Infected

Mice treated with Liposomal Synthetic Pentalinonsterol ...... 71

4.3.4Liposomal Synthetic Pentalinonsterol Treatment Promotes the Formation of

Matured Liver Granulomas in Leishmania donovani Infected Mice ...... 75

4.4 Discussion ...... 77

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Chapter 5: Treatment of Experimental Autoimmune Encephalomyelitis by Co-Delivery of Disease Associated Peptide and Dexamethasone in Acetalated Dextran Microparticles

...... 82

5.1 Introduction ...... 82

5.2 Materials and Methods ...... 86

5.2.1 Materials ...... 86

5.2.2 Animals ...... 86

5.2.3 Cells ...... 87

5.2.4 Synthesis and Analysis of Acetal Coverage of Ac-DEX ...... 87

5.2.5 Preparation of Empty or DXM-Loaded Ac-DEX Microparticles ...... 87

5.2.6 Preparation of MOG or MOG/DXM Co-Encapsulated Ac-DEX MPs ...... 88

5.2.7 Scanning Electron Microscopy (SEM) ...... 88

5.2.8 Quantification of DXM ...... 89

5.2.9 Quantification of Encapsulated MOG ...... 90

5.2.10 In Vitro Release of Dexamethasone ...... 90

5.2.11 Nitrite Analysis ...... 90

5.2.12 Measurement of Il-6 Secreted by Bone Marrow Derived Dendritic Cells ..... 91

5.2.13 Immunization and Treatment of EAE ...... 91

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5.2.14 Measurement of Secreted IL-17 and Granulocyte-Macrophage Colony-

Stimulating Factor ...... 92

5.2.15 Fluorescence Activated Cell Sorting Analysis ...... 92

5.3 Results ...... 93

5.3.1 Particle Formulations and Analysis ...... 93

5.3.2 In Vitro Immunosuppressive Function of Dexamethasone ...... 96

5.3.3 In Vivo Treatment of EAE ...... 96

5.4 Discussion ...... 101

5.5 Conclusions ...... 106

Chapter 6: Particulate-based Tolerogenic Compounds for the Protection Against

Hyperglycemia in NOD Mice ...... 108

6.1 Introduction ...... 108

6.2 Materials and Methods ...... 110

6.2.1 Reagents...... 110

6.2.2 Animals ...... 110

6.2.3 Synthesis and Analysis of Acetal Coverage of Ace-DEX ...... 111

6.2.4 Preparation of Empty Ace-DEX Microparticles ...... 111

6.2.5 Preparation of Insulin Ace-DEX MPs ...... 111

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6.2.6 Preparation of Dexamethasone and Insulin Co-Encapsulated Ace-DEX MPs

...... 112

6.2.7 Preparation of All-Trans Retinoic Acid or Rapamycin and Insulin Co-

Encapsulated Ace-DEX MPs ...... 112

6.2.8 Scanning Electron Microscopy (SEM) ...... 113

6.2.9 Quantification of Encapsulated Dexamethasone ...... 113

6.2.10 Quantification of Encapsulated Rapamycin ...... 113

6.2.11 Quantification of Encapsulated All-Trans Retinoic Acid ...... 114

6.2.12 Quantification of Encapsulated Insulin ...... 114

6.2.13 Prophylactic Protection of NOD Mice ...... 114

6.3 Results ...... 117

6.3.1 Analysis of Particle Formulations Containing Insulin ...... 117

6.3.2 Inhibition of Hyperglycemia by Particles Containing Insulin with either

Rapamycin or Dexamethasone ...... 117

6.4 Discussion ...... 118

Chapter 7: Summary, Conclusions and Future Work ...... 121

7.1 Delivery of TLR-agonists for the Treatment of Infectious Disease ...... 121

7.2 Delivery of Immune Tolerizing Particles for the Treatment of Autoimmunity .... 127

References ...... 130

xvii

Permissions ...... 155

Chapter 2 ...... 155

Chapter 3 ...... 155

Chapter 4 ...... 155

xviii

List of Tables

Table 5.1: Encapsulation Efficiencies for Ac-DEX Particles Containing DXM, MOG, or

Both...... 95

Table 6.1: Encapsulation Efficiencies and Drug Loading for Microparticles Containing

Insulin with or without Tolerogenic Drugs...... 116

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List of Figures

Figure 2.1: Encapsulation and Immunostimulatory Capability of poly I:C in either PLGA or Ac-DEX Microparticles...... 24

Figure 2.2: Cytokine Expression from RAW Macrophages Cultured with Free or

Encapsulated poly I:C...... 27

Figure 2.3: Encapsulation and Immunostimulatory Capability of CpG in either PLGA or

Ac-DEX Microparticles...... 28

Figure 2.4: Macrophage Viability after Culture with Free or Encapsulated CpG...... 30

Figure 2.5: Cytokine Expression from RAW Macrophages Cultured with Free and

Encapsulated CpG...... 33

Figure 3.1: Chemical Structure of Resiquimod...... 41

Figure 3.2: L. donovani units (LDU) in the (a) Liver (b) Spleen (c) and Bone Marrow of

BALB/C Mice Infected with the Parasite...... 49

Figure 3.3: Proliferation of T cells Isolated from the Spleens of BALB/C Mice Infected with L. donovani and Exposed to Antigen...... 50

Figure 3.4: Production of (a) IFN-γ or (b) IL-10 from Splenic Cells Isolated from

BALB/C Mice Infected with L. donovani...... 51

Figure 3.5: Macrophage Viability after being Cultured with Free Resiquimod,

Resiquimod Loaded Liposomes or Empty Liposomes...... 53

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Figure 5.1: Scanning Electron Micrographs of Acetalated Dextran (Ac-DEX)

Microparticles Encapsulating (A) Dexamethasone (DXM) + Myelin Oligodendrocyte

Glycoprotein 35-55 (MOG) (B) DXM or (C) MOG...... 94

Figure 5.2: Nitric Oxide (NO) Release by Macrophages when Cultured with

Lipopolysaccharide plus Free or Encapsulated Dexamethasone (DXM) in Ac-DEX...... 97

Figure 5.3: IL-6 Production in Culture from C57Bl/6 Bone-Marrow Derived Dendritic

Cells...... 98

Figure 5.4: Clinical Scores of 9 Week Old C57Bl/6 Female Mice Immunized with

Experimental Autoimmune Encephalomyelitis (EAE)...... 99

Figure 5.5: Antigen Recall Measurements of (A) IL-17 and (B) GM-CSF production. 100

Figure 6.1: Scanning Electron Micrographs of Microparticles to Prevent Hyperglycemia onset in NOD Mice...... 115

Figure 6.2: Diabetes Incidence in NOD mice...... 120

xxi

Chapter 1: Introduction

1.1 Drug Delivery

It has been documented that patients in hospitals face millions of adverse drug reactions annually (1), many from drug toxicity or hypersensitive responses. To combat adverse events, it is important to develop optimized delivery methods that can provide therapeutic effects while limiting highly toxic doses. One method of optimizing therapeutic effect is through the use of particulate-based delivery systems. Among these are lipid-based (liposomes) or polymer-based particle delivery systems, which can increase solubility and reduce toxicity of compounds, provide depots for sustained release, as well as increase circulation time of small molecules. Increased solubility of compounds is important for pharmaceutical purposes because it directly impacts their bioavailability. Lower solubility compounds require more frequent administration of small doses to reach therapeutically effective plasma levels (2). Retention of compounds, like small molecules, also has a large impact on bioavailability. Liposomes (3) and polymer particles (4) can both be altered to increase circulation and retention time of compounds. Additionally, particles can lower dosing requirements by providing a mechanism of sustained drug release (dose-sparing). Another benefit of particulate

1 platforms is their ability to be passively and readily taken up by antigen presenting cells

(APCs) (5) via phagocytosis. The ability to be phagocytized is beneficial for the treatment of many diseases, including those that require immunomodulation. Many diseases such as cancers and infections require amplified immune responses, while others such as autoimmune diseases, require a dampening of such responses. Much of this immunomodulation can occur through APCs, so targeting them allows for increased efficiency of drug delivery. Particulate vehicles increasing deliverability of drugs, solubility, retention times and release rates make them an exciting platform for the more optimal delivery of therapies.

1.1.1 Liposomes

Liposomes are small lipid-based vesicles and are generally made using batch- processes. One common method is solvent-evaporation and lipid film hydration. During this process, the lipid components are prepared in an organic solution followed by rotary evaporation of the solvent, until a film is formed. This film is reconstituted in an aqueous buffer to form self-assembled vesicles. Generally, hydrophobic drugs can be encapsulated by dispersion in the organic solution while hydrophilic drugs have better encapsulation potential dispersed in the reconstitution buffer. Although this method is easy and widely used, the large heterogeneity of the liposomes requires methods for reduction in liposomal size (6). Probe or bath sonication of liposomes is one method to limit heterogonous populations, however, this method could shear liposomes or contribute to

2 decreased encapsulation efficiency of a drug (7). A commonly used sizing method is extrusion through a polycarbonate membrane with a fixed pore size, resulting in a homogenous population that may have increased drug retention (7, 8). During certain situations, it may be possible to limit the number of steps during batch process liposome formulation by solvent dispersion methods. Two commonly used methods for this are solvent vaporization and ethanol injection. For solvent vaporization, an ether solution is added to an aqueous solution under heat and pressure; however, these conditions may not be ideal for sensitive lipids or cargo. Ethanol injection establishes less harsh conditions, but solutions must be used in excess, making it difficult to remove the resulting large volumes of ethanol from samples (7). Many of these processes contain advantages and disadvantages, so it is important to ensure formulation methods are optimized prior to extensive in vitro and in vivo testing.

1.1.2 Polymer Particles

Recently, polymer-based particle systems have become increasingly popular.

Generally, particles are made via oil-in-water (O/W) and water-in-oil-in-water (W/O/W) emulsions, which provides a way for the encapsulation of both hydrophobic and hydrophilic cargo, respectively. Particles are generated from homogenization or probe sonication, and modification of these processes (e.g. homogenization speed or energy emitted from probe) can directly affect the size of the particles. After emulsification occurs, organic solvents can be removed by solvent evaporation, coacervation or spray

3 drying (9). Solvent evaporation is a simple method where a stabilizer is added to the particle solution, and it is allowed to stir in ambient conditions until solvents are evaporated and the particles harden. Coacervation is a multi-step process where the polymer phase is separated and isolated. Spray drying methods provide a scalable method where particle solutions are exposed to a combination of high temperatures and a stream of air. Another scalable process is the use of electrohydrodynamic spraying

(electrospray). This process allows for aerosolized particles to form through rapid solvent evaporation. The majority of polymeric particle work has been performed using FDA- approved polyesters, such as poly(lactic-co-glycolic acid) (PLGA), poly-L-lactic acid

(PLA) and polycaprolactone (PCL). Along with obtaining FDA-approval for a variety of uses (e.g. sutures and drug delivery), these polyesters are advantageous because of their biodegradability (degradation of PCL is on the order of years (10) and PLGA is on the order of months (11)), low toxicity, and low expense. However, high concentrations and long degradation rates can cause accumulation of byproducts that lower the local pH (11,

12) damaging surrounding tissue or affecting acid-sensitive cargo with hydrolysable bonds (13) or by acylation of peptide primary amines (13-15). Other polymers such as polyanhydrides (e.g. sebacic acid, p-(carboxyphenoxy)propane, or p-

(carboxyphenoxy)hexane) have tunable degradation rates, biodegradability, and have been extensively studied (16, 17); however, they are only able to encapsulate certain classes of drugs (18) and are hydrolytically unstable, resulting in non-degrading polymer at low pH conditions and degradation under basic conditions (19). Although beneficial in certain scenarios, polymers with limited degradation under lysosomal conditions (~ pH 5)

4 are likely not good candidates for passive targeting of APCs and the subsequent delivery within phagolysosomes. Additionally, due to slow degradation of PLGA in lysosomal conditions, it has been proposed that a pH-sensitive polymer capable of releasing cargo upon exposure to acidic conditions may be optimal for delivery to APCs (20). A recently developed polymer acetalated dextran (Ac-DEX) has been formulated from dextran, a of glucose, with acetal groups replacing the hydroxyl groups (21). This novel polymer is very stable at pH 7.4 and quickly degrades upon exposure to pH 5.0 and below. Upon degradation, its pH-neutral byproducts are dextran, , and .

Additionally, because methanol buildup can induce ketosis (22), an even more biocompatible version has been formulated, ethoxy-derivatized acetalated dextran (Ace-

DEX), which replaces the methanol byproduct with ethanol (23). With so many particle formulations available, it is necessary to determine which may be the most effective for disease and therapeutic types.

1.2 Leishmaniasis

Leishmaniasis is caused by several species of the protozoan parasite Leishmania, and is diagnosed in approximately 12 million patients worldwide (24). Leishmania are zoonotic parasites transmitted by sandflies that can affect mammals such as humans and canines. Leishmaniasis, the resulting disease, can manifest as a cutaneous (CL), mucosal

(ML), or visceral (VL) form. CL and ML can be extremely debilitating due to skin sores, ulcers, or scar tissue buildup. Ulcerations of the skin caused by CL or ML infection

5 provide an optimal environment for opportunistic secondary infections, which could exacerbate ulceration and tissue destruction, or pose additional and potentially more severe health problems. In fact, approximately 20% of CL patients are found to have a secondary infection, most commonly Staphylococcus (25). However, each year, close to

50,000 deaths are attributed to Leishmaniasis (24), mainly due to VL. Another emerging problem with Leishmaniasis is the potential for patients co-infected with a previous ailment. A common co-infection that poses a problem to VL patients is the human immunodeficiency virus (HIV). HIV-positive patients are up to 2,300 time more likely to develop VL infection (26) and CD4+ T cell suppression by HIV could complicate treatment of infected patients (27). Unfortunately, in some parts of the globe, up to 30% of VL patients are co-infected with HIV (28). Due to severe complications, including death, resulting from Leishmania infection, it is vital to have access to effective treatments.

1.2.1 Current Therapies for Leishmaniasis

For over 50 years, the primary treatment for VL has been systemic administration of antimonials, however, this treatment can result in severe side-effects such as cardiac arrhythmia and pancreatitis (29-31). Although detrimental side-effects can occur, antimony is a front line therapy due to affordability, ease of administration, and rates of moderate to high success in certain regions of the world (32). Unfortunately, other regions like Bihar, India are facing an epidemic of antimony-resistant VL infections

6

(close to 70% of cases), likely due to poor patient compliance with their drug regimen

(33). In recent decades, to improve upon the shortcomings of antimony, new and emerging treatments for VL have been established. Among these is intravenous delivery of liposomal amphotericin B (AmBisome) (34) and the orally administered miltefosine

(Impavido, Miltex). Liposomal amphotericin B, although functional, is fiscally impractical (35) due to a combination of high costs, hospital admittance for intravenous delivery, and the prevalence of Leishmaniasis in regions of low income. Miltefosine is an oral formulation, which is an ideal delivery route, but some patients may only tolerate it in low doses (36) and it has shown long-term teratogenicity (37). Both of these issues only make miltefosine viable for a limited number of patients. Additionally, cases of drug resistance towards both amphotericin B (38) and miltefosine (39) have already emerged.

To continue combating this disease, it is necessary to develop novel treatments that are well tolerated and limit the chance of resistance.

1.2.2 Parasite Resistance to Therapies

One way to limit the formation of resistance by pathogens, such as Leishmania, is by clearing them through the host pathogen interface. In other words, resistance can be minimized by allowing the host immune system to clear a pathogen, as opposed to directly targeting the pathogen. Upon infection by intracellular pathogens, including

Leishmania, activation of Toll-like Receptors (TLRs) act as a primary line of innate defense (40). Activation of the intracellular TLRs 3, 7, 8 and 9 provides host defense by

7 subsequent secretion of pro-inflammatory factors like cytokines and nitric oxide, which may be vital for clearance of Leishmania (41, 42). Clinically available TLR agonists such as polyinoscinic polycytidylic acid (poly I:C) (TLR 3), resiquimod (TLR7/8), imiquimod

(TLR 7/8), and cytosine bonded to Guanine (CpG) (TLR9), provide mechanisms to stimulate an immune response, which can be beneficial when treating diseases where innate responses from TLRs are down-regulated by pathogens. Previously, CpG (43), poly I:C (44), and imiquimod have shown promising results as a treatment for CL, including clinical usage of imiquimod to treat cases of CL (45). Resiquimod, a sister- compound to imiquimod, may have a significantly higher cytokine response upon usage

(46). One of the most promising pieces of recent data is the ability for TLR agonists to synergize with other therapeutics, and even overcome pathogen resistance to certain treatments. For example, imiquimod cream with antimony has been shown to treat CL patients who were previously unresponsive to antimony treatment (47). Co-treatment with both therapies outperformed treatment with just a single therapy (48). It has been shown that enhancement of a host TH1 response increases the benefits of other traditional treatments such as amphotericin B (49), and it is assumed antimony function requires a host TH1 response (50). Addition of TLR-agonists for the treatment of Leishmaniasis may provide a novel route to enhance the host immune response and decrease parasite burden. Although promising data has shown the clearance of pathogens responsible for

CL, these compounds may have limited use against VL, due to occurrences of side effects after systemic delivery (51, 52). In order to utilize the advantages of TLR agonists to

8 potentially treat systemic intracellular infectious diseases, such as Leishmaniasis, new methods of delivery should be established.

1.2.3 Particulate Delivery Vehicles for the Treatment of Leishmaniasis

Delivery of immunomodulatory compounds using particulate-based platforms provides a number of additional benefits (53). Polymer and lipid-based particulate carriers delivering TLR-agonists could increase their solubility and reduce side-effects, such as flu-like symptoms. Additionally, passive targeting of APCs is important since both targeted TLRs and pathogens like Leishmania reside in the host macrophage (Mϕ)

(54). One issue that needs to be considered with tropical and neglected diseases, like

Leishmaniasis, is cold-chain storage of treatment formulations. With many patients residing in remote areas with limited access to refrigeration, it is imperative to develop new treatments that can withstand long exposure to elevated temperatures. Bulk encapsulation of compounds inside polymer particles is a promising way to bypass the need for cold-chain conditions. A recent study by Kanthamneni et al. showed encapsulation of a model enzyme in a delivery vehicle significantly diminished the need for cold-chain storage, and allowed for long-term storage at 45oC (55). Researchers have previously worked on a variety of treatment and vaccine particulate formulations for

Leishmaniasis. In fact, researchers have compared current treatments of liposomal amphotericin B with polyesters (56-58). PLGA microspheres and liposomes encapsulating soluble Leishmania antigen with CpG have been explored for their

9 potential as a subunit vaccine (59-62). Although a functional vaccine would go a long way to limit the morbidity and mortality of the disease, regions where Leishmaniasis is common have had complications with the implementation of vaccine programs (63).

Individuals continue to become infected and an increasing number of these cases involve pathogens that are resistant to current and commonly used treatments, resulting in increased morbidity and healthcare costs. The development of new therapies that can treat Leishmaniasis, or synergize with current therapies is essential.

1.3 Autoimmunity

Immunomodulatory therapies can also be used to decrease the host immune response towards a disease. This can be important in treating autoimmune diseases such as Rheumatoid Arthritis (RA), Multiple Sclerosis (MS), or Type 1 Diabetes (T1D), which occur when the immune system recognizes self-antigen as foreign, therefore attacking itself. RA is a severely debilitating disease affecting 1.5 million adults (64) and costing approximately $125 billion annually in the United States (65). The National Institutes of

Health report that 80 people are newly diagnosed with T1D in the United States daily, and the disease results in $14.9 billion in annual healthcare costs (66). Additionally, MS impacts more than 2.5 million people world-wide, and domestically direct treatment costs can reach $55 thousand per year per patient (67). Although each autoimmune disease may have unique pathogenic characteristics, one common denominator between RA,

T1D, and MS is cell death due to auto-reactive T cell infiltrates. When these diseases

10 occur, in order to limit the damage caused by these auto-reactive cells, it is necessary to dampen an individual’s immune response. Each of these diseases have established treatments, however, there are still no cures. T1D is relatively well managed with insulin replacement and lifestyle changes, but quality of life is heavily impacted. RA and MS have treatments such as monoclonal antibodies or corticosteroids, but these induce wide spread immune suppression and can have severe side effects including increased risk of cancer (68) and infection (69). To combat total immune suppression, novel therapies must be developed that suppress disease progression in an antigen-specific manner.

1.3.1 Immune Tolerance

Since autoimmune disorders arise because self- are recognized as foreign, it may be possible to directly target these auto-reactive cells. A number of candidate antigens involved in MS (70), RA (71), and T1D (72) have been proposed. Although previous clinical work suggests it is likely that any treatment will need to be equipped to target multiple candidate antigens (73), identification of these provides an opportunity to evaluate their potential use for antigen-specific therapies. Moreover, disease–specific antigens for rodents have been identified, allowing animal models of the diseases to be studied. This is advantageous because it allows for the development of antigen specific treatment platforms, and the subsequent modeling of their mechanisms. These treatments can help tolerize the immune system towards disease antigens, suppressing the pathogenic immune response, while allowing the remainder of the immune system to

11 function normally. Previously, antigen specific tolerance was induced in animal models through mucosal surface interaction using oral, (74, 75) nasal, (76) and sublingual (77) delivery. Mucosal tolerance has proven to be an effective treatment in animal models of

MS, RA, and T1D (78), however, many treatments have been unsuccessful in the clinic due to improper antigens or dosing problems. Particle injections could provide an ideal delivery system for sustained therapeutic windows and to target APCs.

1.3.2 Inducing Tolerance with Particulate based Therapeutics

Administering disease-associated peptides on apoptotic cells has long been thought to be able to ameliorate disease. Recently this has been supported in a phase 1 clinical trial for MS (73). This functionality is likely a result of an inherent mechanism restricting cells from reacting to apoptotic self-debris. However, utilization of cells as delivery vehicles, or ex vivo manipulation of cells, requires costly batch processing per patient, while particulate carriers may be engineered inexpensively to mimic these cells.

In recent years, therapeutics using tolerogenic particles have aided in limiting the progression of disease in animal models of autoimmunity. Getts et al. have shown conjugation disease-associated peptides to either polystyrene or PCL particles induces tolerance in experimental autoimmune encephalomyelitis (EAE), an animal model of MS

(79). Kim et al. have shown bulk encapsulated antigen, not just surface bound, can suppress autoimmune symptoms by using PLGA particles encapsulating collagen to ameliorate symptoms of Collagen-Induced Arthritis, an animal model of RA (80).

12

Synthetic peptides have also shown potent tolerogenic effects in vivo. Tolerance in EAE was accomplished by administration of multiple doses of PLGA microparticles carrying a synthetic peptide (81). In a subsequent study, the same peptide was administered in a single injection of a PLGA, alginate, and chitosan colloidal gel to inhibit symptom onset

(82). Interestingly, injection of dendrimers with conjugated disease-associated peptides also showed protective effects against EAE. Although administering these antigens have shown promising effects against autoimmune models, other recent work has suggested the protective effect could be amplified by co-administration of a tolerogenic drug with the antigen (83, 84). In a promising study, Yeste et al. conjugated a MS disease- associated protein epitope of myelin oligodendrocyte glycoprotein (MOG), with a tolerogenic compound, to gold . These particles were successfully able to limit disease severity in EAE (85). Finally, the use of some tolerogenic natural compounds, with antigen, may be able to limit autoimmune severity. Capini et al. show liposomal encapsulation of curcumin, a natural product with tolerogenic potential, and that an antigen may limit progression of an inflammatory arthritis mouse model (86), while Keselowsky et al. have shown preliminary results suggesting PLGA encapsulating a vitamin D derivative, insulin peptide, and growth factors may protect NOD mice against spontaneous T1D onset (Unpublished, (87)). Although much of this work has shown promising data, it is essential to expand on it. For instance, many studies regarding autoimmunity begin treatment at a clinically irrelevant time (e.g. prior to the onset of symptoms). In fact, Vesterinen et al. showed that only 4% of peer-reviewed EAE publications of the 126 examined, assessed treatment efficacy beyond 2 weeks post

13 induction of EAE (88). Many studies treating before symptom onset are valuable because they propose new models for treatment however, many of them suggest protective mechanisms that may not be accurate in a therapeutic environment. Further development should be performed to find novel delivery methods to treat autoimmune diseases, while taking care to optimize these formulations to increase the likelihood of determining mechanisms and increasing clinical relevance.

1.4 Objective and Research Contribution

Treatment of pathogenic infections and autoimmune diseases using immunomodulatory therapies inducing antigen specific responses could provide a vital advantage over current therapies. For VL, clearing pathogens by targeting the host- pathogen interface may provide a mechanism of parasite clearance that limits the risk of drug resistance. On the other hand, immunomodulatory particles could make it possible to dampen an immune response to a particular antigen, allowing the treatment of autoimmune diseases without suppressing the remainder of the immune system.

Currently, there are no widely used therapies for VL or autoimmune diseases that can accomplish these tasks. In an attempt to treat VL, this work provides novel delivery vehicle formulations of polymer and lipid-based particles containing TLR agonists. This work determines the ability of various formulation methods to encapsulate compounds potentially capable of limiting disease progression, and determine a formulation that displays promising results in clearing the parasite burden in infected organs in a VL

14 murine model. Additionally, particle technology can be altered for the treatment of autoimmune diseases. This work contributes novel findings to a recently emerging field of particulate-based treatments to dampen an immune response in an antigen specific manner. This work provides evidence that using the novel polymers Ac-DEX and Ace-

DEX to make particles encapsulating a disease-associated antigen and a tolerogenic compound is a viable platform for treatment of autoimmunity. This work suggests co- delivery of an antigen and a tolerogenic compound is superior to delivery of antigen alone. Although a mechanism was not identified, this work compares a multitude of formulations containing various tolerogenic compounds in an attempt to identify the optimal therapeutic combination. It is the hope that through comparison of multiple particle formulations and dosing sequences, that a more amplified tolerogenic phenotype will emerge, and through this, a more definitive mechanism can be elucidated. This will determine if particulate-based therapies targeting APCs can modulate the immune response for the treatment of Leishmania infection or autoimmunity.

15

Chapter 2: Efficient Delivery of the TLR-Agonists Poly I:C and CpG to Macrophages by

Acetalated Dextran Microparticles

2.1 Introduction

Treatment of infections at the host-pathogen interface allows for clearance through amplified immune responses. In diseases like Leishmaniasis, innate defenses that recognize pathogens can be diminished by the parasite as a survival defense. Small molecules such as TLR-agonists can work by the upregulation of immune responses through activation of TLRs. This is accomplished through mimicking pathogen- associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), double- stranded RNA (dsRNA), or unmethylated CpG motifs. Poly I:C is another immunostimulatory adjuvant that elicits an immune response via TLR-3. It does so by mimicking dsRNA, commonly found in viral genomes. It has shown to increase the protective capability of an Ebola subunit vaccine (89) and has been studied as a treatment of certain cancers using ex vivo dendritic cell (DC)-activated immunotherapy (90-92).

Additionally, the oligodeoxynucleotide of CpG is an adjuvant that activates TLR-9 by mimicking unmethylated CpG motifs that are uncommon in mammalian cells. CpG has 16 been shown to decrease or eliminate symptoms in asthma (93), enhance the anti-cancer effects of radiation (94) and chemotherapeutics (95), and increase ’ effectiveness towards diseases like Leishmaniasis (96, 97) and Herpes Simplex Virus-2 (98, 99).

Although poly I:C and CpG are useful adjuvants for immunostimulatory treatments, both adjuvants may have significant side-effects if delivered systemically throughout the body.

The side-effects of systemic (e.g. non-encapsulated) administration of poly I:C and CpG are potential systemic toxicity, histological damage, auto-reactive T-cells, and general symptoms of autoimmunity (100-102). These systemic effects are likely the cause of adjuvants like CpG sufficiently causing T cell infiltration into tissues and increasing co- stimulatory signals that are associated with increased inflammatory responses (103).

Directed and efficient delivery of these TLR agonists ensures the positive effects of immunostimulatory compounds while limiting their toxic side-effects. By delivering these agents directly to phagocytic cells, one can focus the drugs’ effect to primarily the immune cells that respond to TLR agonists as well as target the cells that serve as gateways for immune responses. As a result of APCs inherent phagocytizing capability, it is easy to target these cells with particulate-based systems. FDA approved polyesters,

PLGA, PLA and PCL are the most frequently used polymers for particulate delivery systems. The benefits of polyester particulate systems include their biodegradability

(with PLGA degradation on the order of months (11) and PCL on the order of years

(10)), low toxicity and low expense. However, polyesters degrade into acidic by- products that are physiologically present in the body at low concentrations (104). At high concentrations, or if present for extended periods of time, these byproducts can shift the 17 local pH (11, 12), causing toxicity or even granulomas (105). This shift in the pH of the local environment can not only damage surrounding tissue, but also affect acid-sensitive cargo like hydrolysable bonds (13) or acylation of peptide primary amines (13-15).

Stability of the cargo is important for optimization of particles because the better the response a therapy can elicit at the site of antigen presentation, the more likely it will provide long-term protection against a pathogen. For the encapsulation of TLR-specific immunostimulatory agents, polymer and lipid based particles systems have been used for delivering small molecules such as CpG (106), polyuridylic acid (107), poly I:C (108), imidazoquinolines (109-112), and monophosphoryl lipid A (MPLA) (113). Although these polymers are frequently used and have shown some clinical success, new polymers for drug delivery should be developed that are more biologically degradable, are more tunable, and decrease the risk of damage to the cargo from acidic environments.

One such newly developed polymer is Ac-DEX. Ac-DEX is a polysaccharide of glucose with acetal groups replacing the naturally occurring hydroxyl groups (21). Ac-

DEX is an acid sensitive polymer that degrades at lysosomal conditions (pH 5) but is relatively stable at pH 7.4. The length of time needed for Ac-DEX to completely degrade into dextran, acetone, and an is tunable based on the reaction time of dextran with

2-methoxy propene. Due to the slower degradation rates of cyclic , compared to acyclic acetals, and the increase in cyclic coverage with increasing reaction times, it is possible to tune the release of the cargo from Ac-DEX (114). Unlike the previously described polymers, such as PLGA, Ac-DEX does not shift the pH of the local environment, increasing the likelihood of fully functional cargo upon delivery. 18

Furthermore, the degradation rates of Ac-DEX can also be changed by varying the molecular weight of the dextran starting material (5).

In addition to tunable degradation rates that can range from hours to months, Ac-

DEX has numerous other advantages that make it an ideal carrier for vaccine delivery. It displays enhanced MHC I and MHC II presentation when compared to PLGA and other common biomaterial platforms (114). This is important because potential treatments requiring antigens are more feasible with enhanced presentation platforms. It has been shown that Ac-DEX encapsulating the TLR 7/8 agonist imiquimod, significantly increases immunostimulatory capabilities of the drug when compared to free drug (109) in both Mϕ and bone marrow derived dendritic cells (BMDCs). Additionally, it has been shown in previous work that CpG incorporated in an acid-sensitive microparticle hydrogel is safe and effective at stimulating an immune response in vivo (115), which indicated encapsulation of CpG is a viable option for subunit based vaccines. These reasons make Ac-DEX ideal for particle formulation for immunomodulatory therapies

(109).

This study is the first to encapsulate CpG and poly I:C into this novel material and examine if CpG and poly I:C encapsulated Ac-DEX particles have a higher immunostimulatory response when compared to PLGA microparticles or free adjuvant.

Immune stimulation was tested through measurements of nitric oxide (NO) and the ability to stimulate cytokine release in Mϕ.

19

2.2 Materials and Methods

2.2.1. Materials

All reagents were purchased and used unmodified from Sigma-Aldrich (St. Louis,

MO) unless otherwise noted. Water (dd-H2O) for buffers and particle washing steps was purified using a Millipore (Billerica, Massachusetts) Milli-Q Integral water purification system. When used in the presence of acetal-containing materials, dd-H2O was rendered basic (pH 9.0) by the addition of triethylamine (TEA) (approximately 0.01% v/v).

Fluorescence and absorbance measurements were obtained on a Molecular Devices

(Sunnyvale, California) FlexStation 3, courtesy of the Department of Chemistry and

Biochemistry at the Ohio State University.

2.2.2. Cell Lines

RAW 264.7 macrophages were purchased from ATCC (Manassas, VA). Cells were grown and maintained as per guidelines provided by the manufacturer.

2.2.3. Synthesis and Analysis of Acetal Coverage of Ac-DEX

Ac-DEX was synthesized from 10 kDa, 71 kDa, or 150 kDa dextran as described previously (5, 21, 114). Determination of cyclic and acyclic acetals was determined by the NMR protocol of Broaders et al (114).

20

2.2.4. Preparation of Double Emulsion Particles Encapsulating Poly I:C or CpG with

either Ac-DEX or PLGA

Microparticles containing poly I:C or CpG were prepared using a double emulsion method as described previously (116, 117). The weight loading for 71k 5 minute Ac-DEX encapsulating CpG was 0.008 mg and poly I:C was 0.0106 mg.

2.2.5. Extraction and Quantification of Encapsulated Poly I:C and CpG

Similar to what was done by Cohen et al. (118), particles were resuspended in TE buffer (1 mg/ml in 500 μl) and added to dichloromethane (DCM) (1 ml) in a 1.8 ml

Eppendorf tube. The solution was vortexed for one minute, placed in a Burrell’s Wrist

Action Shaker Model 75 (Pittsburgh, PA), and shaken for one hour. The solution was then centrifuged for five minutes at 9,000 RCF. 200 μl of the poly I:C or CpG solution from each sample was removed and placed in a 96-well plate and immediately frozen at -

20°C and stored prior to analyses. Quant-iT OliGreen™ from Life Technologies (Grand

Island, NY) was used to quantify poly I:C and CpG per the manufacturer’s instructions.

2.2.6. Scanning Electron Microscopy (SEM)

Microparticles were characterized by SEM using an FEI (Hillsboro, Oregon)

NOVA nanoSEM. SEM sample preparation and analysis was done as previously described (23).

21

2.2.7. Dynamic Light Scatter

Size of the 71k 5-min poly I:C and CpG microparticles was determined as previously described (111).

2.2.8 Release Profile of Poly I:C Encapsulated in Ac-DEX

1 mg of encapsulated poly I:C particles (in triplicate per pH) was suspended in either PBS (pH 7.4) or 0.3 M sodium acetate buffer (pH 5.0) and shaken moderately on a

VWR Analog Heatblock (Westchester, PA). At each time point, 120 μL of a sample is removed and placed in an Eppendorf centrifuge tube and spun at 9,000 RCF for five minutes. After centrifugation, the supernatant was collected and frozen at -20°C in a 96- well plate for future analysis. Poly I:C concentration was determined by Quant-iT

OliGreen™. Empty particles were degraded at the same conditions described above and the corresponding background signal was subtracted from the particles that encapsulated poly I:C.

2.2.9. Nitrite and Cyotkine Analysis of Macrophages Stimulated by Poly I:C or CpG

Briefly, nitrite concentrations in the supernatants of RAW 264.7 macrophages were determined using the Griess reagent from Promega (Madison, WI). Macrophages were plated in a 96-well plate at 5x104 cells/well, incubated in Thermo Scientific

HyClone DMEM/ High Glucose (Logan, UT) with 5% Fetal Bovine Serum and 1%

22 penicillin/streptomycin (complete media), and left overnight to adhere. The cells were treated with complete media, free adjuvant, Ac-DEX particles encapsulating adjuvant,

PLGA particles encapsulating adjuvant, or blank Ac-DEX particles for 24 hours.

Supernatants were collected after 24 hours and centrifuged at 15,000 rpm for 10 minutes to pellet the remaining particles. The isolated supernatants were collected and analyzed with the Griess reagent in accordance with the manufacturer’s protocol. The supernatants were analyzed for cytokines as previously described in Bachelder et al (109).

2.3 Results

2.3.1. Poly I:C Particle Formulation and Analysis

Figure 2.1A reports Ac-DEX and PLGA encapsulation efficiencies (EEs) for various molecular weights and reaction times of Ac-DEX. Ac-DEX microparticles encapsulating poly I:C are presented in the micrograph given in Figure 2.1B. Particles of poly I:C encapsulated in 71k 5-min Ac-DEX showed an average particle size around 486 nm, which is consistent with the micrograph (Figure 2.1A). PLGA encapsulated an amount of poly I:C greater than both 10k (Figure 2.1A) and 150k (Figure 2.1A) Ac-DEX

(33.5% versus 21.2% and 27.0%, respectively), but both 71k 5-min (53.0%) and 4-hour

(57.0%) Ac-DEX showed increased encapsulation of poly I:C compared to that of PLGA.

Ac-DEX showed an increase in the ability to encapsulate poly I:C (Figure 2.1A). After

24 hours, particles incubated in acidic conditions released approximately 90% of their encapsulated poly I:C, while those incubated at neutral conditions released approximately

40% (Data not shown). 23

Figure. 2.1: Encapsulation and Immunostimulatory Capability of poly I:C in either PLGA or Ac-DEX Microparticles.

(A) Encapsulation of particles containing poly I:C encapsulated by either poly(lactic-co- glycolic acid) (PLGA) or acetalated dextran (Ac-DEX) of various molecular weights and reaction times. Data is presented as average ± standard deviation. (B) Scanning electron micrograph of 71k 5-minute Ac-DEX encapsulating poly I:C. (C) Comparison of nitric oxide (NO) release by macrophages when cultured with either free or encapsulated poly

I:C. Poly I:C was encapsulated by either 71k Ac-DEX or PLGA. Statistical significance with respect to free poly I:C is presented as * p < 0.05, ** p < 0.0005, *** p < 5x10-5, while significance with respect to PLGA is presented as # p < 0.005 and ## p < 5x10-5.

24

2.3.2 Free and Encapsulated poly I:C Stimulation

Figure 2.1C reports nitric oxide (NO) release of Mϕs cultured with free poly I:C

(ranging from 0 to 8 µg/ml), poly I:C encapsulated by Ac-DEX (5 min or 4 hr) or poly

I:C encapsulated by PLGA. With the exception of 8 µg/ml (p < 0.01) and 2 µg/ml (p <

0.05) poly I:C, there was no significant difference between the ability of free poly I:C and poly I:C encapsulated by Ac-DEX (5 min) to stimulate NO release. However, there was a significant decrease in NO production from cells incubated with poly I:C encapsulated by both Ac-DEX (4 hr) and PLGA, when compared to both free poly I:C (p < 5x10-4 and p < 5x10-5 for 8 µg/ml) and poly I:C encapsulated by Ac-DEX (5 min) (p < 0.005 and p <

5x10-5 for 2 µg/ml). Cytokine levels in the poly I:C supernatants are shown in Figure 2.2.

Both TNF-α (p < 0.005) and IL-1β (p < 0.05) show an increase in production with the encapsulated groups over the free poly I:C group. When comparing Ac-DEX (5 min) to both Ac-DEX (4 hr) and PLGA, Ac-DEX (5 min) shows significantly higher production of both TNF-α (p < 5x10-4 and p < 0.05 for 4 and 8 µg/ml, respectively) and IL-1β (p <

0.05 for 2, 4 and 8 µg/ml). IL-6 production was significantly lower for Ac-DEX (4 hr) and PLGA when compared to both free poly I:C (p < 0.005) and Ac-DEX (5 min) (p <

5x10-4), while no difference was seen between free poly I:C and Ac-DEX (5 min), with the exception of 4 µg/ml (p < 0.05). With the exception of 4 µg/ml and 8 µg/ml PLGA, and 8 µg/ml Ac-DEX (4 hr), encapsulated poly I:C increased IFN-γ production over free poly I:C (p < 0.05) while no difference was seen between the encapsulated groups.

Production of IL-2 was significantly higher for poly I:C encapsulated by Ac-DEX (5

25 min) than free poly I:C (p < 5x10-4 for 4 and 8 µg/ml and p < 0.05 for 1 and 2 µg/ml). IL-

2 production by poly I:C encapsulated by Ac-DEX (4 hr) or PLGA was significantly lower than both free poly I:C (p < 0.005 and p < 0.05 for 2µg/ml) and poly I:C encapsulated by Ac-DEX (5 min) (p < 0.05, except p < 5x10-4 for 8 µg/ml Ac-DEX (4 hr) and 1 µg/ml Ac-DEX (4 hr) was insignificant).

Finally, no significant difference of IL-12(p70) production was seen between free poly I:C and poly I:C encapsulated by Ac-DEX (5 min), however there was a significant increase in production when Ac-DEX (4 hr) and PLGA were compared to free poly I:C

(p < 0.05 for 8 µg/ml and p < 0.005 for 1, 2 and 4 µg/ml) and poly I:C encapsulated by

Ac-DEX (5 min) (p < 0.05).

2.3.3. CpG Particle Formulation and Analysis

Figure 2.3A reports Ac-DEX and PLGA EEs, and Ac-DEX (5 min) showed an increase in the ability to encapsulate CpG when compared to PLGA (36.6% versus

3.3%). Ac-DEX (5 min) microparticles encapsulating CpG have an average size of approximately 554 nm, which is consistent with a micrograph of these particles (Figure

2.3B).

26

Figure 2.2: Cytokine Expression from RAW Macrophages Cultured with Free or

Encapsulated poly I:C.

Data is presented as average ± standard deviation. Statistical significance with respect to free poly I:C is presented as * p < 0.05, ** p < 0.005, *** p < 5x10-5. Significance with respect to Ac-DEX (5 min) is presented as # p < 0.05, ## p < 5x10-4.

27

Figure 2.3: Encapsulation and Immunostimulatory Capability of CpG in either PLGA or

Ac-DEX Microparticles.

(A) Encapsulation efficiency of particles containing CpG encapsulated by either poly(lactic-co-glycolic acid) (PLGA) or 71k 5 min acetalated dextran (Ac-DEX). Data is presented as average ± standard deviation. (B) Scanning electron micrograph of CpG encapsulated 71k Ac-DEX (C) Nitric oxide (NO) released by macrophages when cultured with either free or encapsulated CpG. CpG was encapsulated by Ac-DEX.

Statistical significance with respect to free CpG is presented as * p < 0.05, ** p < 5x10-4,

*** p < 5x10-5.

28

2.3.4. Free and Encapsulated CpG Show Increased Macrophage Viability

Figure 2.4 shows the viability of Mϕ exposed to free CpG, encapsulated CpG (1.6

µg/ml to 0.2 µg/ml), and blank Ac-DEX (5 min) particles. The blank particle concentration represents the same amount of Ac-DEX used to encapsulate CpG at the given concentrations. There was no significant difference in cell viability found with increasing blank Ac-DEX (5 min) particle concentrations. A significant increase in cell viability was seen with free CpG, when compared to both CpG encapsulated in Ac-DEX

(5 min) (p < 5x10-5 with exception to p < 5x10-4 for 0.2 µg/ml and p < 0.005 for 1.6

µg/ml) and blank Ac-DEX (5 min) particles (p < 5x10-5 with exception to p < 5x10-4 for

1.6 µg/ml). However, there was also a significant increase in cell viability with CpG encapsulated in Ac-DEX (5 min) when compared to blank Ac-DEX (5 min) particles (p <

0.005 with exception to p < 0.05 for 0.2 µg/ml).

29

Figure 2.4: Macrophage Viability after Culture with Free or Encapsulated CpG.

Comparison of macrophage viability after cells were cultured with either CpG, CpG encapsulated in 71k 5 minute acetalated dextran (Ac-DEX), or blank 71k 5 min Ac-DEX particles. Statistical significance with respect to free CpG is presented as * p < 0.05, ** p

< 5x10-4, *** p < 5x10-5, while significance with respect to CpG encapsulated Ac-DEX particles is presented as # p < 0.05 and ## p < 0.0005.

30

2.3.5 Free and Encapsulated CpG Stimulation of Immune Response in Macrophages

Ability of free CpG, CpG encapsulated in Ac-DEX (5 min) particles, and blank

Ac-DEX (5 min) particles to stimulate NO release in Mϕ is shown in Figure 2.3C. A significant increase in NO was seen in the CpG encapsulated in Ac-DEX (5 min) group when compared to free CpG (p < 5x10-5 for all except p < 0.05 for 0.4 µg/ml and no significance for 0.2 µg/ml). Blank Ac-DEX (5 min) particles also induced a significantly lower NO release compared to the other groups (p < 5x10-5). Analysis of cytokine release by Mϕ incubated with free or encapsulated CpG is displayed in Figure 2.5. Blank

Ac-DEX (5 min) particles showed significantly lower expression of all cytokines when compared to free CpG (p < 0.005 with the exception of p < 5x10-5 for IL-10 and p < 0.05 for GM-CSF) or CpG encapsulated in Ac-DEX (5 min) (p < 0.005 with the exception of p < 5x10-5 for IL-10 and TNF-α). CpG encapsulated in Ac-DEX (5min) significantly increased IL-2 and GM-CSF in concentrations 6.4, 3.2 and 1.6 µg/ml (p < 0.005). CpG encapsulated in Ac-DEX (5min) significantly increased IL-10, IL-12 (p70) and IL-6 release in all CpG concentrations (p < 0.005 with exceptions to p < 0.05 for 0.2 and

0.4µg/ml IL-6, p < 5x10-5 for 1.6 µg/ml IL-6, p < 0.05 for 6.4 µg/ml IL-10, and p < 0.05 for 0.4 and 6.4 µg/ml IL-12 (p70)). TNF-α was also significantly increased by CpG encapsulated in Ac-DEX (5 min) when compared to free CpG (p < 5x10-5 with exceptions to p < 0.005 for 0.2 and 0.4 µg/ml) (Figure 2.5). Finally, IFN-γ (Figure 2.5) was significantly increased in concentrations 0.8 through 6.4 µg/ml by CpG encapsulated

31 in Ac-DEX (5 min), when compared to free CpG (p < 0.05 with exception to p < 0.005 for 3.2 µg/ml).

2.4 Discussion

Poly I:C was initially encapsulated in either PLGA 85:15 or Ac-DEX with varying molecular weights (10k, 71k, or 150k). Ac-DEX (71k) showed a much greater encapsulation efficiency of poly I:C (Figure 2.1A). For all future encapsulation 71k Ac-

DEX polymer was used. Previous work has been performed on the encapsulation of either poly I:C (119) or CpG (120-125) in microparticles. Traditionally, polycationic material is blended with the polymer to enhance the encapsulation efficiency of CpG or poly I:C. Previous work has incorporated diethylaminoethyl dextran (119), cetrimonium bromide CTAB (120), polyetherimide PEI (121), protamine (122), or chitosan (123, 124) in the microparticle. Fisher et al. were able to encapsulate CpG with the incorporation of both protamine and chitosan, and obtained a loading efficiency of 93%. However, without the incorporation of either polycation they were able to achieve a loading efficiency of only 8% (124). When CpG was encapsulated, without the use of polycations, in Ac-DEX and PLGA, EEs of ~36% and ~3% were obtained, respectively.

As Zhang et al. have reported, CpG may be incorporated into PLGA, without the use of a polycation, at the relatively high loading efficiency of 32%; however, their emulsion technique was different compared to these (125).

32

Figure 2.5: Cytokine Expression from RAW Macrophages Cultured with Free and

Encapsulated CpG.

Data is presented as average ± standard deviation. Statistical significance with respect to free CpG is presented as * p < 0.05, ** p < 0.005, *** p < 5x10-5. 33

The differences included using sonication instead of homogenization, co- encapsulating a protein at 10 wt%, and using a high level of CpG. Can et al. have shown with siRNA that the incorporation of protein drastically increases the encapsulation efficiency of the nucleotides (126). Additionally, sonication has been shown to increase the loading efficiency of gentamicin, a hydrophilic antibiotic (127). In these experiments, commercially available PLGA and Ac-DEX were directly compared, and

Ac-DEX was able to incorporate both poly I:C and CpG more effectively. In addition, these results concluded that by using Ac-DEX there is no need to incorporate polycationic material, which has been shown to significantly increase the toxicity of microparticles (128). Toxicity studies performed here (Figure 2.4) also show Mϕs cultured with both free and encapsulated CpG have increased cell viability over empty particle controls, which is possibly due to the ability of CpG to upregulate proliferation- associated genes in Mϕs (129). Future experiments will have to be performed to explore why Ac-DEX is capable of encapsulating these charged immunostimulants more effectively compared to PLGA.

After encapsulating poly I:C in either 5 min or 4 hr Ac-DEX, or PLGA, the NO release of Mϕs cultured with particles over 24 hours was studied. As seen in Figure 2.1, free poly I:C and poly I:C encapsulated in Ac-DEX (5 min) had similar NO release, while poly I:C encapsulated in PLGA or Ac-DEX (4 hr) had much lower NO release.

Similarly, for cytokine secretion, Ac-DEX (5 min) showed a relatively higher cytokine secretion compared to PLGA and Ac-DEX (4 hr), except for IL-12p70 (Figure 2.2). It was hypothesized that due to the pH sensitivity of Ac-DEX (5 min), there is potential to 34 generate a more efficient release of poly I:C to the lysosome of Mϕs that uptake the particle(s). Since TLR3 is expressed in the lysosome (130), the faster degradation of Ac-

DEX (5 min) could potentially release poly I:C quickly, and increase the efficacy of poly

I:C compared to the other two carriers, which degrade more slowly. Additionally, except for IL-6, poly I:C encapsulated in Ac-DEX (5 min) had a higher level of stimulation compared to free poly I:C. Ac-DEX microparticles have been previously shown to encapsulate the TLR7 agonist, imiquimod (109), so higher levels of stimulation can be generated. This has also been shown in other microparticle formulations (123).

After the initial experiments with poly I:C, the efficacy of encapsulated CpG was examined. Due to the low encapsulation efficiency of PLGA (~3%), Ac-DEX (5 min) was the only polymer used for CpG. For both the NO and cytokine data, CpG encapsulated in Ac-DEX was superior to free CpG. As stated previously, the encapsulated CpG and poly I:C are better at stimulating immune cells as compared to free

CpG probably due to the phagocytic properties of the Mϕ. It was previously shown that encapsulated cargo can be internalized preferentially compared to the non-encapsulated form (117). By using Ac-DEX as a carrier, CpG can be incorporated into a microparticle without the use of toxic polycations. The lack of these polycations would potentially decrease or prevent the toxicity of the particles both in vitro and in vivo. In the long term these particles could also incorporate protein for vaccine applications. It has been shown that the encapsulation of CpG with an antigen in a microparticle can enhance the in vivo response against the encapsulated antigen (115, 131). Additionally, the incorporation of

35

CpG in Ac-DEX particles could also be used for immunotherapy applications such as the treatment of cancer (132) or parasitic diseases (111, 133).

2.5 Conclusions

This study shows the EE of poly I:C in Ac-DEX was increased over that of PLGA and this result was also observed for CpG encapsulated in Ac-DEX. One reason this study may not have shown high encapsulation of CpG in PLGA is because a polycation was not incorporated, and these compounds are frequently used to aid in the encapsulation of CpG in PLGA (119, 124). Concurrent with increased encapsulation, poly I:C encapsulated in Ac-DEX is capable of eliciting similar or increased production of immunostimulatory cytokines as poly I:C encapsulated in the FDA-approved polymer

PLGA. Due to a low encapsulation of CpG in PLGA, it was not tested for immunostimulatory responses, however, CpG encapsulated in Ac-DEX has a significant increase in cytokine elicitation when compared to administered free CpG. The ability for

Ac-DEX to encapsulate these adjuvants without the use of potentially cytotoxic polycationic materials while eliciting a higher immune response supports the claim that

Ac-DEX is a viable candidate for the delivery of the TLR-stimulators. Ac-DEX also degrades into biocompatible, non-acidic byproducts, unlike PLGA, making the environment more conducive to the delivery of potentially acid-sensitive cargo such as proteins with antigenic epitopes. Increased immune responses and decreased damage to antigenic cargo mean Ac-DEX is potentially a viable candidate for use in the production

36 of sub-unit vaccines or for immunotherapy against infections like Leishmaniasis.

Subsequent to this work, Duong et al. showed the potential of resiquimod encapsulated in

Ac-DEX to limit the parasite burden of Leishmania (111). However, this delivery platform had limited efficacy in decreasing parasite burden, so further delivery methods should be established.

37

Chapter 3: Liposomal Resiquimod for the Treatment of Leishmania donovani Infection

3.1 Introduction

There are over 30 species of Leishmania which are the causative agent of the zoonotic disease, Leishmaniasis (134). Approximately 12 million people are afflicted with Leishmaniasis worldwide, which results in approximately 50,000 deaths a year, a death-toll for parasitic infection which is only surpassed by malaria (24). Depending on which subspecies infects the patient, the disease can form CL (Leishmania major,

Leishmania tropica and Leishmania Mexicana), ML (Leishmania braziliensis), or VL

(Leishmania donovani “L. donovani” and Leishmania infantum) (135, 136). The most deadly form of the disease is the visceral form. In VL (Kala-azar), L. donovani are transmitted by phlebotomine sand flies (137). Upon introduction into the mammalian host, L. donovani is usually internalized by Mϕs in the dermis, whereupon they lose their flagella and become amastigotes. L. donovani is capable of surviving in the Mϕ’s phagolysosome. It proliferates and eventually spreads through the vascular and lymphatic systems. A high proportion of the parasite will eventually reside in the liver, spleen and bone marrow. If left untreated, VL usually results in death.

38

For the last 70 years, the most common VL treatment is the systemic injection of antimonials such as sodium stibogluconate (SSG) or meglumine antimoniate formulations. Antimonials are highly toxic and have severe adverse side effects including pancreatitis and cardiac arrhythmia (29-31). Additionally, some strains of L. donovani have become resistant to antimonial therapies demanding the development of other VL chemotherapeutics. These therapies include the intravenous injection of liposomal Amphotericin B (AmBisome®) or orally administered Miltefosine (Impavido,

Miltex). Amphoterecin B is highly toxic, requiring liposomal encapsulation for parenteral administration. Moreover, with an increase in Amphotericin B use, there has been an emergence of Leishmania drug-resistance (38). Another therapy, Miltefosine, has been shown in a clinical trial to have an 82% cure rate (138). However, Miltefosine is teratogenic, which prevents its use by pregnant or potentially pregnant women.

Additionally, similar to Amphotericin B, there are now Miltefosine resistant Leishmania strains (39). Due to increased drug resistance and limited application of current therapies, there is a need to develop alternative treatments for VL.

One alternative to conventional therapies, that primarily target the pathogen, is to target the host-pathogen interface (139). By enhancing the host immune response, the pathogen can be effectively cleared while decreasing the potential for development of drug resistance. This strategy has been employed not only for treating typhoid fever

(140), tuberculosis (141), and fungal infections (142), but also Leishmaniasis. Prior research has shown that (143), non-methylated DNA (96), lipid A (144), and other immune adjuvants (145) have been successful in treating Leishmania

39 infections. In particular, imiquimod, a FDA approved toll-like receptor (TLR) 7/8 agonist has been successful in clinical trials for CL (45, 47). Additionally, resiquimod

(Figure 3.1), an imiquimod derivative and a FDA approved molecule for topical delivery, has also shown promise in treating CL infections (46). Both imidazoquolines induce

IFN-α, IL-1β, IL-6 and TNF-α in Mϕs and monocytes (46). Buates et al. (146) have shown that resiquimod decreases the amount of intracellular Leishmania by inducing the production of nitric oxide, yet resiquimod has no direct effect on the parasite. Recently,

Duong et al encapsulated resiquimod in Ac-DEX particles because the hydrophobic nature of resiquimod limits its parental use in vivo (147). The data presented in this manuscript expands on this work. Here, resiquimod is encapsulated in a liposomal carrier for treatment of visceral L. donovani infection. Toxicity of the formulation was measured through a cell viability assay. Also, liposomal resiquimod efficacy was tested in a L. donovani infected murine model by quantification of parasites in infected organs, cytokine responses and quantification of liver enzymes.

3.2 Materials and Methods

3.2.1. Materials

All reagents were purchased and used unmodified from Sigma-Aldrich (St. Louis,

MO, United States), unless otherwise noted. All lipids membranes used for liposome formulation were purchased from Avanti Polar Lipids (Alabaster, Alabama, United

40

Figure 3. 1 Chemical structure of resiquimod.

41

States). Water (dd-H2O) was purified using a Millipore (Billerica, MA, United States)

Milli-Q Integral water purification system. Fluorescence and absorbance measurements were obtained on a Molecular Devices FlexStation 3 (Sunnyvale, CA, United States), courtesy of the Department of Chemistry and Biochemistry at the Ohio State

University,United States. All reagents used for cytokine detection by ELISA were purchased from BioLegend Inc. (San Diego, CA, United States).

3.2.2. Animals

Six to eight week old BALB/c mice were purchased from Jackson Labs (Bar

Harbor, ME, United States). Six to eight week old Syrian Golden Hamsters were purchased from Harlan Labs (Indianapolis, IN, United States). All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of The Ohio

State University, United States.

3.2.3. Preparation of Empty or Resiquimod Loaded Liposomes

Liposomes were formulated by addition of chloroform and methanol (9:1 v/v) to a mixture of hydrogenated (soy)L-a-phosphatidylcholine, 1,2-distearoyl-sn-glycero-3-

[phospho-rac-(1-glycerol)], cholesterol (Ovine Wool), D-alpha-tocopherol (Acros

42

Organics, Morris Plains, NJ, United States), either with or without resiquimod (Enzo Life

Sciences, Farmingdale, NY, United States). The lipid solution was rotary evaporated with a Buchi R-200 rotary evaporator (New Castle, DE, United States) with a Buchi B-

490 water bath set at 60o C. Lipids were reconstituted in dd-H2O for 30 minutes in a 60o

C water bath. Liposomes were freeze-thawed 3 times, followed by extrusion through an

Avanti Mini-Extruder/Heating Block with an 80 nm polycarbonate membrane and filter supports (Alabaster, Al, United States), 11 times before passage through a disposable PD-

10 column (GE Healthcare, Pataskala, OH, United States). Sucrose was added to the liposomes (150 wt %) followed by lyophilization.

3.2.4. Determining Size and Encapsulated Resiquimod in Liposomes

Liposome size was determined by dynamic light scatter (DLS). Liposomes were suspended in dd-H2O and sizing was performed using a NiComp Submicron Particle

Sizer Model 370 (Santa Barbara, CA, United States) courtesy of Dr. Robert Lee, College of Pharmacy, Ohio State University, United States. Encapsulation of resiquimod was determined by dissolving three sets of empty and resiquimod-loaded liposomes in ethanol

(1 mg/mL) and placing the solution into wells of a solvent resistant 96-well plate in triplicate. A calibration curve containing resiquimod dissolved in ethanol was added to the plate. The plate was read at Excitation: 260 nm and Emission: 360 nm. Fluorescence readings for empty liposomes were subtracted from those of resiquimod-loaded

43 liposomes and encapsulation was determined by fluorescence readout comparison to the calibration curve

3.2.5. Infection and Treatment Protocol

L. donovani (Lv82 strain) were grown and maintained in Syrian Golden

Hamsters. BALB/c mice were infected with 1 x 107 amastigotes by tail vein injection.

Mice were treated 2 weeks post infection with a 100 µl tail vein injections of either empty liposomes, resiquimod loaded liposomes (7.6 µg resiquimod/mouse), or SSG

(500mg/kg) (Albert David Ltd, Kolkata India). Mice were sacrificed and analyzed 2 weeks post injection.

3.2.6. Histology and LDU (L. donovani units) in Treated Mice

Parasite loads in the spleen and liver were quantified as previously described

(148). Briefly, the liver and spleen smears were stained using Giemsa (Fisher Scientific,

Waltham, MA, United States) and parasite loads were quantified microscopically. LDU was calculated as the number of amastigotes per 500 nuclei multiplied by the weight (mg) of the liver or spleen. Bone marrow parasite load counts were performed as previously described (149). Briefly, bone marrow was removed from the femurs of BALB/c mice.

Smears of the bone marrow were stained with Giemsa, and bone marrow LDU was quantified as with spleen and liver. Sections of the liver and spleen were fixed in 10% formalin for 24 hours and submitted to the Histology Core at The Ohio State University 44 for Hametoxylin and Eoisin (H&E) staining and further histopathology. Tissue sections from the organs of L. donovani-infected mice were stained using H&E and subjected to histopathology by a pathologist. Liver granulomas were enumerated and scored as follows: 1) no reaction; 2) developing; 3) mature; and 4) empty. Samples were imaged using an Olympus BX41 light microscope (Olympus, Center Valley, PA, United States).

3.2.7. T cell Proliferation and Cytokine ELISA

T cell proliferation was performed as previously described (150). Briefly, 5 × 105 splenic cells were added in quadruplicate to the wells of sterile 96-well, flat-bottom tissue culture plates and stimulated with freeze-thawed L. donovani antigen (20 μg/mL). The proliferation responses were measured by an Alamar blue assay, as previously described

(151). Supernatants were collected after 72 h of incubation at 37 oC and analyzed for the production of IFN-γ and IL-10.

3.2.8. Measurement of Liver Aminotransferase Enzymes

Blood was collected in non-heparinized tubes and allowed to clot overnight at 4 oC. Serum was obtained and submitted to the Hematology Lab of The Ohio State

University Veterinary Hospital for analysis of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) using the Cobas C501 serum analyzer (Roche,

Indianapolis, IN, United States).

45

3.2.9. Cell Viability

Mϕs (RAW 264.7; ATCC, Manassas, VA, United States) were grown and maintained according to the manufacturer’s guidelines. Dulbecco’s Modified Eagle’s

Medium (500 mL; Fischer, Pittsburgh, PA) was supplemented with fetal bovine serum

(50 mL; Hyclone, Pittsburgh, PA, United States), and penicillin-streptomycin (10 mL;

Fischer, Pittsburgh, PA, United States). Mϕs were seeded on a 96-well plate at a concentration of 5 x 104 cells/well and allowed to become confluent. After the cells adhered, media was removed and replaced with media containing free resiquimod, liposomes loaded with resiquimod (0.312% wt/wt), empty liposomes or media alone.

Cell viability was determined after 24 h using 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Briefly, media in each well was replaced with fresh media (150 µl) and MTT solution (5 mg/mL, 20 μL). The plate was incubated until purple formazan crystals formed. Crystals were dissolved in isopropanol.

Absorbance was measured at 560 nm and a background reading was measured at 670 nm.

Viability of cells treated with resiquimod, liposomal resiquimod or empty liposomes were compared to untreated cells.

46

3.3 Results

3.3.1. Liposomal Preparation

Liposomes were formed by lipid film hydration with extrusion. As measured by dynamic light scattering, the resiquimod liposomes were found to be 75.0  30.7 nm in diameter. After extrusion, liposomes were placed in a PD-10 size exclusion column to remove any non-encapsulated resiquimod. Then the liposomes were lyophilized with a cryoprotectant (sucrose). Liposome yield was 60.4%, while the encapsulation efficiency of resiquimod was 7.0% resulting in a weight loading of 1.1% drug per weight liposome.

3.3.2 Treatment of L. donovani Infection

BALB/c mice were infected with L. donovani, and treated two weeks post infection with either empty liposomes, resiquimod loaded liposomes, or SSG, as a positive control. Mice were sacrificed and analyzed two weeks post injection for L. donovani load. Figure 3.2B shows the spleen LDU of mice treated with liposomal resiquimod is lower than those treated with empty liposomes (p-value0.01). Mice treated with SSG, the current standard of treatment, have lower LDUs than both empty liposomes (p-value0.0005) and liposomes encapsulating resiquimod (p-value0.003).

In Figure 3.2A, it can be noted that the liver LDU of mice treated with liposomal resiquimod is lower than those given empty liposomes (p-value0.05). Mice treated with

47

SSG have lower LDUs than both empty liposomes (p-value0.005) and resiquimod liposomes (p-value0.03). In Figure 3.2C, the bone marrow counts of amastigotes per

200 Mϕs are lower in mice receiving liposomal resiquimod than empty liposomes (p- value0.05) and bone marrow amastigote counts are significantly lower in mice treated with SSG when compared to empty liposomes (p-value0.005) or resiquimod liposomes

(p-value0.03).

3.3.3. T cell Proliferation and Cytokines

Splenocytes were isolated and cultured from sacrificed BALB/c mice infected with L. donovani. The cells were pulsed with L. donovani antigen and proliferation was measured after 72 hours (Figure 3.3). Mice treated with encapsulated resiquimod showed a higher level of proliferation compared to empty liposomes (p-value.01), while those treated with the positive control SSG, had a higher proliferation rate compared to empty liposomes (p-value0.0005) and were close to having significantly higher proliferation when compared to resiquimod liposomes (p-value0.06). IFN- levels in mice treated with resiquimod liposomes were significantly higher when compared to those treated with both empty liposomes (p-value0.00005) and SSG (p-value0.00005). Mice receiving resiquimod liposomes also had statistically significant increases in IL-10 production when compared to both empty liposomes (p-value0.0005) and SSG (p- value0.005) (Figure 3.4).

48

Figure 3. 2: L. donovani units (LDU) in the (a) Liver (b) Spleen (c) and Bone Marrow of

BALB/C Mice Infected with the Parasite.

Mice were treated 2 weeks post infection with tail vein injections of either empty liposomes, resiquimod (7.6 µg) loaded liposomes or 500 mg/kg SSG. LDU was determined 2 weeks post injection using number of amastigotes per 500 nuclei. Data are presented as mean with 95% CI. (n = 5 for empty or resiquimod loaded liposomes and n

= 4 for SSG).

49

Figure 3. 3: Proliferation of T cells Isolated from the Spleens of BALB/C Mice Infected with L. donovani and Exposed to Antigen.

Mice were treated 2 weeks post infection with either empty liposomes, resiquimod (7.6

µg/mL) loaded liposomes, or 500 mg/kg sodium SSG. Spleens were removed 2 weeks post injection and pulsed with L. donovani antigen (20 µg/mL) and T cell proliferation was measured after 72 hours. Data are presented as mean with 95% CI. (n = 5 for empty or resiquimod loaded liposomes and n = 4 for SSG).

50

Figure 3. 4: Production of (a) IFN-γ or (b) IL-10 from Splenic Cells Isolated from

BALB/C Mice Infected with L. donovani.

Mice were treated 2 weeks post infection with either empty liposomes, resiquimod (7.6

µg/mL) loaded liposomes, or 500 mg/kg SSG. Spleens were removed 2 weeks post injection and pulsed with L. donovani antigen (20 µg/mL) and cytokine production was measured after 72 hours. Data are presented as mean with 95% CI. (n = 5 for empty or resiquimod loaded liposomes and n = 4 for SSG).

51

3.3.4. In vitro Viability, Histology and Liver Enzyme Activity

RAW Mϕs were cultured with varying doses of resiquimod ranging from 0.04 to

5 μg/mL (Figure 3.5). The viability of Mϕs cultured with free resiquimod or liposomal resiquimod was not significantly different. Free resiquimod at 5 µg/mL show increased viability over empty liposomes (p-value0.05). Mϕs cultured with liposomal resiquimod showed increased viability compared to empty liposomes at the two lowest concentrations, 0.07 and 0.03 µg/mL (p-value0.05). Analysis of the liver enzymes ALT shows significantly lower levels of ALT in both the liposomal resiquimod (p-value0.05) and SSG groups (p-value0.05); however there was no significant difference in AST levels among any of the groups. Additionally, histology was performed on the liver and spleen and no visible differences in inflammation or toxicity were detected in mice treated with empty liposomes, and liposomes encapsulating resiquimod (data not shown).

52

Figure 3. 5: Macrophage Viability after being Cultured with Free Resiquimod,

Resiquimod Loaded Liposomes or Empty Liposomes.

Cells were seeded at 5x104 cells per well and treated for 24 hours before viability was measured with respect to untreated cells. Data are presented ± standard deviation.

Statistical significance with respect to free resiquimod is presented as * p < 0.05 and significance with respect to empty liposomes is presented as † p < 0.05.

53

3.4 Discussion

Immunotherapy is a potential method that could be employed for the treatment of

VL. Previous research has shown that TLR agonists such as CpG can be a possible treatment for VL infections (96). Here, the use of resiquimod, a TLR7/8 agonist, is proposed for the treatment of VL. Previous research has shown that a similar compound, imiquimod, is successful in treating CL (152, 153). Unfortunately, for systemic administration, both imiquimod and resiquimod are highly insoluble in an aqueous environment. It was hypothesized that liposomal resiquimod would allow for intravenous injection of the TLR 7/8 agonist for treatment of VL. This approach has been successful in the formulation of Amphotericin B, a hydrophobic drug that is encapsulated in a liposome (Ambisome®). Liposomal Amphotericin B has shown decreased drug toxicity and facilitates systemic delivery of the compound (154). Here, a formulation similar to that used for Ambisome®, was used and encapsulated resiquimod in liposomes using lipid film hydration. The lipids were sized via extrusion to form particles approximately

~80 nm in diameter, which is roughly the same size as Ambisome® (155). This small liposomal size allows for longer circulation time and substantial penetration into tissues such as the liver and spleen, which are tissue sites where Leishmania resides.

Additionally, resiquimod-loaded liposomes would likely be approved for clinical use due to the formulation similarity between these liposomes and those used in the FDA- approved Ambisome, as well as the FDA-approved status of resiquimod. Duong et al. formulated resiquimod for in vivo applications used electrosprayed microparticles that

54 are roughly 1 μm in diameter (111). Unfortunately, it would be difficult using electrospray technology, or emulsion chemistry to make particles roughly the same size as Ambisome®. Prior research has shown that liposomal formulations larger than 400nm have selective uptake in the bone marrow (156), which can act as an important reservoir for the parasite (157) and may contribute VL relapses after treatment is administered.

Future work will study the size of liposomal formulations on optimizing resiquimod concentration in the liver, spleen, and bone marrow.

For this study, liposomal resiquimod was intravenously injected into mice two weeks post infection. These results indicate that encapsulated resiquimod treatment significantly decreased the number of L. donovani in the liver, spleen, and bone marrow of infected mice compared to empty liposomes. As a positive control, a very high dose

(500 mg/kg) of SSG (traditional dose < 300 mg/kg) was used. By developing an immunotherapy that targets the host interface, the formation of drug resistant strains can be circumvented. This is significant, considering that in places like the Bihar State, India, it has been reported that over 60% of patients with VL do not respond to antimonial treatments. Sane et al. have shown that by treating VL with both CpG and miltefosine, there was a synergetic effect with respect to decreased Leishmania load (96). Future experiments that co-deliver both Amphotericin B and resiquimod, in liposomal forms, for the treatment of drug resistant Leishmaniasis might lead to even a more significant reduction in parasite load.

To further characterize treatment with liposomal resiquimod, spleens from treated mice were cultured with Leishmania antigen and measured for proliferation and cytokine

55 production. In comparing the proliferation of splenocytes from resiquimod treated mice to those given empty liposomes, a slightly higher proliferation was observed, but this difference was not seen between splenocytes from SSG treated mice. For production of

IFN-, splenocytes from mice treated with resiquimod liposomes had a significant increase in production compared to those treated with empty liposomes or SSG. Prior research has shown that systemic treatment with resiquimod enhances the production of

IFN-, which prevented and treated a TH2 allergy response (158). Since liposomal resiquimod treatment induced a high level of IFN-, it was hypothesized that this, in part, is how resiquimod decreased Leishmania load. IFN- has been shown to be essential in clearing VL infections since it has been reported that IFN- knockout mice are highly susceptible to infections (159). Additionally, Scott et al. have shown that mouse strains resistant to VL infection predominately make a substantial amount of IFN-, compared to susceptible mouse strains (160). On the other hand, treatment with encapsulated resiquimod induced a high level of IL-10 in splenocytes isolated from treated mice. Prior research has shown that resiquimod can simultaneously induce both IFN- and IL-10 production (161-163). While Boghdadi et al. have shown that peripheral blood mononuclear cells from patients infected with Schistosoma mansoni make high levels of both IFN- and IL-10 when stimulated with resiquimod (164). IL-10 has been linked to increased pathogenesis of VL infection since IL-10 knockout mice are highly resistant to infections in both C57BL/6 and BALB/C strains (165). However, prior research has also shown that CpG up-regulates IL-10 production in monocytes similar to resiquimod (166,

167). Overall, it seems even though treatment with resiquimod liposomes induced IL-10 56 in mice, the parasite load was still decreased, potentially due to the large up-regulation of

IFN-γ.

Prior research has shown that resiquimod is a highly effective adjuvant in stimulating immune cells, with saturating responses in vitro at ~0.1 μg/mL. When cultured with liposomal resiquimod, RAW Mϕs showed no difference in cell viability up to 5 μg/mL. Therefore it was concluded, at the concentrations necessary to stimulate host cells, resiquimod does not affect the viability of Mϕs. Schön et al. have shown that imiquimod, but not resiquimod, induces apoptosis by the activation of caspases and Bcl-

2-dependent translocation of cytochrome c (168). Oral resiquimod has been used in a

Phase IIa clinical trial for Hepatitis C treatment and no toxicity was shown in the clinical trial with only two patients quitting the clinical trial due to flu like symptoms (51).

Histology studies showed that there were no differences in inflammation and toxicity in mice, when comparing animals treated with empty liposomes to liposomal resiquimod.

Elevated liver enzymes, such as ALT and AST, are indicative of disease and represent possible damage to the liver (169). Although increased levels of these enzymes may represent drug compounds acting on parasites in the liver, it is generally accepted that lowered liver enzyme levels in the serum is indicative of diminished disease (170). The liposomal resiquimod formulation showed equal levels of ALT compared to those treated with SSG and significantly lowered levels compared to those treated with empty liposome. The ALT levels seen in mice treated with SSG and liposomal resiquimod were considered in the normal range for a mouse (171) however, all groups showed higher than normal AST levels (171) and samples from mice treated with SSG or liposomal

57 resiquimod did not differ from empty liposome treatment. Additional studies will be performed to determine if the increased levels of AST seen in treated mice is indicative of progression or diminishment of disease. Interestingly though, the liposomal resiquimod followed the same pattern of liver enzymes as the current standard for treatment, SSG.

3.5 Conclusions

Overall, liposomal resiquimod was shown as a potential treatment for VL.

Treatment with liposomal resiquimod lowered the parasite load of Leishmania in the liver, spleen, and bone marrow of L. donovani infected mice. Furthermore, resiquimod induced the production of IFN- and IL-10 in the splenocytes isolated from infected mice, with antigen recall. In vitro testing reported no noticeable toxicity and the liver enzyme ALT was decreased in mice treated with both resiquimod liposomes and SSG compared to mice treated with empty liposomes. The enzyme levels show decreased liver damage, likely due to lowered parasite levels, and the similar levels seen in mice treated with resiquimod liposomes and SSG indicate the liposomal resiquimod formulation has low in vivo liver toxicity. Future work will be performed to explore in- depth the systemic cytokines that are released during liposomal resiquimod treatment.

58

Chapter 4: Liposomal Synthetic Pentalinonsterol for the Treatment of Leishmania

donovani Infection

4.1 Introduction

Visceral Leishmaniasis is caused by the obligate intracellular parasites

Leishmania donovani, L. infantum, L. chagasi, L. amazonensis, or L. tropica via transmission by a sand fly vector. Half a million people are infected with VL, and over

60,000 succumb to disease annually. The WHO classifies VL as a neglected tropical disease of global health concern. As a potentially life-threatening disease, VL is characterized by parasitic invasion of the blood and reticulo-endothelial system, which affects internal organs like the spleen, liver, and bone marrow (24). Moreover, the disease’s zoonotic potential has been recently escalating in canines in the US. There are currently no licensed vaccines, and chemotherapy is the mainstay to combat the disease.

Generally, treatments utilize antileishmanial drugs such as SSG, AmpB, liposomal

AmpB, miltefosine and others (172). All these drugs suffer from significant drawbacks, including the need for parenteral routes of administration, poor patient compliance due to long treatment lengths and toxicity, and/or high cost, which limits their use in disease endemic regions. For more than 50 years, the most common treatment has been

59 antimony, which has potentially cardiotoxic side effects (173). Additionally, AmpB has been associated with nephrotoxicity (174). The only promising oral treatment, miltefosine

(Impavido, Miltex), is frequently unusable due to its potential teratogenicity (37) and low tolerable doses (36) that are exacerbated by a long half-life. Other anti-leishmanial treatments that are currently under clinical development do not offer new alternatives to patients because they are either reformulations of current antileishmanial drugs, combination therapies, or the result of therapeutic switching. In addition, the emergence of antimonial-resistant strains of VL is rapidly increasing worldwide. Areas like Bihar,

India are confronting an epidemic of antimony-resistant VL infections (close to 70% of cases), likely due to poor patient compliance (33). Additionally, drug resistance has been reported with AmpB (38) and miltefosine (39). Due to emergence of drug resistant parasites, high drug toxicity and lower patient compliance due to treatment costs (35,

175-177), there is an immediate requirement for safe, cost effective, and reliable drugs which can successfully treat VL.

Natural products derived from plants have long proved to be an invaluable resource for antiparasitic drugs and is considered a priority by the WHO (178, 179).

Anti-malarial drugs such as quinine and artemisinin are plant-derived. Also, plants in the families’ Apocynaceae, Araceae, Cycadaceae, Fabaceae, Piperaceae, Solanaceae and

Sapindaceae are commonly used for CL treatment in South America (12-15). The root of

Pentalinon andrieuxii has been used for years by Mayan traditional healers and has shown to exhibit a wide range of biological activity including antileishmanial properties

(180, 181). Recently, the in vitro antileishmanial activity in both the aqueous and organic

60 extracts from the roots of P. andrieuxii has been shown (180). Additionally, pentalinosterol (PEN), a new cholesterol derivative from n-hexane partition of the methanol extracts of the roots of P. andrieuxii, has been isolated (182). PEN exhibited potent antileishmanial activity against L. mexicana promastigotes and intracellular amastigotes (182). Preliminary studies by Dr. Narasimham Parinandi suggest PEN inhibits parasite growth through inhibition of fatty acid synthesis. Further studies exploring the use of PEN to treat in vivo models of VL are desired. However, due to the hydrophobicity of the active extract, more optimized methods for parenteral delivery of this compound are required for the use against VL.

In this study, a novel synthetic form of PEN (sPEN) was utilized to evaluate the therapeutic efficacy of sPEN in both in vitro and in vivo models of VL. To address solubility concerns, in vivo evaluation was performed using a formulation of liposomal sPEN, and the host immune response was evaluated to determine potential host mechanisms of parasite clearance upon L. donovani infection.

4.2 Materials and Methods

4.2.1 Materials

All reagents were purchased from Sigma-Aldrich, and used as purchased, unless otherwise noted. All lipids, membranes, and extruders used for liposome formulation

61 were purchased from Avanti Polar Lipids. All animals used for experiments were purchased from Harlan Laboratories.

4.2.2 Preparation of Empty or Synthetic Pentalinonsterol Loaded Liposomes

Hydrogenated (Soy) L-a-Phosphatidylcholine, 1,2-Distearoyl-sn-Glycero-3-

[Phospho-rac-(1-glycerol)], Cholesterol (Ovine Wool), and D-alpha-Tocopherol (Acros

Organics), with or without sPEN (provided by Dr. Jim Fuchs, The Ohio State University

College of Pharmacy), were placed into a solution of chloroform and methanol (9:1 v/v).

The solution was rotary evaporated with a Buchi R-200 (New Castle, DE, United States) rotary evaporator and Buchi B-490 water bath, courtesy of Dr. Karl Werbovetz, College of Pharmacy, Ohio State University, set at 60oC to make a lipid film. The lipid film was

o reconstituted in dd-H2O for 30 minutes in a 60 C water bath. Liposomes were freeze thawed 3 times, followed by extrusion through an Avanti Mini-Extruder/Heating Block with an 80 nm polycarbonate membrane and filter supports, 11 times before passage through a disposable PD-10 column (GE Healthcare). Sucrose was added to the liposomes (150 wt%) followed by lyophilization.

4.2.3 Determining Size and Encapsulated Synthetic Pentalinonsterol in Liposomes

Liposomes were suspended in dd-H2O and sizing was determined using an

NICOMP Submicron Particle Sizer Model 370 (Santa Barbara, CA, United States) courtesy of Dr. Robert Lee, College of Pharmacy, Ohio State University. Liposomes containing sPEN were determined to be 116.9 nm. Encapsulation efficiency of sPEN in

62 liposomes was 99%, determined by HPLC (Waters, Milford, Massachusetts) in a method adapted from Pan et. al (182). Briefly, liposomes were dissolved in a methanol:ddH2O solution (95:5 v/v) (1 mg/ml) and sPEN concentration was determined using a 150 mm x

4.6 mm, pore size 5 µm, C18 column. Samples were passed through the column at a flow rate of 1 ml min-1 and read at a wavelength of 240 nm and had retention time of 22 minutes.

4.2.4 Animals and Parasites

Syrian gold hamsters (6-8 weeks old) were used to maintain L. donovani (LV 82).

L. donovani (LV82) expressing dsRed were grown and maintained in stat4-/- BALB/c mice (6-8 week old; females). All animals were kept in a sterile facility according to The

Ohio State University Institutional Guidelines. All animal procedures were in accordance with and approved by the Institutional Animal Care and Use Committee (IACUC) of The

Ohio State University.

4.2.5 Promastigote Assay

Log phase dsRed expressing L. donovani (LV82) promastigotes (1x106) were seeded in 1 mL of complete RPMI-1640 medium. Parasite numbers, their mobility and morphology were measured at 48 hours by flow cytometry measurements using

63 experimental and control groups of parasites and with Quillaja saponaria saponin as positive control (183).

4.2.6 Amastigote Assay

Bone marrow-derived macrophages (BMMϕ) (0.5x106) were plated to adherence on round glass coverslips in a 24-well tissue culture plate. Adherent Mϕs were infected overnight with 2.5x106 stationary phase promastigotes of L. donovani parasites (1:5 Mϕs to parasites). Following infection, cells were washed with Hank’s balanced salt solution

(HBSS) and cultured for 48 hours with sPEN. Finally, BMMϕs were stained with Giemsa and infection rates were determined blinded by the number of parasites per 100 macrophages, in triplicate (184).

4.2.7 Cytotoxicity Assay in Non-Infected Macrophages

BMMϕs (0.5x106) from BALB/c mice were cultured in a 96-well plate for 48 hours with sPEN or controls. Following 42 hours, 10% amalar blue was added to each well and the total number of live cells was determined (185).

4.2.8 In Vivo Infection and Treatment Protocol

BALB/c mice were infected with 1 x 107 amastigotes by tail vein injection (i.v.). Two weeks post-infection, mice were treated i.v. with 100 µl PBS, empty liposomes,

64 liposomal sPEN (50 µg sPEN/mouse), or sodium stibogluconate (SSG) (70mg/kg)

(Albert David Ltd, Kolkata India).

4.2.9 Parasite Burden Calculation

Two weeks post-treatment, mice were euthanized and their livers and spleens were harvested, weighed and sectioned to prepare impression smears. Smears were stained with Giemsa to tally the number of parasite load, which were expressed as Leishmania-

Donovan Units (LDU) = Number of amastigotes per 1000 nucleated cells x organ weight

(grams). Additionally, bone marrow parasite counts were performed as previously described (149). Briefly, bone marrow was removed from the femurs of BALB/c mice, smears taken, stained with Giemsa, and total parasite load calculated.

4.2.10 Histopathology

Two weeks post-treatment, tissue sections from the liver of L. donovani-infected mice were stained using H&E stain and histopathological examinations were performed. Liver granulomas were tallied as follows: 1) no reaction; 2) developing; 3) mature; and 4) empty (148). Liver granuloma totals are representative of the average of 4–5 individual mice.

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4.2.11 T cell Proliferation and Cytokine ELISA

T cell proliferation was performed as previously described (150). Briefly, 5 × 105 splenic cells isolated two weeks post-treatment were added to a 96 well flat bottom plate.

An antigen recall assay was performed with L. donovani antigen (20 μg/ml) and proliferation were measured using Alamar blue assay as previously described (151)

Additionally, supernatants were collected at 72 hours of and analyzed for the production of IFN-γ, IL-12p70, IL-13, IL-4, and IL-10 by ELISA (BD Pharmingen).

4.2.12 Quantification of Transcript Levels by RT-PCR

Total RNA was extracted from 50 mg of spleen tissue using TRIZOL Reagent. mRNA was reverse transcribed and cDNA was amplified by real-time PCR as described previously (186). Primers and reaction conditions were obtained from the PRIMER

BANK website (Massachusetts General Hospital. Primer Bank (187-189)). Data were normalized to the housekeeping gene β-actin and presented as fold induction over infected wild-type mice using the delta-delta CT method.

4.2.13 Measurement of Liver Aminotransferase Enzymes

Blood was collected in non-heparinized tubes and allowed to clot overnight at 4 ⁰C.

66

The Hematology Lab of The Ohio State University Veterinary Hospital analyzed serum

ALT and AST using the Cobas C501 serum analyzer (Roche).

4.2.14 Statistical Analysis

Student unpaired t tests were used to determine statistical significance of differences in the values. A value of p < 0.05 was considered significant. The statistical significance of IFN-γ: IL-4 and IFN-γ: IL-10 ratios were determined by non-parametric tests using

Mann-Whitney U- test. Statistical significance among controls and treatments was established by one-way ANOVA with the p value set at ≤ 0.05.

4.3 Results

4.3.1 Antileishmanial Activity of Synthetic Pentalinonsterol against Leishmania donovani

Promastigotes and Intracellular Amastigotes

sPEN was evaluated at different concentrations ranging from 0, 1, 10, 25, 50 and

100µg/mL for its antileishmanial activity against L. donovani promastigotes and intracellular amastigotes. Results show that sPEN treatment of L. donovani promastigotes for 48 hours mediated significant killing which corresponded to nearly 12%, 58% and

90% killing at 25, 50 and 100µg/mL, respectively (p < 0.001) (Figure 4.1A). Similarly, sPEN treatment for 48 hours showed a dose dependent clearance of L. donovani

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Figure 4. 1: In Vitro Antileishmanial Activity of Synthetic Pentalinonsterol.

(A) The percentage killing of dsRed expressing L. donovani promastiogotes following incubation with different doses of sPEN at 48h post incubation was calculated by flow cytometry. (B) The percentage of viable macrophages following treatment with different doses of sPEN at 48h post incubation was calculated by the addition of amalar blue. (C)

The number of intracellular L. donovani amastigotes, following treatment with indicated doses of sPEN at 48h post incubation, was calculated by microscopic counting of the infected macrophages. Data are shown as mean ±SEM of three replicates for each treatment and is a representative of three individual experiments. Statistical significance with respect to DMSO treated cells is presented as ** p < 0.01 and *** p < 0.001.

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Figure 4. 2: Liposomal Synthetic Pentalinonsterol Treatment Renders Protection against

L. donovani Infected Mice.

(A) Liver (B) spleen and (C) bone marrow parasite loads in L. donovani infected BALB/c mice treated with either PBS, empty liposomes, sPEN loaded liposomes (50 µg or 2.5 mg per kg body weight), or sodium stibogluconate SSG (70 mg per kg body weight). Parasite burdens in spleen of and liver were expressed as mean LDU; parasite burden in bone marrow was expressed as number of amastigotes per 200 macrophages. Data is presented as mean ± SE. These data are mean values from four or five individual mice per group at each time point in three independent experiments with similar results. Significance is presented as *** p < 0.001.

69

Figure 4. 3: Effect of Liposomal Synthetic Pentalinonsterol Treatment on Serum

Aminotransferase Enzymes in L. donovani Infected Mice.

Quantification of the liver enzymes (A) Alanine Aminotransferase (ALT) and (B)

Aspartate Aminotransferase (AST) in the serum of mice infected with Leishmania donovani. Mice were treated 2 weeks post infection with either empty liposomes or sPEN loaded liposomes (50 µg or 2.5 mg per kg body weight). Blood was collected 2 weeks post injection for analysis of serum enzymes. Data shown are the mean ± SE of triplicates from four or five individual mice per group and are representative of three individual experiments. Significance with respect to empty liposomes is presented as *p < 0.05.

70 amastigotes from infected Mϕs which corresponded to nearly 40%, 50%, 55%, 70% and

80% intracellular amastigote clearance at 1, 10, 25, 50 and 100µg/mL, respectively (p <

0.01) (Figure 4.1C). None of the concentrations of sPEN used for in vitro amastigote killing were within the range that was cytotoxic to host Mϕs (Figure 4.1B).

4.3.2 Liposomal Synthetic Pentalinonsterol Treatment clears Leishmania donovani from

Infected Mice.

Figure 4.2 displays a nearly 64% hepatic, 83% splenic and 57% bone marrow parasite suppression compared to empty liposomes (p < 0.001). Comparatively, sodium stibogluconate (70 mg/kg) (SSG), a standard antileishmanial drug, showed low clearance similar to that of PBS treated mice (Figure 4.2A-C). Furthermore, liposomal sPEN injection was well tolerated by L. donovani infected mice and showed a decrease in the level of serum AST (non-significant) and ALT (p < 0.05) compared to empty liposome treated infected mice (Figure 4.3A-B).

4.3.3 Induction of Proliferative and Proinflammatory Cytokine Responses in Infected

Mice treated with Liposomal Synthetic Pentalinonsterol

Analysis of T cell responses showed that splenocytes from liposomal sPEN treated mice showed a significantly higher (2-fold) increase in T cell proliferation levels as

71

Figure 4. 4: Effect of Liposomal Synthetic Pentalinonsterol Treatment on T cell

Proliferation in L. donovani Infected Mice.

Proliferation of splenic T cells of BALB/c mice infected with L. donovani in response to

L. donovani antigen. Mice were treated 2 weeks post infection with PBS, empty liposomes, or sPEN loaded liposomes (50 µg or 2.5 mg per kg body weight). Spleens were removed 2 weeks post injection, pulsed with L. donovani antigen (20 µg/ml) and T cell proliferation was measured after 72 hours. Data shown are the mean ± SE of triplicates from four or five individual mice per group and are representative of three individual experiments. Significance with respect to PBS treatment is presented as *** p

<0.001.

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Figure 4. 5: Effect of Liposomal Synthetic Pentalinonsterol Treatment on Cytokine

Release in L. donovani Infected Mice.

(A-D) Th1 and Th2 cytokine production by splenocytes from PBS, empty liposome or sPEN loaded liposomes treated L. donovani infected mice stimulated with 20µg/ml L. donovani antigen. (A) IFN-γ, (B) IL-10, (C) IL-4 and (D) IL-13 were measured by

ELISA. Data shown were the mean ± SE of triplicates from four or five individual mice per group and are representative of three individual experiments. Significance with respect to empty liposomes is presented as ** p < 0.01.

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Figure 4. 6: Effect of Liposomal Synthetic Pentalinonsterol Treatment on Cytokine

Transcription in L. donovani Infected Mice.

(A-F) Th1 cytokine and Th2 mRNA expression by spleen tissue from empty liposome or sPEN loaded liposomes treated L. donovani infected mice. (A) IFN-γ mRNA, (B) IL-

12p35 mRNA, (C) TNF-α mRNA, (D) IL-10 mRNA, (E) IL-4 mRNA and (F) IL-13 mRNA were measured by Real Time PCR. (G-H) Ratios of (G) IFN-γ to IL-10 mRNA and (H) IFN-γ to IL-4 mRNA are also shown. Data shown were the mean ± SE of triplicates from four or five individual mice per group and are representative of three individual experiments. Significance with respect to empty liposomes is presented as ** p

< 0.01.

74 compared to empty liposome treated mice (p < 0.05) (Figure 4.4). Supernatants taken from cultures of re-stimulated splenocytes from infected mice treated with liposomal sPEN showed a 10-fold increase in IFN-γ production, compared to empty liposome treated infected mice (p < 0.01) (Figure 4.5A).However, these mice did not show a significant difference in production of IL-10, IL-4, or IL-13 (Figure 4.5B-D). There were undetectable levels of IL-12 and TNF-α in the supernatants (data not shown), so Real

Time PCR (RT-PCR) was performed to look at transcription of these cytokines. Infected mice treated with liposomal sPEN showed significant increases in the levels of splenic

IFN-γ (p < 0.05), IL-12 (p < 0.05) and TNF-α (p < 0.05), but not IL-10, IL-4 or IL-13 mRNA compared to empty liposome treated mice (Figure 4.6A-F). Furthermore, a significantly higher ratio of IFN-γ to IL-10 mRNA (p < 0.05) and IFN-γ to IL-4 mRNA

(p < 0.05) (Figure 4.6G-H) was seen.

4.3.4Liposomal Synthetic Pentalinonsterol Treatment Promotes the Formation of

Matured Liver Granulomas in Leishmania donovani Infected Mice

Liposomal sPEN treated mice had a significant reduction in the level of immature hepatic granulomas compared to empty liposome treated mice (p < 0.01). Furthermore, there was a significant increase in the number of parasite free mature hepatic granulomas

(p < 0.05) in the liposomal sPEN treated mice compared to empty liposome treated mice

(Figure 4.7A-B). Finally, there was a significant decrease in instances of no cellular response when compared with empty and PBS treated mice (p < 0.01).

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Figure 4. 7: Effect of Liposomal Synthetic Pentalinonsterol Treatment on Hepatic

Granuloma Formation in L. donovani Infected Mice.

Liver sections from PBS, empty liposome or sPEN loaded liposomes treated L. donovani infected mice (A) were scored for the extent of granuloma formation. Results were from one representative experiment of three with similar findings. Data were expressed as mean granuloma number per 10 high-power fields (magnification 400X) ±SE. *, P < 0.05 and **, P < 0.01 compared to compared to empty liposome treated groups. (B)

Histopathology of infected livers from PBS, empty liposome or sPEN loaded liposome treated L. donovani infected mice. Both PBS and empty liposome treated mice had poorly formed granulomas which were highly parasitized (arrow). In contrast, sPEN loaded liposome treated mice had well-formed granuloma which were mostly devoid of parasites

(400X).

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4.4 Discussion

The use of natural products is gaining increased recognition as an effective approach for the treatment of infectious diseases. Previously, the medicinal plant P. andrieuxii, specifically phytosterols (e.g. pentalinonsterol) isolated from it, have shown promising biological activity against Leishmania (181, 182, 190). However, PEN’s effect against

VL, specifically L. donovani, has yet to be shown. sPEN showed a significant level of promastigote killing at higher concentrations (50 and 100 µg/ml) where no cellular toxicity was observed (Figure 4.1B). Interestingly, amastigote killing was much more successful with lower levels of sPEN (e,g 10 and 25 µg/ml) (Figure 4.1C). This is encouraging because amastigote models more correctly represent in vivo mechanisms of drug activity (191). In addition, since sPEN has higher activity against the amastigote form compared to the promastigote form, sPEN’s potential mode of action could also be directed towards the host. These in vitro studies show that potentially clinically relevant concentrations of sPEN are not cytotoxic to host macrophages, but have a strong antileishmanial activity against both L. donovani promastigotes and intracellular amastigotes.

Although initial in vitro assays showed promising results, it is important for potentially clinically relevant compounds to be formulated properly for in vivo administration. Unfortunately, sPEN and other similar sterol compounds are limited in their ability to be delivered systemically. Sterols, a subgroup of steroids, show poor absorption in the intestine (192), limiting their ability to be delivered orally. In addition,

77 water-insoluble compounds, like a majority of sterols (193) and promising antimicrobial compounds extracted from plants (194), show very limited ability to be delivered intravenously. Due to poor systemic delivery and bioavailability, it is necessary to discover novel ways to deliver sterols for antimicrobial therapy. Here, liposomes were used to systemically deliver sPEN for VL treatment. Liposomes have effectively been used clinically as a delivery vehicle for hydrophobic and toxic compounds such as amphotericin B (AmBisome) (195). Using a formulation similar to AmBisome’s, sPEN was incorporated into liposomes with nearly 100% efficiency. Plant sterol compounds such as PEN generally reside in cell membranes (196), which may explain our relatively high encapsulation efficiency. Liposomal encapsulation of therapeutics have been shown to increase the blood circulation of encapsulated drugs (197). Additionally, prior research has shown that by adjusting the size or composition of liposomes, specific sites such as the liver, spleen, or bone marrow can be targeted (198, 199). Future studies will be required to fully analyze the size and tissue distribution of sPEN containing liposomes.

When used to treat infected mice, liposomal sPEN significantly lowered the parasite load in the liver, spleen, and bone marrow of infected mice (Figure 4.2A-C).

Amastigotes present in the spleen and smears of bone marrow aspirate are diagnostic signs of VL. Classically, parasite clearance in the liver and spleen are hallmarks of a successful VL treatment. The dose of SSG used (70 mg / kg) failed to adequately limit parasite burden in the liver, spleen and bone marrow (Figure 4.2A-C). This lack of clearance using this dose is consistent with a previous study by Carter et al. (200). Mullen et al. showed AmBisome increased hepatic suppression of L. donovani when compared to

78 our findings however, the splenic suppression after treatment with sPEN was almost identical to that of AmBisome (201). When compared, we also saw relatively similar suppression in the bone marrow. Clearance in the bone marrow has been shown to play an important role in VL infection, as it can serve as a parasite reservoir (157). In order to determine if liposomal sPEN was capable of inducing an effective T cell-mediated immune response, we performed an antigen recall assay on the spleens of treated mice.

Traditionally, an effective chemotherapy against VL requires a strong T cell response

(202). For example, successful treatment with SSG requires a functional TH1 and TH2 immune response (50). This limits the use of SSG treatment for immunocompromised individuals, such as HIV+ individuals for which the risk of developing VL can be up to

2300 times higher than HIV- individuals (26). Although treatment with liposomal sPEN results in increased in TH1 and TH2 cytokines with antigen recall (Figure 4.5A-D &

Figure 4.6 A-H), the direct effect of promastigote killing observed with sPEN would seem to indicate that it works on both the parasite and host cell.

Active VL is associated with an impaired cell mediated immune response, which is reflected by T cell anergy, even after exposure to Leishmania specific antigens (203,

204). Liposomal sPEN treated mice showed a marked recovery in T cell proliferative responses in comparison to empty liposome treated mice (Figure 4.4). This suggests that liposomal sPEN could effectively enhance the lympho-proliferative responses in

Leishmania infected mice. Additionally, an effective chemotherapy for experimental VL requires a strong Th1 cytokine response, which in-turn could mediate clearance of L. donovani (205). Importantly, liposomal sPEN treatment of infected mice also induced a

79 significant increase in the production of IFN-γ (Figure 4.5A & 4.6A) which has been shown to be essential for the clearance of experimental Leishmaniasis (159). L. donovani infected IFN-γ knockout mice are not responsive to antimonial based chemotherapy (50).

Thus, induction of IFN-γ by liposomal sPEN treatment enhances the antileishmanial activity of L. donovani infected mice. The production of IFN-γ by Th1 cells has been shown to be dependent on IL-12 production from DCs, Mϕs, and B cells (206). In this study, liposomal sPEN treatment induced the expression of splenic IL-12 and TNF-α mRNA in infected mice (Figure 4.6B & C). It is evident that in addition to its direct antileishmanial activity, sPEN enhances host protective immune responses resulting in eradication of the parasite.

On the other hand, both liposomal sPEN and empty liposome treated infected mice showed comparable levels of anti-inflammatory cytokines like IL-10 and IL-4 (Figure 4.5

B-C & Figure 4.6 D-E). Thus, sPEN does not appear to modulate the levels of IL-10 and

IL-4 during infection. Interestingly, there was an increase in the ratio of IFN-γ: IL-10 and

IFN-γ: IL-4 mRNA in infected mice (Figure 4.6G-H) treated with liposomal sPEN compared to empty liposomes, which suggests that this predominant Th1 response is a possible contributing factor to its in vivo antileishmanial activity (207).

Liposomal sPEN treatment resulted in increased IFN-γ release from infected mice which has been shown to be associated with the formation matured granulomas required for killing amastigotes (208). Liposomal sPEN treatment of VL infected mice resulted in the formation of matured and parasite free hepatic granulomas (Figure 4.7A & B). This is not surprising as IFN-γ and IL-12 have been shown to be essential in promoting

80 granuloma formation, activation of Mϕs, and induction of anti-parasitic immune responses (209-211). These data taken together suggest liposomal sPEN promotes clearance of L. donovani, at least partially, through the formation of hepatic granulomas.

To evaluate the effect of liposomal sPEN toxicity, the concentration of liver enzymes were evaluated. Elevated liver enzymes, such as ALT and AST, are indicative of disease, and represent possible damage to the liver (169). This study revealed serum levels of

ALT enzyme in liposomal sPEN treated mice were significantly lower than empty liposome treated mice (Figure 4.3A). The ALT levels seen in mice treated with liposomal sPEN were considered to be within the range of an un-infected mouse (171), indicating that liposomal sPEN was well tolerated by the mice.

Overall, this study highlights the immunotherapeutic potential of sPEN for the treatment of VL using both in vitro and in vivo models. Liposomal sPEN successfully rescued the impaired immune response of the L. donovani infected host by mediating a strong pro-inflammatory cytokine response. Additionally, it cleared parasite load in the liver, spleen, and bone marrow significantly better than controls, including the currently used clinical treatment, SSG. The results of this study strongly suggest that sPEN could be developed as a therapy for the treatment of VL.

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Chapter 5: Treatment of Experimental Autoimmune Encephalomyelitis by Co-Delivery of Disease Associated Peptide and Dexamethasone in Acetalated Dextran Microparticles

5.1 Introduction

Multiple sclerosis (MS) is a chronic inflammatory disease in the central nervous system (CNS) affecting approximately 2.5 million people world-wide (212). MS is thought to be induced by Mϕ and T cell infiltrates to localized areas, causing demyelination of axonal regions (213), possibly in a myelin-specific manner (214), T cells secreting IFN-γ, IL-17, or TH17 cells have been determined to exacerbate the disease (215, 216). New and emerging treatments for MS have successfully targeted this subset of cells responsible for inflammation in the CNS, however most treatments have serious side-effects.

Current common treatments for MS are generally effective at decreasing relapses; however there are serious concerns due to their non-specific suppression of the immune system. Natalizumab (Tysabri®) is a monoclonal antibody treatment that blocks leukocyte migration into the CNS (217), but can lead to immunosuppressive related diseases such as progressive multifocal leukoencephalopathy (PML) (218). Another treatment, beta IFN (Betaseron®; Extavia®, Avonex, and Rebif), creates neutralizing 82 antibodies towards endogenous IFN (219), and has been shown to decrease relapse rates, but it does not stop overall disease progression (220). Fingolimod (GILENYATM) is an oral treatment that inhibits migration of naïve T cells out of the peripheral lymph nodes

(221), but side-effects related to immunosuppression have been seen clinically (222).

Long-term treatment with immunosuppressive drugs can lead to increased risk of cancer

(68) and infection (223), illustrating the need to develop new therapies that limit not only relapse rates and disease progression, but also provide antigen specific immunosuppression.

Therapies have been developed to treat in an antigen specific manner, therefore limiting suppression of the entire immune system. Antigen specific tolerance has been accomplished through interaction with mucosal surfaces by oral (74, 75), nasal (76), and sublingual (77) delivery to treat animal models. Additionally, tolerance has been achieved through ex vivo antigen pulsing of dendritic cells (224). The mechanism of how antigen specific tolerance forms varies, but usually involves the generation of either regulatory CD4 T cells that can inhibit cellular inflammatory responses (225), or inflammatory cell anergy (226). Even though these methods have been successful pre- clinically, they have failed once they have reached the clinics, likely due to an inaccurate choice of antigen or issues with dosing quantities or timing (227). Based on this lack of success, new methods for treatment that can be applied to delivery of a broad array of antigens or allow for sustained release of antigens are desired.

83

Recently, Kang et al. have shown that injecting both an immunodominant peptide of insulin with dexamethasone (DXM), they were able to prevent the onset of Type 1

Diabetes in a regulatory T cell manner (83). By immunizing with an antigen and an

“immune tolerizing” adjuvant, Kang et al. were able to generate immune tolerance towards a self-antigen. Others have built on this success, by using biomaterial-based antigen-specific immunomodulatory formulations that protect mice from experimental autoimmune encephalomyelitis (EAE) (79, 81, 82, 85, 228), a model of MS, and inflammatory arthritis via antigen specific t-regulatory cell activation (86). Studies using potential treatments for EAE have rarely examined outcome after administration at a clinically relevant time point. A recent review by Vesterinen et al. showed approximately 4% of EAE papers, out of the 126 studied, examine treatment efficacy beyond 2 weeks post induction of EAE (88). Unfortunately, this implies the vast majority of studies begin treatment prior to symptom onset. Two recent studies use particle-based post-induction treatments to ameliorate a relapsing and remitting form of EAE. Yeste et al. utilized non-biodegradable gold nanoparticles injected intraperitoneally to treat EAE and Getts et al. induced tolerance through intravenous injection of microparticles with surface conjugation of an encephalogenic peptide (79, 85). Although both studies are promising, alternate methods optimizing particle formulation and function should be explored. The goal of this work was to build on this existing research to formulate a clinically relevant antigen specific therapy utilizing a biodegradable polymer, FDA- approved immunosuppressive drug and a disease-associated antigen. Traditionally, only phagocytic cells such as APCs can internalize microparticles (5). By encapsulating an

84 immunosuppressive drug and an antigen in a microparticle, only antigen presenting cells

(APCs) can internalize the particles, which then could induce an adaptive immune response that results in tolerance against the autoimmune antigen.

Expanding on the previous work formulating microparticles with the novel acid sensitive polymer, Ac-DEX (21, 109, 114). Ac-DEX is derived from dextran by modifying the hydroxyl groups with pendant acetals. Since acetals are sensitive to acidic conditions, in low pH conditions present in the lysosome of APCs, Ac-DEX microparticles degrade releasing their cargo inside the lysosome. Previous nanoparticulate formulations (79, 85) used surface bound proteins which may be exposed to degradation in vivo, such as low pH or enzymatic degradation. The proposed Ac-DEX formulation can be an improvement upon this because not only is the protein or peptide encapsulated but it has shown enhanced protein stability across a broad range of temperatures when a protein is encapsulated in Ac-DEX (55). Here, Ac-DEX is used to formulate a first of its kind polymer-based particulate delivery vehicle co-encapsulating a major MS target antigen, Myelin Oligodendrocyte Glycoprotein (MOG), and an immunosuppressive drug, DXM in an effort to treat MS. Efficiency of encapsulated

DXM delivery to APCs was determined in vitro using nitric oxide (NO) and cytokine measurements. In order to determine the protective properties of the particles in vivo, they were administered as a treatment to C57Bl/6 mice with EAE, and their effect on IL-

17 and GM-CSF production by these mice was examined through an antigen recall assay.

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5.2 Materials and Methods

5.2.1 Materials

All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Water (dd-H2O) for buffers was purified using a Millipore (Billerica,

MS) Milli-Q Integral water purification system, which was made basic by addition of triethylamine (TEA) (0.01% v/v). DXM (98%) was purchased from Alfa Aesar (Ward

Hill, MS) and MOG35-55 peptide was purchased from CS Bio Co. (Menlo Park, CA).

Antibodies used for ELISA and FLOW Cytometry were acquired from BD biosciences

(San Jose, CA), unless otherwise specified. Fluorescence measurements were detected using a Molecular Devices (Sunnyvale, CA) FlexStation 3, courtesy of the Department of

Chemistry and Biochemistry at the Ohio State University and Molecular Devices

SoftMax Pro Software (Sunnyvale, CA).

5.2.2 Animals

Mice used for experiments were 10 week old C57Bl/6 females purchased from

Taconic Farms (Hudson, NY). All animals were kept in a sterile facility according to

The Ohio State University Institutional Guidelines. All animal procedures were in

86 accordance with and approved by the Institutional Animal Care and Use Committee

(IACUC) of The Ohio State University.

5.2.3 Cells

RAW 264.7 macrophages were purchased from ATCC (Manassas, VA) and cultured according to the manufacturer specifications. BMDCs were prepared as previously described (74).

5.2.4 Synthesis and Analysis of Acetal Coverage of Ac-DEX

Ac-DEX was synthesized using 71 kDa dextran as previously described (21).

Cyclic acetal coverage was determined to be 51.9% by nuclear magnetic resonance as previously described (114).

5.2.5 Preparation of Empty or DXM-Loaded Ac-DEX Microparticles

To formulate MPs containing DXM, Ac-DEX (100mg) and DXM were dissolved in chloroform and ethanol (95:5 v/v respectively) and mixed with 3% PVA (MW~13-23 kg/mol, 87-89% hydrolyzed) in phosphate buffered saline (PBS). This solution was probe sonicated (Branson Sonifier 450, Branson, Los Angeles, CA) in an ice bath with a flat tip for 30 seconds with max energy 30W. MPs were stirred for 2 hours in 0.3% PVA

87 in PBS and were washed and collected by centrifugation at 18,000 rpm for 16 minutes on a Beckman Coulter Avanti J-E centrifuge (Brea, CA). Empty MPs (/MPs) were formulated in the same manor, excluding the addition of DXM.

5.2.6 Preparation of MOG or MOG/DXM Co-Encapsulated Ac-DEX MPs

DXM (1 mg) and Ac-DEX (100 mg) were dissolved in chloroform and ethanol

(95:5 v/v respectively), and MOG peptide (1 mg) in PBS was added. The mixture was probe sonicated in an ice bath with a flat tip at with max energy at 30W for 30 seconds and 3% PVA in PBS (2 ml) was added and the mixture was probe sonicated for a second time. MPs were stirred for 2 hours in 0.3% PVA in PBS and were washed and collected by tangential flow filtration using a mPES MidiKros Filter Module (500 kD pore size,

235cm2 surface area) (Spectrum Labs, Rancho Dominguez, CA). MOG MPs

(MOG/MPs) were formulated by the same mechanism without the addition of DXM.

5.2.7 Scanning Electron Microscopy (SEM)

Microparticles size and morphology were characterized by SEM using an FEI

NOVA nanoSEM (Hillsboro, OR). SEM sample preparation and analysis was done as previously described (5).

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5.2.8 Quantification of DXM

Quantification of DXM for in vitro assays was performed by liquid chromatography-mass spectrometry (LCMS) with a Thermo Scientific Accela Pump and

Finnigan TSQ Quantum Discovery Max and analyzed using LCquan software. MPs were dissolved in acetonitrile (1mg/ml) and purified by centrifugation for 5 minutes at 15,000g and passed through a 0.2 µm filter. Samples were run in a mobile phase of acetonitrile:ddH2O:formic acid (50:49.9:0.1 v/v) through a Thermo Scientific 2.5 µm x

100 x100 C18 column with a C8 guard column in isocratic mode with a flow rate of 0.2 ml/min. Desoxymethasone, the internal standard, and dexamethasone were quantified using ion reaction monitoring at 25% collision energy with ion transitions m/z 377 

339 and m/z 393  237 respectively.

Quantification of DXM in DXM/MPs and DXM/MOG/MPs was determined by high-performance liquid chromatography (HPLC) with a method adapted from Zhang et al.(229). MPs were dissolved in a methanol:ddH2O solution (80:20 v/v) (1 mg/ml) and

DXM concentration was determined using an Agilent 1100 series HPLC (Santa Clara,

California) with a Thermo Scientific 150 mm x 4.6 mm, pore size 5 µm, Aquasil C18 column (Waltham, MS). Samples were passed through the column at a flow rate of 1

ml/min in a mobile phase of methanol:ddH2O (80:20 v/v). DXM was detected at a wavelength of 240 nm. Sample peaks were compared to a standard curve and analyzed using Aglient Chemstation software. DXM/MP encapsulation was 3.5% and

DXM/MOG/MP was 1.5%. 89

5.2.9 Quantification of Encapsulated MOG

Samples of either MOG MPs (MOG/MP), MOG or /MPs (1 mg) were suspended in 990 µl PBS and the pH was lowered with 5 µl 50% formic acid (v/v). Samples were incubated on a 37oC shaker plate overnight and the solution was returned to neutral pH using 13.25M sodium hydroxide. Encapsulation efficiency was determined using a fluorescamine assay per the manufacturer’s instructions.

5.2.10 In Vitro Release of Dexamethasone

DXM/MPs were suspended in either sodium acetate buffer (pH5.0) or PBS

(pH7.4) at a concentration of 1 mg/ml. Samples were incubated on a 37oC shaker plate and aliquots were withdrawn at each time point, centrifuged (15,000g for 5 minutes), and supernatant collected and freeze dried. DXM quantification was performed using LCMS

(above).

5.2.11 Nitrite Analysis

Nitrite concentrations in supernatants from RAW 264.7 Mϕs cultured with LPS and DXM were determined using Griess reagent from Promega (Madison, WI). Mϕs

90 were seeded in a 96-well plate at 5x104 cells/well with Thermo Scientific HyClone

DMEM/High Glucose (Logan, UT) with 5% Fetal Bovine Serum and 1% penicillin/streptomycin (complete media), and left overnight to adhere. Cells were cultured with LPS (10 µ/ml) for 24 hours, then treated with empty MPs or varying concentrations of DXM (0 – 0.1 µM), in the form of DXM/MPs or free DXM, for 24 hours. Supernatants were collected after 24 hours and centrifuged at 15,000 rpm for 10 minutes to remove residual particles and cells, then analyzed with Griess reagent in accordance with the manufacturer’s protocol.

5.2.12 Measurement of Il-6 Secreted by Bone Marrow Derived Dendritic Cells

BMDCs were seeded at a concentration of 5 x 104 cells/well in a 96-well plate, stimulated with LPS (1µg/ml) for 24 hours, then treated with various concentrations of

DXM or DXM/MPs (0-0.5 µM). Supernatants were collected after 24 hours and centrifuged at 15,000 rpm for 10 minutes to pellet residual cells and particles. The level of IL-6 was measured by ELISA per the manufacturer’s specifications.

5.2.13 Immunization and Treatment of EAE

Mice were immunized with a Complete Freund’s Adjuvant (CFA) and MOG peptide emulsion along with pertussis toxin, purchased from Hooke Laboratories

91

(Lawrence, MS), per the manufacturer’s suggestions. After immunization, mice were given clinical scores as previously described (230) and treated 18, 21 and 24 days post immunization with 100 µl injections of either PBS, /MPs, DXM/MPs (8 µg DXM),

MOG/MPs (17.6 µg MOG), DXM/MOG/MPs (8 µg DXM and 17.6 µg MOG) or free

DXM (8 µg) with free MOG (17.6 µg). On day 32 post immunization mice were euthanized and their spinal cord, spleens, and inguinal lymph nodes removed.

5.2.14 Measurement of Secreted IL-17 and Granulocyte-Macrophage Colony-Stimulating

Factor

Splenocytes from treated mice were plated at 5x106 cells/well in a 12 well plate and stimulated for 24 hours with 2 µg/ml MOG. Supernatants were isolated and an

ELISA was performed on Immulon 2 plates (Fisher Scientific) to determine the levels of

IL-17 and GM-CSF as previously described (231).

5.2.15 Fluorescence Activated Cell Sorting Analysis

Cells from the spinal cord, spleen, and inguinal lymph node of the in vivo mouse study were seeded at approximately 5x106 cells/well in a 12 well plate and stimulated with 2 µg/ml MOG for 24 hours. Approximately 1x106 cells were removed and placed in a 96-well plate for staining of intracellular IL-17, IFN-γ, and FoxP3. Cells were treated with FACS Buffer (1X PBS, 1% Ethylenediaminetetraacetic acid (EDTA), and 0.2% heat

92 shocked sterile FBS) and FC-receptor blocker (BD Biosciences), followed by addition of surface antibodies. Cells were fixed and permeabilized with Cytofix/Cytoperm (BD

Biosciences) for 30 min at 4°C, then stained for IL-17, IFN-γ, and FoxP3. Flow cytometry was performed with a FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo.

5.3 Results

5.3.1 Particle Formulations and Analysis

Figure 5.1 shows electron micrographs of DXM/MOG/MPs (Figure 5.1A),

DXM/MPs (Figure 5.1B), and MOG/MPs (Figure 5.1C). Table 4.1 reports encapsulation efficiencies of DXM, MOG, or both in Ac-DEX MPs as well as quantity of drug per milligram of particles. DXM was loaded with higher efficiency into DXM/MPs compared with DXM/MOG/MPs (3.5% versus 1.5%) and MOG/MPs showed increased encapsulation of MOG compared with DXM/MOG/MPs (48% versus 21.8%).

93

Figure 5. 1: Scanning Electron Micrographs of Acetalated Dextran (Ac-DEX)

Microparticles

Encapsulating (A) Dexamethasone (DXM) + Myelin Oligodendrocyte Glycoprotein 35-

55 (MOG) (B) DXM or (C) MOG.

94

Table 5.1: Encapsulation Efficiencies for Ac-DEX Particles Containing DXM, MOG, or

Both.

95

5.3.2 In Vitro Immunosuppressive Function of Dexamethasone

Figure 5.2 shows the effect of DXM on nitric oxide (NO) release from Mϕ stimulated with LPS. Mϕ were cultured with ranging doses of DXM (0-0.1 µM) in the form of free DXM or DXM/MPs. DXM/MPs significantly decreased the amount of NO compared with free DXM (p < 0.05 except p < 0.005 for 0.05 and 0.1 µM). Inhibition of

IL-6 production (Figure 5.3) in LPS stimulated C57Bl/6 BMDCs was significantly decreased at 0.01 and 0.1 µM with DXM/MPs when compared with free DXM (p < 0.05 and p < 0.01, respectively).

5.3.3 In Vivo Treatment of EAE

Figure 5.4 shows average clinical score data for mice treated with either PBS,

/MPs, DXM/MPs, MOG/MPs, or DXM/MOG/MPs 18 days post immunization. Mice receiving injections of DXM/MOG/MPs had significantly lower clinical scores compared to mice receiving any other treatment type. As analyzed with an ELISA, splenocytes stimulated with 2 µg/ml MOG peptide after removal from DXM/MOG/MPs treated mice showed significantly lower levels of IL-17 (Figure 5.5A) when compared with mice treated with /MPs (p < 0.005), DXM (p < 0.05), DXM/MPs (p < 0.5) or MOG/MPs (p <

0.05). Also, with the exception of DXM, splenocytes from mice treated with

DXM/MOG/MPs also had significantly lower levels of GM-CSF (Figure 5.5B), when compared with mice treated with PBS (p < 0.05), /MPs (p < 0.005), DXM/MPs (p < 0.05) or MOG/MPs (p < 0.05). 96

Figure 5. 2: Nitric Oxide (NO) Release by Macrophages when Cultured with

Lipopolysaccharide plus Free or Encapsulated Dexamethasone (DXM) in Ac-DEX.

Significance with respect to free DXM is presented as * p < 0.01 and ** p < 0.001. Data is presented as average ± standard deviation.

97

Figure 5. 3: IL-6 Production in Culture from C57Bl/6 Bone-Marrow Derived Dendritic

Cells.

Cells were cultured in the presence of lipopolysaccharide and with free dexamethasone

(DXM) or DXM encapsulated in Ac-DEX microparticles. Significance with respect to free DX M encapsulated in Ac-DEX microparticles. Significance with respect to free

DXM is presented as * p < 0.05 and ** p < 0.005. Data is presented as average ± standard deviation.

98

Figure 5. 4: Clinical Scores of 9 Week Old C57Bl/6 Female Mice Immunized with

Experimental Autoimmune Encephalomyelitis (EAE).

Mice were treated after symptoms began by subcutaneous injections of PBS, empty microparticles (/MP), dexamethasone (DXM) MPs (DXM/MP), DXM with myelin oligodendrocyte glycoprotein peptide (MOG), MOG MPs (MOG/MP) or

DXM/MOG/MP on days 18, 21, and 24 post-immunization (as indicated with arrows).

Treatments with DXM contained 8 µg DXM and treatments with MOG contained 17.6

µg MOG. Statistical significance with respect to DXM/MOG/MP is presented as * p <

0.05. Data is presented as average ± standard deviation.

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Figure 5. 5: Antigen Recall Measurements of (A) IL-17 and (B) GM-CSF production.

Cytokines were measured from splenocytes isolated from C57Bl/6 mice immunized for experimental autoimmune encephalomyelitis (EAE) and treated with PBS, empty microparticles (/MP), dexamethasone MPs (DXM/MP), free DXM and myelin oligodendrocyte glycoprotein (MOG) MPs (MOG/MP) or DXM/MOG/MPs on days 18,

21, and 24 post-immunization. Significance with respect to DXM/MOG/MP is presented as * p < 0.05 and ** p < 0.005. Data is presented as average ± standard deviation.

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5.4 Discussion

Scanning electron micrographs show the particles encapsulating MOG, DXM, or

DXM and MOG display relatively spherical morphology and heterogeneity (Figure 5.1).

EE’s for DXM/MPs and MOG/MPs were greater than when the therapeutics were co- encapsulated (DXM/MOG/MPs; Table 5.1). Variability with amount of therapeutics encapsulated in polymeric particles has been shown to affect EE. Uchida et al. showed that the EE of OVA in PLGA microparticles increased proportionally with theoretical drug loading, however after a certain point, large loading attempts resulted in decreased efficiencies (232). Additionally, Fan et al. were able to encapsulate DXM in poly(D,L- lactic acid) (PLA) microparticles with efficiencies near 70%, but as attempts at increased drug loading were made, the efficiencies also decreased (233). This data suggests there may potentially be a maximum loading, which could explain the diminished loading of

DXM and MOG in DXM/MOG/MPs compared with DXM/MPs and MOG/MPs.

MP size seems to have little effect on DXM EE. Hickey et al. were able to encapsulate DXM (8 µg / mg PLGA) in PLGA microspheres ranging from 1-50 µm, however they still only achieved 3-4% EE (234). Krishnan et al. used nanoprecipitation to formulate copolymer particles using poly(ethylene glycol) (PEG) and poly(ε- caprolactone) (PCL). Although DXM encapsulation did slightly increase the particle size upon loading (111 nm to 127 nm), the particles displayed a high EE of DXM (52.6%), while still maintaining small size and dispersion (235). It is clear that DXM loading 101 efficiencies and particle sizing vary greatly based on polymer usage, formulation technique, and drug loading quantity, therefore further work should be performed to optimize formulation methods used in this paper.

To evaluate the bioactivity of DXM/MPs, the release of pro-inflammatory nitric oxide (NO) in Mϕ stimulated with LPS was monitored in vitro. Mϕs cultured with

DXM/MPs had significantly lower NO production compared to those cultured with free

DXM (Figure 5.2). DXM/MP treatment also diminished the response of LPS-stimulated

BMDC’s in vitro, which was illustrated by decreasing levels IL-6 (Figure 5.3). Inhibition of IL-6 has been shown to have a protective effect in mice immunized with EAE, possibly through the inhibition of TH17-mediated CNS infiltration of auto-reactive cells

(236). Increased DXM levels were able to decrease the amount of IL-6 produced by

BMDC’s; however, only at 0.01 and 0.1 µM were DXM/MPs able to significantly inhibit

IL-6 production compared to free DXM. This trend suggests MPs containing DXM may inhibit IL-6 more efficiently at high levels.

This study found mice therapeutically treated with DXM/MOG/MPs at 18, 21, and 24 days post immunization, displayed significantly decreased mean clinical scores compared to mice receiving PBS, /MPs, DXM/MPs, MOG/MPs or free DXM with free

MOG (Figure 5.4). Since diagnostics recognizing MS prior to symptom onset have yet to be developed, the MP treatment was administered after the mean clinical score for each group was approximately 3.5. A clinical score of 4 represents mice that no longer have hind limb function, while clinical scores of 3 indicate severely deficient motor function of

102 the hind limbs. Mice receiving DXM/MOG/MPs improved to a mean clinical score of

1.8, 16 days after the final injection, and maintained this clinical score throughout the remainder of the trial (Figure 5.4). A clinical score between 1 and 2 indicates the mice have limpness in their tails, but their hind limb function ranges from normal to slightly inhibited. This improvement in clinical score was significantly lower than all experimental groups, showing MPs loaded with MOG and DXM are superior at ameliorating disease compared with the other groups. In particular, DXM/MOG/MPs were superior to DXM/MPs, suggesting antigen is required in amelioration of the disease.

This possibly occurs through inhibition of disease-associated cytokine production. These data indicate that MOG stimulated splenocytes from EAE immunized mice treated with

DXM/MOG/MPs had significantly lower production of both IL-17 and GM-CSF (Figure

5.5A & B), however, these mice did not have a significant difference in intracellular splenocytes IFN-γ production (data not shown). Additionally, there was no significant difference in FoxP3+ T-regulatory cells, or intracellular IFN-γ and IL-17 in the spinal cord or inguinal lymph nodes (data not shown). Other previous studies have indicated that FoxP3+ T-regulatory cells have a primary role in autoimmune treatment, when the tolerogenic adjuvant and antigen are given prophylactically or at the time of disease induction. Kang et al. previously showed that administration of unencapsulated DXM and protein antigen allowed for protection against delayed–type hypersensitivity and diabetes onset (83). With prophylactic administration of the compounds, induction of tolerogenic DCs and FoxP3+ T-regulatory cells occurred (83). Fissolo et al. reported that a MOG-based DNA vaccine administered prophylactically to EAE immunized mice had

103 a significant protective effect, decreasing the overall clinical score through FoxP3+ T- regulatory cell expansion, and concomitant decreases in IL-17 and IFN-γ expression

(237). Although there were no changes in regulatory T cell populations with treatment, interestingly, the DXM/MOG/MPs induced regulatory T cell formation and limited the progression of EAE, when given prophylactically (data not shown). Previous work with biomaterials and an immunomodulating peptide has shown both prophylactic EAE protection (82) and protection after immunization, but prior to EAE symptom onset (81).

Administration of gold-based nanoparticles containing a tolerogenic small molecule and an epitope of MOG resulted in decreased IFN-γ, IL-6 and IL-17, as well as increased levels of FoxP3+ T-regulatory cells (85). As previously stated, there was no significant change in FoxP3+ populations with this treatment study and the mechanism of treatment has not been previously reported with a similar study. Therapeutic treatment with the aforementioned MOG-based DNA vaccine at 10 and 24 days after disease immunization displayed significant inhibition of EAE progression and diminished symptoms; however

Fissolo et al. make no mention of mechanisms with regard to therapeutic action (237).

Although both prophylactic and therapeutic treatments have been shown to work, very little has been elucidated on a therapeutic mechanism. Wegmann et al. showed dendrimers containing a synthetic peptide therapeutically protected mice against experimental allergic encephalomyelitis however, unlike this study they showed an increase in IL-17 generated by splenocytes (228). In a recent promising study, Getts et al. have suggested that encephalogenic peptide-based EAE therapies work through an increase in T cell anergy (79). T cell anergy is a process where T cells are inactivated in

104 the periphery, possibly through co-stimulatory inhibition between APCs and T cells

(238). Co-stimulatory inhibition has been associated with DXM (238). Classically, anergeric cells do not respond to antigen, however, Figure 5.5 shows that with antigen stimulation significant levels of cytokines are produced. This antigen specific response indicates that perhaps anergy might not be the mechanism of action, but further work would need to be performed to fully rule-out anergy as a mechanism of action for therapeutic-based EAE treatments.

Here it is reported that one possible contribution to the protective effects of

DXM/MOG/MPs is the significant decrease in production of IL-17 and GM-CSF from antigen stimulated splenocytes (Figure 5.5A & B). IL-17 is an important cytokine in autoimmune disorders, and Yan et al. have recently shown CNS-specific inhibition of IL-

17 protects against EAE without diminishing the entire immune system (239). A study by

Komiyama et al. also suggests IL-17 may play a more significant role in EAE than that of

IFN-γ (240). Additionally, GM-CSF has functioned to increase activation of CNS- associated APCs (241), to enhance the survival of Th17 cells (242), and may possibly function in IL-17 and IFN-γ independent roles to exacerbate disease (243). Although this study suggests IL-17 and GM-CSF playing a possible role in the protective functions of

DXM/MOG/MPs in EAE treatment, there are other possible mechanisms of tolerance that need to be explored.

Further mechanism could include expression of Transforming Growth Factor β

(TGF-β); a cytokine that has previously been shown to inhibit the trafficking of disease-

105 causing effector cells (244). Also, it is possible that a less well-studied regulatory T cell subset could be involved, such as CD8+FoxP3- cells, which suppress EAE through TGF-β mediated mechanisms (245). Other ways that T-effector cell function may be inhibited is by T cell anergy (vide supra) or T cell deletion. Glucocorticoids, such as DXM, have been shown to induce thymocyte death (246). Repetitive exposure to antigen has also been shown to induce T cell deletion (247). Due to the significantly lowered mean clinical score of the DXM/MOG/MPs group and the lowered mean clinical score of the

MOG/MPs group, it is possible that repetitive MOG or DXM exposure may result in some level of T cell deletion. Future work should be performed to try and elucidate through what mechanisms therapeutic treatments act in order to provide protection from autoimmune disorders such as MS.

5.5 Conclusions

Here it is reported that encapsulation of MOG peptide and DXM into Ac-DEX

MPs in order to therapeutically treat EAE, a model of MS. These data show encapsulated

DXM was more efficient at decreasing in vitro immune responses to LPS using both Mϕ and primary BMDCs. Furthermore, mice immunized for EAE were treated with Ac-DEX

MPs co-encapsulating DXM and MOG peptide. This therapy significantly reduced disease clinical score, and expression of IL-17 and GM-CSF, two inflammatory and disease-associated cytokines. Additionally, DXM/MOG/MPs were superior to free DXM

106 and MOG at ameliorating disease which indicated encapsulation of these compounds provides for more efficient delivery to the desired cellular populations, in vivo.

Treatment of EAE after symptom onset by sub-cutaneous injection of DXM and disease- associated peptide in a polymeric delivery vehicle provides promising new possibilities for the treatment of MS.

107

Chapter 6: Particulate-based Tolerogenic Compounds for the Protection Against

Hyperglycemia in NOD Mice

6.1 Introduction

Type 1 diabetes (T1D) is an autoimmune disease resulting in the destruction of insulin producing pancreatic β-cells. According to the National Institutes of Health, approximately 80 people are diagnosed in the United States daily, and the disease is reported to account for $14.9 billion in annual healthcare costs (66). Destruction of β- cells in T1D occurs through T cell-mediated destruction, which subsequently results in insulin deficiency (248). Although therapies like injectable insulin exist, there is no current cure for T1D. Since symptom onset occurs while functional β-cells still exist

(249) development of a long-lasting immunomodulatory therapy, and subsequent remission from disease, is possible.

Immunomodulation therapies can function in a disease-specific manner, allowing suppression of a disease without fully suppressing the immune system. One mechanism for immunomodulation is through antigen tolerance. Previously, for T1D, clinical

108 evaluation of insulin tolerance through oral (250) or intranasal (251) delivery was performed. In an effort to inhibit disease onset, patients with high risk of disease were administered insulin; however, both studies failed to have an effect on T1D onset.

Although clinical data indicates antigen-specific therapies will likely not work, it is possible these studies, and others, failed due to poor dosing concentrations and regimens

(252). Therefore, it is important to evaluate more optimal tolerogenic treatments and their delivery methods.

One approach for antigen-specific tolerance, suggested by Kang et al., is co- delivery of DXM, a corticosteroid, and a T1D antigen (83). In theory, addition of an immunosuppressive drug could amplify populations of cells with a tolerogenic phenotype. However, prolonged usage of corticosteroids, like DXM can result in bone loss, increased risks of infection and other adverse events (253), so it is important to optimize delivery methods so long-term side-effects of the therapy are reduced. One of approach to antigen-specific therapy for T1D would be to translate the work previously performed for MS (Chapter 4) into an Ace-DEX microparticulate treatment for T1D.

To better optimize the method developed with the MS model, three known FDA- approved tolerogenic drugs were evaluated with insulin as the auto-antigen in the non- obese diabetic (NOD) mouse model. In accordance with the MS study, the corticosteroid

DXM was evaluated. The second drug, all-trans retinoic acid (ATRA), a derivative of vitamin A, has shown to be effective at reducing hyperglycemia, therefore incidence of

T1D by an increase in regulatory T cells and inhibition of inflammatory cytokines (254).

Finally, sirolimus, or rapamycin (rapa), has shown to be effective at limiting

109 hyperglycemia, when administered prophylactically (255). Rapa has also been shown to help maintain insulin independence after islet cell transplants in T1D patients (256), suggesting its ability to alter disease progression while functional insulin producing cells are available. Here, the ability of Ace-DEX microparticulate-based therapy co-delivering insulin, with either DXM, ATRA, or rapa to delay the onset of hyperglycemia in the

NOD mouse model was evaluated.

6.2 Materials and Methods

6.2.1 Reagents

All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. Water (dd-H2O) for buffers was purified using a Millipore (Billerica,

MS) Milli-Q Integral water purification system, which was made basic by addition of triethylamine (TEA) (0.01% v/v). DXM (98%) was purchased from Alfa Aesar (Ward

Hill, MS) and Rapa from LC Laboratories (Woburn, MA). Fluorescence measurements were detected using a Molecular Devices (Sunnyvale, CA) FlexStation 3, courtesy of the

Department of Chemistry and Biochemistry at the Ohio State University and Molecular

Devices SoftMax Pro Software (Sunnyvale, CA).

6.2.2 Animals

Mice used for the prophylactic study were 8 week old NODs purchased from

Jackson Labs (Bar Harbor, ME). All animals were kept in a sterile facility in accordance 110 with The Ohio State University Institutional Guidelines. All animal procedures were in accordance with and approved by the Institutional Animal Care and Use Committee

(IACUC) of The Ohio State University.

6.2.3 Synthesis and Analysis of Acetal Coverage of Ace-DEX

Ace-DEX was synthesized using 71 kDa dextran as previously described (23).

Cyclic acetal coverage was determined to be 51.9% by nuclear magnetic resonance as previously described (114).

6.2.4 Preparation of Empty Ace-DEX Microparticles

To formulate MPs, Ace-DEX (100mg) was dissolved in dichloromethane (2 ml) and mixed with 3% PVA (MW~13-23 kg/mol, 87-89% hydrolyzed) in PBS. This solution was homogenized (IKA T25 Digital Ultra-Turrax, Cole Parmer, Vernon Hills,

IL) at 18,500 RPM. MPs were stirred for 2 hours in 0.3% PVA in PBS and were washed

(3 times) and collected by centrifugation at 10,000 rpm for 10 minutes on a Beckman

Coulter Avanti J-E centrifuge (Brea, CA). MPs were re-suspended in basic water for washes.

6.2.5 Preparation of Insulin Ace-DEX MPs

Ace-DEX (100 mg) was dissolved in dichloromethane (2 ml) and Insulin (2 mg) in PBS was added. The mixture was homogenized at 18,500 RPM and 3% PVA in PBS

111

(2 ml) was added, and the mixture was homogenized for a second time. MPs were stirred for 2 hours in 0.3% PVA in PBS and were washed (3 times) and collected by centrifugation at 10,000 rpm for 10 minutes. MPs were re-suspended in basic water for washes.

6.2.6 Preparation of Dexamethasone and Insulin Co-Encapsulated Ace-DEX MPs

Ace-DEX (100 mg) and DXM (2 or 4 mg) were dissolved in chloroform and ethanol (95:5 v/v respectively) and insulin (1 mg) in PBS was added. The mixture was homogenized at 18,500 RPM and 3% PVA in PBS (2 ml) was added, and the mixture was homogenized for a second time. MPs were stirred for 2 hours in 0.3% PVA in PBS and were washed (3 times) and collected by centrifugation at 10,000 rpm for 10 minutes.

MPs were re-suspended in basic water for washes.

6.2.7 Preparation of All-Trans Retinoic Acid or Rapamycin and Insulin Co-Encapsulated

Ace-DEX MPs

Ace-DEX (100 mg) and ATRA (2 mg) or rapa (1.5-2.5 mg) were dissolved in dichloromethane and insulin (1-2 mg) in PBS was added. The mixture was homogenized at 18,500 RPM and 3% PVA in PBS (2 ml) was added, and the mixture was homogenized for a second time. MPs were stirred for 2 hours in 0.3% PVA in PBS and were washed (3 times) and collected by centrifugation at 10,000 rpm for 10 minutes.

MPs were re-suspended in basic water for washes.

112

6.2.8 Scanning Electron Microscopy (SEM)

Microparticles size and morphology were characterized by SEM using an FEI

NOVA nanoSEM (Hillsboro, OR). SEM sample preparation and analysis was done as previously described (5).

6.2.9 Quantification of Encapsulated Dexamethasone

DXM quantification was determined by high-performance liquid chromatography

(HPLC) with a method adapted from Zhang et al (229). MPs (1 mg/ml) were dissolved in a methanol:ddH2O solution (80:20 v/v) (1 mg/ml) and DXM concentration was determined using an Agilent 1100 series HPLC (Santa Clara, California) with a Thermo

Scientific 150 mm x 4.6 mm, pore size 5 µm, Aquasil C18 column (Waltham, MS).

Samples were passed through the column at a flow rate of 1 ml min-1 in a mobile phase of methanol:ddH2O (80:20 v/v). DXM was detected at a wavelength of 240 nm. Sample peaks were compared to a standard curve and analyzed using Aglient Chemstation software.

6.2.10 Quantification of Encapsulated Rapamycin

Rapa quantification was determined by HPLC. MPs (1 mg/ ml) were dissolved in acetonitrile:ddH2O solution (55:45 v/v) (1 mg/ml) and rapa concentration was determined using an Agilent 1100 series HPLC (Santa Clara, California) with a Thermo Scientific

113

150 mm x 4.6 mm, pore size 5 µm, Aquasil C18 column (Waltham, MS). Samples were passed through the column at a flow rate of 1 ml min-1 in a mobile phase of acetonitrile:ddH2O (55:45 v/v)and detected at 278 nm. Sample peaks were analyzed using Aglient Chemstation software.

6.2.11 Quantification of Encapsulated All-Trans Retinoic Acid

ATRA quantification was determined by suspending MPs (1 mg / ml) in DMSO and measuring in a plate reader 350 nm.

6.2.12 Quantification of Encapsulated Insulin

Samples containing Insulin (1 mg) were suspended in 990 µl PBS and the pH was lowered with 5 µl 50% formic acid (v/v). Samples were incubated on a 37oC shaker plate overnight and the solution was returned to neutral pH using 13.25M sodium hydroxide.

Encapsulation efficiency was determined using a fluorescamine assay per the manufacturer’s instructions.

6.2.13 Prophylactic Protection of NOD Mice

Mice were treated, prior to disease onset, beginning in week 9 with MPs, or what is termed a late-stage prophylactic T1D model. Mice received 3 total injections, 3 days apart. Mice were bled by tail prick with a 21 gauge needle. Glucose was measured using an OptiumEZ meter and test strips (Abbott Laboratories, Abbott Park, IL). Incidence of

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Figure 6. 1: Scanning Electron Micrographs of Microparticles to Prevent Hyperglycemia onset in NOD Mice.

Scanning electron micrographs were taken of (A) empty microparticles (E/ MP), microparticles containing (B) insulin (Insulin/ MP) or Insulin co-encapsulated with (C)

All-Trans Retinoic Acid (ATRA/ Insulin/ MP) (D) Dexamethasone (DXM/ Insulin/ MP) or Rapamycin (Rapa/ Insulin/ MP).

115

Table 6.2: Encapsulation Efficiencies and Drug Loading for Microparticles Containing

Insulin with or without Tolerogenic Drugs.

116 the disease was defined as 2 consecutive weeks of a blood glucose level over 250 mg/dl.

Upon incidence of disease, mice were euthanized.

6.3 Results

6.3.1 Analysis of Particle Formulations Containing Insulin

Table 6.1 reports encapsulation efficiencies of Insulin/ MPs or MPs containing insulin with ATRA (ATRA/ Insulin/ MPs), DXM (DXM/ Insulin/ MPs), or rapa (Rapa/

Insulin/ MPs). Additionally, Table 6.1 reports the amount of insulin and drug per milligram of these particle sets. Scanning electron micrographs showing morphology of these particles are shown in Figure 6.1A-E.

6.3.2 Inhibition of Hyperglycemia by Particles Containing Insulin with either Rapamycin

or Dexamethasone

Figure 6.2 depicts weekly incidence of NOD mice, as determined by blood- glucose readings, taken after prophylactic treatment using either PBS, E/ MPs, Insulin/

MPs (15 µg insulin), ATRA/ Insulin/ MPs (121 µg ATRA), Rapa/ Insulin/ MPs (51 µg rapa) or DXM/ Insulin/ MPs (13.76 µg DXM). Readings of blood glucose indicated a disease incidence of 100% by week 28 for mice treated with ATRA/ Insulin/ MPs. This incidence was higher than any other treatment group in the study including final

117 incidence rates for the PBS (70%) and E /MP (90%) controls. Insulin /MPs had an incidence rate equivalent to PBS. Finally, Rapa/ Insulin/ MPs and DXM/ Insulin/ MPs had the lowest incidence rates of any group with 30% and 33% respectively. These incidence rates were significantly lower than those of ATRA/ Insulin/ MPs (p < 0.005), E

/MPs (p < 0.01), PBS (p < 0.5) and Insulin/ MPs (p < 0.05).

6.4 Discussion

Scanning electron micrographs of particle sets show a heterogeneous population of MPs (Figure 6.1 A-E). Encapsulation efficiencies show the encapsulation of each drug is highly variable, when compared to the other compounds used (Table 6.1). DXM had an encapsulation efficiency that was considerably lower than that of both rapa and

ATRA. Initial drug loading of DXM was established by quantities previously used in autoimmune mouse models (83) and this level was able to be achieved in this study.

Also, doses of rapa with bioavailability’s similar to those used previously in NOD mice were accomplished (255). In contrast, levels of ATRA were not able to achieve therapeutic loading levels, because of limitations with increased loading attempts resulting in diminishing encapsulation efficiencies. ATRA has been shown to be effective in high doses. For example, it have been effective against EAE and hyperglycemia in NOD mice at 300 µg (257) and 500 µg (254), respectively. However, due to limited encapsulation capabilities, each injection for this experiment contained only 121 µg of drug. Although ATRA showed a relatively high encapsulation efficiency

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(90% at 2 mg loading), attempts to load more compound resulted in drastic decreases in encapsulation efficiency and poor particle morphology (data not shown). Previous work has shown that attempts to increase loading efficiency in MPs will eventually result in diminished encapsulation efficiencies (232). These particle sets containing drug were evaluated for their ability to prophylactically protect against the onset of hyperglycemia in NOD mice.

The protective effect of these particles, shown in Figure 6.2, indicates, as in EAE,

DXM is capable of protecting against autoimmunity in mouse models. However, treatment with Rapa/ Insulin/ MPs was comparable in the ability to protect against disease onset. ATRA/ Insulin/ MPs were statistically the worst group at delaying disease onset, and was the only group, including controls, to reach 100% disease incidence.

However, with the inability to formulate particles containing higher doses of ATRA, it is hard to determine if this effect is due to poor particle efficacy or sub-optimal doses of the drug. Both DXM/ Insulin/ MPs and Rapa/ Insulin/ MPs significantly limited hyperglycemia onset when compared with PBS, E/ MPs, ATRA/ Insulin/ MPs and

Insulin /MPs, showing that, of these particle sets, co-encapsulated MPs are optimal for inhibiting disease onset. Although both DXM and rapa seem to have similar capability in protecting against autoimmunity in mouse models, rapa’s ease of encapsulation could make it a more ideal compound to formulate therapies in Ace-DEX. Therefore, further work utilizing particulate-based therapies for the treatment of autoimmunity should be performed using rapa. Future studies will determine an optimized dosing sequence and drug quantity for the treatment of diseases such as T1D and MS.

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Figure 6. 2: Diabetes Incidence in NOD mice.

Mice were treated with injections of either PBS alone, empty Ace-DEX microparticles

(/MP), insulin (15 µg) in Ace-DEX MPs (Insulin/ MP) or Ace-DEX MPS containing insulin (15µg) with the drugs dexamethasone (13.76 µg) (DXM/ Insulin/ MP), all-trans retinoic acid (121 µg) (ATRA/ Insulin/ MP), or rapamycin (51 µg)(Rapa/ Insulin/ MP).

Mice received injections 3 days prior to week 10, on the 10 week mark, and 3 days after the 10 week mark. Statistical significance with respect to Rapa/ Insulin/ MP is presented as * p < 0.05, **p < 0.005, and *** p ≤ 0.0005.

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Chapter 7: Summary, Conclusions and Future Work

Particulate delivery systems are frequently used because of their ability to efficiently deliver cargo to targeted cells, increase drug solubility, reduce drug toxicity, provide depot for sustained release, and increase drug circulation time. By efficiently delivering and allowing sustained release of compounds, smaller quantities of potentially toxic drugs can be utilized. Finding ways to decrease harmful side-effects can increase patient quality of life, decrease medical expenses, and likely increase patient compliance.

Through the use of Ac-DEX, Ace-DEX, and lipid-based delivery systems, increased bioactivity of compounds and the potential for dose-sparing delivery of drugs was shown.

Most importantly, it was shown that these particulate systems can potentially be used as vehicles to both activate or tolerize the immune system. This capability leads to a number a potential uses to combat either infectious diseases or autoimmunity.

7.1 Delivery of TLR-agonists for the Treatment of Infectious Disease

The immunostimulatory nature of adjuvants provides for unique advances in both the field of vaccines and for treatment of infection at the host pathogen interface.

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Currently, novel treatments combating infectious disease are essential because of the increasing failures of long-used first line therapies. For instance, antimony, the mainstay chemotherapeutic for the disease Leishmaniasis, has an epidemic of resistant infections in portions of India (WHO). Generally, therapies conducive to inducing resistance directly target the pathogen. Therefore, by amplifying a host immune response, it is possible to clear an infection without targeting the pathogen. Both CpG and poly I:C are TLR- agonists commonly used to activate an immune response however, they may have significant side-effects if delivered systemically in there unencapsulated form. The side- effects of systemic (e.g. non-encapsulated) administration of poly I:C and CpG are potential systemic toxicity, histological damage, auto-reactive T-cells, and general symptoms of autoimmunity (100-102). Therefore, it is necessary to find a delivery method such as particulate systems to optimize delivery.

In order to have high encapsulation efficiencies of CpG and poly I:C, most formulations require the blending of polymer and potentially harmful polycationic materials. However, encapsulation of these compounds was shown to be relatively high in Ac-DEX, without the addition of polycations. Interestingly, poly I:C in Ac-DEX had minimal differences in immunostimulatory function when compared with PLGA however, the increased biocompatibility of the formulation supported further exploration with Ac-DEX. Conversely, the high incorporation of CpG into Ac-DEX MPs compared with PLGA showed for some adjuvants, Ac-DEX is a more optimal platform. When compared with free drug, CpG encapsulated in Ac-DEX showed significantly higher cytokine and nitric oxide production. These data are supported by Bachelder et al., who

122 previously showed nearly 100% encapsulation of immiquimod in Ac-DEX, significantly increased pro-inflammatory cytokines over free drug, and also showed encapsulation without a polycation (109). Currently, topical immiquimod is approved for the use against CL, which is why Ac-DEX particles containing its more potent derivative resiquimod, were formulated by Duong et al. and subsequently used to treat a VL mouse model (111). Although resiquimod–loaded electrosprayed microparticles did significantly decrease L. donovani parasite load, the decrease was not overwhelming. One potential reason for limited suppression of the parasite was the particle size (≥1 µm). It has been reported that particles smaller than 20 nm, or 1 µM and higher, have increased clearance rates from circulation (258), as well as particles larger than 400 nm have selective uptake in the bone marrow (156). Therefore, the assumption was made that a particulate size distribution between 20 and 400 nm could provide for a more optimal delivery of resiquimod in a VL mouse model.

To examine if smaller sized particles could increase efficacy, a homogenous population of liposomes, approximately 75 nm in size, encapsulating resiquimod was formulated. Additionally, liposomes of the same size were formulated containing the novel molecule pentalinonsterol. Both of these liposome formulations were compared, in their ability to treat an animal model of VL, with intravenous therapy of PBS or a dose of

SSG. Both liposomal resiquimod and liposomal pentalinonsterol significantly decreased parasite burden in the liver, spleen, and bone marrow compared with PBS. They also increased overall T cell proliferation over PBS, which is important because Leishmania infection causes T cell anergy leading to worse infections. Both induced production of

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IFN-γ, showed increased IFN-γ to IL-10 ratios, and pentalinonsterol showed an increased

IFN-γ to IL-4 ratio, suggesting a shift from a TH2 to a TH1 immune response, which is vital for clearing intracellular infections. Finally, results of in vitro toxicity and in vivo liver enzyme analysis suggested the liposomal resiquimod and liposomal pentalinonsterol therapies were capable of inducing anti-parasitic effects without damaging host cells.

Although delivery of resiquimod in Ac-DEX microparticles showed a valuable proof of concept, it seems incorporation into a more ideal carrier enhanced protection. This delivery vehicle allowed a compound associated with CL to, for the first time, be repackaged and administered for the treatment of VL. Although this data is promising, future work should evaluate the benefits of liposomal resiquimod, liposomal pentalinonsterol and the potential uses for alternative adjuvants.

Further evaluation of should be performed to assess its ability to decrease parasite burden in humans. For example, the treatment should be evaluated in a Syrian Gold

Hamster model, as the host immunological response and parasite activity more closely represents that of humans and canines, than does a mouse model (259). Additionally a new model has been recently developed utilizing sandflies and vectors for induction of a more clinically accurate model of disease pathogenicity (260). It is important to evaluate these treatments under conditions with sandflies, specifically salivary antigens, which have been well documented to have an effect on disease severity (261). While the in vivo data was promising, one thing that limited the study was a low encapsulation of resiquimod (~ 7%). Due to intravenous administration of the treatment, an isotonic solution was needed, thus limiting the amount of liposomes capable of being injected. An

124 increase in encapsulation efficiency may allow higher doses of resiquimod to be evaluated. One possible way to accomplish this is through a process called remote loading. Remote loading uses a transmembrane ion gradient, generally induced by a change in pH, to diffuse a compound and subsequently protonate it inside the lipids.

Protonation inside the liposome will trap the compound, therefore increasing encapsulation efficiency. The amine group located on resiquimod allows this drug to be loaded via remote loading. Aside from encapsulation efficiency issues, liposomal therapies can carry large costs, so scalable formulations are necessary. One process capable of producing large quantities of homogenous particles in short periods of time is the electrospray process used to produce resiquimod Ac-DEX MPs for VL. Previously,

Wu et al. showed lipoplex particle formulation via electrospray techniques (262), so future work should be done to determine if electrospray can be used to formulate liposomes containing resiquimod, with higher encapsulation efficiency, while maintaining the optimal size distribution for treatment of VL. Finally, one other avenue of future work is the evaluation of low dose liposomal resiquimod to synergize in the treatment of drug resistant strains causing VL. Arevalo et al. were able to treat antimony- resistant CL with low dose imiquimod and antimony (47). The synergy of these two compounds increased the efficacy of a resistant strain of VL. Here, systemic delivery of resiquimod is shown, and it provides a novel platform to deliver synergistic therapies to patients, like the more than 70% in Bihar State with an antimony resistant infection.

Further work should be performed using animal models of VL infected with antimony-

125 resistant strains and treated with various doses of both liposomal resiquimod and antimony.

Particle size of Ac-DEX microparticles seems to be sub-optimal for intravenous delivery of a systemic parasite infection however, their ability to encapsulate immunomodulatory compounds without polycations provides an ideal platform for additional uses. One other potential use for Ac-DEX is in the development of subunit vaccines. Unlike the commonly used attenuated and heat-inactivated vaccines, which are cost-efficient but have the potential to pose significant safety risks for diseases such as

HIV+/AIDS (263, 264), tularemia (265), or avian influenza (266), subunit vaccines can provide a safe an widely usable platform for delivery. Subunit vaccines provide a safer alternative because they use only a portion of an antigenic protein, not a whole pathogen.

However, a subunit antigen alone is poor at generating a protective immune response. To enhance efficacy, subunit vaccines for tuberculosis (267), influenza (268), and

HIV+/AIDS (269) have been coupled with adjuvants, which increase the activation of the immune response. In fact, Ac-DEX has been previously used for the co- encapsulation of an adjuvant and a protein (115, 270). Additional studies should be performed co-encapsulating proteins or peptides with CpG or poly I:C Ac-DEX MPs.

Expanding this area of work could result in a broad platform capable of being utilized for prophylactic prevention of a variety of pathogens. These MPs should be evaluated for the ability to produce things like pro-inflammatory cytokines (e.g. GM-CSF, IFN-γ, IL-

12p70, TNF-α) and an antibody response. Additionally, particle sets should be optimized for dosing patterns and drug/protein concentrations, with a subsequent challenge with

126 lethal doses of relevant pathogens. As with resiquimod liposomes, experiments should also be performed to find methods of scaling production. Ac-DEX MPs show promise for the use of vaccines towards infectious disease, but much work should also be done to explore how Ac-DEX and Ace-DEX MPs can be used for protection against autoimmunity.

7.2 Delivery of Immune Tolerizing Particles for the Treatment of Autoimmunity

Autoimmune diseases such as MS and T1D are due to the immune system recognizing self-antigens as foreign. To combat these diseases, a number of therapies suppressing the immune response have been developed; however, these therapies generally suppress a wide-range of immune functions leaving patients open to increased risks of further problems such as cancers and increased rates of infection. Development of novel therapies which suppress immune responses towards disease-specific antigens, while leaving immune responses towards all other antigens functional, could drastically increase patient quality of life. Here, development of tolerogenic MPs was established through co-encapsulation of a tolerogenic compound and a disease-associated antigen.

Initial studies were performed using DXM/ MOG/ MPs to treat EAE, an animal model of MS. Although these particles were successful in therapeutically ameliorating symptoms, there was no solid indication to a potential mechanism of action. Prophylactic treatment with these particles did induce regulatory T cell responses (data not shown), but analysis after therapeutic treatment showed protection after symptom onset was likely not

127 due to FOXp3+ regulatory cell presence. In addition, cytokine profiles of these mice were altered; mainly decreased IL-17 and GM-CSF, but it is unclear whether this is a contributing factor to the limiting of disease-associated symptoms. Although no mechanism was confirmed, in vivo suppression of EAE was significant. Further examination of formulation methods could yield particles that enhance disease suppression. Additionally, enhanced functionality could help to amplify and isolate a protective mechanism.

In an effort to begin evaluate enhanced particle formulations, alternative compounds were evaluated on the NOD mouse model. The NOD model has symptoms of spontaneous onset of T1D, like hyperglycemia. Here, Rapa/ Insulin/ MPs showed similar efficacy compared with DXM/ Insulin/ MPs however, increased encapsulation efficiencies show rapa has a more malleable formulation profile. Due to increased ability to alter formulations, thus altering dosing concentrations, it is likely rapa is more promising than DXM for tolerogenic particles.

Future work using rapa to determine a mechanism of action for tolerogenic particles must begin examining administration routes and schedules. Ideally, a clinically relevant administration route will be identified through particle trafficking studies. Here, trafficking of fluorescent particles to lymph nodes and other organs can be evaluated after delivery through sub-cutaneous, intraperitoneal, intradermal, intravenous or oral routes.

Additional studies should also be performed testing high and low doses, as well as proper dosing regimens. By testing a wide range of dose concentrations and schedules on disease models, it is possible to hypothesize dosing ranges and protocols that may have clinical

128 relevance. Subsequent to proper dosing and injection schedule studies, it is important to re-examine the potential of these particles in disease models. Further EAE and NOD studies should be performed using particle sets with enahnced dosing and injections.

Therapeutic studies on these animal models will also include evaluation of regulatory cells, cytokines, and cell phenotypes. It will be important to evaluate many pathways and cell subsets to determine a mechanism of how formulations used in this work are ameliorating disease.

Particulate-based delivery systems are valuable tools for the creation of novel therapies. They increase solubility and decrease toxicity of compounds, allowing delivery of drugs previously unable to be used for systemic therapies. The wide range of polymers, lipids and formulation methods provides extensive possibilities to treat both infectious and autoimmune diseases.

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